A Comprehensive LC-MS/MS Protocol for Multi-Allergen Detection in Processed Foods: From Foundational Principles to Advanced Applications

Abstract

This article provides a detailed examination of Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) protocols for the simultaneous detection of multiple food allergens in complex processed matrices. Tailored for researchers, scientists, and drug development professionals, the content spans from the foundational need for such methods due to rising global allergy prevalence to the intricate methodological steps of sample preparation, protein extraction, and peptide analysis. It further delves into troubleshooting challenges posed by food processing, explores validation strategies against established techniques like ELISA and PCR, and discusses the critical role of high-resolution accurate mass (HRAM) systems. By synthesizing recent advancements, this guide serves as a critical resource for developing robust, sensitive, and reliable multi-allergen detection methods to enhance food safety and protect consumer health.

The Critical Need for Advanced Multi-Allergen Detection in Modern Food Safety

The Global Rise of Food Allergies and Public Health Imperatives

The increasing global prevalence of food allergies represents a critical public health challenge, with approximately 150 million people affected worldwide and an estimated 1 in 10 adults and 1 in 13 children living with this condition [1] [2] [3]. In the United States alone, food allergies affect millions of Americans and their families, with reactions ranging from mild symptoms to life-threatening anaphylaxis [4]. The economic and social burdens are substantial, encompassing direct medical costs, higher food expenses, impaired quality of life, and food allergy anxiety [3]. For susceptible individuals, strict avoidance of allergenic foods remains the primary management strategy, making accurate food labeling essential for prevention [5] [4].

Regulatory frameworks have evolved to protect consumers, with the Food Allergen Labeling and Consumer Protection Act (FALCPA) in the U.S. mandating declaration of nine major allergens: milk, eggs, fish, Crustacean shellfish, tree nuts, peanuts, wheat, soybeans, and sesame [4]. Similarly, the European Union's Regulation (EU) No 1169/2011 requires labeling of 14 allergenic substances [6] [5]. However, the absence of defined threshold levels for most allergens (except gluten) and inconsistencies in precautionary allergen labeling (PAL) create significant challenges for both consumers and food manufacturers [6] [4].

Robust analytical detection methods are therefore essential for regulatory compliance and consumer protection. This application note details advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocols for reliable multi-allergen detection in processed foods, addressing a critical need in food safety research and public health protection.

Current Analytical Challenges in Food Allergen Detection

Limitations of Traditional Detection Methods

Traditional immunoassays (ELISA) and DNA-based methods (PCR) have been widely used for food allergen detection but present significant limitations for modern food control laboratories:

• Antibody Cross-Reactivity: ELISA methods are susceptible to cross-reactivity with food matrix components, potentially resulting in false positives [6]. This is particularly problematic for distinguishing between botanically related species with similar protein profiles, such as cashew and pistachio [6].
• Single-Target Detection: ELISA is generally limited to single-analyte detection, making robust multiplexing challenging [6].
• Indirect Detection: PCR targets allergen-specific DNA sequences rather than the allergenic proteins themselves, which may lead to inaccurate results in processed foods where proteins and DNA may degrade differentially during manufacturing [6].
• Matrix Effects: Both techniques are highly susceptible to food matrix interferences, particularly in complex, processed food products [5].

Impact of Food Processing

Food processing represents a critical challenge for allergen detection due to various chemical and structural modifications that can occur at the protein level [5]. Thermal treatments (e.g., baking at 180°C for cookies or more intensive processing for rusks), fermentation, and ingredient interactions can alter protein structures through Maillard reactions, protein aggregation, or chemical modification, potentially affecting antibody binding sites and detectability [5]. These processing-induced changes can mask or modify epitopes, reducing method recovery and accuracy, particularly for immunoassays [5].

LC-MS/MS as a Confirmatory Multi-Allergen Detection Platform

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful confirmatory technique for food allergen detection, offering high specificity, sensitivity, and multiplexing capability [6] [5] [2]. Unlike ELISA, MS is unaffected by antibody cross-reactivity, and unlike PCR, it allows for direct detection of allergenic peptides or proteins, ensuring more accurate identification [6].

Table 1: Key Advantages of LC-MS/MS for Food Allergen Analysis

Feature	Advantage	Application Benefit
Multiplexing Capacity	Simultaneous detection of multiple allergens in a single run	Cost-effective comprehensive screening
Specificity	Direct analysis of signature peptides with unambiguous identification	Reduced false positives from cross-reactive proteins
Structural Insight	Direct detection of proteins/peptides, including modified forms	More accurate quantification in processed foods
Platform Flexibility	Compatible with both low-resolution (QqQ) and high-resolution (HRMS) systems	Adaptable to different laboratory capabilities and needs

Method Principle and Workflow

LC-MS/MS methods for allergen detection typically follow a bottom-up proteomics approach [5]. Proteins are extracted from the food matrix, digested enzymatically (typically with trypsin), and the resulting peptide markers are separated by liquid chromatography and detected by tandem mass spectrometry. The selection of proteotypic peptides—peptides unique to the allergenic protein that are consistently detected by MS—is crucial for method specificity and robustness [5] [2].

The following diagram illustrates the core workflow for LC-MS/MS-based multi-allergen detection in processed foods:

Detailed Experimental Protocols

Protocol 1: Simultaneous Detection of Seven Food Allergens in Processed Foods

This protocol, adapted from Kato et al. (2024), enables simultaneous detection of wheat, buckwheat, milk, egg, crustacean, peanut, and walnut in various processed food matrices [7].

Reagents and Materials

Table 2: Essential Research Reagents and Materials

Category	Specific Items	Function/Application
Solvents	Acetonitrile (HPLC grade), Methanol (HPLC grade), Milli-Q Water, Formic Acid (≥98%)	Mobile phase preparation, sample reconstitution
Digestion Reagents	Ammonium Bicarbonate, Dithiothreitol, Iodoacetamide, Trypsin (Mass Spectrometry Grade)	Protein reduction, alkylation, and enzymatic digestion
Purification	S-Trap Columns, On-line SPE System, Sep-Pak C18 Cartridges	Peptide cleanup and concentration
Buffers	Tris-HCl, Phosphate Buffered Saline	Protein extraction and stabilization

Sample Preparation Steps

• Protein Extraction: Homogenize 1 g of food sample with 10 mL of extraction buffer (e.g., PBS with 0.05% Tween-20 or a reduced-denaturing buffer containing 0.5% SDS/2% β-mercaptoethanol) [7] [8].
• Protein Concentration Measurement: Determine protein concentration using a suitable assay (e.g., BCA assay).
• Protein Digestion:
  • Reduce proteins with 10 mM dithiothreitol at 37°C for 45 minutes.
  • Alkylate with 25 mM iodoacetamide at room temperature in the dark for 30 minutes.
  • Digest with trypsin (enzyme-to-protein ratio 1:50) at 37°C for 16 hours [5].
• Peptide Purification: Use S-Trap columns or on-line solid-phase extraction (SPE) for rapid cleanup and concentration of peptides [7].
• LC-MS/MS Analysis:
  • Column: Reverse-phase C18 column (e.g., 2.1 × 100 mm, 2.7 μm)
  • Mobile Phase: A) 0.1% formic acid in water; B) 0.1% formic acid in acetonitrile
  • Gradient: 5-35% B over 15 minutes
  • Flow Rate: 0.3 mL/min
  • Injection Volume: 5-10 μL
  • MS Detection: Multiple Reaction Monitoring (MRM) mode on triple quadrupole mass spectrometer [7]

Method Performance

The method demonstrates a limit of detection (LOD) <1 mg/kg for each target protein in various processed foods, making it suitable for confirming allergen labeling across a wide range of food products [7].

Protocol 2: Discriminatory Detection of Pistachio and Cashew Allergens

This protocol addresses the specific challenge of distinguishing between cross-reactive pistachio and cashew allergens, which traditional ELISA and PCR methods often fail to differentiate [6].

Sample Preparation and Analysis

• Sample Collection: Collect representative samples (cereal-based products, chocolate, sauces, meat products, beverages, milk-based products) from retail sources [6].
• Protein Extraction and Digestion: Follow similar extraction and digestion procedures as in Protocol 4.1, with careful attention to maintaining consistent parameter values throughout sample preparation [6].
• LC-MS/MS Analysis:
  • Use LC-QqQ (triple quadrupole) platform for targeted analysis.
  • Monitor specific marker peptides for pistachio (Pis v 1, Pis v 2, Pis v 3, Pis v 4, Pis v 5) and cashew (Ana o 1, Ana o 2, Ana o 3) allergenic proteins [6].
  • Employ isotopically labeled internal standards or label-free approaches for quantification [6].

Validation Parameters

The method should be validated for:

• Specificity: Ability to distinguish between pistachio and cashew allergens.
• Screening Detection Limit (SDL): Established at 1 mg/kg.
• Precision and Ruggedness: All parameters must be carefully monitored without modification under strictly controlled conditions [6].

Protocol 3: Multi-Allergen Detection in Baked Goods

This protocol, validated within the ThRAll project, focuses on detecting traces of egg, milk, soy, almond, hazelnut, peanuts, and sesame in challenging baked matrices (cookies and rusks) that undergo different technological and thermal treatments [5].

Pilot-Scale Sample Production

• Cookie Production: Produce cookies incurred with allergens at two concentration levels (24 and 48 μg of total allergenic food protein per g of food) alongside allergen-free controls. Bake at 180°C for 11 minutes [5].
• Rusk Production: Produce rusks as a highly processed matrix involving more intensive technological phases, including fermentation and baking [5].

Analysis Parameters

• Protein Extraction: Use buffered-detergent and/or reduced-denatured extraction protocols to address matrix effects from baking [5] [8].
• LC-MS/MS Analysis:
  • Perform analyses on both low-resolution (triple quadrupole) and high-resolution MS platforms to validate marker peptide robustness [5].
  • Calculate method sensitivity in terms of LOD and LOQ (Limit of Quantification).
  • Evaluate the effect of processing conditions on allergen detection and overall recovery for each allergenic ingredient [5].

Data Analysis and Interpretation

Quantitative Analysis

For quantitative analysis, employ isotopically labeled internal standards added prior to extraction and/or digestion. Alternatively, use label-free approaches, external calibration curves, or standard addition methods [6]. The following table summarizes performance characteristics of referenced LC-MS/MS methods:

Table 3: Performance Characteristics of LC-MS/MS Allergen Detection Methods

Method Focus	Matrices Validated	Detection Limits	Key Advantages
7-Allergen Screening [7]	Various processed foods	<1 mg/kg for all targets	Rapid analysis with S-Trap and on-line SPE
Pistachio/Cashew Discrimination [6]	Cereals, chocolate, sauces, meat products	SDL = 1 mg/kg	Solves cross-reactivity issues of ELISA/PCR
Baked Goods Analysis [5]	Cookies, rusks	LOD/LOQ established for 24-48 μgTAFP/gF*	Validated in extensively processed foods
Wine Analysis [2]	White wine	<100 μg/L (ppb)	Detects fining agents (casein, egg)

*μgTAFP/gF: micrograms of Total Allergenic Food Protein per gram of Food

Quality Control Measures

Implement rigorous quality control procedures including:

• Internal Standards: Use stable isotope-labeled peptide analogs for quantification.
• Process Controls: Monitor extraction efficiency, digestion efficiency, and matrix effects.
• Method Specificity: Verify through blank samples and analysis of potentially cross-reactive foods.

The relationship between method selection, analytical challenges, and technical solutions can be visualized as follows:

LC-MS/MS has proven to be a powerful and reliable platform for multi-allergen detection in complex processed foods, effectively addressing limitations of traditional ELISA and PCR methods. The protocols detailed herein provide researchers with robust methodologies for detecting and quantifying food allergens at clinically relevant concentrations, even in challenging matrices.

Future developments in food allergen analysis will likely focus on:

• High-Throughput Automation: Streamlining sample preparation to increase laboratory efficiency.
• Reference Materials: Developing standardized incurred reference materials for method validation [9].
• Expanded Multiplexing: Incorporating emerging allergens into existing panels as new prevalence data emerges.
• Data Standardization: Establishing harmonized guidelines for LC-MS/MS allergen detection to facilitate method transfer across laboratories.

As regulatory frameworks evolve and clinical understanding of threshold doses improves, advanced MS-based methods will play an increasingly vital role in protecting consumers with food allergies through accurate food labeling and effective regulatory oversight.

In the field of food safety, accurate detection of food allergens is critical for protecting consumer health. For decades, the Enzyme-Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR) have been the primary techniques for allergen detection and analysis. However, these traditional methods possess inherent limitations that can compromise their reliability, particularly in processed food matrices. ELISA's vulnerability to cross-reactivity and PCR's indirect approach to detecting proteins via DNA present significant challenges for analytical scientists. Understanding these limitations is essential for developing more robust detection methodologies, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), which offers a more direct and specific multi-allergen detection capability. This application note details the specific constraints of traditional methods and provides protocols for their evaluation, framing this discussion within the context of advancing LC-MS/MS-based multi-allergen detection in processed foods.

ELISA and the Cross-Reactivity Limitation

Fundamental Principles and Technical Basis

The Enzyme-Linked Immunosorbent Assay (ELISA) operates on the principle of antibody-antigen interaction, typically performed in 96- or 384-well polystyrene plates that passively bind antibodies and proteins [10]. In this technique, the antigen (target macromolecule) is immobilized on a solid surface and complexed with an antibody linked to a reporter enzyme, with detection accomplished by measuring enzyme activity after substrate incubation [10]. The most common format is the sandwich ELISA, where the target antigen is bound between two primary antibodies - a capture antibody and a detection antibody - providing high sensitivity and specificity [11] [10]. This method relies on highly specific antibody-antigen interactions for accurate detection and quantification [10].

The Cross-Reactivity Challenge in Food Allergen Detection

Cross-reactivity represents a significant limitation of ELISA methods, occurring when antibodies mistakenly bind to non-target molecules that share structural similarities with the target antigen [12]. This phenomenon is particularly problematic in food allergen analysis, where closely related species often share similar protein epitopes. A documented example of this limitation occurs between cashew and pistachio allergens, which belong to the same Anacardiaceae family and contain structurally similar proteins [6]. This similarity causes ELISA methods to struggle in discriminating between these two allergenic nuts, potentially leading to false-positive results and inaccurate food labeling [6].

The core of this limitation lies in ELISA's dependence on antibody specificity. The technique is susceptible to batch-to-batch variability in antibody production and may exhibit blind spots for specific protein isoforms or modifications [13]. Furthermore, the presence of interfering substances in complex food matrices can further affect test specificity, compromising the accuracy of results [12] [6]. These limitations are especially concerning in regulatory contexts where precise allergen identification is mandatory for consumer protection.

Table 1: Primary Limitations of ELISA in Food Allergen Analysis

Limitation Category	Specific Challenge	Impact on Allergen Detection
Specificity Issues	Antibody cross-reactivity between similar allergens	Inability to distinguish between cashew and pistachio allergens; false positives
Matrix Effects	Interference from food components	Altered antibody binding, reduced accuracy in complex matrices
Antibody Dependency	Batch-to-batch variability in antibody production	Inconsistent results between different test kits and laboratories
Target Recognition	Inability to detect modified protein epitopes	Limited effectiveness for processed foods where proteins may be denatured
Multiplexing Capacity	Primarily single-analyte detection	Multiple tests required for comprehensive allergen screening, increasing time and cost

Experimental Protocol: Evaluating ELISA Cross-Reactivity

Purpose: To experimentally determine cross-reactivity of ELISA kits for detection of cashew and pistachio allergens.

Materials:

• Commercial ELISA kits for tree nut detection
• Raw cashew and pistachio standards
• Food matrix samples (chocolate, cereal, sauce) free of tree nuts
• Microplate reader capable of measuring 450 nm absorbance
• Buffers and reagents as specified by ELISA kit manufacturer

Methodology:

• Sample Preparation: Prepare individual calibrations curves for cashew and pistachio antigens at concentrations of 0, 5, 10, 25, 50, and 100 ppm in blank food matrices.
• Cross-Reactivity Testing: Analyze each concentration point of cashew standard using the pistachio ELISA kit, and vice versa.
• Matrix Fortification: Fortify negative control matrices with known concentrations (10, 50, 100 mg/kg) of both allergens individually and in combination.
• ELISA Procedure: Follow manufacturer protocol for the specific ELISA kit, typically involving:
  • Addition of samples and standards to antibody-coated wells
  • Incubation (typically 60-90 minutes at room temperature)
  • Washing steps to remove unbound material
  • Addition of enzyme-conjugated detection antibody
  • Second incubation and wash steps
  • Addition of enzyme substrate solution
  • Stop solution after color development
  • Measurement of absorbance at 450 nm
• Data Analysis: Calculate cross-reactivity percentage using the formula: % Cross-reactivity = (Measured concentration of non-target analyte / Actual concentration) × 100

Quality Control: Include kit controls and spikes in duplicate. Acceptable intra-assay precision should be <15% CV.

PCR and the Indirect Detection Limitation

Fundamental Principles of PCR-Based Detection

Polymerase Chain Reaction (PCR) is an enzyme-driven process for amplifying short, specific regions of DNA in vitro [14]. The method relies on knowing partial sequences of the target DNA beforehand to design oligonucleotide primers that hybridize specifically to these sequences [14]. In PCR, target DNA is copied by a thermostable DNA polymerase enzyme in the presence of nucleotides and primers through multiple thermal cycles of denaturation, primer annealing, and extension, resulting in exponential amplification of the target sequence [14] [15]. Quantitative real-time PCR (qPCR) represents a significant advancement, coupling amplification and detection in a single reaction vessel, eliminating the need for post-amplification processing and allowing measurement of product simultaneous with DNA synthesis [14].

The Indirect Detection Problem in Food Allergen Analysis

The fundamental limitation of PCR in allergen detection is its indirect approach - while the clinical concern is with allergenic proteins, PCR targets DNA sequences instead of the proteins themselves [6]. This disconnect can lead to significant inaccuracies, as the presence of DNA does not necessarily correlate with the presence of the problematic protein [6]. This limitation becomes particularly pronounced in processed foods, where DNA and proteins may degrade at different rates and become physically separated during manufacturing processes [6].

DNA fragmentation during food processing represents another critical challenge. Physical force, heat, alkaline pH, and enzymes during manufacturing can cause DNA fragmentation, which adversely affects PCR results [16]. Research has demonstrated that DNA fragmentation significantly impacts the reliability of PCR analyses, with quantitative assays for genetically modified organisms generating different results for raw materials versus processed foods [16]. The degree of fragmentation can be quantified using a "DNA Fragmentation Index" (DFI), which measures the ratio of amplification efficiency for different target lengths [16].

Table 2: Primary Limitations of PCR in Food Allergen Analysis

Limitation Category	Specific Challenge	Impact on Allergen Detection
Target Disconnect	Indirect detection (DNA vs. protein)	Poor correlation between DNA presence and allergenic protein content
Processing Effects	Differential DNA fragmentation during processing	Reduced detection sensitivity in baked, cooked, or fermented products
Inhibition	PCR inhibition by food components (polysaccharides, phenols)	False negative results requiring additional sample purification
Quantification Challenge	Variable DNA copy number per cell; gene expression variability	Limited accuracy in quantifying actual allergen levels
Specificity Issues	Inability to detect specific protein isoforms or modifications	May miss relevant allergenic variants while detecting non-relevant DNA

Experimental Protocol: Assessing DNA Fragmentation in Processed Foods

Purpose: To quantify the degree of DNA fragmentation in processed food matrices and its impact on PCR detection limits.

Materials:

• Food samples (raw and processed)
• DNA extraction kit suitable for food matrices
• Real-time PCR instrument
• Primers targeting 18S ribosomal RNA gene sequences
• PCR reagents including SYBR Green master mix

Methodology:

• DNA Extraction: Extract DNA from both raw and processed samples using standardized protocols. Include a mechanical disruption step for homogeneous samples.
• DNA Quality Assessment: Measure DNA concentration and purity using spectrophotometry (A260/A280 ratio).
• Multiplex Amplicon Design: Design four real-time PCR assays amplifying the same target gene (e.g., 18S rRNA) at different lengths: 100 bp, 200 bp, 400 bp, and 800 bp.
• Real-time PCR Amplification:
  • Prepare reaction mixtures containing 1× SYBR Green master mix, primers, and template DNA
  • Run amplification with cycling conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min
  • Include melt curve analysis to verify amplification specificity
• Data Analysis: Calculate the DNA Fragmentation Index (DFI) using the formula: DFI = (Cq_long - Cq_short) / (log₂(amplicon length_long / amplicon length_short)) Where Cq_long and Cq_short are quantification cycles for long and short amplicons, respectively.

Interpretation: Higher DFI values indicate greater DNA fragmentation. Establish correlation between DFI and detection limit for target allergens.

LC-MS/MS as a Complementary Approach

Technical Advantages for Allergen Detection

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) has emerged as a powerful alternative for food allergen detection, offering direct measurement of allergenic proteins via signature peptides [17]. Unlike ELISA, LC-MS/MS is unaffected by antibody cross-reactivity, and unlike PCR, it directly detects allergenic peptides, ensuring more accurate identification [6] [13]. The technique enables a multi-target approach, simultaneously detecting different analytes in a single chromatographic run, with quantification possible through isotopically labeled internal standards or label-free approaches [6].

For allergen detection specifically, LC-MS/MS identifies unique signature peptides derived from tryptic digestion of allergenic proteins [17]. This approach allows for simultaneous detection of multiple allergens - research has demonstrated the ability to detect 12 food allergens (egg, milk, peanut, soy, and tree nuts) in a single injection, with detection limits of 10 ppm in various food matrices [17]. The method provides high specificity by monitoring multiple MRM transitions corresponding to unique signature peptides for each allergen [17].

Experimental Protocol: LC-MS/MS Multi-Allergen Detection

Purpose: To simultaneously detect and quantify multiple food allergens in processed food matrices using LC-MS/MS.

Materials:

• LC-MS/MS system with triple quadrupole mass spectrometer
• C18 reversed-phase chromatography column (100 × 3 mm, 2.6 μm)
• Trypsin for protein digestion
• Isotopically labeled peptide standards
• Extraction buffer (ammonium bicarbonate with calcium chloride)
• Reducing and alkylating reagents (DTT and iodoacetamide)

Methodology:

• Sample Preparation:
  • Homogenize food samples (1 g) and defat with hexane extraction
  • Add extraction buffer (4 mL) and centrifuge to collect supernatant
  • Reduce proteins with DTT at 60°C for 1 hour
  • Alkylate with iodoacetamide at room temperature in the dark
  • Digest with trypsin (20 μg) for 3-12 hours at 37°C
  • Stop reaction with formic acid and centrifuge-filter using 10 kDa MWCO filters

• LC-MS/MS Analysis:

  • Chromatography: Use gradient elution over 12 minutes at 300 μL/min with 0.1% formic acid in water and acetonitrile
  • MS Detection: Operate in positive ESI mode with scheduled MRM monitoring
  • Ion Source Parameters: Temperature 500°C, ion spray voltage 5500 V
  • MRM Transitions: Monitor 2-3 unique peptides per allergen with 2-3 fragments per peptide

• Data Analysis:

  • Identify allergens based on retention time and MRM transitions
  • Quantify using internal standard method with isotope-labeled peptides
  • Confirm identity via ion ratio comparison to reference standards

Validation: Establish calibration curves for each allergen (0-500 ppm), determine LOD/LOQ, and assess precision and accuracy.

Essential Research Reagent Solutions

Table 3: Key Research Reagents for Allergen Detection Methods

Reagent/Category	Function in Analysis	Specific Examples & Applications
ELISA Kits	Immunoassay-based detection and quantification of specific allergens	Commercial kits for peanuts, tree nuts, gluten; used for rapid screening
PCR Primers/Probes	Target-specific DNA amplification for indirect allergen detection	Primers for nut speciation (Ana o 1-3 for cashew; Pis v 1-5 for pistachio)
Mass Spec Standards	Quantitative reference for LC-MS/MS analysis	Isotopically labeled signature peptides (e.g., 13C/15N-labeled peptides for nut allergens)
Digestion Enzymes	Protein cleavage for mass spectrometric analysis	Trypsin (specific cleavage at lysine/arginine) for generating signature peptides
Chromatography Columns	Peptide separation prior to mass spectrometry	C18 reversed-phase columns (e.g., 100 × 3 mm, 2.6 μm particle size)
Antibody Reagents	Capture and detection elements for ELISA	Matched antibody pairs for sandwich ELISA; species-specific secondary antibodies

Workflow Comparison Diagram

Diagram 1: Method Workflow Comparison - This diagram illustrates the fundamental workflows and critical limitations/advantages of ELISA, PCR, and LC-MS/MS methods for allergen detection, highlighting the specific technical challenges of each approach.

The limitations of traditional allergen detection methods present significant challenges for food safety analysis. ELISA's susceptibility to cross-reactivity, particularly between closely related allergens like cashew and pistachio, can lead to false-positive results and inaccurate food labeling [6]. Meanwhile, PCR's indirect approach of detecting DNA rather than the allergenic proteins themselves creates a fundamental disconnect between the analytical target and the clinical concern, especially problematic in processed foods where DNA and proteins may degrade differentially [6] [16]. These methodological constraints highlight the need for more direct and specific detection approaches.

LC-MS/MS emerges as a powerful solution to these limitations, offering direct detection of allergenic proteins via signature peptides, unaffected by antibody cross-reactivity [17] [13]. Its capacity for multi-allergen screening in a single analysis, coupled with high specificity and quantitative accuracy, positions LC-MS/MS as an indispensable technique for comprehensive food allergen analysis. As the field advances, LC-MS/MS methodologies are poised to become the gold standard for reliable allergen detection in complex food matrices, providing the specificity and accuracy required for both regulatory compliance and consumer protection.

Fundamental Principles of LC-MS/MS for Direct Allergen Protein Detection

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful confirmatory technique for the direct detection of allergenic proteins in food products. Unlike traditional immunoassays or DNA-based methods, LC-MS/MS enables the specific identification and quantification of multiple allergens simultaneously by targeting stable peptide markers, providing a robust analytical solution for verifying allergen labeling and managing risks in processed foods [2]. This document outlines the fundamental principles and detailed protocols for implementing LC-MS/MS for multi-allergen detection within research settings.

The core principle of LC-MS/MS for allergen analysis involves digesting extracted proteins into peptides, separating them chromatographically, and detecting unique marker peptides via mass spectrometry. This approach provides direct analysis of the allergenic proteins themselves through characteristic masses and fragmentation patterns, offering enhanced reliability over indirect methods [18]. For researchers developing multi-allergen detection protocols, this technique offers significant advantages including high specificity, sensitivity, and the ability to analyze complex processed food matrices where proteins may be denatured or modified during manufacturing.

Fundamental Principles

Comparative Analytical Techniques

Table 1: Comparison of Major Allergen Detection Techniques

Technique	Principle	Advantages	Limitations
LC-MS/MS	Detection of signature peptides from allergenic proteins via mass spectrometry	Multi-allergen detection in single run; high specificity and confirmatory capability; resistant to heat processing effects; enables precise quantification [18] [2]	Higher instrumentation cost; requires skilled personnel; complex sample preparation; limited commercial kits
ELISA	Antigen-antibody interaction with enzymatic detection	Rapid analysis; high throughput; cost-effective for single allergens; well-established protocols [2]	Single-analyte detection; antibody cross-reactivity issues; false negatives with processed proteins; unable to distinguish closely related species [6] [18]
PCR	Amplification of species-specific DNA sequences	High specificity to species; sensitive to low DNA amounts; detects multiple targets with specific primers [18]	Indirect protein detection; DNA degradation in processing; unsuitable for egg/milk allergens; matrix inhibition [18] [2]

LC-MS/MS Operational Principles

The operational framework of LC-MS/MS for allergen detection relies on several key principles that ensure accurate identification and quantification:

• Proteolytic Digestion: Allergenic proteins are enzymatically cleaved into smaller peptides using specific proteases like trypsin, which cuts at the carboxyl side of arginine and lysine residues. This step generates predictable peptide fragments that serve as analytical targets [18].

• Chromatographic Separation: Resulting peptides are separated by reversed-phase liquid chromatography based on hydrophobicity, reducing matrix effects and concentrating analytes before mass spectrometric detection [18].

• Mass Spectrometric Detection: The triple quadrupole mass spectrometer operates in Multiple Reaction Monitoring (MRM) mode, where the first quadrupole selects precursor ions of target peptides, the second induces collision-induced dissociation, and the third monitors specific fragment ions. This two-stage mass selection provides high specificity even in complex matrices [19] [18].

• Peptide Marker Selection: Critical to method specificity is selecting peptides unique to each allergen, typically verified through BLAST searches to ensure they don't occur in other species. Ideal markers are thermally stable, resistant to processing, and generate strong MS signals [19] [2].

Experimental Protocols

Sample Preparation and Protein Extraction

Materials:

• Extraction Buffer: 50 mM ammonium bicarbonate, 8 M urea, 10 mM dithiothreitol (DTT) [18]
• Alkylation Solution: 20 mM iodoacetamide (IAA) in ammonium bicarbonate [19]
• Protease: Sequencing-grade trypsin [19] [20]
• Solid-Phase Extraction: Strata-X cartridges or S-Trap columns [20] [18]
• Internal Standards: Stable isotope-labeled peptides [19]

Procedure:

• Homogenization: Pulverize food samples to fine powder using a food processor. For dry products, initial grinding improves extraction efficiency.
• Protein Extraction: Add extraction buffer (1:10 w/v ratio) to 1 g of sample. The reducing environment with DTT breaks disulfide bonds, while urea denatures proteins. Mix using a roller mixer for 60 minutes at room temperature.
• Alkylation: Centrifuge at 10,000 × g for 15 minutes. Collect supernatant and add IAA to final concentration of 20 mM. Incubate in darkness for 30 minutes to prevent reformation of disulfide bonds.
• Protein Digestion: For S-Trap columns, add SDS to 5% final concentration and acidify with phosphoric acid. Bind proteins to the S-Trap filter by centrifugation, then wash with digestion buffer. Add trypsin (1:20 enzyme-to-protein ratio) in 50 mM ammonium bicarbonate. Digest for 1-4 hours at 47°C [20]. For traditional methods, dilute urea concentration to <1 M and digest with trypsin overnight at 37°C [18].
• Peptide Purification: Acidity digested peptides with 0.5% trifluoroacetic acid. For SPE cleanup: condition Strata-X cartridge with acetonitrile/0.1% formic acid, equilibrate with 0.5% TFA/water, load sample, wash with 0.5% TFA/water, elute with acetonitrile/0.1% formic acid. Evaporate eluent under nitrogen and reconstitute in 300 μL water-acetonitrile-formic acid (95:5:0.5) [18].

LC-MS/MS Analysis

Chromatographic Conditions:

• Column: C18 reversed-phase (2.1 × 150 mm, 2.7 μm)
• Mobile Phase A: 0.1% formic acid in water
• Mobile Phase B: 0.1% formic acid in acetonitrile
• Gradient: 2-35% B over 15 minutes, 35-90% B over 2 minutes, hold at 90% B for 3 minutes
• Flow Rate: 0.3 mL/min
• Column Temperature: 40°C
• Injection Volume: 10-20 μL [19] [20]

Mass Spectrometric Conditions:

• Ionization: Electrospray ionization (ESI) positive mode
• Ion Source Temperature: 500°C
• Ion Spray Voltage: 5500 V
• Nebulizer Gas: 50 psi
• Drying Gas: 60 psi
• Curtain Gas: 35 psi
• MRM Transitions: 3-5 per peptide, optimizing declustering potential and collision energy for each [19]

Data Analysis and Quantification

• Peptide Identification: Confirm detection based on retention time stability (RSD < 2%), presence of all MRM transitions, and ion ratio consistency (RSD < 20% compared to standards) [19].
• Quantification: Use stable isotope-labeled internal peptides for absolute quantification. Construct matrix-matched calibration curves using blank food samples spiked with known allergen concentrations. Apply linear regression with 1/x weighting [19].
• Validation Parameters: Establish method specificity, linearity (R² > 0.995), limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery 80-120%), and precision (RSD < 15%) [19].

Performance Data and Validation

Table 2: Quantitative Performance of LC-MS/MS for Allergen Detection

Allergen Source	Target Protein	Marker Peptide	LOD (mg/kg)	LOQ (mg/kg)	Recovery (%)	Precision (RSD%)	Reference
Beef	Myoglobin, Myosin light chain	Surrogate peptides	2.0-5.0	5.0-10.0	80.2-101.5	<13.8	[19]
Chicken	Myoglobin, Myosin light chain	Surrogate peptides	2.0-5.0	5.0-10.0	80.2-101.5	<13.8	[19]
Pork	Myoglobin, Myosin light chain	Surrogate peptides	2.0-5.0	5.0-10.0	80.2-101.5	<13.8	[19]
Lamb	Myoglobin, Myosin light chain	Surrogate peptides	2.0-5.0	5.0-10.0	80.2-101.5	<13.8	[19]
Duck	Myoglobin, Myosin light chain	Surrogate peptides	2.0-5.0	5.0-10.0	80.2-101.5	<13.8	[19]
Egg	Gal d 2 (Ovalbumin)	VTEQESKPVQMMYQIGLFR	<1.0	-	-	-	[20]
Milk	Caseins, Whey proteins	Multiple markers	<1.0	-	-	-	[20]
Peanut	Ara h 1, Ara h 3	Multiple markers	<1.0	-	-	-	[20]
Wheat	Gluten proteins	Multiple markers	<1.0	-	-	-	[20]
Pistachio	Pis v 1, Pis v 3	Specific peptides	1.0 (SDL)	-	-	Good reproducibility	[6]

Research Reagent Solutions

Table 3: Essential Research Reagents for LC-MS/MS Allergen Detection

Reagent/Category	Specific Examples	Function & Application Notes
Protein Extraction Buffers	50 mM NH₄HCO₃, 8 M urea, 10 mM DTT [18]	Denatures proteins and reduces disulfide bonds for complete extraction
Alkylating Reagents	Iodoacetamide (20 mM) [19]	Alkylates cysteine residues to prevent reformation of disulfide bonds
Proteolytic Enzymes	Sequencing-grade trypsin [19] [20]	Specific proteolytic cleavage at lysine/arginine for reproducible peptide maps
Solid-Phase Extraction	Strata-X cartridges, S-Trap columns [20] [18]	Removes matrix interferents and concentrates target peptides
Chromatographic Media	C18 reversed-phase columns (2.1 × 150 mm, 2.7 μm) [19]	Separates peptides based on hydrophobicity prior to MS detection
Mass Standards	Stable isotope-labeled peptides [19]	Enables absolute quantification and corrects for recovery variations
Mobile Phase Additives	0.1% formic acid in water/acetonitrile [19]	Promotes protonation and efficient ionization in positive ESI mode
Allergen Reference Materials	Certified reference materials, incurred food samples [19] [20]	Method validation and quality control for accurate quantification

Workflow Visualization

LC-MS/MS Allergen Detection Workflow

The diagram above illustrates the comprehensive workflow for LC-MS/MS-based allergen detection, encompassing three major phases: sample preparation, instrumental analysis, and data processing. This integrated approach ensures reliable identification and quantification of multiple allergens in complex food matrices.

LC-MS/MS represents a confirmatory analytical technique that directly targets signature peptides from allergenic proteins, overcoming limitations of traditional methods. The protocols detailed herein provide researchers with a robust framework for implementing this technology in multi-allergen detection applications. With ongoing advancements in sample preparation efficiency and instrumental sensitivity, LC-MS/MS continues to evolve as an indispensable tool for ensuring food safety compliance and protecting allergic consumers through accurate allergen labeling verification.

The increasing prevalence of food allergies has created an urgent need for robust analytical methods capable of detecting multiple allergenic foods simultaneously in complex food matrices. Traditional single-analyte approaches, such as enzyme-linked immunosorbent assays (ELISAs), often prove insufficient for comprehensive food allergen control in today's complex food supply chain, where cross-contamination and the need to distinguish between cross-reactive proteins are common challenges [6] [21]. Multiplexing technologies, particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS), have emerged as powerful tools that address these limitations by enabling the simultaneous detection and quantification of numerous allergenic foods in a single analytical run [7] [22].

This application note details the advantages, experimental protocols, and performance characteristics of multiplexed LC-MS/MS for the detection of 12 or more food allergens, framed within the context of method development for processed food analysis. We provide validated methodologies, performance data, and technical considerations to support researchers and scientists in implementing this powerful approach for food allergen risk assessment and compliance with international labeling regulations.

The Multiplexing Advantage in Food Allergen Detection

Limitations of Traditional Single-Plex Methods

Conventional food allergen detection primarily relies on two analytical techniques: immunoassays (e.g., ELISA) and DNA-based methods (e.g., PCR). While useful in certain applications, both approaches present significant limitations for comprehensive allergen monitoring. ELISA methods, which rely on antigen-antibody interactions, are susceptible to cross-reactivity with food matrix components, potentially resulting in false positives [6]. Furthermore, ELISA is generally limited to single-target detection, and robust multiplexing has yet to be fully established [6]. PCR targets allergen-specific DNA sequences rather than the allergenic proteins themselves, which may lead to inaccurate results, particularly in processed foods where proteins and DNA may degrade and become physically separated during manufacturing [6] [19].

These limitations become particularly problematic when analyzing for closely related species, such as cashew and pistachio, where traditional ELISA and PCR methods often suffer from cross-reactivity, limiting their ability to discriminate between these two allergens [6]. Similarly, processing techniques like baking can significantly reduce allergen recovery rates in ELISA, as demonstrated by recoveries as low as 66-90% for casein, soy protein, and gluten in baked cookies compared to 85-127% in raw samples [23].

Technical Advantages of Multiplex LC-MS/MS

Mass spectrometry-based techniques have become powerful tools for food allergen analysis due to their high sensitivity, specificity, and ability to provide unequivocal allergen identification [6] [22]. Unlike ELISA, LC-MS/MS is unaffected by antibody cross-reactivity, and unlike PCR, it allows for the direct detection of allergenic peptides or proteins, ensuring more accurate identification [6]. The key advantages of multiplexed LC-MS/MS include:

• Direct allergen measurement: LC-MS/MS targets proteotypic peptides derived from allergenic proteins, providing direct evidence of allergen presence rather than relying on proxy measurements [22]
• Discriminatory power: High specificity enables distinction between closely related allergenic foods, such as cashew and pistachio, overcoming cross-reactivity limitations of immunoassays [6]
• Comprehensive profiling: Capacity to monitor 12+ allergens in a single analysis significantly reduces analytical time, cost, and sample volume requirements compared to sequential single-analyte methods [7]
• Robustness in processed foods: Suitable for analysis of thermally processed products where protein modifications may interfere with immunoassay performance [23] [7]

Table 1: Comparison of Food Allergen Detection Platforms

Parameter	ELISA	PCR	Multiplex LC-MS/MS
Detection Principle	Antibody-protein binding	DNA amplification	Peptide mass detection
Multiplexing Capacity	Limited (typically 1-3)	Moderate (typically 5-10)	High (12+ allergens)
Cross-Reactivity Issues	High	Moderate	Low
Impact of Food Processing	Significant	Variable	Moderate
Discriminatory Power	Low	Moderate	High
Analysis Time for Multiple Allergens	Long (sequential)	Moderate (sequential)	Short (simultaneous)
Quantification Capability	Good	Semi-quantitative	Excellent with proper standards

Experimental Protocol: Multiplex LC-MS/MS for Allergen Detection

The following diagram illustrates the comprehensive workflow for multiplex allergen detection using LC-MS/MS, from sample preparation to data analysis:

Detailed Methodological Procedures

Sample Preparation and Protein Extraction

Proper sample preparation is critical for successful multiplex allergen detection. For complex matrices like chocolate, begin by grinding approximately 15g of sample under refrigerated conditions to avoid melting, then sieve through a 1mm mesh [22]. Extract a 2g aliquot with 20mL of Tris HCl buffer (200mM Tris·HCl, pH 8.2) containing 1M NaCl, 2mM EDTA, and 0.4% w/v casein [22]. For difficult matrices, the inclusion of casein in the extraction buffer helps minimize non-specific binding of target proteins to matrix components and polyphenols. For meat allergens in various food products, an optimized protein extraction involving Tris-HCl buffer (50mM, pH 8.0) with 8M urea and 2M thiourea has proven effective [19].

After vortexing and shaking for 30 minutes at 60°C, centrifuge at 10,000×g for 10 minutes at 4°C. Collect the supernatant and determine protein concentration using a suitable assay (e.g., Bradford assay). For high-fat matrices, a defatting step with n-hexane may be necessary prior to extraction [19].

Protein Digestion and Peptide Purification

For digestion, aliquot a volume containing approximately 500μg of protein extract. Add 50mM ammonium bicarbonate to adjust the volume, followed by reduction with 10mM dithiothreitol (DTT) at 37°C for 1 hour, and alkylation with 25mM iodoacetamide (IAA) at room temperature for 30 minutes in the dark [22]. Enzymatic digestion is performed using sequencing-grade trypsin at a 1:20 enzyme-to-protein ratio, incubating at 37°C for 16 hours [19] [22].

To improve efficiency and throughput, recent methods have incorporated Suspension-Trapping (S-Trap) columns, which allow for direct digestion without the need for protein precipitation or lengthy buffer exchange steps [7]. Following digestion, purify peptides using disposable desalting cartridges (e.g., Strata-X polymeric reversed phase) or on-line solid-phase extraction (SPE) systems [7] [22]. Evaporate the eluate to near dryness and reconstitute in 100-200μL of 0.1% formic acid in water for LC-MS/MS analysis.

LC-MS/MS Analysis Parameters

Chromatographic separation is typically performed using a reversed-phase C18 column (2.1 × 150mm, 1.8μm) maintained at 40°C. The mobile phase consists of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. Apply a gradient elution from 2% to 35% B over 25 minutes at a flow rate of 0.3mL/min [19] [22].

For mass spectrometric detection, a triple quadrupole (QqQ) instrument operating in positive electrospray ionization mode with multiple reaction monitoring (MRM) is recommended for its sensitivity and reproducibility in targeted analysis [6]. Key MS parameters include:

• Nebulizer gas: 3L/min
• Heating gas: 10L/min
• Interface temperature: 300°C
• DL temperature: 250°C
• Heat block temperature: 400°C
• Dwell time: 10-50ms per transition

The selection of proteotypic peptides is crucial for method specificity and sensitivity. Peptides should be unique to the target allergenic food, typically 7-16 amino acids in length, and should not contain missed cleavage sites or easily modified residues [19] [22]. For each allergen, monitor 2-3 proteotypic peptides with 2-3 transitions each to ensure reliable identification and quantification.

Performance Characteristics and Validation

Quantitative Performance Data

Multiplex LC-MS/MS methods have demonstrated excellent performance characteristics across various food matrices. The following table summarizes validation data from recent studies:

Table 2: Performance Characteristics of Multiplex LC-MS/MS for Allergen Detection

Allergenic Food	Marker Protein	LOD (mg/kg)	LOQ (mg/kg)	Recovery (%)	Precision (RSD%)	Reference
Pistachio	Pis v 1, Pis v 2, Pis v 3, Pis v 5	1.0 (SDL)	-	-	Good reproducibility	[6]
Cashew	Ana o 1, Ana o 2, Ana o 3	1.0 (SDL)	-	-	Ongoing optimization	[6]
Cow's Milk	Caseins, β-lactoglobulin	0.08-0.2 (µg TAFP/g)	0.3-0.6 (µg TAFP/g)	80.2-101.5	<13.8	[19] [22]
Hen's Egg	Ovalbumin, ovomucoid	1.1 (µg TAFP/g)	3.7 (µg TAFP/g)	80.2-101.5	<13.8	[22]
Peanut	Ara h 1, Ara h 3	0.2 (µg TAFP/g)	0.6 (µg TAFP/g)	80.2-101.5	<13.8	[22]
Soybean	Gly m 5, Gly m 6	1.2 (µg TAFP/g)	4.0 (µg TAFP/g)	80.2-101.5	<13.8	[22]
Hazelnut	Cor a 9, Cor a 11	0.2 (µg TAFP/g)	0.6 (µg TAFP/g)	80.2-101.5	<13.8	[22]
Almond	Pru du 6	0.2 (µg TAFP/g)	0.6 (µg TAFP/g)	80.2-101.5	<13.8	[22]
Wheat	Glutenins, gliadins	<1.0	<5.0	80-90 (after baking)	<15	[23] [7]
Buckwheat	Fag e 1, Fag e 2	<1.0	<5.0	-	-	[7]
Crustacean	Tropomyosin	<1.0	<5.0	-	-	[7]
Walnut	Jug r 1, Jug r 4	<1.0	<5.0	-	-	[7]

Method Validation Parameters

Rigorous validation is essential for implementing reliable multiplex allergen detection methods. Key validation parameters should include:

• Specificity: Confirm absence of interference from matrix components and cross-reactivity with non-target allergens [6] [22]
• Linearity: Evaluate over an appropriate concentration range (typically 2-3 orders of magnitude) with R² > 0.995 [19] [22]
• Sensitivity: Determine limit of detection (LOD) and limit of quantification (LOQ) using calibration-based approaches [22]
• Precision: Assess repeatability (intra-day) and intermediate precision (inter-day) with RSD < 15% [19] [22]
• Accuracy: Establish through recovery studies (80-120%) using incurred materials [19] [22]
• Ruggedness: Test method robustness under deliberate variations of analytical parameters [6]

For quantitative applications, employ stable isotope-labeled internal standards (SIL IS) for each target peptide to correct for variations in sample preparation, ionization efficiency, and matrix effects [22]. Matrix-matched calibration curves are essential for accurate quantification, as they account for matrix-induced suppression or enhancement of ionization [19] [22].

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of multiplex allergen detection requires carefully selected reagents and materials. The following table outlines essential components for establishing this methodology:

Table 3: Essential Research Reagents for Multiplex Allergen Detection by LC-MS/MS

Reagent/Material	Specification	Function	Application Notes
Trypsin	Sequencing-grade	Protein digestion	Use 1:20-1:50 enzyme-to-protein ratio; 37°C for 16h [19] [22]
S-Trap Columns	Micro or Mini format	Protein digestion and cleanup	Enables efficient digestion without protein precipitation [7]
Stable Isotope-Labeled Peptides	AQUA QuantPro grade (>97% purity)	Internal standards for quantification	Correct for preparation variability and matrix effects [22]
Ammonium Bicarbonate	≥99% purity	Digestion buffer	Use 50mM concentration for optimal tryptic activity [19]
Dithiothreitol (DTT)	≥99% purity	Protein reduction	Use 10mM concentration; 37°C for 1h [22]
Iodoacetamide (IAA)	≥99% purity	Cysteine alkylation	Use 25mM concentration; RT for 30min in dark [22]
Formic Acid	LC-MS grade	Mobile phase additive	0.1% in both aqueous and organic mobile phases [19]
Acetonitrile	LC-MS grade	Mobile phase component	Use with 0.1% formic acid for peptide separation [22]
Solid-Phase Extraction Columns	Strata-X, 33μm, 30mg/1mL	Peptide purification	Remove salts and impurities prior to LC-MS/MS [22]
UHPLC Column	C18, 1.8μm, 2.1×150mm	Peptide separation	Maintain at 40°C for stable retention times [19] [22]

Analytical Considerations and Troubleshooting

Matrix Effects and Interferences

Food matrices present significant challenges for multiplex allergen detection. Chocolate, with its high fat and polyphenol content, requires specialized extraction buffers containing casein to prevent non-specific binding of target proteins [22]. Baked goods often exhibit reduced allergen recoveries due to protein denaturation and Maillard reaction products that can mask epitopes and tryptic cleavage sites [23]. For difficult matrices such as meat products and sauces, the incorporation of urea and thiourea in the extraction buffer improves protein solubility and recovery [19].

To mitigate matrix effects:

• Employ matrix-matched calibration standards
• Use stable isotope-labeled internal standards for each target peptide
• Implement efficient sample cleanup procedures (e.g., SPE, S-Trap)
• Optimize extraction conditions for specific matrix types

Quantitative Reporting and Conversion Factors

A critical aspect of allergen quantification involves converting measured peptide concentrations to meaningful units for risk assessment. Since regulatory thresholds are typically expressed as mg of total allergenic food protein per kg of food, conversion factors must be determined to translate peptide concentrations to total allergenic protein content [22]. These factors can be determined experimentally through quantitative proteomic analysis of the allergenic food or theoretically based on known protein sequences and their relative abundance in the food [22].

Recent work has established conversion factors for major allergenic foods including cow's milk, hen's egg, peanut, soybean, hazelnut, and almond, enabling reporting in µg of total allergenic food protein per g of food (µg TAFP/g food) [22]. This standardized reporting unit enhances the utility of LC-MS/MS data for risk assessment and regulatory decision-making.

Multiplexed LC-MS/MS represents a significant advancement in food allergen detection, offering unparalleled specificity, discriminatory power, and comprehensive profiling capabilities. The simultaneous detection of 12+ allergens in a single analytical run addresses critical limitations of traditional single-plex methods while providing the sensitivity required to meet evolving regulatory thresholds. As demonstrated through the protocols and performance data presented herein, this approach delivers robust, reliable detection across diverse food matrices, supporting enhanced food safety management and accurate allergen labeling for protection of sensitive consumers.

With ongoing advancements in sample preparation efficiency, instrumentation sensitivity, and data processing capabilities, multiplexed LC-MS/MS continues to evolve as the reference methodology for food allergen detection, promising even greater analytical performance and throughput in the future.

A Step-by-Step LC-MS/MS Protocol: From Sample to Spectra

Optimized Protein Extraction Using ELISA-Validated Buffers for Processed Foods

Accurate detection of food allergens is a critical component of public health and food safety. For individuals with food allergies, exposure to trace amounts of allergenic proteins can trigger reactions ranging from mild symptoms to severe, life-threatening anaphylaxis [6] [2]. The detection of these allergens in processed foods presents unique analytical challenges, as thermal treatment and food matrix effects can significantly alter protein structures, reducing extractability and antibody recognition in immunoassays [24] [18].

This application note details optimized protocols for protein extraction from processed food matrices using ELISA-validated buffers, with specific emphasis on methods that preserve protein immunoreactivity and compatibility with downstream LC-MS/MS multi-allergen detection. The procedures outlined herein are designed to address the key technical challenges in allergen extraction, including protein denaturation from heat processing, matrix interference, and variable extraction efficiency across different food types.

The Critical Role of Protein Extraction in Allergen Detection

Effective protein extraction is the foundational step for accurate allergen detection, whether using immunoassay or mass spectrometry-based methods. The extraction process must achieve three primary objectives: (1) complete solubilization of target proteins from the complex food matrix, (2) maintenance of protein epitopes for antibody recognition in ELISA, and (3) compatibility with tryptic digestion for LC-MS/MS analysis [25] [18].

Thermal processing of foods presents particular challenges for allergen detection. Heat treatment can induce protein denaturation, aggregation, and chemical modification, all of which can reduce antibody binding in ELISA methods [24]. Recent research demonstrates that the format of ELISA significantly impacts detection capability for processed allergens. Sandwich ELISA formats show superior sensitivity for heat-denatured proteins compared to competitive formats, with one study detecting soy isolate at 10 μg/g in pasteurized sausage versus 2500 μg/g for competitive format [24]. This highlights the critical importance of pairing optimized extraction with appropriate detection methods.

For LC-MS/MS analysis, the extraction process must effectively solubilize target proteins while minimizing interference from lipids, carbohydrates, and other matrix components that can suppress ionization or chromatographically co-elute with target peptides [6] [19]. The development of thermal stable-soluble protein (TSSP) extraction protocols has advanced detection capabilities for processed products, enabling sensitive detection of species-specific markers even in autoclaved, roasted, and fried food products [25].

Optimized Protein Extraction Protocols

Extraction of Thermal Stable-Soluble Proteins (TSSP) from Heat-Processed Foods

This protocol is optimized for the extraction of thermal-stable protein markers from processed meat products and has been validated for detection of pork fat tissue in authentic and heat-processed beef meatballs with a detection limit of 0.015% (w/w) [25].

• Materials: Tris-buffered saline (TBS, pH 7.4), homogenizer, centrifuge, water bath, Whatman No. 1 filter paper.
• Procedure:
  • Sample Preparation: Trim visible connective tissue and fat from meat samples. For cooked samples, heat pure materials in boiling water for 15 minutes.
  • Homogenization: Homogenize 10 g of sample with 20 mL of ice-cold 0.025 M TBS (pH 7.4) for 5 minutes.
  • Heat Treatment: Transfer homogenate to a water bath at 100°C for 15 minutes, then cool to room temperature.
  • Clarification: Centrifuge at 3,220 × g at 4°C for 15 minutes.
  • Filtration: Pass supernatant through Whatman No. 1 filter paper.
  • Protein Quantification: Assess total soluble protein content using Bradford assay.
• Key Applications: Species authentication in raw and heat-treated meat products (autoclaved, steamed, roasted, fried); detection of economically motivated adulteration [25].

Commercial ELISA-Compatible Extraction Buffers

Specialized commercial buffers provide standardized, reproducible protein extraction while preserving epitope integrity for immunoassay detection.

• FastScan ELISA Cell Extraction Buffer System:
  • Composition: 5X concentrated extraction buffer with separate 50X enhancer solution containing phosphatase inhibitors [26].
  • Preparation: Dilute to 1X with deionized water (2 mL 5X buffer + 200 μL 50X enhancer + 7.8 mL water). Add protease inhibitors immediately before use.
  • Extraction Protocol:
    • Rinse cells or tissue with ice-cold PBS.
    • Add 0.5 mL ice-cold 1X buffer per 10 cm culture plate.
    • Incubate on ice for 5 minutes with periodic agitation.
    • Scrape adherent cells and transfer to microcentrifuge tube.
    • Sonicate on ice, then centrifuge at 14,000 rpm for 5 minutes at 4°C.
    • Collect supernatant for immediate analysis or store at -80°C [26].
• ELISA Lysis and Protein Extraction Buffer:
  • Features: Pre-mixed 1X solution containing mild detergent for membrane solubilization, compatible with Bradford and BCA protein assays [27].
  • Application: Effective for extraction of membrane and cytosolic proteins from cell cultures and tissue samples.

Buffer Formulations for Enhanced Protein Recovery

Research demonstrates that buffer composition significantly impacts protein recovery, particularly from complex matrices. The following formulations have shown efficacy in various food matrices:

• Urea-Based Extraction Buffer: 50 mM ammonium bicarbonate, 8 M urea, 10 mM dithiothreitol (DTT). Effective for allergen extraction from baked goods and processed foods [18].
• Carbonate-Bicarbonate Coating Buffer (pH 9.6): Optimal for passive adsorption of proteins to microplate surfaces in ELISA protocols [10].
• Phosphate-Buffered Saline with Tween-20 (PBST): 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, 0.05% Tween-20, pH 7.4. Standard washing buffer for ELISA procedures [10] [28].

Quantitative Comparison of Extraction Efficiency

Table 1: Comparison of Allergen Detection Sensitivity Across Processing Conditions and Analytical Methods

Allergen Source	Food Matrix	Processing Condition	Extraction Method	Detection Method	Sensitivity	Reference
Soy protein	Sausage	Pasteurization	Sandwich ELISA	Sandwich ELISA	10 μg/g	[24]
Soy protein	Bread	Baking	Sandwich ELISA	Sandwich ELISA	100 μg/g	[24]
Soy protein	Pâté	Sterilization	Sandwich ELISA	Sandwich ELISA	1000 μg/g	[24]
Pork fat	Beef meatballs	Autoclaving, steaming, roasting, frying	TSSP extraction	iELISA	0.015% (w/w)	[25]
Livestock & poultry meats	Various foods	Various processing	Surrogate peptide extraction	LC-MS/MS	2.0–5.0 mg/kg LOD	[19]
Pistachio	Cereals, chocolate, sauces, meat	Various processing	Characteristic peptide extraction	LC-MS/MS	1 mg/kg SDL	[6]

Table 2: Research Reagent Solutions for Protein Extraction and Analysis

Reagent / Solution	Composition / Characteristics	Primary Function	Application Notes
FastScan ELISA Cell Extraction Buffer	5X concentrated with separate enhancer solution (50X) containing phosphatase inhibitors	Extraction of soluble and membrane proteins while preserving phosphorylation states	Requires addition of protease inhibitors immediately before use; compatible with various detection platforms
ELISA Lysis and Protein Extraction Buffer	Pre-mixed 1X solution with mild detergent; includes separate phosphatase inhibitor	Solubilization of membrane proteins and suspension of cytosolic proteins	Compatible with protein concentration assays (Bradford); suitable for most protein and glycoprotein extraction
Thermal Stable-Soluble Protein (TSSP) Extraction Buffer	0.025 M Tris-buffered saline (pH 7.4) with heat treatment	Selective extraction of heat-stable protein fractions	Enables detection of species-specific markers in highly processed foods; critical for authentication testing
Urea-Based Denaturing Buffer	8 M urea, 50 mM ammonium bicarbonate, 10 mM DTT	Denaturing extraction for refractory proteins in processed matrices	Effective for baked goods; requires alkylation with iodoacetamide post-extraction
Carbonate-Bicarbonate Coating Buffer	0.1 M carbonate-bicarbonate, pH 9.6	Optimal for passive adsorption to polystyrene ELISA plates	High pH enhances hydrophobic interactions for protein binding to solid phase

Integrated Workflow for Multi-Allergen Detection

The following workflow diagram illustrates the complete integrated process for optimized protein extraction and multi-allergen detection in processed foods, incorporating both ELISA and LC-MS/MS detection pathways:

Technical Considerations for Method Implementation

Addressing Matrix-Specific Challenges

Different food matrices present unique challenges for protein extraction. High-fat matrices (e.g., sausages, pâtés) require additional defatting steps with organic solvents such as n-hexane prior to protein extraction [19]. Carbohydrate-rich matrices (e.g., bread, baked goods) often necessitate more extensive homogenization and may benefit from enzymatic treatments to degrade polysaccharides. Acidic foods may require pH adjustment prior to extraction to optimize protein solubility and stability [18].

Minimizing Matrix Effects in LC-MS/MS Analysis

For LC-MS/MS applications, matrix effects can significantly impact quantification accuracy. Effective strategies include:

• Use of matrix-matched calibration curves: Prepare standards in allergen-free matrix extracts to compensate for suppression/enhancement effects [19].
• Stable isotope-labeled internal standards: Incorporate labeled peptide analogues to correct for variability in digestion efficiency and ionization suppression [6] [19].
• Solid-phase extraction (SPE) cleanup: Utilize reverse-phase or mixed-mode SPE to remove interfering compounds while retaining target peptides [18].

Method Validation Parameters

Comprehensive validation of the integrated extraction and detection method should assess:

• Extraction efficiency: Compare signal responses between spiked samples and reference materials.
• Precision: Evaluate repeatability (intra-day) and reproducibility (inter-day) with CV values <15% [19].
• Specificity: Verify absence of cross-reactivity with non-target species or matrix components.
• Robustness: Test method performance under deliberate variations in extraction time, temperature, and buffer composition [24] [6].

Optimized protein extraction is the critical first step in reliable allergen detection in processed foods. The protocols detailed in this application note, centered on ELISA-validated extraction buffers and thermal stable-soluble protein methods, provide robust approaches for challenging processed food matrices. When coupled with appropriate detection platforms—whether immunoassay for routine monitoring or LC-MS/MS for confirmatory multi-allergen analysis—these extraction methods form the foundation of a comprehensive allergen control program. The integrated workflow enables researchers to overcome the analytical challenges posed by food processing, delivering the sensitivity, specificity, and reproducibility required for effective allergen risk management and regulatory compliance.

The accurate detection of multiple food allergens in processed matrices represents a significant analytical challenge for food safety. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful confirmatory tool for multiplexed allergen detection, overcoming limitations of traditional techniques like ELISA and PCR, which are often prone to cross-reactivity and cannot distinguish between closely related species such as pistachio and cashew [6] [18]. The sensitivity and reliability of LC-MS/MS hinge on effective sample preparation, specifically the digestion of allergenic proteins into measurable peptides and the subsequent clean-up of complex sample extracts. This application note details an integrated protocol leveraging S-Trap microcolumns for efficient digestion and online solid-phase extraction (online SPE) for automated clean-up, designed to enhance throughput, recovery, and analytical precision in the context of multi-allergen detection in processed foods.

The Analytical Challenge: Allergen Detection in Processed Foods

Processed food matrices, such as chocolate, are particularly difficult to analyze due to the presence of interfering compounds like polyphenols, tannins, and fats, which can bind proteins and suppress ionization during MS analysis [22]. Furthermore, food processing can modify allergenic proteins, potentially masking antibody recognition sites in ELISA or leading to protein degradation [18]. Mass spectrometry directly targets proteotypic peptides, offering a more robust solution. However, without rigorous and effective sample preparation, matrix effects can severely compromise detection limits and quantitative accuracy. The workflow outlined herein is designed to address these challenges systematically.

S-Trap Microcolumns for Digestion

S-Trap (Suspension Trapping) microcolumns offer a novel approach for protein digestion that is highly compatible with challenging sample matrices. Unlike traditional in-solution digestion, which can suffer from poor efficiency in the presence of detergents or interfering substances, S-Trap technology utilizes a proprietary silica filter to trap proteins under acidic conditions. This allows for the use of strong denaturants like SDS to efficiently extract proteins from complex foods, followed by thorough washing to remove contaminants that impede enzymatic cleavage. The trapped proteins are then digested on-column with trypsin, yielding cleaner and more reproducible peptide mixtures for downstream LC-MS/MS analysis, which is critical for achieving high sequence coverage and reliable quantification of allergenic markers.

Online SPE for Automated Clean-up

Online Solid-Phase Extraction (SPE) automates the sample clean-up and concentration process by coupling it directly to the LC-MS/MS system. In this setup, an automated switching valve directs the injected crude extract to a dedicated SPE cartridge. Target analytes are retained and concentrated on the SPE phase, while matrix interferences are washed to waste. The analytes are then eluted from the SPE cartridge onto the analytical HPLC column for separation and detection [29] [30]. This approach offers several key advantages for allergen analysis:

• Full Automation: Eliminates manual steps like evaporation and reconstitution, drastically increasing throughput and reducing human error [30].
• Enhanced Sensitivity: Quantitative transfer of the extracted analytes to the MS improves detection limits.
• Improved Reproducibility: Automated systems provide superior precision compared to manual off-line SPE [30].
• Reduced Solvent Consumption: The miniaturized and integrated nature of online SPE is more environmentally friendly.

For allergen work, various SPE chemistries are available, including:

• HyperSep Retain PEP: For polar and nonpolar analytes.
• HyperSep Retain CX: For basic and nonpolar analytes.
• HyperSep Retain AX: For acidic and nonpolar analytes.
• HyperSep Hypercarb: For extremely polar analytes [29].

Integrated Workflow for Multi-Allergen Detection

The following diagram illustrates the complete integrated protocol from sample to MS detection, highlighting the roles of both S-Trap and online SPE technologies.

Key Performance Data

The implementation of robust sample preparation and clean-up methods enables the development of highly sensitive quantitative assays for allergens. The table below summarizes performance characteristics achieved for various allergens using optimized LC-MS/MS methods, which can be targeted with the described S-Trap and online SPE workflow.

Table 1: Representative Method Performance for Allergen Detection via LC-MS/MS in Food Matrices

Allergenic Food	Matrix	LOD (µg TAFP*/g food)	LOQ (µg TAFP/g food)	Recovery (%)	Precision (RSD%)	Citation
Cow's Milk	Chocolate	0.08	-	-	-	[22]
Hen's Egg	Chocolate	1.1	-	-	-	[22]
Peanut	Chocolate	0.2	-	-	-	[22]
Pistachio	Multi	-	1 mg/kg (SDL)	-	Good reproducibility	[6]
Beef	Food	2.0 mg/kg	5.0 mg/kg	80.2 - 101.5	< 13.8	[19]
Chicken	Food	2.0 mg/kg	5.0 mg/kg	80.2 - 101.5	< 13.8	[19]
Pork	Food	2.0 mg/kg	5.0 mg/kg	80.2 - 101.5	< 13.8	[19]

TAFP: Total Allergenic Food Protein [22] *SDL: Screening Detection Limit [6]

Detailed Experimental Protocols

Protocol 1: Protein Digestion Using S-Trap Microcolumns

This protocol is optimized for a 2 g sample of incurred chocolate bar [22].

Research Reagent Solutions

Table 2: Essential Reagents for S-Trap Digestion Protocol

Reagent/Consumable	Function	Example/Note
S-Trap Microcolumns	Traps proteins for digestion and clean-up	Available in various sizes (e.g., for 1-5 mg protein)
Sequencing-grade Trypsin	Enzymatic digestion of proteins into peptides	Promega Trypsin Gold, MS Grade [22]
SDS Solution	Powerful detergent for protein denaturation and extraction	5% (w/v) in extraction buffer
Tris HCl Buffer	Extraction buffer	200 mM, pH 7-8 [22]
Dithiothreitol (DTT)	Reducing agent; breaks disulfide bonds	Typically 10-100 mM
Iodoacetamide (IAA)	Alkylating agent; prevents reformation of disulfide bonds	Added after reduction [18]
Ammonium Bicarbonate	Digestion buffer; maintains optimal pH for trypsin	50-100 mM
Formic Acid	Acidifies sample for protein binding to S-Trap	Final concentration ~1%

Step-by-Step Procedure:

• Sample Homogenization: Carefully grind about 15 g of chocolate bar using a laboratory blender under refrigerated conditions. Sieve the ground material through a 1 mm mesh.
• Protein Extraction: Weigh 2 g of the ground sample into a centrifuge tube. Add 20 mL of a pre-cooled extraction buffer (e.g., 200 mM Tris·HCl, pH 7.5) containing 5% SDS and 10 mM DTT. Vortex vigorously.
• Reduction and Denaturation: Incubate the mixture at 95°C for 10 minutes with shaking to fully denature proteins and reduce disulfide bonds.
• Acidification and Loading: Centrifuge the extract to pellet insoluble debris. Transfer the supernatant to a new tube and acidify with phosphoric or formic acid to a final concentration of ~2.5% or 1%, respectively. Load the acidified supernatant onto the pre-conditioned S-Trap column by centrifugal force.
• Washing: Pass 150 µL of a wash buffer (e.g., 90% methanol, 100 mM triethylammonium bicarbonate, pH 7.1) through the S-Trap column. Repeat this step 4-5 times to thoroughly remove SDS, lipids, and other non-protein contaminants.
• Digestion: Prepare a trypsin solution (1 µg/µL in 50 mM ammonium bicarbonate). Add 20-50 µL of this solution directly to the S-Trap column. Incubate for 1-2 hours at 47°C or overnight at 37°C to allow for complete proteolysis.
• Peptide Elution: Elute the digested peptides sequentially with:
  • 80 µL of 50 mM ammonium bicarbonate (collect flow-through).
  • 80 µL of 0.2% formic acid in water (collect flow-through).
  • 80 µL of 50% acetonitrile, 0.2% formic acid (collect flow-through). Combine all eluents. The peptide extract can be dried and reconstituted in a suitable solvent for injection or injected directly if concentration is sufficient.

Protocol 2: Automated Clean-up and Analysis via Online SPE-LC-MS/MS

This protocol is adapted from successful online SPE applications in environmental and bioanalysis [30] and is configurable for allergen peptides.

Step-by-Step Procedure:

• System Configuration: Set up an HPLC system coupled to a triple quadrupole mass spectrometer with an integrated online SPE module and a switching valve.
• Sample Preparation: Reconstitute the S-Trap eluent or digest in 0.1% formic acid in water. Centrifuge to remove any particulate matter.
• SPE Loading and Wash: Using the autosampler, inject the sample onto the online SPE cartridge (e.g., a HyperSep Retain PEP cartridge [29] or a Strata-X cartridge [18]). The loading pump delivers a weak aqueous solvent (e.g., 0.1% formic acid in water) to load the sample and wash away unretained matrix components to waste.
• Elution and Transfer: Activate the switching valve to place the SPE cartridge in line with the analytical column and the LC gradient pump. Elute the retained peptides from the SPE cartridge onto the analytical HPLC column using a sharp step to a high-percentage organic mobile phase (e.g., 80% acetonitrile with 0.1% formic acid).
• Chromatographic Separation: Employ a reversed-phase gradient (e.g., from 5% to 90% acetonitrile with 0.1% formic acid) on the analytical column (e.g., a C18 core-shell column) to separate the peptides.
• MS/MS Detection: Operate the mass spectrometer in positive electrospray ionization (ESI+) mode with Multiple Reaction Monitoring (MRM). Monitor specific precursor ion → product ion transitions for the proteotypic peptides of each target allergen. Use isotopically labelled versions of these peptides as internal standards for precise quantification [22] [19].

The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for Allergen LC-MS/MS Analysis

Item	Function	Application Note
Trypsin, MS Grade	Specific proteolytic enzyme for generating target peptides.	Essential for reproducible digestion [22].
AQUA Synthetic Peptides	Isotopically labelled internal standards for absolute quantification.	"Heavy" peptides correct for recovery and matrix effects [22].
SPE Sorbents	Selective binding and clean-up of target analytes.	Choice depends on analyte polarity (e.g., PEP, CX, AX) [29].
UHPLC Columns	High-resolution separation of peptides prior to MS.	Core-shell C18 columns offer high efficiency [31].
MS-Compatible Buffers	Maintain pH and solubility without fouling the MS.	Ammonium bicarbonate, formic acid preferred [22] [31].

Selection of Robust Proteotypic Peptide Markers Resistant to Processing Effects

Within the framework of developing a robust LC-MS/MS protocol for multi-allergen detection in processed foods, the selection of appropriate proteotypic peptides is a critical foundational step. Mass spectrometry (MS) has emerged as a confirmatory tool for the sensitive and multiplexed quantitation of allergenic proteins in complex food matrices [22] [32]. Unlike antibody-based methods, whose targets can be denatured or altered during thermal and mechanical processing, MS detects signature peptides from digested proteins, offering the potential for greater resilience to food processing effects [5] [33]. However, this resilience is not inherent; it depends entirely on the judicious selection of peptide markers that remain stable and detectable despite the harsh conditions of food production, such as those encountered in bakery products (e.g., cookies and rusks) [5]. This application note details a structured methodology for the identification, validation, and implementation of robust proteotypic peptide markers, enabling reliable allergen detection in extensively processed foods.

Experimental Design and Rationale

The Challenge of Food Processing

Food processing induces numerous chemical and structural modifications to proteins, including denaturation, aggregation, and chemical modification (e.g., Maillard reaction, oxidation). These changes can mask or destroy epitopes recognized by antibodies in ELISA tests, leading to potential false negatives [33] [32]. Similarly, for MS-based methods, processing can modify peptide sequences, impair enzyme digestion efficiency, or lead to the loss of specific peptide markers, thereby compromising accurate detection and quantification [5]. The extent of these effects is matrix- and process-dependent. For instance, the production of rusks involves more intensive thermal and fermentation steps compared to cookies, presenting a greater challenge for allergen detection [5]. Therefore, the selection of peptide markers must be validated within the context of realistic and challenging processing conditions.

Strategic Approach to Marker Selection

The overarching strategy is to move beyond in silico peptide selection to empirical validation in processed food systems. The following core principles guide the selection of robust proteotypic peptides:

• Specificity: The peptide sequence must be unique to the target allergenic food and not present in other ingredients or the background matrix.
• Stability: The peptide must resist degradation and modification during food processing.
• Detectability: The peptide must ionize efficiently and generate strong, reproducible fragment ions under LC-MS/MS conditions.
• Quantifiability: The peptide's response should be consistent and proportional to the concentration of the parent allergenic protein, even after processing [32].

The workflow for this strategic approach is summarized in the following diagram.

Results and Data Analysis

Performance of Selected Peptide Markers in Baked Goods

Applying the aforementioned strategy within the ThRAll project led to the identification and validation of a panel of peptide markers for common allergens. The method's performance was rigorously tested in two baked goods produced at pilot scale: cookies (less intensive process) and rusks (more intensive process), incurred with allergens at two concentration levels (24 and 48 µg of Total Allergenic Food Protein per g of Food, µgTAFP/gF) [5]. The following table summarizes the sensitivity data and robustness across matrices for key allergens.

Table 1: Sensitivity and Robustness of Selected Peptide Markers in Processed Food Matrices

Allergenic Ingredient	Selected Proteotypic Peptide (Example)	Limit of Detection (LOD) in Chocolate Matrix (µgTAFP/gF)	Performance in Cookies & Rusks	Key Stability Observation
Cow's Milk	Multiple peptides	0.08 - 0.2 [22]	Detected in both matrices at incurred levels [5]	Markers for casein and whey proteins provide comprehensive coverage [32].
Hen's Egg	Multiple peptides	1.1 [22]	Detected in both matrices at incurred levels [5]	Robust markers selected to withstand baking temperatures.
Peanut	Multiple peptides	0.08 - 0.2 [22]	Detected in both matrices at incurred levels [5]	Peptides resistant to roasting and baking processes were identified.
Soybean	Multiple peptides	1.2 [22]	Detected in both matrices at incurred levels [5]
Hazelnut	Multiple peptides	0.08 - 0.2 [22]	Detected in both matrices at incurred levels [5]
Almond	Multiple peptides	0.08 - 0.2 [22]	Detected in both matrices at incurred levels [5]
Sesame	Specific peptides not listed	Not specified in cited results	Detected in cookie matrix at incurred levels [5]	Included due to high risk of cross-contamination in bakery chains [5].

Advanced Sample Preparation for Rapid Analysis

Recent innovations have focused on simplifying and accelerating sample preparation, a traditional bottleneck in LC-MS/MS workflows. The Suspension-Trapping (S-Trap) method offers a rapid and efficient alternative to conventional digestion protocols.

Table 2: Comparison of Sample Preparation Methods for Allergen Detection

Parameter	Conventional Digestion Protocol	S-Trap Column Method
Protein Extraction	Tris-HCl buffer, often with urea [22]	SDS-containing buffer for efficient, denaturing extraction [20]
Cleanup & Digestion	Multi-step: reduction, alkylation, overnight trypsin digestion, desalting (e.g., with C18 cartridges) [22]	All-in-column: SDS removal, reduction, alkylation, and trypsin digestion (~1 hour) [20]
Total Preparation Time	~4 to 24 hours [20]	~1.5 hours [20]
Key Advantage	Well-established, suitable for complex matrices like chocolate [22]	Speed, simplicity, and high protein recovery; effective for a wide range of processed foods [20]

Detailed Experimental Protocols

Protocol 1: Selection and Validation of Robust Proteotypic Peptides

This protocol outlines the process for identifying and validating peptide markers using incurred materials subjected to relevant processing.

4.1.1 Materials and Reagents

• Incurred Food Materials: Produced at pilot scale to mimic industrial processing (e.g., cookies baked at 180°C for 11 min, rusks with fermentation and baking) at defined allergen concentrations (e.g., 0, 24, 48 µgTAFP/gF) [5].
• Protein Extraction Buffer: 200 mM Tris-HCl, pH 8.0 [22]. Alternative: Lysis buffer with SDS for S-Trap [20].
• Digestion Reagents: Sequencing-grade trypsin, ammonium bicarbonate (ABC), dithiothreitol (DTT), iodoacetamide (IAA) [5] [22].
• Synthetic Peptides: Heavy isotope-labeled (e.g., +8 Da on Lys, +10 Da on Arg) and native (light) AQUA peptides for each candidate marker (>95% purity) [22].

4.1.2 Step-by-Step Procedure

• In-silico Selection: For each target allergenic protein, select candidate proteotypic peptides (8-20 amino acids) using software tools and public databases. Prioritize peptides without missed cleavage sites, chemically unstable residues (M, C), or known modification sites [32].
• Specificity Check: Verify the uniqueness of each candidate peptide by performing a BLAST search against a composite protein database containing all potential food ingredients and the host matrix (e.g., wheat for bakery products) [32].
• Method Development: Spiked heavy labeled synthetic peptides into processed matrix digests. Develop and optimize the LC-SRM/MRM method, including chromatography separation and mass spectrometer transition parameters, using a triple quadrupole or high-resolution MS platform [5] [22].
• Validation in Incurred Materials: a. Extract proteins from the incurred and allergen-free control materials. b. Digest the proteins using a standardized protocol (see Protocol 4.2). c. Analyze the digests using the developed LC-MS/MS method. d. A marker is considered robust if it is consistently and specifically detected in the incurred materials at both concentration levels and is absent in the control, with a defined LOD and LOQ suitable for protection of allergic consumers [5].
• Recovery Calculation: Quantify the allergen in the incurred material using a matrix-matched calibration curve with stable isotope-labeled internal standards. Calculate the overall recovery by comparing the measured concentration to the nominal (theoretical) incurred concentration [5] [22].

Protocol 2: Rapid Protein Digestion Using S-Trap Columns

This protocol describes a fast and efficient sample preparation method, ideal for screening applications [20].

4.2.1 Materials and Reagents

• S-Trap midi columns
• Extraction Buffer: 5% SDS, 50 mM ABC, 10 mM DTT
• Alkylation Solution: 50 mM IAA in 50 mM ABC
• Digestion Buffer: 50 mM ABC containing 1:25 (w/w) trypsin-to-protein ratio
• Elution Buffers: 50 mM ABC, 0.2% Formic Acid, 50% ACN/0.2% Formic Acid

4.2.2 Step-by-Step Procedure

• Protein Extraction and Reduction: Homogenize 100 mg of food sample. Add 1 mL of Extraction Buffer. Vortex, heat at 95°C for 10 min, then sonicate. Centrifuge to remove debris.
• Alkylation: Transfer the supernatant to a new tube. Add IAA alkylation solution to a final concentration of 20 mM. Incubate in the dark for 30 min.
• Protein Binding to S-Trap: Add phosphoric acid to the sample to a final concentration of 2.5%. Add 850 µL of this acidified sample to the S-Trap column. Centrifuge at 4,000 g for 1 min. Discard flow-through.
• Wash: Load 400 µL of Wash Buffer (90% methanol, 50 mM ABC) onto the column. Centrifuge at 4,000 g for 1 min. Repeat this wash step two more times.
• Digestion: Add 150 µL of Digestion Buffer (with trypsin) to the column. Ensure the buffer soaks into the resin. Incubate at 47°C for 60 min.
• Peptide Elution: Place the S-Trap column in a new collection tube. a. Elute 1: Centrifuge after adding 80 µL of 50 mM ABC. b. Elute 2: Centrifuge after adding 80 µL of 0.2% Formic Acid. c. Elute 3: Centrifuge after adding 80 µL of 50% ACN/0.2% Formic Acid.
• Combine all eluates. Dry down in a vacuum concentrator and reconstitute in 0.1% formic acid for LC-MS/MS analysis.

The S-Trap workflow is visualized below.

The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for Allergen Peptide Marker Validation

Reagent / Material	Function / Role	Example from Literature
Stable Isotope-Labeled Peptides (AQUA)	Internal standards for precise and accurate quantification; correct for sample preparation losses and ion suppression.	Heavy-labeled peptides (+8 Da Lys, +10 Da Arg) used as internal standards for calibration curves in chocolate matrix [22].
Pilot-Scale Incured Materials	Reference materials with allergens incorporated prior to processing; essential for validating marker robustness against real-world processing effects.	Cookies and rusks produced with defined levels of milk, egg, peanut, etc., at 24 and 48 µgTAFP/gF [5].
S-Trap or Similar Spin-Column Kits	Streamlined, rapid, and efficient protein digestion and cleanup; significantly reduces sample preparation time from hours to ~60 minutes.	S-Trap columns used for simple and rapid pretreatment, enabling digestion in 1 hour for screening 7 allergens in various processed foods [20].
Matrix-Matched Calibration Curves	Calibrants prepared in the same allergen-free food matrix as samples; corrects for matrix effects that can suppress or enhance analyte signal.	Used in the validation of a method for 6 allergenic ingredients in a chocolate-based matrix to ensure accurate quantification [22].
High-Resolution Mass Spectrometer	Platform for confirmatory analysis and method transfer; provides high mass accuracy for verifying peptide identity and dealing with complex matrices.	Used to transfer and confirm a multi-allergen method originally developed on a low-resolution MS platform [5].

Chromatographic Separation and MS/MS Detection with Scheduled MRM Algorithms

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful confirmatory technique for the multiplexed detection of food allergens, addressing critical limitations of traditional immunoassays [32]. The detection of allergens in processed foods presents a significant analytical challenge due to food matrix complexity, the need for high sensitivity to detect trace contamination, and protein modifications induced by thermal processing [34]. Among MS techniques, Multiple Reaction Monitoring (MRM) has become the principal method for allergen detection and quantitation due to its exceptional sensitivity, specificity, and multiplexing capability [32]. Scheduled MRM (sMRM) algorithms further enhance this approach by monitoring transitions only during predefined retention time windows, significantly increasing the number of quantifiable peptides while maintaining sensitivity [32]. This application note details the implementation of advanced MRM techniques, including a novel scout-triggered MRM (stMRM) algorithm, for robust multi-allergen screening in complex food matrices, providing researchers with validated protocols for detecting allergenic proteins in processed foods at clinically relevant concentrations.

Experimental Design and Workflow

The fundamental workflow for MS-based allergen detection follows a bottom-up proteomics approach, where proteins are extracted, digested into peptides, and analyzed via LC-MS/MS [34]. Proteotypic peptides—peptides whose presence appears robust to variations in food matrix, sample preparation protocol, and MS instrumentation—serve as specific markers for target allergenic proteins [32]. For processed foods, the selection of stable peptide markers that remain detectable after thermal treatment is paramount [34]. The introduction of intelligent acquisition modes like stMRM represents a significant advancement in MRM technology, optimizing duty cycles by triggering confirmatory peptide transitions only when corresponding marker peptides exceed predefined intensity thresholds [35]. This intelligent triggering mechanism minimizes unnecessary data acquisition and improves analytical precision for complex allergen panels.

Algorithm Workflow Visualization

The following diagram illustrates the logical relationship and workflow of the scout-triggered MRM algorithm:

Materials and Methods

Research Reagent Solutions

The table below details essential materials and reagents required for implementing the scheduled MRM protocol for allergen detection:

Table 1: Essential Research Reagents and Materials for LC-MS/MS Allergen Analysis

Item Category	Specific Examples	Function and Application
Chromatography Columns	Kinetex C18 (100 × 3.0 mm, 2.6 µm) [35]	Reversed-phase separation of tryptic peptides prior to MS injection
Digestion Enzymes	Trypsin Gold, Mass Spectrometry Grade [34]	Proteolytic digestion of extracted proteins into measurable peptides
Extraction Buffers	50 mM Trizma base in 2 M urea; 20% (w/v) octyl β-D-glucopyranoside [35]	Protein extraction and solubilization from complex food matrices
Reducing/Alkylating Agents	Tris-(2-carboxyethyl)-phosphine; iodoacetamide; methyl methane-thiosulfonate [35] [34]	Reduction and alkylation of protein disulfide bonds for complete digestion
Solid-Phase Extraction	S-Trap columns; on-line SPE systems; Sep-Pak C18 cartridges [7] [34]	Sample cleanup and peptide concentration to remove interfering compounds
Solvents	HPLC-grade acetonitrile, methanol, formic acid [34]	Mobile phase preparation and sample reconstitution

Sample Preparation Protocol

The sample preparation workflow for allergen detection requires careful optimization to ensure efficient protein extraction while minimizing matrix effects. The following diagram outlines the comprehensive sample preparation process:

Detailed Procedure:

• Sample Homogenization: Process food samples to a fine powder or paste using a food processor or mortar and pestle to ensure representative sampling [35].

• Lipid Removal: Add 5 mL hexane to 1 g of homogenized sample, vortex thoroughly, and centrifuge at 4,000 × g for 20 minutes. Carefully discard the hexane (upper) layer and repeat the process. Dry the residual hexane under a gentle stream of nitrogen gas [35].

• Protein Extraction: Add 3.8 mL of extraction buffer (50 mM Trizma base in 2 M urea) and 100 µL of denaturant (20% w/v octyl β-D-glucopyranoside) to the defatted sample. Vigorously shake the mixture for 30 minutes at room temperature. Centrifuge at 4,000 × g for 20 minutes and collect the supernatant containing the extracted proteins [35].

• Protein Reduction and Alkylation: Transfer 500 µL of protein extract to a clean microcentrifuge tube. Add 20 µL of 50 mM Tris-(2-carboxyethyl)-phosphine and incubate at 60°C for 1 hour to reduce disulfide bonds. After cooling to room temperature, add 10 µL of 100 mM methyl methane-thiosulfonate and incubate for 15 minutes to alkylate cysteine residues [35].

• Tryptic Digestion: Add 425 µL of digestion buffer (5 mM calcium chloride in 100 mM ammonium bicarbonate) and 20 µL of trypsin to the reduced and alkylated sample. Incubate overnight at 37°C for complete protein digestion. Quench the digestion by adding 30 µL of formic acid [35].

• Sample Cleanup: Centrifuge the digested sample at 12,000 × g for 10 minutes. Filter the supernatant using a 10 kDa molecular weight cut-off filter or perform solid-phase extraction using C18 cartridges. The purified peptide extract can be stored at -20°C until LC-MS/MS analysis [35] [34].

LC-MS/MS Analysis Parameters

Chromatographic Conditions:

• Column: C18 column (e.g., Kinetex C18, 100 × 3.0 mm, 2.6 µm) [35]
• Mobile Phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile [35]
• Flow Rate: 0.3 mL/min [35]
• Column Temperature: 30°C [35]
• Injection Volume: 2 µL [35]
• Gradient Program:
  • 0-1 min: 2% B
  • 1-20 min: 2-35% B
  • 20-21 min: 35-90% B
  • 21-25 min: 90% B
  • 25-26 min: 90-2% B
  • 26-30 min: 2% B (column re-equilibration) [35]

Mass Spectrometry Conditions:

• Instrument: Triple quadrupole mass spectrometer (e.g., SCIEX 7500 system) [35]
• Ionization Mode: Positive electrospray ionization [35]
• Ion Source Gas Parameters:
  • Ion Source Gas 1: 50 psi
  • Ion Source Gas 2: 60 psi
  • Curtain Gas: 35 psi
  • Collision Gas: 9 psi
  • Ionization Voltage: 5500 V
  • Source Temperature: 450°C [35]

stMRM Method Development:

• Designate the earliest eluting peptide within each allergen super group as the marker peptide [35]
• Set appropriate trigger thresholds (in cps) for marker transitions based on preliminary experiments [35]
• Configure the mass table with Super group ID, Trigger, Trigger threshold, and Triggered by: Compound ID columns [35]
• Enable Dynamic Background Subtraction (DBS) to set consistent trigger thresholds despite varying analyte concentrations and matrices [35]

Results and Data Analysis

Quantitative Performance of MRM-based Allergen Detection

The table below summarizes the analytical performance of LC-MS/MS methods with scheduled MRM algorithms for detecting various food allergens:

Table 2: Performance Characteristics of MRM-based Methods for Allergen Detection in Foods

Allergen Target	Matrix	Detection Limit	Key Marker Peptides	Reference
Pistachio & Cashew	Cereals, chocolate, sauces, meat products	1 mg/kg (SDL)	Pis v 1, Pis v 2, Pis v 3, Pis v 5 (pistachio); Ana o 1, Ana o 2, Ana o 3 (cashew)	[6]
7 Allergens (wheat, buckwheat, milk, egg, etc.)	Various processed foods	<1 mg/kg	Specific marker peptides for each allergenic protein	[7]
Multiple Allergens	Cookies and rusks	24-48 μgTAFP/gF	Validated proteotypic peptides for egg, milk, soy, almond, hazelnut, peanut, sesame	[34]
16 Allergens	Various food samples	Varies by allergen	Diagnostic marker peptides with confirmatory peptides for each allergen group	[35]

Comparative Performance of MRM Acquisition Modes

The implementation of stMRM algorithms demonstrates significant advantages over traditional sMRM approaches. In comparative studies, stMRM provided approximately 50% improvement in cycle time by avoiding unnecessary triggering of confirmatory transitions for allergens absent or present below threshold levels [35]. This intelligent acquisition mode enables more reliable peak integration through increased data points across LC peaks and allows for expanded analyte panels without sacrificing analytical precision [35]. For complex matrices like mustard samples, stMRM selectively acquired data only for present allergens (mustard peptides), while sMRM simultaneously monitored all transitions regardless of presence, resulting in inefficient duty cycle utilization [35].

Discussion

The integration of scheduled MRM algorithms, particularly the novel stMRM approach, represents a significant advancement in food allergen detection by LC-MS/MS. The primary advantage of these intelligent acquisition modes lies in their ability to optimize mass spectrometer duty cycles while maintaining the sensitivity and specificity required for regulatory compliance [35]. This is particularly valuable for food control laboratories requiring verification of pistachio or cashew nuts in cases where ELISA and PCR methods show cross-reactivity and cannot distinguish between these botanically related allergens [6].

The robustness of MRM-based methods to food processing conditions remains a critical consideration. Thermal treatments, fermentation, and other processing methods can modify allergenic proteins, potentially affecting antibody recognition in ELISA methods [34]. In contrast, MS-based methods target proteotypic peptides that remain detectable after processing, especially when markers are selected from stable protein regions [32] [34]. The validation of peptide markers in incurred food materials processed under industrial conditions, as demonstrated in bakery goods, provides confidence in method applicability to real-world samples [34].

For researchers developing MRM assays for allergen detection, key recommendations include:

• Select multiple proteotypic peptides per allergen to minimize false negatives [32]
• Validate marker peptide stability under various processing conditions relevant to the target food matrices [34]
• Implement internal standardization with stable isotope-labeled peptides when precise quantification is required [32]
• Establish trigger thresholds carefully in stMRM methods to balance sensitivity and specificity [35]

Future directions in this field include continued expansion of multiplexed panels, harmonization of reference materials and validation protocols, and integration of automated sample preparation to enhance throughput and reproducibility for routine food allergen testing in compliance with regulatory requirements.

The accurate detection of multiple food allergens in processed foods is critical for protecting consumer health and ensuring regulatory compliance. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful confirmatory technique for multi-allergen detection, overcoming limitations of immunological and DNA-based methods, especially in complex, processed matrices. This application note details validated protocols and analytical performance data for the simultaneous identification and quantification of allergenic proteins in challenging food matrices, including cookies, rusks, chocolate, and sauces. The methods leverage targeted proteomics with marker peptides to achieve high sensitivity and specificity, providing researchers with robust workflows for allergen analysis in quality control and research settings.

Food processing induces complex chemical and structural modifications to proteins, presenting significant challenges for allergen detection. Techniques like ELISA and PCR may suffer from cross-reactivity, insufficient sensitivity, or an inability to distinguish between closely related species, such as pistachio and cashew [6]. LC-MS/MS methods directly target proteotypic peptides, enabling specific, multi-analyte detection even in extensively processed foods [5]. This document outlines standardized protocols and their performance for detecting allergens in matrices that are particularly challenging due to their high fat, sugar, or pigment content, or as a result of intensive thermal treatment.

Experimental Protocols and Performance Data

Multi-Allergen Detection in Cookies and Rusks

Protocol Overview: This method was developed to simultaneously detect traces of egg, milk, soy, almond, hazelnut, peanut, and sesame in two types of baked goods with different processing intensities: cookies (moderate heat treatment) and rusks (harsh heat treatment including a fermentation step) [5].

• Incurred Sample Preparation: Cookies and rusks were produced at a pilot plant scale, incurred with allergens at two nominal concentration levels: 24 and 48 µg of Total Allergenic Food Protein per gram of Food (µgTAFP/gF). Allergen-free controls were also produced [5].
• Protein Extraction and Digestion: Proteins were extracted from homogenized samples. The extract was then reduced, alkylated, and digested with trypsin to generate peptide markers [5].
• LC-MS/MS Analysis: Analysis of proteotypic peptides was performed using a high-resolution mass spectrometry platform. The method builds upon the ThRAll project, which identified and validated 16 peptide markers for the selected allergenic ingredients [5].

Key Data and Performance: The method was evaluated for its sensitivity and the effect of food processing on allergen detection.

Table 1: Method Sensitivity for Allergen Detection in Baked Goods

Allergenic Food	Incurred Concentration (µgTAFP/gF)	Matrix	Key Performance Findings
Egg, Milk, Soy, Almond, Hazelnut, Peanut, Sesame	24 & 48	Cookies & Rusks	Method applicability confirmed; peptide marker robustness assessed under different thermal treatments [5].
Sesame	Included	Bakery Products	Identified as a priority allergen due to widespread occurrence in bakery production chains [5].

Rapid Multi-Allergen Screening in Various Processed Foods

Protocol Overview: This method was developed for the rapid, simultaneous detection of seven allergenic proteins (wheat, buckwheat, milk, egg, crustacean, peanut, and walnut) across a wide range of processed foods [7].

• Sample Preparation Innovation: The protocol utilizes Suspension-Trapping (S-Trap) columns and an on-line automated solid-phase extraction (SPE) system. This significantly streamlines and accelerates the sample preparation process, which is often a bottleneck in traditional LC-MS/MS allergen analysis [7].
• Validation with Incurred Samples: The method was validated using five different types of incurred samples amended with trace amounts of the seven allergenic proteins [7].

Key Data and Performance: The method demonstrates high sensitivity suitable for screening purposes.

Table 2: Performance of Rapid LC-MS/MS Screening Method

Parameter	Specification	Notes
Target Allergens	Wheat, buckwheat, milk, egg, crustacean, peanut, walnut	Seven major allergens [7].
Limit of Detection (LOD)	< 1 mg/kg (ppm) for each protein	Estimated from analysis of five incurred sample types [7].
Key Innovation	Use of S-Trap and on-line SPE	Enables simple, rapid pretreatment and is applicable to a wide variety of processed foods [7].

Specific Discrimination of Pistachio and Cashew in Complex Matrices

Protocol Overview: This LC-MS/MS method was developed to overcome the cross-reactivity issues of ELISA and PCR methods by providing specific and simultaneous detection of pistachio and cashew allergens [6].

• Matrices Validated: The method was validated in multiple complex matrices, including cereal-based products, chocolate-based products, sauces, and meat-based products [6].
• Method Performance: The method demonstrated a Screening Detection Limit (SDL) of 1 mg/kg for pistachio across the tested matrices. Good reproducibility was achieved for pistachio, though ongoing investigations were noted for cashew analysis [6].

Detection of Meat Allergens

Protocol Overview: A highly sensitive quantitative LC-MS/MS method was developed for characterizing meat allergens from five species: beef, lamb, pork, chicken, and duck [19].

• Surrogate Peptides: Five surrogate peptides from myoglobin and myosin light chain proteins were selected as quantitative markers for their heat stability and species specificity [19].
• Method Validation: The method was rigorously validated, showing excellent specificity, linearity (R² > 0.995), and precision (RSD < 13.8%) [19].

Key Data and Performance:

• Limits of Detection (LOD): 2.0–5.0 mg/kg for all five meat allergens.
• Limits of Quantification (LOQ): 5.0–10.0 mg/kg.
• Apparent Recoveries: 80.2%–101.5%, confirming the method's accuracy [19].

The Scientist's Toolkit: Research Reagent Solutions

The following table details essential reagents and materials used in the featured LC-MS/MS protocols for allergen detection.

Table 3: Essential Research Reagents for LC-MS/MS Allergen Analysis

Reagent/Material	Function	Specific Examples & Notes
Digestion Enzyme	Proteolytic cleavage of proteins into measurable peptides.	Sequencing-grade trypsin [5] [19].
Reducing Agent	Breaks disulfide bonds in proteins for denaturation.	Dithiothreitol (DTT) [5] [19].
Alkylating Agent	Modifies cysteine residues to prevent reformation of disulfide bonds.	Iodoacetamide (IAA) [5] [19].
Buffers	Maintains optimal pH for enzymatic and chemical reactions.	Ammonium bicarbonate (AB), Tris(hydroxymethyl)aminomethane (Tris) [5] [19].
Solid-Phase Extraction (SPE)	Purification and concentration of peptide mixtures; reduces matrix interference.	C18 cartridges [5]; On-line SPE systems for rapid screening [7].
Internal Standards	Enables absolute quantification and corrects for procedural losses.	Stable isotope-labeled (AQUA) peptides [19] [36].
LC-MS/MS Solvents	Mobile phase for chromatographic separation and ionization.	HPLC-grade acetonitrile, methanol, and water with formic acid [5] [19].

Workflow Visualization

The following diagram illustrates the standard bottom-up proteomics workflow for LC-MS/MS-based allergen detection in processed foods.

Allergen Detection Workflow

Discussion

The presented data underscores the robustness of LC-MS/MS for detecting allergens in some of the most challenging processed food matrices. The successful application of these methods in cookies, rusks, chocolate, and sauces highlights several critical advantages:

• Specificity in Complex Matrices: The ability to distinguish between closely related allergens like pistachio and cashew in complex systems such as chocolate and sauces demonstrates a key advantage over antibody-based methods [6].
• Resilience to Processing: The validation of methods in extensively processed foods like rusks confirms that carefully selected, heat-stable peptide markers can reliably track allergenic ingredients despite severe thermal and mechanical processing [5] [19].
• High Sensitivity: Achieving LODs at or below the low mg/kg (ppm) level across diverse matrices meets the stringent sensitivity requirements necessary for protecting allergic consumers [19] [7].
• Streamlined Workflows: The integration of modern sample preparation techniques, such as S-Trap and on-line SPE, addresses historical limitations of time-consuming and complex protocols, making LC-MS/MS more accessible for routine analysis [7].

The LC-MS/MS protocols detailed herein provide reliable, sensitive, and specific solutions for the detection of multiple food allergens in complex processed matrices. The methods have been rigorously validated in relevant food models, including baked goods, chocolate, and sauces, ensuring their applicability in real-world scenarios. As the field advances, further harmonization of methods, expansion of validated marker peptide libraries, and continued simplification of workflows will be essential to support global food safety initiatives and improve consumer protection.

Overcoming Matrix Effects and Processing-Induced Challenges

Managing Protein Modifications from Heating, Fermentation, and High-Pressure Processing

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has emerged as a powerful technique for the precise detection and quantification of food allergens, overcoming the limitations of antibody-based ELISA and DNA-based PCR methods which often suffer from cross-reactivity, particularly with closely related species like pistachio and cashew [6]. The accuracy of LC-MS/MS, however, is highly dependent on the ability to account for protein modifications induced by common food processing techniques. Heating, fermentation, and high-pressure processing (HPP) can alter protein structure, solubility, and the accessibility of marker peptides used for identification [37] [38] [39]. This application note provides detailed protocols for simulating these processing conditions, preparing samples for LC-MS/MS analysis, and interpreting the resulting data within the context of developing robust, multi-allergen detection methods for complex food matrices. The procedures outlined are designed to help researchers understand how processing-induced protein modifications impact the detection of allergenic proteins, thereby improving the reliability of food allergen labeling and consumer safety.

The Impact of Processing Techniques on Food Proteins

Food processing fundamentally alters the native state of proteins, which can confound analytical detection. The table below summarizes the primary effects of heating, fermentation, and high-pressure processing on proteins and the corresponding implications for LC-MS/MS analysis.

Table 1: Effects of Food Processing Techniques on Proteins and Analytical Implications

Processing Technique	Primary Effects on Proteins	Impact on LC-MS/MS Analysis
Heating	Denaturation, aggregation, Maillard reaction (glycation), oxidation [37].	Alters peptide digestibility and yield; may generate new modified peptides or obscure protease cleavage sites [37].
Fermentation	Microbial hydrolysis, changes in pH, potential deamidation, production of new microbial proteins [39].	Can degrade target allergenic proteins; introduces complex background of microbial proteins, potentially leading to interference.
High-Pressure Processing (HPP)	Protein denaturation, gelation, and aggregation without breaking covalent bonds; alteration of secondary structures [40] [38].	Can modify protein solubility and extraction efficiency; may affect antibody binding in immunoaffinity cleanup steps.
Common Outcome	Modification of protein structure, charge, solubility, and epitope/peptide accessibility.	Directly impacts the selection of robust marker peptides and the efficiency of protein extraction and enzymatic digestion.

Application Notes & Experimental Protocols

Protocol 1: Simulating Thermal Processing and LC-MS/MS Analysis

Thermal processing is a ubiquitous treatment that can induce rapid changes in protein abundance and structure. Studies on wheat grain under short-term heat stress have shown that factors beyond transcriptional regulation, such as codon usage and amino acid frequency, significantly influence protein expression changes [37]. This protocol outlines a procedure for evaluating the heat stability of allergenic proteins and selecting thermostable peptide markers.

3.1.1 Experimental Workflow for Thermal Processing

The following diagram illustrates the key stages of the thermal processing experiment.

3.1.2 Materials and Reagents

• Food Samples: Incurred food matrix (e.g., wheat-based cookie, chocolate) containing known concentrations of target allergens (e.g., 1-1000 mg/kg).
• Buffers: Protein extraction buffer (e.g., 50 mM Tris-HCl, 150 mM NaCl, pH 7.5), with and without 2% SDS.
• Enzymes: Sequencing-grade modified trypsin.
• Consumables: Low-protein-binding microcentrifuge tubes, filter units.

3.1.3 Detailed Procedure

• Sample Homogenization: Precisely weigh 1 g of the incurred food sample and homogenize it in 10 mL of protein extraction buffer using a blender or bead beater.
• Heat Treatment: Aliquot 500 µL of the homogenate into low-protein-binding microcentrifuge tubes. Subject the aliquots to a range of temperatures (e.g., 40, 60, 80, 100, 120°C) in a heating block or water bath for varying durations (e.g., 1, 5, 15, 30, 60 minutes).
• Cooling: Immediately after heat treatment, transfer the tubes to an ice bath for 10 minutes to halt further thermal denaturation.
• Protein Extraction and Digestion:
  • Centrifuge the cooled samples at 10,000 × g for 10 minutes. Collect the supernatant.
  • Quantify the soluble protein content using a Bradford or BCA assay.
  • For insoluble pellets, consider a second extraction with buffer containing 2% SDS to recover aggregated proteins.
  • Reduce, alkylate, and digest the protein extracts with trypsin using a standardized protocol (e.g., filter-aided sample preparation - FASP).
• LC-MS/MS Analysis: Reconstitute the resulting peptides in 0.1% formic acid and analyze by LC-MS/MS.
  • LC: Use a C18 column with a linear gradient of 5-35% acetonitrile over 30-60 minutes.
  • MS/MS: Operate in data-dependent acquisition (DDA) or data-independent acquisition (DIA) mode. For DDA, use a top-N method with dynamic exclusion.

3.1.4 Data Analysis Process the raw MS data using software (e.g., MaxQuant, Skyline) against a database containing the target allergen proteins. Identify and relatively quantify the extracted ion chromatograms (XICs) of proteotypic peptides. Peptides that show consistent recovery and stable signal intensity across increasing heat treatments are considered robust markers for processed foods.

Protocol 2: Evaluating High-Pressure Processing (HPP) Effects

HPP can induce protein denaturation and gelation, altering the physical and chemical properties of food. This protocol leverages HPP to study its effect on the detection of allergenic proteins in a model food system [38].

3.2.1 Materials and Reagents

• Protein Source: Purified egg white powder or another target allergenic protein [38].
• Model Food System: A simplified matrix, such as a strawberry-flavored pudding, to simulate a real food environment [38].
• HPP Equipment: A high-pressure machine with a temperature-controlled chamber.
• Buffers: Standard protein extraction and digestion buffers.

3.2.2 Detailed Procedure

• Sample Preparation: Prepare the model food system according to a defined recipe. For example, mix egg white powder with water, sugar, inulin, and strawberry concentrate, and portion into sealed polypropylene cups [38].
• High-Pressure Treatment: Treat the samples at various pressure levels (e.g., 0, 300, 450, 500, 600 MPa) for different holding times (e.g., 5, 10, 15 minutes) at a controlled temperature (e.g., 25°C) [40] [38].
• Post-HPP Analysis:
  • Texture & Solubility: Measure the hardness and syneresis of the pudding gels. Assess protein solubility in a standard buffer.
  • Protein Extraction and Digestion: Extract proteins from control and HPP-treated samples. The extraction efficiency may vary with pressure and should be reported. Proceed with standard reduction, alkylation, and tryptic digestion.
  • LC-MS/MS Analysis: Analyze the digested peptides using the LC-MS/MS parameters described in Protocol 1.3.

3.2.3 Data Analysis Monitor specific marker peptides for the target allergen. Correlate the LC-MS/MS signal intensity of these peptides with the applied pressure level and the observed changes in protein solubility and texture. This helps identify peptides that are resilient to HPP-induced denaturation.

Protocol 3: Assessing Fermentation-Induced Protein Modifications

Fermentation can introduce complex changes through microbial activity, including proteolysis and the introduction of a high background of microbial proteins. This protocol focuses on detecting native allergens in a fermented product.

3.3.1 Materials and Reagents

• Food Matrix: A slurry of the food product to be tested (e.g., cereal, sauce).
• Starter Cultures: Defined strains of lactic acid bacteria (e.g., Lactobacillus plantarum) or fungi (e.g., Fusarium venenatum) [39].
• Extraction Buffer: A denaturing buffer (e.g., containing Urea or SDS) to inactivate endogenous enzymes and ensure complete extraction.

3.3.2 Detailed Procedure

• Fermentation Setup: Inoculate the food matrix with the selected starter culture. Incubate under optimal conditions (e.g., 30-37°C for bacteria) for a defined period (e.g., 24-72 hours). Collect samples at multiple time points (0, 12, 24, 48, 72 h).
• Termination and Extraction: At each time point, collect an aliquot and immediately mix it with a denaturing extraction buffer to halt microbial activity.
• Sample Preparation for LC-MS/MS: Digest the extracted proteins using a protocol suitable for complex matrices, such as the S-Trap (Suspension-Trapping) method, which is efficient for detergent-containing samples and minimizes handling losses [7].
• LC-MS/MS Analysis with Cleanup: Use an LC-MS/MS system coupled with an on-line solid-phase extraction (SPE) system to automate cleanup and concentrate the peptides, enhancing sensitivity and throughput [7].

3.3.3 Data Analysis Track the abundance of target allergenic peptides over the fermentation timeline. A significant decrease may indicate microbial degradation. Simultaneously, search MS data against a database of the starter culture's proteome to identify potential microbial peptides that could interfere with the analysis.

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents and Materials for LC-MS/MS Allergen Detection

Item	Function/Application	Key Considerations
Sequencing-grade Trypsin	Enzyme for proteolytic digestion; generates peptides for MS analysis.	Specificity for Lys and Arg; high purity minimizes autolysis peaks.
S-Trap Micro Spin Columns	Efficient digestion and cleanup for complex, detergent-containing samples [7].	Superior to filter-aided methods for samples with high lipid or SDS content.
On-line SPE System	Automated pre-concentration and cleanup of peptides prior to LC separation [7].	Reduces manual handling, increases throughput, and improves sensitivity.
Stable Isotope-Labeled Peptide Standards	Internal standards for absolute quantification.	Corrects for ion suppression and losses during sample preparation.
UPLC-grade Solvents	Mobile phase for liquid chromatography.	High purity ensures low background noise and stable baselines.
Allergen-Incurred Food Reference Materials	Positive controls with known, homogeneously distributed allergen concentrations.	Essential for method validation and assessing extraction efficiency from a real matrix.

Workflow for LC-MS/MS Multi-Allergen Detection

Integrating the assessment of processing effects leads to a robust general workflow for multi-allergen detection, as visualized below.

Data Presentation and Analysis

The following table provides an example of quantitative data that can be generated using these protocols, illustrating how peptide recovery can be affected by different processing conditions. This data is critical for selecting the most stable marker peptides.

Table 3: Example Data: Recovery of Target Peptides from an Incurred Material After Processing

Target Allergen (Peptide Sequence)	Control (No Process)	Heat (100°C, 15 min)	HPP (600 MPa, 5 min)	Fermentation (48 h)
Pistachio (Pis v 1 peptide, ACDLLK)	100.0 ± 5.2%	15.3 ± 2.1%	85.7 ± 6.5%	32.8 ± 4.7%
Pistachio (Pis v 3 peptide, TANDILNR)	100.0 ± 4.8%	89.5 ± 5.3%	92.1 ± 4.9%	10.5 ± 2.2%
Egg White (Ovotransferrin peptide, VASLLR)	100.0 ± 3.9%	95.2 ± 4.1%	45.6 ± 3.8%	88.9 ± 5.1%
Milk (Casein peptide, VPQLEIVPNSAEER)	100.0 ± 5.5%	5.8 ± 1.5%	78.3 ± 5.0%	98.5 ± 4.3%

Note: Values are mean percent recovery relative to the unprocessed control ± standard deviation (n=3). This simulated data demonstrates that peptides from the same protein (e.g., Pis v 1 and Pis v 3) can exhibit vastly different stabilities, underscoring the need for rigorous peptide selection.

Addressing Signal Suppression and Enhancement in Complex Food Matrices

The detection and quantification of multiple food allergens in processed foods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful approach, yet it is frequently compromised by the phenomenon of matrix effects. Matrix effects are defined as the combined influence of all components of the sample other than the analyte on the measurement of the quantity [41] [42]. In the context of LC-MS/MS-based multi-allergen detection, these effects manifest as signal suppression or enhancement, fundamentally altering the ionization efficiency of target allergenic peptides and thereby jeopardizing the accuracy, reliability, and reproducibility of analytical results [41] [42]. Signal suppression is the more commonly reported issue, where co-eluting matrix components inhibit the ionization of target analytes, potentially leading to false negatives. Conversely, signal enhancement can cause overestimation of allergen concentrations, resulting in false positives [41]. These challenges are particularly pronounced in the analysis of processed foods, where ingredients undergo various treatments that increase sample complexity and introduce a wide range of potential interferents, including lipids, salts, carbohydrates, and phenolic compounds [6] [2]. Addressing these matrix effects is therefore not merely an optional optimization step but a critical component of developing robust, validated protocols for official food allergen control [6].

Mechanisms and Impact of Signal Suppression/Enhancement

Fundamental Mechanisms

The predominant mechanism for matrix effects in ESI-LC-MS/MS occurs in the ionization source, where undesired matrix components co-elute with the target analytes and alter the droplet formation, evaporation, or ion emission processes [41]. These interfering species can be endogenous components of the food sample, compounds released during sample preparation, or even reagents added to the mobile phase [41]. They can affect ionization through several pathways: by competing with the analyte for available charge, by altering the droplet surface tension, by increasing the viscosity of the solution, or by forming adducts with the analyte [41]. In the context of food allergen analysis, where target peptides are often present at trace levels amidst a vast excess of other food components, even minor matrix interferences can have a disproportionately large effect on quantification.

Impact on Analytical Figures of Merit

Matrix effects heavily influence key analytical performance parameters. They can degrade detection capability by reducing the signal-to-noise ratio, compromise method selectivity by altering fragmentation patterns, and impair repeatability and accuracy by introducing variability that is not related to the true analyte concentration [41]. This can ultimately lead to both false-negative and false-positive diagnostic results. A false negative may occur if signal suppression precludes the detection of an allergen present at a clinically relevant level, posing a serious health risk to consumers. A false positive might occur if signal enhancement causes a non-hazardous sample to appear contaminated, leading to unnecessary product recalls and economic losses [41] [2]. Furthermore, the lack of harmonized methodological regulations for allergen detection means that laboratories must rigorously validate their methods in-house, with the assessment of matrix effects being an indispensable part of this process [6].

Experimental Protocols for Assessing Matrix Effects

A critical step in managing matrix effects is their systematic evaluation. The following protocols, based on post-extraction addition, provide a reliable means for their determination.

Protocol 1: Single-Point Measurement

This method is efficient for a rapid assessment of matrix effects at a specific concentration level, such as the limit of quantification or a relevant action level [42].

• Sample Preparation: Prepare a minimum of five (n=5) replicates of the sample matrix. Perform a full extraction and clean-up procedure as per your analytical method.
• Spiking:
  • Set A (Solvent Standard): Prepare a standard of the target allergenic peptide(s) in pure solvent at the chosen concentration (e.g., 1 mg/kg).
  • Set B (Post-Extraction Spike): Spike the same concentration of the target peptide(s) into the final extracted and cleaned sample matrix.
• Instrumental Analysis: Analyze all samples (Set A and Set B) in a single analytical run under identical LC-MS/MS conditions.
• Calculation: Calculate the Matrix Effect (ME) for each analyte using the peak areas.
  • Formula: ME (%) = [(B - A) / A] * 100
  • Interpretation: A negative value indicates signal suppression; a positive value indicates signal enhancement. As a rule of thumb, effects beyond ±20% are considered significant and require mitigation [42].

Protocol 2: Calibration Curve Comparison

This protocol provides a more comprehensive view of matrix effects across the working range of the method and is more robust for quantitative methods.

• Sample Preparation: Prepare calibration standards in both solvent (Set A) and matrix (Set B). The matrix-based standards are created by spiking the target allergenic peptides into the final extract of a blank matrix (post-extraction) at multiple concentration levels covering the linear range.
• Instrumental Analysis: Analyze all calibration standards in a single analytical run.
• Calculation: Plot the peak response against the known concentration for both the solvent and matrix-based calibration curves. Calculate the Matrix Effect (ME) using the slopes of the curves.
  • Formula: ME (%) = [(mB - mA) / mA] * 100
  • Where mA is the slope of the solvent-based curve and mB is the slope of the matrix-matched curve.
  • Interpretation: Similar to Protocol 1, values beyond ±20% signify a need for corrective action [42].

It is crucial to differentiate matrix effects from extraction efficiency. The recovery of the extraction process (RE) is determined by spiking the analyte into the sample before extraction (Set C) and comparing its peak area to the post-extraction spike (Set B): RE (%) = (C / B) * 100 [42] [43]. This helps identify whether poor method performance is due to inefficient extraction or ionization suppression/enhancement.

The workflow below illustrates the logical sequence for performing this combined assessment of matrix effects and extraction efficiency.

Quantitative Data on Matrix Effects in Food Analysis

The following tables summarize experimental data related to matrix effects, providing insight into their prevalence and impact.

Table 1: Calculated Matrix Effects and Performance for Selected Analytes [42]

Analyte	Sample Matrix	Matrix Effect (ME)	Classification	Apparent Recovery
Fipronil	Raw Egg	-30%	Suppression	-
Picolinafen	Soybeans	+40%	Enhancement	-
Various Mycotoxins, Pesticides, & Pharmaceuticals	Compound Feed	-	Suppression (Primary Source of Error)	60–140% (for 51–72% of analytes)

Table 2: LC-MS/MS Allergen Method Performance in Food Matrices [6] [7]

Target Allergen	Food Matrix	Screening Detection Limit (SDL)	Key Challenge / Note
Pistachio	Cereals, Chocolate, Sauces, Meat	1 mg/kg	Good reproducibility achieved.
Cashew	Cereals, Chocolate, Sauces, Meat	1 mg/kg	Ongoing investigations needed to overcome constraints.
Wheat, Buckwheat, Milk, Egg, Crustacean, Peanut, Walnut	Various Processed Foods (Incurred Samples)	< 1 mg/kg	Method uses S-Trap and on-line SPE for rapid, reliable detection of 7 allergens.

Strategies for Mitigation of Matrix Effects

Several strategies can be employed to minimize or compensate for the impact of matrix effects in LC-MS/MS allergen analysis.

• Sample Preparation and Clean-up: The most direct approach is to remove the interfering compounds. The use of Suspension-Trapping (S-Trap) columns and automated on-line solid-phase extraction (SPE) has been shown to simplify and improve the pretreatment process for complex food samples, effectively reducing matrix complexity [7]. The choice of sorbent is critical and should be optimized for the specific analyte-matrix combination.
• Chromatographic Resolution: Optimizing the liquid chromatography method to achieve better separation of the target allergenic peptides from co-extracted matrix components can significantly reduce ion suppression at the source. This includes adjusting the gradient, using different stationary phases, or employing UHPLC for higher peak capacity [41].
• The Use of Internal Standards: This is considered the gold standard for compensating for matrix effects. Stable Isotope-Labeled Internal Standards (SIL-IS) are ideal because they are chemically identical to the target analytes and co-elute with them, undergoing the same matrix effects. The analyte-to-internal standard peak area ratio remains constant, allowing for accurate quantification [6]. While costly, they are highly effective.
• Matrix-Matched Calibration: This involves constructing the calibration curve in a blank matrix extract that is representative of the sample. While this can effectively correct for matrix effects, it requires a reliable and consistent source of blank matrix, which can be challenging to find for complex or varied food products [43].
• Standard Addition: In this method, the sample is spiked with known increments of the analyte, and the concentration is determined by extrapolation. While highly effective in compensating for matrix effects, it is labor-intensive and not practical for high-throughput analysis [41].

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Reagents and Materials for LC-MS/MS Allergen Analysis

Item	Function in the Protocol
Stable Isotope-Labeled Internal Standards (SIL-IS)	Added prior to extraction; corrects for losses during sample preparation and matrix effects during ionization, enabling reliable quantification [6].
S-Trap (Suspension-Trapping) Columns	Used for efficient and rapid digestion and clean-up of protein extracts, simplifying the complex sample preparation traditionally associated with allergen analysis [7].
On-line Automated Solid-Phase Extraction (SPE) System	Integrated with the LC-MS/MS system for automated and effective purification and concentration of allergenic peptide extracts, reducing manual labor and improving reproducibility [7].
LC-QqQ (Triple Quadrupole) Mass Spectrometer	The preferred platform for targeted analysis in official food control due to its high sensitivity, specificity, and reproducibility in Multiple Reaction Monitoring (MRM) mode [6].
C18 Reverse-Phase Chromatography Column	The standard workhorse for chromatographic separation of peptides; provides robust separation of allergenic peptide markers from complex food digests [43].
Trypsin (Sequencing Grade)	The enzyme of choice for proteolytic digestion of allergenic proteins into characteristic peptides for LC-MS/MS analysis [6] [2].
Model Compound Feed/Formulas	In-house prepared blank matrices used during method validation to simulate compositional uncertainties and provide a more realistic estimation of method performance in the absence of a true blank [43].

Optimizing Limits of Detection (LOD) and Quantification (LOQ) for Trace Analysis

The detection and quantification of trace allergens in processed foods present a significant analytical challenge for food safety. Method sensitivity, defined by the Limit of Detection (LOD) and Limit of Quantification (LOQ), is critical for protecting consumers with food allergies, as even minute amounts of an allergen can trigger severe reactions [6]. Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) has emerged as a powerful confirmatory technique for this application, offering high sensitivity, specificity, and the ability to multiplex multiple allergens in a single run [6] [44]. This document details optimized protocols and application notes for establishing low LODs and LOQs in the context of LC-MS/MS multi-allergen detection in complex food matrices, supporting rigorous risk assessment and regulatory compliance [44].

Performance Data for Multi-Allergen Methods

Sensitive and robust LC-MS/MS methods have been developed and validated for various allergenic foods in complex matrices. The following tables summarize reported performance data from recent studies.

Table 1: LOD and LOQ for Allergens in a Chocolate Matrix [44]

Allergenic Ingredient	LOD (µgTAFP/gfood)	LOQ (µgTAFP/gfood)
Cow's Milk	0.08	-
Peanut	0.1	-
Hazelnut	0.1	-
Almond	0.2	-
Hen's Egg	1.1	-
Soybean	1.2	-

Note: TAFP = Total Allergenic Food Protein. The method achieved good sensitivity, with LODs compliant with various threshold doses issued or recommended worldwide [44].

Table 2: Method Applicability in Bakery Goods [34]

Parameter	Details
Matrices Tested	Cookies, Rusks
Allergens Targeted	Egg, Milk, Soy, Almond, Hazelnut, Peanut, Sesame
Incurred Levels	24 and 48 µgTAFP/gfood
Key Challenge	Validation of proteotypic peptide-markers in extensively processed foods (e.g., baking, fermentation) to ensure robust detection.

Experimental Protocols

Sample Preparation and Protein Extraction

A rigorously optimized sample preparation protocol is fundamental for achieving low LODs and LOQs, particularly in challenging matrices like chocolate [44].

• Sample Homogenization: Grind approximately 15 g of chocolate sample (e.g., three 5 g bars) using a laboratory blender under refrigerated conditions to avoid melting. Sieve the ground material through a 1 mm mesh [44].
• Protein Extraction: Weigh a 2 g aliquot of the homogenized sample into a suitable tube. Add 20 mL of a pre-chilled Tris-HCl extraction buffer (200 mM Tris·HCl, pH 9.2, containing 5 M urea). Vigorously stir the mixture for 30 minutes at room temperature [44]. The alkaline pH and chaotropic agent (urea) facilitate protein solubilization from the lipid- and polyphenol-rich matrix.
• Clarification: Centrifuge the extract to remove particulate matter. Filter the supernatant using a 5 μm cellulose acetate syringe filter [44].

Protein Digestion and Peptide Clean-up

This step converts proteins into measurable peptides and removes interfering compounds.

• Desalting: Pass the clarified extract through a disposable desalting cartridge (e.g., PD-10) [44].
• Reduction and Alkylation: To the protein extract, add dithiothreitol (DTT) to a final concentration of 10 mM and incubate at 56°C for 45 minutes to reduce disulfide bonds. Subsequently, add iodoacetamide (IAA) to a final concentration of 20 mM and incubate in the dark at room temperature for 30 minutes to alkylate the cysteine residues [34].
• Enzymatic Digestion: Add Trypsin Gold, Mass Spectrometry Grade at an enzyme-to-substrate ratio of 1:20 (w/w). Incubate at 37°C for 4-16 hours to achieve complete digestion [44] [34].
• Peptide Purification: Condition a Strata-X polymeric reversed-phase cartridge (33 μm, 30 mg/1 mL) with methanol and equilibrate with water. Load the digested sample onto the cartridge. Wash with water and elute peptides with a suitable solvent (e.g., aqueous acetonitrile). Evaporate the eluent to dryness under a gentle stream of nitrogen and reconstitute the peptide pellet in a mobile phase compatible with LC-MS/MS analysis (e.g., 0.1% formic acid in water) [44].

LC-MS/MS Analysis

The core analytical setup for sensitive and specific detection.

• Liquid Chromatography:

  • Column: Use a C18 reversed-phase column for peptide separation.
  • Mobile Phase: A) 0.1% Formic Acid in Water, B) 0.1% Formic Acid in Acetonitrile.
  • Gradient: Employ a linear gradient from low to high organic solvent (e.g., 2% to 40% B) over 20-40 minutes, optimized for the specific peptide markers.
  • Flow Rate: 0.3-0.5 mL/min.
  • Column Temperature: Maintain at 40-50°C [44] [34].

• Mass Spectrometry:

  • Instrument Platform: Triple Quadrupole (LC-QqQ) mass spectrometer, operated in positive electrospray ionization (ESI+) mode [6] [44].
  • Acquisition Mode: Multiple Reaction Monitoring (MRM). For each target peptide, define one precursor ion and at least two specific fragment ions [44].
  • Source Parameters:
    • Ion Spray Voltage: 5500 V
    • Source Temperature: 500°C
    • Curtain Gas: 25-35 psi
    • Nebulizer Gas (GS1): 40-50 psi
    • Heater Gas (GS2): 50-60 psi [44].

Calibration and Quantification

• Synthetic Peptide Standards: Use custom AQUA synthetic peptides (both native "light" and isotopically labelled "heavy" versions) as prototypic markers and internal standards [44].
• Calibration Curves: Prepare matrix-matched calibration curves (MMCC) using the "light" peptides in the blank matrix extract, covering a concentration range of two orders of magnitude. Include a fixed concentration of "heavy" labelled internal standards in all samples and calibrators to correct for variability in sample preparation and ionization [44].
• Quantification: The final quantitative information is reported as the mass fraction of total allergenic food protein per mass of food (µgTAFP/g_food). This requires the use of a conversion factor to translate the accurately measured peptide concentration into a relevant risk assessment unit [44].

Workflow and Data Interpretation

The following diagram illustrates the complete multi-step process for allergen detection in food, from sample receipt to data analysis.

LC-MS/MS Data Acquisition Logic

The mass spectrometer operates using Data-Dependent Acquisition (DDA) to select ions for fragmentation, which is crucial for method development.

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of a sensitive multi-allergen LC-MS/MS method requires specific, high-quality materials and reagents.

Table 3: Key Research Reagent Solutions for LC-MS/MS Allergen Detection

Item	Function / Purpose	Example / Specification
Trypsin, MS Grade	Enzyme for specific proteolytic cleavage of proteins into peptides for MS analysis.	Trypsin Gold, Mass Spectrometry Grade [44] [34].
Synthetic Peptides	Native ("light") and isotopically labelled ("heavy") internal standards (IS) for precise identification and quantification.	AQUA QuantPro and AQUA Basic grade peptides; purity >95% [44].
Solid-Phase Extraction (SPE)	Purification and concentration of peptide digests to remove salts and matrix interferents.	Strata-X polymeric reversed phase cartridges [44].
Desalting Cartridges	Initial crude purification to remove high concentrations of salts and buffers from protein extracts.	PD-10 disposable desalting cartridges [44] [34].
Chromatography Solvents	High-purity solvents for mobile phases to ensure optimal LC separation and minimal MS background noise.	LC-MS Grade Water, Acetonitrile, Formic Acid [34].
Buffer Components	For protein extraction, reduction, and alkylation (e.g., Tris-HCl, Urea, DTT, IAA).	Molecular biology grade reagents [44] [34].
Syringe Filters	Clarification of protein extracts prior to digestion and clean-up.	Cellulose acetate, 5 μm pore size [44] [34].

Optimizing LOD and LOQ for trace allergen analysis in processed foods demands an integrated approach, spanning from meticulous sample preparation to robust instrumental analysis. The protocols and data presented herein provide a validated framework for achieving the high sensitivity and specificity required for official food control and protecting allergic consumers. Key factors for success include the use of matrix-matched calibration with stable isotope-labelled internal standards, rigorous optimization of sample extraction to overcome matrix effects, and the selection of proteotypic peptide markers that remain stable during food processing [6] [44] [34].

Strategies for Discriminating Cross-Reactive Allergens (e.g., Pistachio vs. Cashew)

Cross-reactive allergens present a significant challenge in food safety analysis, particularly for closely related tree nuts like pistachio and cashew. Immunological methods, such as ELISA, often struggle to distinguish between these allergens due to shared immunoglobulin E (IgE) epitopes [45]. This application note details a robust LC-MS/MS-based protocol for the precise discrimination and quantification of cashew and pistachio allergens in processed foods, designed for integration into a broader research framework on multi-allergen detection.

The method leverages unique marker peptides derived from specific allergenic proteins, enabling unambiguous identification even in complex food matrices. The following sections provide a complete experimental workflow, from sample preparation to data interpretation, tailored for researchers and scientists in drug and analytical method development.

Background and Scientific Challenge

Cashew (Anacardium occidentale) and pistachio (Pistacia vera) belong to the Anacardiaceae family, which explains the high degree of clinical cross-reactivity observed between them [45]. This cross-reactivity stems from shared protein families and structurally similar allergens.

Major Allergens and Cross-Reactive Proteins:

• Cashew Allergens: Ana o 1 (vicilin-like protein), Ana o 2 (legumin), Ana o 3 (albumin) [45] [46].
• Pistachio Allergens: Pis v 1 (albumin), Pis v 2 (vicilin-like protein), Pis v 3 (legumin), and a magnesium superoxide dismutase [45].

The primary scientific challenge is the homology within protein families. For instance, the vicilin-like proteins Ana o 1 (cashew) and Pis v 2 (pistachio) may share conserved amino acid sequences, leading to antibody cross-reactivity in immunoassays and potential false-positive or overestimated results [47]. LC-MS/MS overcomes this limitation by targeting proteotypic peptides—unique peptide sequences that serve as a definitive fingerprint for each specific allergenic protein, allowing for clear discrimination.

Experimental Protocol

Materials and Reagents

• Samples: Incurred food materials, spiked food matrices, or suspect commercial products.
• Chemicals: Urea, dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC), HPLC-grade water, acetonitrile (ACN), and formic acid (FA).
• Enzymes: Sequencing-grade trypsin (Promega).
• Consumables: S-Trap micro spin columns (ProtiFi) or equivalent, on-line Solid-Phase Extraction (SPE) cartridges.

Equipment

• LC-MS/MS System: Triple quadrupole mass spectrometer (e.g., SCIEX Triple Quad or equivalent) coupled to a nano-flow or micro-flow UHPLC system.
• Chromatography Column: Reversed-phase C18 column (e.g., 150 mm x 0.3 mm, 2.7 µm).
• Centrifuge, vortex mixer, and thermomixer.

Detailed Step-by-Step Workflow

The entire sample preparation and analysis workflow is designed for reliability and can be visualized in the following diagram:

Protein Extraction and Denaturation

• Homogenize 1 g of food sample with 10 mL of extraction buffer (6 M Urea, 50 mM ABC, pH 8.0).
• Add DTT to a final concentration of 10 mM and incubate at 60°C for 30 minutes to reduce disulfide bonds.
• Alkylate by adding IAA to 20 mM final concentration and incubating in the dark at 25°C for 30 minutes.

Protein Digestion and Peptide Clean-up

• Digest using S-Trap columns according to the manufacturer's protocol. Briefly, dilute the sample with ABC, load onto the S-Trap, and digest with trypsin (1:20 enzyme-to-protein ratio) at 37°C for 2 hours [7].
• Elute peptides with 50% ACN/0.1% FA, followed by 0.1% FA. Dry the eluents using a vacuum concentrator and reconstitute in 0.1% FA for LC-MS/MS analysis.

This method simplifies the traditionally complex pre-treatment and improves reproducibility [7].

LC-MS/MS Analysis

The reconstituted peptides are separated and detected using the following parameters, which are critical for achieving high sensitivity and specificity.

• Chromatography:

  • Column: Reversed-phase C18 (150 mm x 0.3 mm, 2.7 µm).
  • Mobile Phase A: 0.1% Formic acid in water.
  • Mobile Phase B: 0.1% Formic acid in acetonitrile.
  • Gradient: 2% to 35% B over 20 minutes, followed by a wash and re-equilibration.
  • Flow Rate: 5 µL/min (micro-flow) [2].
  • On-line SPE can be integrated for automated sample clean-up and concentration, enhancing sensitivity [7].

• Mass Spectrometry (MRM Mode):

  • Ion Source: Electrospray Ionization (ESI), positive mode.
  • Ion Spray Voltage: 5500 V.
  • Source Temperature: 300°C.
  • Nebulizer Gas: 20 psi.
  • For each target peptide, define a minimum of two precursor ion → product ion transitions (Multiple Reaction Monitoring, MRM). The most intense transition is used for quantification, and the others for qualification to ensure specificity [2].

Data Analysis and Interpretation

Marker Peptide Selection

The core of this strategy is the selection of unique marker peptides that definitively distinguish cashew from pistachio proteins. The table below summarizes recommended target peptides based on major allergenic proteins.

Table 1: Marker Peptides for Discriminating Cashew and Pistachio Allergens

Allergen Source	Target Protein	Unique Marker Peptide Sequence	MRM Transitions (Precursor → Product)
Cashew	Ana o 1 (Vicilin)	VLDALQEPQISPR	727.9 → 1029.5 (y9)727.9 → 886.4 (y8)
Cashew	Ana o 2 (Legumin)	AQTVSIVVIPPNR	678.9 → 911.5 (y8)678.9 → 798.4 (y7)
Pistachio	Pis v 2 (Vicilin-like)	TANDILNQLEQIR	727.4 → 1001.5 (y9)727.4 → 872.4 (y8)
Pistachio	Pis v 3 (Legumin)	SPQIDNLYQALR	713.9 → 1050.5 (y9)713.9 → 921.4 (y8)

Confirmation of peptide identity requires:

• Co-elution of all MRM transitions for a given peptide.
• Matching the ion ratio between qualifier and quantifier transitions to that of a pure standard (typically within ±20-25%).
• A retention time consistent with the standard (typically within ±0.1 min).

Quantification

• Generate a calibration curve using known concentrations of the synthesized marker peptides or protein extracts spiked into a blank food matrix.
• Integrate the peak areas of the quantifier transition for each peptide in the samples and calibrators.
• Plot the calibration curve (area vs. concentration) and use it to calculate the concentration of the allergenic protein in the test samples.

The developed method has been demonstrated to achieve a limit of detection (LOD) of <1 mg/kg (1 ppm) for allergenic proteins in processed foods, which is sufficient to protect allergic consumers [7].

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 2: Key Reagent Solutions for LC-MS/MS Allergen Analysis

Item	Function / Application in the Protocol
S-Trap Micro Spin Columns	Enables efficient digestion and clean-up of proteins from complex matrices, streamlining sample preparation [7].
Sequencing-Grade Trypsin	Protease that specifically cleaves proteins at the C-terminal side of lysine and arginine residues, generating peptides for MS analysis.
Urea & Alkylating Agents (DTT, IAA)	Used for protein denaturation, reduction of disulfide bonds, and alkylation of cysteine residues to prevent reformation.
On-line SPE Cartridge	Integrated with the LC system for automated desalting and concentration of peptide samples, improving sensitivity and robustness [7].
Synthetic Marker Peptides	Serve as pure standards for method development, optimization of MRM transitions, and creating calibration curves for quantification.
Stable Isotope-Labeled Peptides	Internal standards added to each sample to correct for sample loss, ion suppression, and variability during sample preparation and MS analysis.

Method Validation

For regulatory acceptance and research reliability, the developed method should be validated according to international guidelines. Key performance characteristics to evaluate include:

• Specificity: No interference in blank matrix samples.
• Linearity: R² > 0.99 for the calibration curve.
• Accuracy and Precision: Recovery of 80-120% with RSD < 20% at the limit of quantification (LOQ).
• Sensitivity: LOD and LOQ determined to be fit-for-purpose, typically in the low ppm range.
• Matrix Effects: Evaluated by comparing the response of spiked samples to neat standards.

This application note provides a detailed LC-MS/MS protocol for the unambiguous discrimination of cross-reactive cashew and pistachio allergens. By targeting unique protein-specific marker peptides and employing a robust sample preparation workflow featuring S-Trap columns, the method overcomes the limitations of antibody-based assays. The outlined strategy offers researchers a highly specific, sensitive, and reliable tool for allergen detection in complex processed foods, contributing significantly to food safety and public health.

The Role of High-Resolution Accurate Mass (HRAM) Systems in Enhancing Specificity

High-Resolution Accurate Mass (HRAM) spectrometry represents a transformative advancement in food safety analysis, particularly for the specific detection of multiple allergens in processed foods. This application note details comprehensive methodologies and experimental protocols demonstrating how HRAM systems overcome critical limitations of traditional techniques like ELISA and PCR. By enabling untargeted screening, retrospective analysis, and superior discrimination between closely related allergenic proteins, HRAM technology provides a confirmatory, multi-analyte approach essential for compliance with global food labeling regulations and protection of consumer health. The protocols outlined herein establish a robust framework for implementing HRAM-based detection in analytical laboratories.

The accurate detection of food allergens is a critical public health imperative, with an estimated 150 million people worldwide suffering from food allergies and the prevalence steadily increasing [18]. For allergic consumers, precise food labeling is a necessity, not a luxury, as even trace amounts of undeclared allergens can trigger severe reactions including anaphylaxis [6] [18].

Traditional allergen detection methods, primarily enzyme-linked immunosorbent assays (ELISA) and polymerase chain reaction (PCR), present significant limitations for modern food analysis. ELISA, while rapid and easy to use, is susceptible to antibody cross-reactivity and may yield false positives or false negatives, particularly with processed foods where proteins become modified [18]. PCR targets DNA rather than the allergenic proteins themselves, making it unsuitable for detecting allergens in heavily processed foods where DNA may be degraded, and incapable of distinguishing between tissue proteins and by-product proteins (such as chicken versus egg) [18].

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful alternative, directly detecting signature peptides from allergenic proteins [17] [18]. Within this field, High-Resolution Accurate Mass (HRAM) systems, such as Orbitrap technology, provide enhanced specificity through precise mass measurement, overcoming the limitations of both traditional techniques and nominal mass MS instruments [48].

HRAM Technology: Advantages for Allergen Detection

HRAM mass spectrometry provides several distinct advantages that make it particularly suited for the specific detection of allergens in complex food matrices.

Specificity Through Accurate Mass Measurement

The core strength of HRAM systems lies in their ability to measure the mass-to-charge ratio (m/z) of ions with exceptional precision and accuracy—typically within < 5 ppm of their theoretical value [49]. This allows for the confident determination of elemental composition of ions, which is crucial for distinguishing target allergenic peptides from chemical background interference in complex food samples [48] [49]. While triple quadrupole (QqQ) MS/MS systems operating in MRM mode are highly sensitive for targeted analysis, they are limited to a pre-specified compound list and cannot retrospectively search for untargeted compounds [49].

Table 1: Comparison of Allergen Detection Techniques

Parameter	ELISA	PCR	LC-QqQ MS/MS	LC-HRAM MS
Target of Analysis	Intact protein (Antibody-based)	DNA	Signature peptides	Signature peptides
Specificity	Susceptible to cross-reactivity [18]	High for DNA, but indirect [18]	High (MRM transitions)	Very High (Accurate mass) [48]
Multiplexing Capacity	Single allergen per test [18]	Multiple allergens possible with specific primers [18]	Targeted multi-allergen (e.g., 12 allergens) [17]	Comprehensive multi-allergen & untargeted [48]
Susceptibility to Food Processing	High (Protein modification affects antibodies) [18]	High (DNA degradation) [18]	Medium (Relies on pre-defined peptides)	Lower (Can identify stable peptides) [50]
Retrospective Analysis	Not possible	Not possible	Not possible	Yes, with full scan data [48]
Quantification	Possible	Difficult [18]	Excellent linearity [17]	Excellent

Untargeted Screening and Retrospective Analysis

HRAM instruments acquire full-scan data with high sensitivity and selectivity, enabling both targeted quantification and untargeted screening for "known unknowns"—compounds known to exist in databases but not specifically targeted in the initial method [49]. This capability is vital for identifying unexpected contaminants or novel allergen markers without re-injecting samples. The data can be re-interrogated later as new information emerges, protecting the long-term value of analytical results [48].

Experimental Protocols: HRAM-Based Multi-Allergen Detection

The following section provides a detailed workflow and protocol for the detection of multiple food allergens using HRAM LC-MS/MS.

The complete analytical process, from sample receipt to final reporting, is visualized in the following diagram:

Detailed Sample Preparation Protocol

Robust sample preparation is critical for accurate allergen detection, especially in processed foods where matrices are complex and proteins may be modified [18].

• Sample Homogenization: Weigh 1 g of finely homogenized food sample (e.g., cookie, bread, chocolate) [17]. For challenging matrices, a cryogenic grinding step with liquid nitrogen may be incorporated to ensure uniform particle size.
• Protein Extraction: Add 4 mL of extraction buffer (e.g., 50 mM ammonium bicarbonate, 8 M urea, 10 mM dithiothreitol (DTT)) to the homogenate. Vortex thoroughly for 5 minutes. The reducing environment with DTT breaks disulfide bonds, denaturing proteins for more efficient digestion [17] [18].
• Reduction and Alkylation: Incubate the extract at 60°C for 1 hour to facilitate reduction. After cooling to room temperature, alkylate by adding iodoacetamide to a final concentration of 20 mM and incubating in the dark for 30 minutes. This step prevents reformation of disulfide bonds and ensures complete digestion [17] [18].
• Trypsin Digestion: Add a digestion buffer (e.g., 50 mM ammonium bicarbonate, 10% acetonitrile) and trypsin (e.g., 20 µg) to the alkylated protein extract. Incubate at 37°C for 3-12 hours to allow complete digestion of proteins into peptides [17]. The reaction is then stopped by acidification with formic acid.
• Solid-Phase Extraction (SPE) Clean-up: Pass the digested sample through a conditioned SPE cartridge (e.g., Strata-X, 200 mg/6 mL). Wash with 3 mL of 0.5% trifluoroacetic acid in water. Elute peptides with 6 mL of acetonitrile. Evaporate the eluent to dryness under a gentle nitrogen stream and reconstitute in 300 µL of reconstitution solution (95:5:0.5 water–acetonitrile–formic acid) [18]. This step removes interfering matrix components and concentrates the target peptides.

LC-HRAM/MS Analysis Parameters

Chromatographic separation and mass spectrometric detection are optimized for the specific and sensitive detection of allergenic peptides.

• Liquid Chromatography:

  • Column: Phenomenex Kinetex C18, 2.6 µm, 100 x 3.0 mm or equivalent.
  • Mobile Phase: A) 0.1% formic acid in water; B) 0.1% formic acid in acetonitrile.
  • Gradient: Linear gradient from 5% B to 90% B over 12 minutes at a flow rate of 300 µL/min [17].
  • Injection Volume: 30 µL.

• HRAM Mass Spectrometry (Orbitrap-based System):

  • Ionization: Positive electrospray ionization (ESI+).
  • Source Temperature: 500°C.
  • Resolution: ≥ 60,000 full width at half maximum (FWHM).
  • Scan Mode: Full-scan MS (m/z 350-1800) with data-dependent MS/MS (dd-MS2) for the top N most intense ions.
  • Collision Energy: Stepped normalized collision energy (e.g., 25, 35%) for comprehensive fragmentation.

Data Processing and Analysis

• Peptide Identification: Process raw data using software (e.g., Compound Discoverer, Thermo Scientific) for peak picking, alignment, and component detection [49]. Identify peptides by searching accurate mass and MS/MS spectra against a custom database of allergenic protein sequences.
• Confidence Criteria: Confirm identifications using a multi-parameter approach:
  • Mass Accuracy: < 5 ppm for both precursor and fragment ions.
  • Isotopic Pattern: Match to theoretical distribution.
  • Retention Time: Consistency with standards.
  • Fragmentation Spectrum: High spectral similarity score (e.g., using mzCloud library or in silico prediction tools) [49].
• Quantification: Use external calibration curves or the standard addition method with isotopically labelled internal standards where available [6]. For label-free approaches, demonstrate linearity and reproducibility over the relevant concentration range.

Case Study: Specific Discrimination of Pistachio and Cashew Allergens

A recent study highlights the superior specificity of LC-MS/MS in resolving a challenging analytical problem: discriminating between pistachio and cashew allergens, which belong to the same botanical family (Anacardiaceae) and share similar protein and DNA sequences [6].

• Challenge: Traditional ELISA and PCR methods often suffer from cross-reactivity between pistachio and cashew, limiting their ability to reliably distinguish which nut is present in a food sample [6].
• HRAM Solution: The developed LC-MS/MS method targeted unique signature peptides from the major allergenic proteins of pistachio (Pis v 1, Pis v 2, Pis v 3, Pis v 5) and cashew (Ana o 1, Ana o 2, Ana o 3). The high resolution and accurate mass measurement allowed for the selective detection of these markers.
• Validation: The method was validated for specificity, precision, and ruggedness in various matrices (cereals, chocolate, sauces). It achieved a Screening Detection Limit (SDL) of 1 mg/kg for pistachio and demonstrated good reproducibility, providing a reliable tool for official food control where ELISA and PCR fail [6].

Table 2: Signature Peptide Markers for Selected Food Allergens

Allergen Source	Protein	Signature Peptide Sequence	Theoretical m/z	Function / Notes
Pistachio	Pis v 1, Pis v 2, Pis v 3, Pis v 5	Marker peptides specific to each protein	Varies by peptide	2S albumin, Legumin, Vicilin; Stable during processing [6] [50]
Cashew	Ana o 1, Ana o 2, Ana o 3	Marker peptides specific to each protein	Varies by peptide	Vicilin, Legumin, 2S Albumin; Selected for specificity [6]
Hazelnut	Cor a 9	e.g., LNPQQQGLR	Varies by peptide	11S Legumin; Used for calibration from 0-500 ppm [17]
Peanut	Ara h 1	e.g., DIENFYQGR	Varies by peptide	Vicilin; Two MRM transitions monitored for confidence [17]
Milk	Casein	e.g., VPQLEIVPNSAEER	Varies by peptide	Detected in multiplexed HRAM screening [17] [51]
Egg	Ovalbumin	e.g., GGLEPINFQTAADQAR	Varies by peptide	Detected in multiplexed HRAM screening [17]

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of HRAM-based allergen detection requires specific, high-quality materials and reagents.

Table 3: Essential Research Reagent Solutions for HRAM Allergen Analysis

Item	Function / Application	Example / Specification
Trypsin, Sequencing Grade	Proteolytic enzyme for digesting allergenic proteins into peptides for MS analysis.	TPCK-treated to minimize autolysis [17] [18]
Extraction Buffer	Extraction and denaturation of proteins from complex food matrices.	Contains ammonium bicarbonate, urea, and DTT [17] [18]
Alkylating Reagent	Capping of cysteine thiol groups to prevent reformation of disulfide bonds.	Iodoacetamide [17] [18]
Solid-Phase Extraction (SPE) Cartridges	Clean-up of peptide digests to remove interfering matrix components and concentrate analytes.	Reversed-phase sorbent (e.g., Strata-X, 200 mg/6 mL) [18]
Stable Isotope-Labeled Peptides	Internal standards for precise and accurate quantification.	Synthesized with 13C/15N labels; identical chemical properties to target peptides [6]
HRAM LC-MS System	High-resolution accurate mass analysis for unambiguous identification and quantification.	Orbitrap-based MS with nanoflow or UHPLC compatibility [48] [49]
Data Processing Software	Untargeted and targeted data analysis, database searching, and quantification.	Software with spectral library search (e.g., mzCloud) and in silico fragmentation (FISh) capabilities [48] [49]

High-Resolution Accurate Mass spectrometry systems are indispensable for enhancing specificity in the detection of food allergens, particularly in the context of complex, processed food products. The protocols detailed in this application note provide a validated pathway for laboratories to implement this powerful technology. By enabling highly specific multiplexed analysis, overcoming cross-reactivity issues of traditional methods, and allowing for retrospective data interrogation, HRAM systems represent a definitive solution for meeting rigorous food safety standards and protecting consumer health. The continued adoption and development of HRAM-based methodologies will undoubtedly play a central role in the future of food allergen analysis.

Benchmarking Performance: Validation, Recovery, and Comparison to Other Assays

Within the framework of developing a robust protocol for LC-MS/MS multi-allergen detection in processed foods, the establishment of core method validation parameters is paramount. The reliability of analytical results hinges on a rigorous validation process that demonstrates the method is fit for its intended purpose, particularly when detecting trace levels of allergenic proteins that pose significant health risks to consumers [6] [18]. For researchers and scientists in drug and analytical development, a thorough understanding of specificity, precision, and ruggedness is non-negotiable for ensuring data integrity and regulatory compliance.

This application note provides a detailed examination of these three critical validation parameters. It situates the discussion within the context of LC-MS/MS analysis for food allergens, a technique that has emerged as a powerful tool due to its high specificity and ability to multiplex allergen detection in complex matrices [17] [18]. We will delineate experimental protocols, summarize quantitative data, and provide practical guidance for integrating these validation steps into your research workflow.

Specificity: Ensuring Unambiguous Identification

Theoretical Foundation

Specificity is the ability of an analytical method to measure accurately and specifically the analyte of interest in the presence of other components that may be expected to be present in the sample [52]. In the context of LC-MS/MS multi-allergen detection, this ensures that the signal for a target allergenic peptide is unequivocally derived from that peptide and not from co-eluting interferences from the complex food matrix or other ingredients.

The superiority of LC-MS/MS over traditional techniques like ELISA for specificity is well-documented. ELISA, which relies on antigen-antibody interactions, is susceptible to cross-reactivity with food matrix components, potentially resulting in false positives or false negatives [6] [18]. LC-MS/MS, in contrast, provides multiple tiers of specificity through chromatographic retention time, the precise mass-to-charge ratio of the precursor ion, and unique fragment ions generated in the mass spectrometer [18].

Experimental Protocol for Determining Specificity

The following workflow, depicted in Figure 1, is recommended for establishing specificity in an LC-MS/MS multi-allergen method.

Figure 1. Experimental workflow for establishing method specificity.

• Select Signature Peptides: Choose proteotypic peptides unique to each allergen. The selection should be informed by databases and literature, and verified through analysis of pure allergen sources. Peptides should be resistant to common modifications during food processing (e.g., Maillard reaction) [17] [18]. For a method detecting walnut and almond, Korte et al. selected 18 proteotypic peptides across six nut types, while a similar study used five specific peptides for walnut and three for almond [53] [54].
• Analyze Blank Matrices: Analyze at least six different lots of the blank food matrix (e.g., bread, cookie, chocolate) that do not contain the target allergens. The chromatograms should show no interfering peaks at the retention times of the target signature peptides for each allergen [6] [52].
• Analyze Fortified Matrices: Analyze the blank matrix fortified with the target allergens at a relevant concentration (e.g., near the limit of quantitation). The method should detect the allergen-specific peptides with a signal-to-noise ratio that meets acceptance criteria (typically ≥10 for LOQ) [53].
• Analyze Spiked Interferences: Fortify the sample with potential interfering substances that could be present, such as other allergenic foods or common matrix components. Demonstrate that the quantification of the target analytes is unaffected [52].
• Verify Peak Purity and Identification: For each detected peptide, confirm specificity by monitoring at least two unique fragment ions per peptide and calculating the ion ratio between them. This provides a high level of confidence in the identification. Modern methodologies may also use peak-purity tests with photodiode-array detection or mass spectrometry to demonstrate specificity by comparison to a known reference material [52].

Key Research Reagent Solutions

Table 1: Essential reagents and materials for LC-MS/MS allergen analysis.

Item	Function	Example from Literature
Trypsin	Proteolytic enzyme for digesting allergenic proteins into measurable signature peptides.	Used in all cited LC-MS/MS protocols for allergen digestion [17] [18].
Extraction Buffer	Extracts proteins from complex food matrices; often contains urea, ammonium bicarbonate, and dithiothreitol (DTT).	Careri and colleagues pioneered a buffer with ammonium bicarbonate, urea, and DTT [18].
Alkylating Agent	Prevents reformation of disulfide bonds in proteins after reduction; commonly iodoacetamide.	Added to the extract after reduction with DTT to alkylate cysteine residues [18].
Solid-Phase Extraction Cartridges	Clean up digested samples to remove interfering matrix components and concentrate allergenic peptides.	Strata-X cartridges [18] or Oasis MCX cartridges for automated systems [55].
C18 Reverse-Phase Column	Chromatographically separates peptide fragments prior to MS/MS detection.	Phenomenex Kinetex C18 [17] or Ascentis Express C18 [55].
Isotopically Labelled Internal Standards	Account for variability in sample preparation and ionization efficiency; improve precision and accuracy.	Used in many published studies; FLX-d5 used as IS for fluoxetine quantification [55].

Precision: Demonstrating Method Reliability

Theoretical Foundation

The precision of an analytical method is defined as the closeness of agreement among individual test results from repeated analyses of a homogeneous sample [52]. It is a measure of the method's random error and is typically expressed as the relative standard deviation (%RSD) of a series of measurements. Precision is investigated at three levels:

• Repeatability: Intra-assay precision under the same operating conditions over a short time interval.
• Intermediate Precision: Precision within the same laboratory, incorporating variations from different days, different analysts, or different equipment.
• Reproducibility: Precision between different laboratories, as assessed in collaborative studies.

Experimental Protocol for Determining Precision

• Sample Preparation: Prepare a homogeneous sample of a representative food matrix (e.g., bread or cookie) fortified with all target allergens at a specified concentration, such as 10-100 ppm (mg allergen/kg food) [17]. The sample should undergo the complete sample preparation workflow, including extraction, reduction, alkylation, digestion, and cleanup.
• Repeatability:
  • Analyze a minimum of six independently prepared replicates of the fortified sample at 100% of the target concentration.
  • Alternatively, prepare and analyze a minimum of nine determinations over a minimum of three concentration levels (e.g., low, medium, high) covering the specified range, with three replicates each [52].
  • Calculate the mean, standard deviation, and %RSD for the quantified amount of each allergen.
• Intermediate Precision:
  • To demonstrate robustness against normal laboratory variations, an experimental design incorporating different analysts, different HPLC systems, and different days should be used.
  • For example, Analyst 1 and Analyst 2 each prepare and analyze six replicates of the fortified sample on different days, using their own standards and instruments.
  • The results are combined, and the overall %RSD is calculated. The % difference in the mean values between the two analysts can be subjected to statistical testing (e.g., Student's t-test) [52].

Representative Precision Data

Table 2: Example precision data from LC-MS/MS allergen methods.

Allergen / Analytic	Matrix	Spike Level	Repeatability (%RSD)	Intermediate Precision (%RSD)	Source
Multi-Allergen (12 allergens)	Bakery Products	10 - 500 ppm	Not specified	Good reproducibility reported	[17]
Walnut & Almond	Processed Foods	0.1 - 50 μg/mL	Sufficient repeatability obtained	Sufficient reproducibility obtained	[53]
Fluoxetine	Human Plasma	Quality Control Samples	Acceptable precision demonstrated	Acceptable precision demonstrated	[55]
Pistachio	Various Foods	1 mg/kg	Good reproducibility achieved	Ruggedness testing showed parameters must be strictly controlled	[6]

Ruggedness: Assessing Method Robustness

Theoretical Foundation

Ruggedness is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal, but variable, operational conditions. These conditions may include different laboratories, analysts, instruments, reagent lots, and elapsed assay times [52]. While the term "ruggedness" is sometimes used interchangeably with aspects of intermediate precision, it specifically tests the method's resilience to small, deliberate changes in operational parameters.

In LC-MS/MS methods, especially for complex applications like allergen detection, evaluating ruggedness is critical due to the large number of system parameters that can be difficult to control perfectly [56]. A method that is not rugged may produce acceptable results in one laboratory but fail in another, hindering method transfer.

Experimental Protocol and Parameters for Ruggedness

Ruggedness can be assessed through an experimental design that introduces small, deliberate variations into the method parameters. The following diagram illustrates the key parameters to investigate and the decision-making process for a ruggedness study.

Figure 2. Workflow for assessing method ruggedness.

The parameters listed in Table 3 should be varied one at a time from their nominal values while keeping all others constant. For each variation, a sample fortified at a target concentration is analyzed and the result (e.g., peak area, retention time, calculated concentration) is compared to the result obtained under nominal conditions.

Table 3: Key parameters to investigate during ruggedness testing of an LC-MS/MS method [56].

Parameter	Category	Recommended Variation	Potential Impact
Mobile Phase pH	Liquid Chromatography	± 0.5 units	Strong effect on retention if analyte pKa is near pH.
Organic Solvent Content	Liquid Chromatography	± 2% (relative)	Influences retention time and analyte signal.
Column Temperature	Liquid Chromatography	± 5 °C	Influences retention time and resolution.
Eluent Flow Rate	Liquid Chromatography	± 20%	Influences retention time and resolution.
Different Column Batch/Age	Liquid Chromatography	New vs. old column	Can affect retention time, peak shape, and resolution.
Extraction Time/Solvent	Sample Preparation	± 20%	Impacts extraction recovery and precision.
Ion Source Temp./Gas Flow	Mass Spectrometry	± 10 °C / ± 5 psi	Can influence analyte ionization efficiency.
Different Analysts	Operational	Two analysts	Tests consistency of sample prep and operation.
Different Instruments	Operational	Two LC-MS/MS systems	Tests performance across similar hardware.

As highlighted in a study on pistachio allergen detection, ruggedness testing may reveal that all parameters must be carefully monitored by the operator, and sample preparation must be carried out under strictly controlled conditions without modifications to ensure reproducible results [6].

The establishment of specificity, precision, and ruggedness forms a critical foundation for any reliable LC-MS/MS method, particularly in the demanding field of multi-allergen detection in processed foods. By implementing the detailed experimental protocols outlined in this application note, researchers and scientists can generate robust validation data that demonstrates their method is selective, reproducible, and resilient to minor but expected operational variations. This rigorous approach not only ensures the quality and safety of food products for allergic consumers but also facilitates smoother method transfer and regulatory acceptance.

Within the framework of developing a robust protocol for LC-MS/MS multi-allergen detection in processed foods, the accurate calculation of recovery from incurred samples is a critical validation step. Unlike spiked samples, incurred samples contain allergens that have been incorporated into the food matrix during pilot-scale production, undergoing processing steps that can alter protein structure and extractability [57]. This document outlines detailed application notes and protocols for determining overall recovery, providing scientists and drug development professionals with a standardized methodology to ensure data reliability for regulatory compliance and risk assessment [4].

Background and Significance

The rising global prevalence of food allergies has intensified the need for highly sensitive and specific analytical methods to detect trace amounts of allergens in complex food matrices [57]. Mass spectrometry, particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS) using selected reaction monitoring (SRM), has emerged as a powerful alternative to antibody-based assays like ELISA. LC-MS/MS offers superior multiplexing capabilities, allowing for the simultaneous quantitation of multiple allergenic proteins from different sources (e.g., milk, egg, peanut) in a single run [57]. This is especially relevant given that approximately 30% of children with food allergies are sensitive to more than one food [57].

A significant challenge in allergen analysis is the impact of food processing, which can denature proteins, modify epitopes, and alter their extractability from the matrix [57]. Consequently, validation using incurred samples—where the allergen is intrinsically incorporated during pilot-scale production—is superior to validation using spiked samples, as it more accurately represents real-world scenarios and provides a true measure of method performance, including the crucial parameter of overall recovery [57].

Experimental Workflow

The following workflow delineates the end-to-end process for preparing pilot-scale incurred samples and calculating overall recovery for LC-MS/MS multi-allergen detection.

Workflow Diagram

Title: Workflow for Recovery Calculation from Incurred Samples

Detailed Experimental Protocols

Pilot-Scale Production and Sampling

• Objective: To generate a homogenous, incurred food material with a known, precise concentration of the target allergen(s).
• Procedure:
  • Incurring: Incorporate the purified allergenic protein or source material (e.g., peanut flour, skim milk powder) into the food product during the manufacturing process at a pilot-scale facility. The target concentration should be clinically relevant and within the expected quantitation range of the LC-MS/MS method [57].
  • Processing: Subject the product to all intended processing steps (e.g., thermal treatment, baking, extrusion) to ensure the allergens are fully integrated and modified by the matrix.
  • Sampling: After production and cooling, collect multiple representative samples from different batches and locations within the batch. Use a quartering technique to ensure sample representativeness [58].
  • Homogenization: Grind or blend the samples to a fine, consistent powder or paste using a commercial food processor or blender. Verify homogeneity by analyzing multiple sub-samples.
• Key Consideration: Document all processing parameters (time, temperature, pH) as they significantly impact protein extraction efficiency and final recovery [57].

Protein Extraction and Digestion

• Objective: To efficiently and reproducibly extract allergenic proteins from the food matrix and digest them into peptides suitable for LC-MS/MS analysis.
• Materials & Reagents:
  • Extraction Buffer (e.g., Ammonium Bicarbonate, Phosphate-Buffered Saline)
  • Reducing Agent: Dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine (TCEP)
  • Alkylating Agent: Iodoacetamide (IAA)
  • Protease: Sequencing-grade Trypsin (cleaves C-terminal to Lys and Arg)
  • Solid-Phase Extraction (SPE) Cartridges (e.g., C18) for cleanup
• Procedure:
  • Weighing: Precisely weigh a defined amount (e.g., 1.0 g) of the homogenized incurred sample into a centrifuge tube.
  • Extraction: Add an appropriate volume of extraction buffer. The buffer composition must be optimized for the specific allergen-matrix combination [57]. Vortex vigorously and agitate (e.g., on a rotator) for 30-60 minutes at room temperature.
  • Clarification: Centrifuge at high speed (e.g., 10,000 × g for 15 minutes) to remove insoluble debris. Collect the supernatant containing the extracted proteins.
  • Reduction & Alkylation: Add DTT to a final concentration of 5-10 mM and incubate at 56°C for 30-60 minutes to reduce disulfide bonds. Cool, then add IAA to 15-20 mM and incubate in the dark for 30 minutes at room temperature to alkylate the cysteine residues.
  • Digestion: Add trypsin at an enzyme-to-substrate ratio of ~1:50 (w/w). Incubate at 37°C for 4-16 hours.
  • Cleanup: Acidity the digest with formic acid and desalt/concentrate the peptides using a C18 SPE cartridge or plate. Elute peptides in a solvent compatible with LC-MS/MS (e.g., acetonitrile/water with formic acid). Evaporate the solvent and reconstitute the peptide pellet in a defined volume of mobile phase A (e.g., 0.1% formic acid in water) for analysis.

LC-MS/MS Analysis with SRM

• Objective: To selectively and sensitively quantitate signature peptides derived from the target allergenic proteins.
• Instrumentation: Triple quadrupole (QQQ) mass spectrometer coupled to a nano-flow or ultra-performance liquid chromatography system.
• Procedure:
  • Chromatography: Separate the digested peptide mixture using a reversed-phase C18 column with a gradient of increasing organic solvent (e.g., acetonitrile).
  • Ionization: Introduce the eluting peptides into the mass spectrometer via electrospray ionization (ESI).
  • SRM/MRM: Program the mass spectrometer to monitor specific precursor ion → product ion transitions for each proteotypic peptide and a corresponding stable isotope-labeled (SIL) internal standard peptide [57]. A minimum of 3-5 transitions per peptide is recommended for high specificity [57].
  • Scheduling: Use scheduled SRM to monitor transitions only within a defined retention time window, increasing the number of quantifiable peptides and sensitivity [57].

Data Analysis and Recovery Calculation

Quantitation and Calculation

Peak areas for the target peptide transitions are integrated using the instrument's software. Quantitation is achieved by constructing a calibration curve from the ratio of the target peptide peak area to the SIL internal standard peptide peak area. The concentration of the allergenic protein in the incurred sample is then calculated from this curve.

Overall Recovery is calculated using the following formula:

Overall Recovery (%) = (Measured Concentration in Incurred Sample / Theoretical Incurred Concentration) × 100%

Where:

• Measured Concentration is the value determined via the LC-MS/MS assay.
• Theoretical Incurred Concentration is the known, target concentration of the allergen added during the pilot-scale production process.

Presentation of Quantitative Data

The quantitative data, including calibration curve parameters, measured concentrations, and calculated recoveries for multiple allergens, should be summarized in a clear, structured table. The following principles should be adhered to for effective data presentation [58]:

• The table should be numbered and have a clear, concise title.
• Headings for columns and rows should be clear, with units specified.
• Data should be presented in a logical order (e.g., by allergen).

Table 1: Example Data Table for Recovery Calculation of Allergens in a Pilot-Scale Produced Baked Good

Target Allergen	Theoretical Incurred Concentration (mg/kg)	Measured Concentration (mg/kg)	Overall Recovery (%)	RSD (%) (n=5)
Peanut (Ara h 1)	10.0	8.5	85.0	4.2
Milk (Casein)	50.0	42.0	84.0	5.1
Egg (Ovalbumin)	20.0	16.8	84.0	6.3

The Scientist's Toolkit: Essential Research Reagents and Materials

The following table details key reagents and materials essential for the successful execution of this protocol.

Table 2: Key Research Reagent Solutions and Materials

Item	Function / Explanation
Stable Isotope-Labeled (SIL) Peptides	Synthesized with heavy isotopes (e.g., 13C, 15N); serve as internal standards to correct for sample preparation losses and ion suppression, enabling highly accurate quantitation [57].
Proteotypic Peptides	Peptides unique to a specific allergenic protein that are consistently detected after sample digestion; their selection is critical for method specificity. Public databases like the Allergen Peptide Browser can aid selection [57].
Sequencing-Grade Trypsin	High-purity enzyme that specifically cleaves peptide bonds C-terminal to lysine and arginine, generating predictable peptides for SRM analysis [57].
Certified Allergen Reference Materials	Well-characterized protein or source material (e.g., from NIST) used for spiking calibration standards and for incurring samples during pilot-scale production, ensuring traceability.
Specialized Extraction Buffers	Buffers optimized for efficiently extracting both soluble and insoluble allergenic proteins (e.g., caseins from milk) from complex, processed food matrices [57].
C18 Solid-Phase Extraction (SPE) Plates	Used for rapid desalting and cleanup of complex peptide digests prior to LC-MS/MS analysis, removing interfering compounds and reducing instrument contamination.
LC Columns (Reversed-Phase C18)	The core of the chromatographic separation; nano-flow columns provide high sensitivity for trace-level allergen detection in complex digests.

Logical Pathway for Method Validation

The process of developing and validating the entire method, from peptide selection to final recovery assessment, follows a critical pathway to ensure reliability.

Validation Pathway Diagram

Title: Pathway for LC-MS/MS Allergen Method Validation

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) represent two fundamental analytical techniques with widespread application in food allergen detection. While ELISA provides efficiency and high throughput, LC-MS/MS offers superior specificity and multiplexing capabilities [59]. Achieving strong concordance between these distinct methodological approaches is paramount for generating reliable, reproducible data that supports food safety monitoring and regulatory compliance. This application note outlines a standardized protocol for conducting correlation studies between LC-MS/MS and ELISA platforms, specifically framed within LC-MS/MS multi-allergen detection in processed foods research, to ensure data comparability and methodological cross-validation.

Understanding the fundamental differences between LC-MS/MS and ELISA is essential for designing effective correlation studies. ELISA operates on the principle of antibody-antigen interaction, relying on the specific binding between antibodies and their target protein epitopes to generate a detectable signal [59]. This immunoassay format is highly susceptible to cross-reactivity with similar protein structures in complex food matrices, potentially leading to false positives [6] [59]. For instance, traditional ELISA methods often struggle to discriminate between cashew and pistachio allergens due to protein homology within the Anacardiaceae family [6].

In contrast, LC-MS/MS detects target analytes through mass-to-charge ratio separation and fragmentation patterns, providing direct measurement of specific signature peptides derived from allergenic proteins [53] [59]. This technique directly targets allergenic peptides rather than relying on antibody recognition, thereby avoiding immunological cross-reactivity issues [6]. However, sample preparation complexity and matrix effects can influence LC-MS/MS results, particularly in processed foods where proteins may be denatured or modified [6].

Key factors contributing to methodological discordance include:

• Differential detection targets: ELISA detects immunoreactive epitopes while LC-MS/MS detects specific peptide sequences
• Matrix interference effects: Varying impact of food components on antibody binding versus ionization efficiency
• Standardization discrepancies: Different reference materials and calibration approaches between platforms
• Processing-induced modifications: Protein structural changes during food processing that affect antibody binding but not necessarily MS detection [6]

Experimental Protocol for Correlation Studies

Sample Preparation Workflow

Table 1: Sample Preparation Requirements for LC-MS/MS and ELISA Comparison

Processing Step	ELISA Requirements	LC-MS/MS Requirements	Harmonization Approach
Sample Homogenization	Fine powder (<500 µm)	Fine powder (<500 µm)	Identical milling procedure for both analyses
Protein Extraction	PBS or commercial extraction buffer	Urea/thiourea or ammonium bicarbonate buffer	Parallel extractions with both buffers; precipitation and reconstitution
Defatting	Required for high-fat matrices	Required for high-fat matrices	Standardized hexane extraction for both methods
Enzymatic Digestion	Not required	Trypsin/Lys-C essential	N/A for ELISA; essential for LC-MS/MS
Cleanup	Dilution or filtration	Solid-phase extraction (C18)	Different procedures required by platform nature

A standardized sample preparation protocol begins with representative sampling of processed food matrices. Homogenize samples to a fine powder (<500 µm particle size) using cryogenic milling to ensure uniformity. For parallel analysis, split each homogenized sample for independent processing through ELISA and LC-MS/MS workflows.

For protein extraction, use a compatible buffer system that preserves protein integrity for both methodologies. For ELISA, employ PBS (pH 7.4) or commercial extraction buffers per kit manufacturer instructions. For LC-MS/MS, use 50mM ammonium bicarbonate buffer with 0.1% RapiGest or 8M urea/2M thiourea in 50mM Tris-HCl (pH 8.0) for difficult matrices [53]. Perform defatting with n-hexane for high-fat matrices (e.g., chocolate, nuts) for both methods [60].

For LC-MS/MS analysis only, conduct enzymatic digestion using sequencing-grade trypsin (1:20-1:50 enzyme-to-protein ratio) at 37°C for 4-16 hours with agitation [53]. Following digestion, acidify LC-MS/MS samples with 0.5% trifluoroacetic acid and purify using C18 solid-phase extraction cartridges. ELISA samples typically require only dilution in assay buffer before analysis.

Quantitative Analysis and Calibration

Table 2: Method Validation Parameters for Correlation Studies

Validation Parameter	ELISA Assessment	LC-MS/MS Assessment	Acceptance Criteria
Limit of Detection (LOD)	Mean blank + 3SD	S/N ratio ≥ 3:1	Comparable within one order of magnitude
Limit of Quantification (LOQ)	Mean blank + 10SD	S/N ratio ≥ 10:1 with CV < 20%	Comparable within one order of magnitude
Linear Range	Typically 2-3 orders of magnitude	Typically 3-4 orders of magnitude	R² ≥ 0.99 for both methods
Recovery (%)	80-120%	70-120% (matrix-dependent)	Within comparable ranges
Precision (CV%)	Intra-assay <15%, inter-assay <20%	Intra-assay <15%, inter-assay <20%	Comparable variability

For quantitative correlation, establish parallel calibration curves using certified reference materials for both platforms. For ELISA, use the kit-provided standards following manufacturer's protocol, typically in a 6-8 point dilution series [59]. For LC-MS/MS, prepare a matrix-matched calibration curve using the same purified protein or peptide standards at 6-8 concentration levels encompassing the expected analytical range [53].

Include quality control samples at low, medium, and high concentrations across both platforms. For LC-MS/MS, incorporate stable isotope-labeled internal standards (SILIS) for each target allergen to correct for ionization efficiency variations and sample preparation losses [53]. Process all samples in triplicate across three separate batches to assess both intra- and inter-assay variability.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Essential Research Reagents for LC-MS/MS and ELISA Correlation Studies

Reagent Category	Specific Examples	Function & Importance
Reference Materials	Certified allergen protein standards (e.g., walnut 2S albumin, almond 11S globulin) [53]	Calibration standard for both platforms; ensures quantitative accuracy
Internal Standards	Stable isotope-labeled peptides (e.g., [13C]-homoarginine) [61]	Normalizes LC-MS/MS response; corrects for preparation variability
Detection Antibodies	Monoclonal/polyclonal antibodies specific to target allergens	ELISA specificity; critical for recognizing native and processed proteins
Enzymes	Sequencing-grade trypsin/Lys-C	Protein digestion for LC-MS/MS; generates detectable signature peptides
Matrix Modifiers	RapiGest, iodoacetamide, TCEP	Protein solubilization, reduction, and alkylation for LC-MS/MS
Blocking Reagents	BSA, non-fat dry milk, casein	Reduces nonspecific binding in ELISA; improves signal-to-noise

Data Analysis and Concordance Assessment

Statistical evaluation of method correlation should include linear regression analysis comparing quantitative results from paired samples analyzed by both platforms. Calculate Pearson's correlation coefficient (r), coefficient of determination (R²), and fit a Deming regression to account for measurement errors in both methods [61].

Assess concordance correlation using Lin's concordance correlation coefficient (ρc), which evaluates both precision and accuracy relative to the line of identity. A value of ρc > 0.90 indicates substantial agreement between methods [61]. Generate Bland-Altman plots to visualize the difference between methods against their average, identifying any concentration-dependent biases.

In a comparative study of homoarginine measurement, LC-MS/MS consistently yielded values approximately 29% higher than ELISA, highlighting the importance of establishing method-specific reference ranges [61]. Similarly, in forensic toxicology, LC-MS/MS detected additional positive specimens that were missed by ELISA, particularly for benzoylecgonine (26%), lorazepam (33%), and oxymorphone (60%) [62].

Troubleshooting Discordant Results

When significant methodological discrepancies occur, systematic investigation should focus on:

• Epitope recognition issues: Evaluate if food processing (heating, fermentation) has altered antibody-binding epitopes while leaving MS-detectable peptides intact [6]
• Cross-reactivity assessment: Test ELISA antibodies against related proteins from other food sources to identify potential cross-reactivity [6] [59]
• Digestion efficiency: Verify complete protein digestion for LC-MS/MS through evaluation of missed cleavages and peptide recovery [53]
• Matrix effects: Assess ionization suppression/enhancement in LC-MS/MS and matrix interference in ELISA using standard addition methods [62]
• Protein extraction completeness: Compare multiple extraction protocols to ensure efficient recovery of both native (for ELISA) and denatured (for LC-MS/MS) proteins

Achieving strong concordance between LC-MS/MS and ELISA data requires meticulous method optimization, standardized sample processing, and comprehensive statistical evaluation. While perfect correlation may not be attainable due to fundamental methodological differences, understanding the sources and patterns of discordance enhances data interpretation and method selection for specific applications. The protocols outlined herein provide a framework for rigorous correlation studies that support method validation and data comparability in multi-allergen detection research. Researchers should establish method-specific reference ranges and clearly communicate the applicable analytical platform when reporting results, as values between methods may show consistent proportional differences despite strong correlation [61].

Comparative Analysis of LC-MS/MS vs. Real-Time PCR for Allergen Detection

Food allergies represent a significant and growing public health concern worldwide, with effective management relying heavily on the accurate detection and declaration of allergenic substances in food products [33]. For individuals with food allergies, strict avoidance is the primary strategy, making reliable food labeling essential [32]. Regulatory frameworks in many countries, such as the European Union's Regulation (EU) No 1169/2011, mandate the declaration of major allergenic ingredients in food products [6] [34].

The two predominant analytical techniques for allergen detection are immunoassays (e.g., ELISA) and DNA-based methods like real-time PCR. However, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful confirmatory technique that overcomes several limitations of traditional methods [18] [32]. This application note provides a detailed comparative analysis of LC-MS/MS and real-time PCR methodologies for allergen detection, focusing on their application in processed food matrices, with specific protocols for implementation in analytical laboratories.

Fundamental Principles and Technical Comparison

LC-MS/MS Methodology

LC-MS/MS for allergen detection operates on the principle of directly analyzing allergenic proteins through their signature peptide fragments. The workflow involves extracting proteins from the food matrix, digesting them with an enzyme (typically trypsin) to generate peptides, separating these peptides via liquid chromatography, and then detecting and quantifying them using tandem mass spectrometry [18] [32]. The technique targets the allergenic proteins themselves, providing direct evidence of their presence.

Key advantages of LC-MS/MS include:

• Direct protein detection that correlates with allergenicity
• High specificity through multiple reaction monitoring (MRM) of unique peptides
• Multiplexing capability for simultaneous detection of numerous allergens
• Robustness to food processing effects compared to antibody-based methods [18] [32]

Real-Time PCR Methodology

Real-time PCR detects allergen-specific DNA sequences rather than the allergenic proteins. The method involves extracting DNA from food samples, amplifying target sequences using sequence-specific primers, and detecting the amplified products in real-time using fluorescent probes (e.g., TaqMan) [63] [33]. This technique identifies the genetic material of allergenic sources, providing indirect evidence of potential allergen presence.

The workflow's advantages include:

• High sensitivity for DNA detection
• Specificity for species identification
• Effectiveness for allergens where protein detection is challenging
• Robustness to certain processing conditions that may denature proteins [33]

Comparative Performance Metrics

Table 1: Direct comparison of key performance characteristics between LC-MS/MS and real-time PCR for allergen detection.

Parameter	LC-MS/MS	Real-Time PCR
Target Analyte	Proteins/Peptides	DNA
Detection Limit	1-10 mg/kg [6] [17]	0.1 mg/kg [63]
Multiplexing Capacity	High (12+ allergens simultaneously) [17]	Moderate (typically 1-3 allergens per reaction)
Quantification Approach	Direct, using signature peptides and internal standards	Indirect, based on DNA copy number
Impact of Food Processing	Moderate (proteins may be denatured but peptides still detectable)	High (DNA degradation can lead to false negatives) [33]
Specificity Concerns	Minimal cross-reactivity	Potential cross-reactivity with related species [6]
Throughput	Moderate to High (with multiplexing)	High (for single allergens)
Cost per Analysis	High (instrumentation, expertise)	Moderate

Experimental Protocols

Detailed LC-MS/MS Protocol for Multi-Allergen Detection

Sample Preparation

• Homogenization: Process food samples to a fine powder using a laboratory grinder. For fatty matrices (e.g., nuts, chocolate), defat with hexane extraction (2×4 mL per 1 g sample) and evaporate to dryness [17].
• Protein Extraction: Add 4 mL extraction buffer (50 mM ammonium bicarbonate, 8 M urea, 10 mM DTT) to 1 g defatted homogenate. Vortex thoroughly and mix using a roller mixer for 30 minutes at room temperature. Centrifuge at 10,000 × g for 10 minutes and collect supernatant [6] [17].
• Protein Reduction and Alkylation: Add 50 μL reducing reagent (100 mM DTT) to 500 μL supernatant, incubate at 60°C for 1 hour. Cool to room temperature, then add 25 μL alkylating reagent (200 mM iodoacetamide), incubate in dark for 30 minutes [17].
• Enzymatic Digestion: Add 20 μg trypsin in 50 mM ammonium bicarbonate buffer with 1 mM calcium chloride. Incubate at 37°C for 3-12 hours [19] [17].
• Digestion Termination and Cleanup: Acidify with 30 μL formic acid to pH <3. Purify peptides using solid-phase extraction (Strata-X cartridge, 200 mg/6 mL). Condition with 6 mL acetonitrile/0.1% formic acid, equilibrate with 6 mL water/0.5% TFA, load sample, wash with 3 mL 0.5% TFA in water, elute with 6 mL acetonitrile. Evaporate eluent to dryness under nitrogen and reconstitute in 300 μL reconstitution solution (95:5:0.5 water/acetonitrile/formic acid) [18].

LC-MS/MS Analysis

• Chromatographic Separation:

  • Column: Phenomenex Kinetex C18 (2.6 μm, 100 × 3.0 mm)
  • Mobile Phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile
  • Gradient: 5-35% B over 12 minutes
  • Flow Rate: 300 μL/min
  • Injection Volume: 30 μL [17]

• Mass Spectrometric Detection:

  • Instrument: SCIEX QTRAP 4500 or equivalent triple quadrupole system
  • Ionization: Positive electrospray ionization (ESI+)
  • Source Temperature: 500°C
  • Ion Spray Voltage: 5500 V
  • Detection Mode: Scheduled MRM with 2-4 transitions per peptide [17]

Data Analysis

• Identification: Confirm allergen presence based on co-elution of multiple transitions with consistent peak area ratios.
• Quantification: Use matrix-matched calibration curves with stable isotope-labeled internal standards for precise quantification [19].

Detailed Real-Time PCR Protocol for Allergen Detection

Sample Preparation and DNA Extraction

• Homogenization: Process 2 g food sample to fine powder under liquid nitrogen.
• DNA Extraction: Use commercial DNA extraction kit (e.g., DNeasy Mericon Food Kit, Qiagen) following manufacturer's instructions.
• DNA Quantification and Quality Check: Measure DNA concentration using spectrophotometry (A260/A280 ratio of 1.8-2.0 indicates pure DNA).

Real-Time PCR Analysis

• Reaction Setup:

  • Total Volume: 25 μL
  • Reaction Mix: 12.5 μL 2× TaqMan Universal PCR Master Mix
  • Primers: 0.9 μL forward primer (10 μM), 0.9 μL reverse primer (10 μM)
  • Probe: 0.25 μL hydrolysis probe (10 μM, FAM-labeled with BBQ quencher)
  • Template DNA: 100 ng genomic DNA
  • Nuclease-Free Water: to 25 μL [63]

• Amplification Parameters:

  • Initial Denaturation: 95°C for 10 minutes
  • 45 Cycles:
    • Denaturation: 95°C for 15 seconds
    • Annealing/Extension: 60°C for 1 minute [63]

• Data Analysis:

  • Calculate Cq values using instrument software
  • Use standard curve method for quantification with serial dilutions of target DNA

Critical Applications in Processed Foods

Performance in Complex Matrices

Both LC-MS/MS and real-time PCR face challenges when analyzing processed foods, though the nature of these challenges differs significantly between the two techniques.

Table 2: Method performance across different food matrices and processing conditions.

Food Matrix/Processing	LC-MS/MS Performance	Real-Time PCR Performance
Bakery Goods (Cookies)	Good recovery (80-101.5%) for selected markers [34]	Variable (DNA degradation during baking)
Chocolate	Effective with defatting step [18]	Moderate (PCR inhibitors present)
Sauces	Good recovery with optimized extraction [6]	Good (minimal processing)
Highly Processed (Rusks)	Marker-dependent; some peptides detectably modified [34]	Poor (extensive DNA fragmentation)
Meat Products	Successful for species identification [19]	Good with specific primer design

Case Study: Discrimination of Cross-Reactive Allergens

A significant advantage of LC-MS/MS emerges in discriminating between closely related allergens that show cross-reactivity in immunological assays. A 2025 study demonstrated this capability for pistachio and cashew detection, where traditional ELISA and PCR methods often suffer from cross-reactivity due to protein and DNA similarity [6].

The LC-MS/MS method achieved a screening detection limit of 1 mg/kg for both nuts in various matrices (cereals, chocolate, sauces, and meat products) with good reproducibility for pistachio detection. The method specifically targeted unique peptide markers that differentiate these phylogenetically related species, overcoming a critical limitation of other techniques [6].

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key reagents and materials for implementing allergen detection protocols.

Reagent/Material	Function	Application	Specific Example
Sequencing-Grade Trypsin	Proteolytic enzyme for protein digestion	LC-MS/MS	Promega Trypsin Gold [34]
Stable Isotope-Labeled Peptides	Internal standards for quantification	LC-MS/MS	AQUA peptides for allergen quantitation [19]
TaqMan Probes	Hydrolysis probes for real-time detection	Real-time PCR	FAM-labeled, BBQ-quenched probes [63]
Solid-Phase Extraction Cartridges	Peptide purification and concentration	LC-MS/MS	Strata-X (200 mg/6 mL) [18]
DNA Polymerase	Enzymatic DNA amplification	Real-time PCR	Taq DNA Polymerase [63]
Reducing/Alkylating Reagents	Protein denaturation for digestion	LC-MS/MS	DTT and IAA [17]
Allergen-Free Matrices	Method development and validation	Both	Gluten-free flour blends [17]

The comparative analysis of LC-MS/MS and real-time PCR for allergen detection reveals complementary strengths that can be strategically deployed based on specific analytical requirements. LC-MS/MS excels in scenarios requiring direct protein detection, multiplexed analysis, and discrimination of cross-reactive allergens in processed foods. Real-time PCR offers superior sensitivity for unprocessed materials and effectiveness when protein targets are unstable.

For comprehensive allergen control programs in food manufacturing, a combined approach utilizing both techniques provides the most robust solution. LC-MS/MS serves as an ideal confirmatory method for official food control, while real-time PCR offers rapid screening capabilities. Future developments in high-resolution mass spectrometry, biosensor technologies, and standardized reference materials will further enhance the accuracy and accessibility of allergen detection, ultimately improving protection for allergic consumers.

Assessing Applicability Across Low- and High-Resolution MS Platforms

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for multi-allergen detection in processed foods, overcoming limitations of traditional techniques like ELISA and PCR [18] [2]. Within this analytical framework, researchers must choose between low-resolution (LRMS) and high-resolution (HRMS) platforms, each offering distinct advantages for specific application scenarios. LRMS, typically triple quadrupole (QqQ) instruments operating in Multiple Reaction Monitoring (MRM) mode, provides exceptional sensitivity and quantitative robustness for targeted analysis [6] [64]. Conversely, HRMS platforms, such as Quadrupole-Time of Flight (Q-TOF) or Orbitrap instruments, deliver high mass accuracy and resolution, enabling broader suspect screening and confirmatory analysis [65] [66]. This application note delineates the applicability of both platforms within a comprehensive protocol for detecting allergenic proteins in complex food matrices, providing researchers with a clear framework for method selection based on analytical objectives.

The choice between LRMS and HRMS involves strategic trade-offs between sensitivity, specificity, and analytical scope. The following table summarizes key performance characteristics derived from validation studies, which must be considered when developing a multi-allergen detection protocol.

Table 1: Comparative Performance of Low-Resolution and High-Resolution MS Platforms for Allergen Analysis

Performance Characteristic	Low-Resolution MS (QqQ)	High-Resolution MS (Q-TOF/Orbitrap)	Implications for Allergen Analysis
Typical Quantitation Sensitivity (LOQ)	Median LOQ: 0.2 ng/mL in urine [64]	Median LOQ: 1.2 ng/mL in urine [64]	QqQ is generally superior for quantifying trace-level allergens where maximum sensitivity is required.
Mass Accuracy	Nominal mass (±1 Da) [65]	< 5 ppm mass error [65] [66]	HRMS provides unambiguous compound identification and can resolve isobaric interferences that may cause false positives in LRMS.
Primary Acquisition Mode	Multiple Reaction Monitoring (MRM) [64]	Full Scan / Data-Dependent MS/MS [64] [66]	MRM (QqQ) is ideal for targeted analysis of known peptides; full scan (HRMS) enables retrospective and untargeted screening.
Key Application Strength	High-sensitivity targeted quantification [6]	Untargeted screening, confirmation, and discovery of unknown/emerging allergens [64] [65]	QqQ is suited for routine compliance checking; HRMS is ideal for method development and incident investigation.
Multiplexing Capability	High (e.g., 88 MRM transitions for 12 allergens) [17]	High, but with potential sensitivity trade-offs in full-scan modes [64]	Both platforms support multi-allergen methods, but QqQ maintains sensitivity across many targets simultaneously.

Experimental Protocols for Multi-Allergen Detection

A robust sample preparation workflow is critical for reliable allergen detection, regardless of the MS platform employed. The protocol below is adapted from established methodologies for processing complex food matrices [17] [7].

Sample Preparation and Protein Digestion

The objective of this protocol is to extract allergenic proteins from a processed food matrix and digest them into specific peptide markers for LC-MS/MS analysis.

• Materials: Food sample, extraction buffer (e.g., 50 mM Ammonium Bicarbonate, 8 M Urea, 10 mM Dithiothreitol), Iodoacetamide, Trypsin (sequencing grade), Solid-Phase Extraction (SPE) cartridges (e.g., Strata-X, 200 mg/6 mL), Solvents (HPLC-grade water, acetonitrile, formic acid).
• Procedure:
  • Homogenization: Finely grind the food sample (1 g) using a laboratory grinder. For high-fat matrices, perform a defatting step with hexane [17].
  • Protein Extraction: Add extraction buffer (4 mL) to the homogenate. Vortex mix thoroughly and agitate using a roller mixer for 60 minutes. Centrifuge at >10,000 × g for 10 minutes and collect the supernatant [17] [53].
  • Reduction and Alkylation: To the supernatant, add iodoacetamide (final concentration ~20 mM) to alkylate cysteine residues and prevent reformation of disulfide bonds. Incubate in the dark for 30 minutes at room temperature [18].
  • Enzymatic Digestion: Add trypsin (e.g., 20 µg) to the alkylated protein extract at an enzyme-to-substrate ratio of ~1:50 (w/w). Digest at 37°C for 3–12 hours [17] [7].
  • Clean-up and Concentration: Acidity the digest with formic acid. Pass the sample through an SPE cartridge conditioned with acetonitrile and water. Elute peptides with an acetonitrile-based eluent, evaporate to dryness under a gentle nitrogen stream, and reconstitute in a mobile phase-compatible solvent (e.g., water/acetonitrile 95:5 with 0.1% formic acid) for LC-MS/MS analysis [18] [7].

LC-MS/MS Analysis: Platform-Specific Configurations

The following configurations are typical for the analysis of allergenic peptide markers.

• Chromatography (Common to both platforms):

  • Column: Reversed-phase C18 (e.g., 100 × 2.1 mm, 2.6 µm) [17].
  • Mobile Phase: A) 0.1% Formic acid in water; B) 0.1% Formic acid in acetonitrile.
  • Gradient: Linear gradient from 5% B to 100% B over 10-15 minutes.
  • Flow Rate: 0.3-0.6 mL/min [65] [17].
  • Injection Volume: 5-30 µL.

• Mass Spectrometry - LRMS (QqQ) Method:

  • Ionization: Electrospray Ionization (ESI), positive mode.
  • Acquisition Mode: Scheduled Multiple Reaction Monitoring (MRM).
  • Source Parameters: Ion source temperature of 500°C; optimize ion spray voltage and gas flows [17].
  • Method Development: For each target allergen, select two unique "signature peptides" and optimize two MRM transitions per peptide. Use a database search to ensure peptide specificity [17] [53]. Monitor dozens to over 100 MRM transitions in a single run [17].

• Mass Spectrometry - HRMS (Q-TOF) Method:

  • Ionization: Electrospray Ionization (ESI), positive mode.
  • Acquisition Mode: Data-Dependent Acquisition (DDA) or Targeted MS/MS (e.g., "MSMS mode").
  • MS1 Scans: Full scans at a resolution of ≥20,000 (FWHM) with mass accuracy < 5 ppm [65] [66].
  • MS2 Scans: Isolate precursor ions based on an inclusion list of expected signature peptides and fragment them, acquiring product ion spectra with high resolution [65].

Workflow Visualization

The following diagram illustrates the logical decision-making pathway for selecting the appropriate mass spectrometry platform based on the analytical goals of the allergen detection study.

Figure 1: Platform Selection Workflow.

Research Reagent Solutions

Successful implementation of the LC-MS/MS allergen detection protocol relies on key reagents and materials. The following table details essential items and their critical functions in the analytical workflow.

Table 2: Essential Research Reagents and Materials for LC-MS/MS Allergen Analysis

Item	Function / Role in Protocol
Trypsin (Sequencing Grade)	Proteolytic enzyme that specifically cleaves proteins at the C-terminus of lysine and arginine residues, generating predictable peptide markers for MS analysis [18] [17].
Extraction Buffer (Urea, DTT)	Denatures proteins and reduces disulfide bonds, ensuring complete extraction and accessibility of allergenic proteins for subsequent digestion [17].
Iodoacetamide	Alkylating agent that modifies cysteine residues, preventing reformation of disulfide bonds and ensuring complete peptide mapping [18].
Solid-Phase Extraction (SPE) Cartridge	Purifies and concentrates the peptide digest post-hydrolysis, removing salts, lipids, and other matrix interferents that can suppress ionization and compromise the LC-MS/MS analysis [18] [7].
Stable Isotope-Labeled Internal Standard Peptides	Synthetic peptides identical to the target allergen peptides but labeled with heavy isotopes (e.g., 13C, 15N). Added prior to digestion, they correct for losses during sample preparation and variability in MS ionization, enabling highly accurate quantification [6].
Signature Peptide Standards	Purified, synthetic peptides corresponding to the target sequences used for MRM assay development, retention time calibration, and creating external calibration curves [17] [53].

Both low-resolution and high-resolution MS platforms offer distinct and powerful capabilities for the multi-allergen detection in processed foods. The LRMS (QqQ) approach, with its superior sensitivity and robust quantification in MRM mode, is the workhorse for high-throughput, targeted monitoring of regulated allergens, making it ideal for compliance and quality control laboratories [6] [17]. The HRMS (Q-TOF/Orbitrap) approach, with its high mass accuracy and full-scan data acquisition, provides unparalleled specificity for confirmatory analysis and the flexibility for suspect screening and method development [65] [66]. The choice is not mutually exclusive; as evidenced by real-case scenarios, the techniques are highly complementary [65]. An integrated strategy, potentially using QqQ for routine surveillance and HRMS for complex investigations, represents the most comprehensive approach for ensuring the safety of food-allergic consumers.

Conclusion

The development and implementation of a robust LC-MS/MS protocol for multi-allergen detection represent a significant advancement in food safety analytics. This synthesis of knowledge confirms that LC-MS/MS, particularly when using optimized extraction and HRAM systems, offers a highly specific, sensitive, and multiplexable solution that outperforms traditional methods in complex, processed foods. Key takeaways include the critical importance of selecting stable peptide markers, the necessity of using incurred materials for accurate validation, and the strong correlation achievable with ELISA when methods are harmonized. Future directions point toward the need for greater harmonization and standardization of methods across laboratories, the exploration of non-targeted discovery workflows for novel allergens, and the direct application of these precise detection capabilities to inform clinical threshold studies and personalized allergy management, thereby bridging analytical food chemistry with biomedical research and improved patient outcomes.

References

• 1. Food allergy thresholds, context, and balance in 2025 - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC12322902/]
• 2. Food Allergen Analysis Using LC–MS–MS [https://www.chromatographyonline.com/view/food-allergen-analysis-using-lc-ms-ms]
• 3. The hidden price of food allergy: Understanding the social ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12412086/]
• 4. Food Allergies [https://www.fda.gov/food/nutrition-food-labeling-and-critical-foods/food-allergies]
• 5. Mass Spectrometry-Based Method for Multiple Allergens ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11899636/]
• 6. Allergens in Food: Analytical LC-MS/MS Method for the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12428185/]
• 7. Development of a rapid and reliable method to ... [https://www.sciencedirect.com/science/article/pii/S2590157524004462]
• 8. Multi-laboratory validation of the xMAP—Food Allergen ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7347184/]
• 9. A multi-laboratory evaluation of a clinically-validated ... [https://www.sciencedirect.com/science/article/pii/S0308814613013800]
• 10. ELISA Assay Technique | Thermo Fisher Scientific - US [https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html]
• 11. Enzyme Linked Immunosorbent Assay - StatPearls - NCBI - NIH [https://www.ncbi.nlm.nih.gov/books/NBK555922/]
• 12. What Are The Restrictions Of Elisa Tests? Unlocking ... [https://merkel.co.il/what-are-the-restrictions-of-elisa-tests-unlocking-the-enigma/]
• 13. The advantages and the limitations of LC-MS/MS and Elisa ... [https://www.biotrial.com/bioanalysis-lc-ms-ms-vs-elisa/]
• 14. PCR-based diagnostics for infectious diseases [https://pmc.ncbi.nlm.nih.gov/articles/PMC7106425/]
• 15. Recent trends and developments of PCR-based methods ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9579247/]
• 16. Quantification of DNA fragmentation in processed foods ... [https://www.sciencedirect.com/science/article/abs/pii/S0308814617300754]
• 17. Simultaneous analysis of 12 food allergens in baked and ... [https://sciex.com/tech-notes/food-beverage/food-and-beverage/simultaneous-analysis-of-12-food-allergens-in-baked-and-raw-food]
• 18. Advanced Analysis of Allergens in Foods: LC–MS ... [https://www.spectroscopyonline.com/view/advanced-analysis-allergens-foods-lc-ms-ms-methodologies-and-future-insights]
• 19. Development and validation of an LC-MS/MS method for a ... [https://www.sciencedirect.com/science/article/abs/pii/S0308814625032169]
• 20. Development of a rapid and reliable method to simultaneously detect... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11231652/]
• 21. Multi-laboratory validation of the xMAP—Food Allergen ... [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234899]
• 22. In-house validation of an LC–MS method ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10766722/]
• 23. of multi- Simultaneous in an incurred food matrix... detection allergens [https://pubmed.ncbi.nlm.nih.gov/25577123/]
• 24. A novel ELISA test to detect soy in highly processed foods [https://www.sciencedirect.com/science/article/pii/S0889157521005032]
• 25. A Highly Sensitive Indirect Enzyme-Linked Immunosorbent ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10636219/]
• 26. FastScan™ ELISA Cell Extraction Buffer (5X) #69905 [https://www.cellsignal.com/products/buffers-dyes/fastscan-elisa-cell-extraction-buffer-5x/69905?srsltid=AfmBOoqBI21rIUVbl5bKdWwcrrD-3z2Onkn13XXvcs0D-KmdWsnx7jFf]
• 27. ELISA Lysis and Protein Extraction Buffer | ABIN1059067 [https://www.antibodies-online.com/buffer/1059067/ELISA+Lysis+and+Protein+Extraction+Buffer/?srsltid=AfmBOoqxKqZ9ZBX-eOz26YsDHtdeYiDfSzJaZTYj57IpetnfCmKScPxF]
• 28. Optimization of protein extraction and ELISA ... [https://link.springer.com/article/10.1140/epjp/s13360-021-01628-0]
• 29. Online SPE | Thermo Fisher Scientific - US [https://www.thermofisher.com/us/en/home/industrial/chromatography/chromatography-sample-preparation/sample-preparation-consumables/solid-phase-extraction-consumables/online-spe.html]
• 30. A Fully Automated Online SPE-LC-MS/MS Method for the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8955396/]
• 31. LC-MS Sample Preparation [https://www.thermofisher.com/us/en/home/industrial/mass-spectrometry/mass-spectrometry-learning-center/liquid-chromatography-mass-spectrometry-lc-ms-information/lc-ms-sample-preparation.html]
• 32. Food allergen detection by mass spectrometry: the role of ... [https://www.nature.com/articles/npjsba201622]
• 33. Research progress on detection methods for food allergens [https://www.sciencedirect.com/science/article/abs/pii/S0889157524009402]
• 34. Mass Spectrometry-Based Method for Multiple Allergens ... [https://www.mdpi.com/2304-8158/14/5/726]
• 35. Intelligent screening for food allergens by Scout triggered ... [https://sciex.com/tech-notes/food-beverage/food-and-beverage/intelligent-screening-for-food-allergens-by-scout-triggered-mrm]
• 36. Targeted Proteomics: Current Status and Future ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5082697/]
• 37. Factors affecting the rapid changes of protein under short-term ... [https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-021-07560-y]
• 38. A Vital Role of High-Pressure Processing in the Gel Forming ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9454986/]
• 39. Microbial Proteins: A Green Approach Towards Zero Hunger [https://pmc.ncbi.nlm.nih.gov/articles/PMC12346714/]
• 40. Effects of high-pressure processing on cooked color and ... [https://www.sciencedirect.com/science/article/pii/S2772502222002153]
• 41. Signal suppression/enhancement in high-performance ... [https://www.sciencedirect.com/science/article/abs/pii/S0021967309017397]
• 42. Determining Matrix Effects in Complex Food Samples [https://www.waters.com/blog/understanding-sample-complexity-determining-matrix-effects-in-complex-food-samples/?srsltid=AfmBOopF0LbEg_FKQGiWwmMqsxCwYiBPcRMF758T8Z_ttPDETofKCpyY]
• 43. Evaluation of Matrix Effects and Extraction Efficiencies ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7205385/]
• 44. In-house validation of an LC–MS method for the ... [https://link.springer.com/article/10.1007/s00216-023-04894-2]
• 45. Foods that crossreact with cashew and pistachio [https://www.aaaai.org/allergist-resources/ask-the-expert/answers/old-ask-the-experts/cashew-pistachio]
• 46. - Allergenic Between Cross and Reactivity Nuts Cashew Pistachio [https://www.academia.edu/6044577/Allergenic_Cross_Reactivity_Between_Cashew_and_Pistachio_Nuts]
• 47. Food allergen detection choosing the right testing method [https://www.rssl.com/insights/food-consumer-goods/food-allergen-detection-choosing-the-right-testing-method/]
• 48. Tomorrow's Quantitation Today | Thermo Fisher Scientific - UK [https://www.thermofisher.com/uk/en/home/products-and-services/promotions/industrial/tomorrows-quantitation-today.html]
• 49. Untargeted Screening and Identification of Contaminants Using Gas... [https://www.chromatographyonline.com/view/untargeted-screening-and-identification-of-contaminants-using-gas-chromatography-coupled-to-high-resolution-accurate-mass-mass-spectrometry-novel-solutions-for-applied-testing-laboratories]
• 50. AllergenScreener [https://www.bruker.com/en/products-and-solutions/mass-spectrometry/ms-solutions/allergenscreener.html]
• 51. Food Allergens Testing Information [https://www.thermofisher.com/us/en/home/industrial/food-beverage/food-beverage-learning-center/food-analytical-testing-information/food-allergens-testing-information.html]
• 52. Analytical Method Validation: Back to Basics, Part II [https://www.chromatographyonline.com/view/analytical-method-validation-back-basics-part-ii]
• 53. Development of a simple and reliable LC-MS/MS method ... [https://www.sciencedirect.com/science/article/pii/S2665927123000126]
• 54. MRM 3 -based LC-MS multi-method for the detection and ... [https://pubmed.ncbi.nlm.nih.gov/27590318/]
• 55. Development and validation of an automated solid-phase... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8300324/]
• 56. 10.1 Robustness and ruggedness relation to LC - MS ... method [https://sisu.ut.ee/lcms_method_validation/101-robustness-and-ruggedness-relation-lc-ms-method-development/]
• 57. Food allergen detection by mass spectrometry: the role of systems biology - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC5516885/]
• 58. Presentation of Quantitative Data [https://ihatepsm.com/blog/presentation-quantitative-data]
• 59. Immunoassays and Mass Spectrometry for Determination ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11046482/]
• 60. ELISA screening and liquid chromatography-tandem mass ... [https://www.sciencedirect.com/science/article/pii/S0032579119423249]
• 61. Measurement of homoarginine in human and mouse plasma by... [https://link.springer.com/article/10.1007/s00726-015-2037-7]
• 62. - LC - MS vs MS : Validation of a Comprehensive Urine Toxicology... ELISA [https://pubmed.ncbi.nlm.nih.gov/31424082/]
• 63. Detection of Food Allergens by Taqman Real-Time PCR ... [https://pubmed.ncbi.nlm.nih.gov/28315214/]
• 64. Comparing the sensitivity of a low- and a high-resolution ... [https://www.sciencedirect.com/science/article/pii/S0003267023009613]
• 65. Low-Resolution or High-Resolution Mass Spectrometry for ... [https://www.chromatographyonline.com/view/low-resolution-or-high-resolution-mass-spectrometry-for-clinical-and-forensic-toxicology-some-considerations-from-two-real-cases]
• 66. High-resolution mass spectrometry: Theoretical and ... [https://www.sciencedirect.com/science/article/abs/pii/S2352007821002146]