United States

2025 AOAC INTERNATIONAL Annual Meeting &amp; Exposition

AOAC SPIFAN Topics: Heavy Metals and Trace Elements Update

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This multi-faceted “idea incubator” focuses on regulatory changes and emerging food safety issues. A plenary and two breakout sessions feature thought-provoking ideas to identify and meet analytical needs.

The 2025 AOAC INTERNATIONAL Annual Meeting & Exposition was held in person in San Diego, USA.

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2025 AOAC INTERNATIONAL Annual Meeting & Exposition

The AOAC Annual Meeting & Exposition provides unparalleled professional development, networking, and collaboration in methods-based science.

Food analytical methods are classified as either continuous or categorical based on their response type. When a qualitative method is proposed as a standard assay, it must be validated to demonstrate its suitability for the intended purpose. This validation process depends on the availability of standard reference materials or an established reference method. While international standards provide guidance for this process, misinterpretations of performance characteristics—such as the limit of detection, level of detection, relative limit of detection, and probability of detection, in combination with statistical models ranging from the normal and Poisson distributions to the beta-binomial distribution and beyond —have led to inconsistencies. Adding to this complexity, different validation standards use different validation criteria. As a result, there is a growing need for harmonization to ensure comparability across methods. In this context, the potential application of Bayesian methods also arises. The potential of a Bayesian approach for a practical equivalence procedure has never been developed to provide an equivalence estimate in cases where results from similar matrices are compared. This Workshop will review the goals of categorical testing, current standard methods for validation and determining equivalence and the possibilities for potentially harmonizing or interchanging results from them. 

Binary Methods: Goals, Performance Statistics, Validation, and Future Harmonization

More than a decade ago, the analytics of mineral oil saturated and aromatic hydrocarbons (MOSH/MOAH) found its way into contract laboratories, official authorities, and food producers all over the world. As of today, legally binding limits are still not established. Originally developed in Switzerland, this type of analytics quickly gained attention anyhow.
The regulative upper limits discussed by the European Commission will be challenging for a broad variety of matrices. To that end, a lot of harmonization work was done in last years resulting in a standardized method for vegetable oils and fats (ISO 20122) generally providing good inter-laboratory comparability. Nevertheless, official methods for other type of matrices are not available.
Beside the hyphenation of HPLC and GC-FID needed for the quantitation of MOSH and MOAH, automation of the highly complex and variable sample preparation protocol can help to improve precision, robustness, and sample throughput.
The presented work discusses all required sample preparation steps in terms of necessity, performance, and automatability. A generic sample preparation protocol derived from the ISO method is shown and its applicability for a broad range of food matrices subjected to HPLC-GC-FID afterward. 
Additionally, examples for unusual findings in foodstuffs will be outlined and which tools are available to deal with possible limitations and obstacles.

Reliable Determination of MOSH/MOAH: A Conjunction of Multidimensional Chromatography, Organic Chemistry, and Automation

Wheat is critical to Canada’s economy, contributing approximately $10.2 billion annually, with 80% of wheat exported. However, ergot contamination caused by Claviceps purpurea poses a significant food safety risk due to the presence of toxic ergot alkaloids (EAs). This study aimed to evaluate the performance of an enzyme-linked immunosorbent assay (ELISA) for EA detection compared to ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Additionally, an ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method was developed and validated to improve the accuracy and sensitivity of EA detection.
The ELISA kit was validated using standard and in-house reference materials. CR was determined as the ratio of IC50 values of individual alkaloids to ergotamine. Spiked solvent and wheat matrix were analyzed using ELISA and UHPLC-MS/MS, followed by regression analysis and Bland-Altman plots. A UHPLC-HRMS method was developed and validated for enhanced EA quantification.
ELISA showed a strong correlation with UHPLC-MS/MS (r=0.8793) but underestimated total EA concentrations by a factor of two. CR varied widely, with ergometrine exhibiting the highest CR in solvent and wheat matrix, indicating substantial matrix effects. UHPLC-HRMS provided superior resolution, sensitivity, and mass accuracy, enabling comprehensive EA profiling and identification of previously undetected alkaloids.
While ELISA offers a rapid screening approach, its accuracy is highly influenced by matrix effects and CR variability. The validated UHPLC-HRMS method enhanced analytical capabilities by providing high sensitivity and specificity, complementing UHPLC-MS/MS for regulatory compliance and improved food safety in wheat monitoring, ensuring more reliable and comprehensive analysis.

Comparison of ELISA, UHPLC-MS/MS, and Development of UHPLC-HRMS Method for Ergot Alkaloid Quantification in Wheat

Ethylene oxide (EO) and 2-chloroethanol (2-CE) are hazardous pesticide residues that pose significant risks to human health. EO is a carcinogenic compound linked to cancer, genetic mutations, and neurotoxicity, while 2-CE, a degradation product of EO, exhibits toxicity and may damage internal organs such as the liver and kidneys. These compounds are commonly used as fumigants in the food industry to eliminate microbes; however, their residues can persist in products, particularly in complex matrices like spice blends. Analyzing EO and 2-CE residues in such matrices is highly challenging due to matrix interference and sample preparation difficulties.  
This study successfully developed and validated a method for analyzing EO and 2-CE residues in solid spice-based seasonings using GC-MS/MS with Headspace technology, without requiring matrix-matched standards. The method demonstrated excellent performance, with limits of detection (LODs) of 0.000095 mg/kg for EO and 0.0040 mg/kg for 2-CE, and limits of quantification (LOQs) of 0.00019 mg/kg for EO and 0.0060 mg/kg for 2-CE. Linearity was highly robust, with correlation coefficients (r) of 0.9999 for EO and 0.9998 for 2-CE. Recovery rates ranged from (81.19–95.35)% for EO and (80.75–87.31)% for 2-CE, complying with international validation guidelines such as SANTE/11312/2021.  
This method eliminates matrix-matched standards while maintaining high accuracy, offering a cost-effective solution for EO and 2-CE analysis in complex spice matrices, with significant implications for global food safety regulations.
 

Overcoming Matrix Challenges: A Sensitive GC-MS/MS Headspace Method for Ethylene Oxide and 2-Chloroethanol Residues in Multicomponent Spice Seasonings

Introduction

ASF Session Introduction

Quantitative analysis of food components is essential for optimizing food formulation, enhancing the effectiveness of clinical trials, informing nutrition policy, and improving AI applications in the food domain. To support these goals, a suite of advanced analytical tools has been developed for the two most abundant food components: carbohydrates and proteins. These tools significantly improve upon current methodologies by offering high sensitivity and quantitative precision across a broad range of molecular sizes—from monomers to large polymers.
For protein analysis, the platform encompasses metabolomic (amino acids and dipeptides), peptidomic (peptides), and proteomic (whole proteins) approaches. Similarly, carbohydrate analysis spans monosaccharides, oligosaccharides, and polysaccharides, corresponding to metabolomic and glycomic levels. Carbohydrates present unique challenges due to their structural complexity and diverse linkage patterns, which often lead to misclassification in nutritional databases and inaccuracies in clinical interpretations.
To address these issues, a comprehensive multi-glycomic platform has been developed. Carbohydrates are fractionated into alcohol-soluble and alcohol-insoluble components. The soluble fractions are analyzed using advanced liquid chromatography–mass spectrometry (LC-MS) to identify free mono-, di-, and oligosaccharides. The insoluble fractions undergo three specialized LC-MS workflows that enable detailed monosaccharide quantification, linkage analysis (covering over 100 types), and polysaccharide identification and quantification.
These multi-omics methods are rapid, automated, and highly quantitative, allowing for in-depth structural characterization of both whole and processed foods, as well as their interactions with the digestive tract and the gut microbiome. The resulting data not only inform clinical trials and nutrition policy but also enhance AI models by providing detailed structural-functional insights into key food components.

Awards Ceremony & Keynote Address

Choline, a vital nutrient essential for cognitive functions, including memory, executive functions, and learning. Humans obtain choline from two sources: dietary intake and hepatic biosynthesis. Choline's biosynthesis is regulated by estrogen, which diminishes with ageing, impacting postmenopausal women. However, the recent NHANES shows only 2% of postmenopausal women in the U.S. meet the adequate intake (AI) of choline, highlighting the critical need for a novel ingredient to increase choline consumption. We hypothesize Whey Protein Phospholipid Concentrate (WPPC), a byproduct of cheese processing, is a bioavailable and safe source of choline because of its richness of choline precursors, phosphatidylcholine, and unique triple-layered phospholipid structure. To test this hypothesis, I will conduct a single-blinded parallel-designed randomized control trial in postmenopausal women. The changes in serum choline metabolites after WPPC intervention will be measured by UHPLC-HRMS/MS as the primary outcome. The safety of WPPC consumption will be determined by dissecting the changes in detrimental metabolites of choline, trimethylamine (TMA) and its oxidized product (TMAO), using UHPLC-HRMS/MS, and alteration of oxidative stress and proinflammatory biomarkers, such as 8-hydroxydeoxyguanosine, IL-1, IL-6, TNF-a, and hsCRP by utilizing ELISA and immunoturbidimetry. Lastly, the abundance and diversity of gut microbes will be measured using 16S rRNA sequencing to explore the impacts of WPPC on cognition-related microbe populations. Our findings will demonstrate the safety and bioavailability profile of WPPC and its application in supplementing choline intake in postmenopausal women. The clinical data will support the establishment of a Recommended Daily Allowance (RDA) for choline.

Assessing Choline Bioavailability and Metabolism from a New Dairy Ingredient (WPPC) for Post-Menopausal Women: A Randomized Control Trial

The “Paralytic Shellfish Poisons” (PSP), which can be produced by various marine dinoflagellates, mainly of the genus Alexandrium, and other freshwater algae and cyanobacteria. Due to the accumulation of these neurotoxins during filter feeding, shellfish (mussels, clams, oysters and scallops) can often become contaminated in areas when harmful algal blooms (HABs) are present. When shellfish tissue levels exceed a safe action level, typically 80 ug Saxitoxin diHCl (STX diHCl) equivalent /100 g tissue (800 ppb), the shellfish beds are closed to harvesting.
There are over 30 different analogues of STX identified and classified by their different degrees of toxicity. Saxitoxin, Neosaxitoxin, gonyautoxin- (GTX1& 4, GTX 2 & 3) and decarbamoyl-STX (dc-STX) are the more acutely toxic based upon their i.p. toxicity in mice. 
Beacon developed a quantitative immunoassay, which capable of detecting both Saxitoxin and Neosaxitoxin.  Also, GTX1&4, GTX 2&3 can be converted to STX and Neosaxitoxin by L-cysteine treated sample extract. This quantitative ELISA assay can detect the most toxic PSP congeners in shellfish.

Paralytic Shellfish Toxins (PSTs) Immunoassay Plate Kit

Background & Objective: Wheat is critical to Canada’s economy, contributing approximately $10.2 billion annually, with 80% of wheat exported. However, ergot contamination caused by Claviceps purpurea poses a significant food safety risk due to the presence of toxic ergot alkaloids (EAs). This study aimed to evaluate the performance of an enzyme-linked immunosorbent assay (ELISA) for EA detection compared to ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Additionally, an ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method was developed and validated to improve the accuracy and sensitivity of EA detection.  
Methods: The ELISA kit was validated using standard and in-house reference materials. CR was determined as the ratio of IC50 values of individual alkaloids to ergotamine. Spiked solvent and wheat matrix were analyzed using ELISA and UHPLC-MS/MS, followed by regression analysis and Bland-Altman plots. A UHPLC-HRMS method was developed and validated for enhanced EA quantification.  
Results: ELISA showed a strong correlation with UHPLC-MS/MS (r=0.8793) but underestimated total EA concentrations by a factor of two. CR varied widely, with ergometrine exhibiting the highest CR in solvent and wheat matrix, indicating substantial matrix effects. UHPLC-HRMS provided superior resolution, sensitivity, and mass accuracy, enabling comprehensive EA profiling and identification of previously undetected alkaloids.  
Conclusion: While ELISA offers a rapid screening approach, its accuracy is highly influenced by matrix effects and CR variability. The validated UHPLC-HRMS method enhanced analytical capabilities by providing high sensitivity and specificity, complementing UHPLC-MS/MS for regulatory compliance and improved food safety in wheat monitoring, ensuring more reliable and comprehensive analysis.

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Binary Methods: Goals, Performance Statistics, Validation, and Future Harmonization

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RESOURCES

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