United States

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&#39;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.

2025 AOAC INTERNATIONAL Annual Meeting &amp; Exposition

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

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.

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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.

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.

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

A rapid and sensitive method for the confirmation and quantification of sports performance pharmaceutical adulterants in dietary supplements using high-performance liquid chromatography coupled to tandem mass spectrometry was developed and validated. The  method presented here is applicable to the determination of  21 sports performance pharmaceutical adulterants. The sample preparation includes a methanol extraction and a dilution step. Chromatographic separation was performed on an InfinityLab Poroshell 120 Chiral-V column. The method was validated and proved to be linear, accurate, robust, and precise over the range 20 to 400 ng/ml (5µg/g to 100µg/g)  and with the detection limit of 2 µg/g for all compounds in different types of dietary supplements.  

Determination of Sports Performance Pharmaceutical Adulterants in Dietary Supplements by LC-MS/MS

Taraxacum kok-saghyz (TKS) is a sustainable industrial crop traditionally cultivated for natural rubber production. However, its pharmaceutical potential remains underexplored. In this study, we present an integrated green analytical platform to simultaneously recover natural rubber and pharmacologically active secondary metabolites from TKS roots and leaves. Using UPLC-MS/MS, we identified 1,813 metabolites, including caffeoylquinic acids, flavonoids, and lipid derivatives. Multivariate analyses (PCA, OPLS-DA) confirmed tissue-specific metabolic specialization, revealing 964 differentially expressed compounds between roots and leaves. Notably, leaf tissues were enriched in cynarin, chicoric acid, and luteolin derivatives—compounds known for antioxidant, hepatoprotective, and neuroprotective effects.
We implemented an ethanol–aqueous biphasic extraction to optimize compound preservation and co-extraction efficiency, validated via KEGG pathway mapping and mass spectrometric quantification. KEGG enrichment highlighted active biosynthesis of flavonoids and amino acid metabolites, supporting the nutraceutical relevance of TKS leaves. Root-specific enrichment of quinones and alkaloids suggests stress adaptation and antimicrobial potential.
This study demonstrates the feasibility of developing a dual-use biomanufacturing platform for botanical APIs and clean latex from U.S.-grown TKS. It contributes to the standardization of metabolite recovery from non-model plants and supports domestic sourcing of high-value nutraceutical ingredients. Our green extraction methodology enhances reproducibility and analytical rigor in dietary supplement development.

Integrated Extraction of Botanical Actives and Natural Rubber from Taraxacum kok-saghyz Using Green Analytical Techniques

Mycotoxins are naturally occurring toxins formed by fungi and have been shown to cause a variety of human health effects. Recent studies have shown that mycotoxins are occurring in areas of the world that historically have not had contamination, such as Aflatoxin in European wheat, presumably due to climate change. In this study, a method was developed using a high-end LC-MS/MS system to quantify multiple mycotoxins in cereals and food products at lower limits of detection. This is important because mycotoxin regulations have been lowered in some countries and regions. In addition, novel mycotoxins not routinely monitored have emerged, necessitating the expansion of the analyte panel. The method was applied to the analysis of various globally sourced toddler snacks.Preliminary data from the analysis of 30 toddler food samples showed that 94% were positive for at least one mycotoxin. Most of the samples contained mycotoxins from the Enniatin class with 47 positives found for this group. In addition, Beauvericin and the aflatoxins were also very prominent in the majority of the samples. The preliminary study was expanded to include toddler food samples from various regions around the world. These global trends will be presented as well as the results from various cereal grains.
 
 

Multi-Mycotoxin Analysis in Cereals and Toddler Food Using a High-End Triple Quadrupole Mass Spectrometer

Total ORAC 5 (Oxygen Radical Absorbance Capacity) is a series of five test methods that are used for the individual measurement of 5 different antioxidant capacities against its respective radical in functional ingredients and foods. These radicals are hydroxyl, peroxyl, peroxynitrite, singlet oxygen (1O2), and superoxide anion (O2-). Each of these test methods works on the principle of a fluorescent probe’s fluorescence being either oxidized or reduced by an active free radical species that was not inhibited by an antioxidant. The analysis takes place in a 96 well plate with various reference standards used and the final results being reported as umol Trolox equivalents per gram of material (umol TE/g) . These reactions are mixed, incubated, monitored and recorded using a fluorescence platereader. The degree of which the probe fluoresces is used to indicate the antioxidant capacity of the material. Each of the five antioxidant capacity test methods were individually validated and then combined into a single standard operating procedure and referred to as Total ORAC 5. The five methods can be referred to as ORAC (Hydrophilic Oxygen Radical Absorbance Capacity), HORAC (Hydroxyl Radical Absorbance Capacity, NORAC (Peroxynitrite Scavenging Activity Assay), SORAC (Singlet Oxygen Scavenging Activity Assay) and SOAC (Superoxide Scavenging Activity Assay). The validation incorporated method linearity/range, precision, accuracy, ruggedness, robustness and LOD/LOQ to demonstrate the method is fit for purpose.

A Method Validation for the Quantitative Analysis of the Antioxidant Capacity in Functional Ingredients and Foods by Total ORAC 5

Tang Kuei (Angelica sinensis) shares similar phytochemical profiles with other Angelica species, including A. archangelica, A. dahurica, A. decursiva, and A. acutiloba. These similarities—comprising volatile oils, polysaccharides, organic acids, and flavonoids—pose challenges for species identification using conventional methods like HPLC and HPTLC, especially in the absence of sufficient reference standards. To overcome these limitations, Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-TOF-MS) was employed, leveraging its high-resolution accurate mass (HRAM) capabilities to detect subtle differences in component profiles. Chromatograms of the test samples were compared with a botanical reference material (BRM) of A. sinensis, revealing two distinct peaks absent in the BRM. UPLC-Q-TOF-MS analysis identified the two peaks as decursin and its isomer, which are known markers predominantly associated with A. decursiva or specific subspecies of A. sinensis. This method integrates database searches with authenticated reference standards, allowing for precise identification of unknown compounds and confirmation of their botanical origins. It establishes a reliable framework for detecting deviations from the typical chemical profile of A. sinensis, effectively identifying potential contamination or adulteration by other Angelica species.

Identification of Angelica sinensis and differentiation of A. sinensis from other close species from Angelica Species Using UPLC-Q-TOF-MS

Most mycotoxins are chemically stable and survive food processing. Several hundred different mycotoxins have been identified, but the most observed mycotoxins that present a concern to human health and livestock include aflatoxins, ochratoxin A, patulin, fumonisins, zearalenone and nivalenol/deoxynivalenol. Most current reference methods for mycotoxin analysis still rely on non-confirmatory techniques such as TLC, HPLC-UV, and HPLC-FLD, with IAC cleanup often used to improve specificity. Regulatory bodies have only recently emphasized the need for standardized LC-MS methods, prompting the CEN to develop European standards under the M/520 mandate. Mycotoxins are typically extracted using liquid–liquid extraction for liquids (milk, wine, juices) and solid–liquid extraction for solids (cereals, dried fruits, spices), often with acidic buffers to enhance efficiency. The procedure enables the quantitative LC-MS/MS determination of regulated mycotoxins. Mass spectrometric parameters were optimized using Shimadzu’s Lab Solutions Connect software and get the best sensitivity, selectivity, and precision. This study presents a simple, high-throughput method for the determination of multiple mycotoxins in animal feed using the Shimadzu LCMS-8050RX system. Key steps included precursor ion selection and MRM optimization at different collision energies (CE), leading to a segmented MRM method with gradient elution. The mobile phase and column conditions were optimized using a GIST Shim-pack C18 AQ column to achieve better separation and peak shape. A commercially available animal feed sample was extracted, and a matrix blank was spiked with a standard mycotoxin mixture. The matrix-matched calibration curve ranged from 0.5 to 50 ppb, with accuracy between 80–120%. Spiked sample recoveries at 5 and 10 ppb were within 70–120%, in compliance with SANTE guidelines.

Quantitation of Multi-Mycotoxins in Animal Feed by Using Ultra-Fast LCMSMS

Herbal skincare (Ayurveda) products (HSPs) are used daily by many people around the world. Quality control of these products is immensely important to consumer safety, as the botanical ingredients used in HSPs are at risk of containing heavy metals (HMs) that can have adverse health effects. The Ministry of Ayush in India regulates HM levels in raw material (RM) and finished goods (FG). Given the wide range of fatty matter (0–100%) in HSPs, this study aimed to set up a robust method to quantitate HMs (As, Cd, Pb Hg, Sb, and Ni) in RM and FGs using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), with tailored digestion programs for low and high-fat matrices.  Limit of detection (LOD) was determined by analyzing 20 reagent blanks resulting as 0.014 mg/kg for Hg and 0.001 to 0.067 mg/kg for other analytes. Limit of quantification (LOQ) for Hg, Ni, and other analytes was found to be 0.050, 0.203 and 0.100 mg/kg respectively. Repeatability (% RSDr) and reproducibility (%RSDR) at LOQ was established by analyzing six spike samples, resulting in 0.8879% to 13.35% and 1.66% to 19.69% respectively. Accuracy was demonstrated by spiking at 1x, 3x and 5x LOQ, and results ranged from 82.73% and 104.32% for RM and 81.75% to 125.85% for FG. This study emphasizes the need for rigorous quality control and regulatory monitoring in the HSPs industry to minimize consumer exposure to HMs and promote the development of safer and compliant products.

Simultaneous estimation of arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg), antimony (Sb) and nickel (Ni) in herbal skincare products using ICP-MS

This study aims to the optimization of High-Performance Liquid Chromatography (HPLC) conditions for the separation of distinct peaks in a complex mixture using Design of Experiments (DOE) strategy. The objective was to evaluate and improve the resolution between Quercetin and Luteolin peaks by considering factors such as mobile phase composition, flow rate and column temperature. The design was employed systematically to study the impact of these variables on separation efficiency. The interaction effects of the factors were analyzed to determine the optimal conditions that would minimize peak overlap and enhance resolution. The results demonstrated the effectiveness of the DOE approach in identifying the critical factors influencing peak separation and provided insights into the optimal HPLC conditions for achieving distinct, well-resolved peaks. The approach also highlighted the importance of method and data analysis in optimizing HPLC method for complex sample analysis.

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