United States

Non-targeted analysis (NTA) is a type of analytical technique with the purpose of identifying chemicals in a material without the use of a priori knowledge regarding the chemical composition of the material. As novel methods using NTA approaches rapidly emerge, the Best Practices for Non-Targeted Analysis (BP4NTA) Working Group formed to address the need for harmonization across research communities. The presentation will discuss all facets of BP4NTA, from formation in 2017 to its current goals. Over the past 8 years, BP4NTA has become an international, nonprofit organization consisting of members from academia, government (state, federal US, and non-US), and industry with a range of NTA experience. Initial efforts within BP4NTA aimed at harmonization of language and methods through the creation of the informative website with NTA Reference Content. Numerous other outcomes have been generated, ranging from manuscripts and tools to engagement activities with varied stakeholders involved in the development of NTA workflows and use of the data. Examples include the NTA Study Reporting Tool, guidance on understanding performance metrics for non-targeted analysis and proposing a roadmap for the high-impact research directions for NTA. In addition, BP4NTA has identified opportunities fulfill an educational in training and supporting researchers that are new to NTA. Moving forward BP4NTA endeavors to identify new collaborative opportunities with other scientific communities using non-targeted approaches, develop new tools that further enhance transparency and reproducibility of non-targeted analysis, and to establish best practices for scientists using non-targeted approaches.

2025 AOAC INTERNATIONAL Annual Meeting &amp; Exposition

The Best Practices for Non-Targeted Analysis (BP4NTA) Working Group

#### The AOAC INTERNATIONAL Annual Meeting: A Unique Analytical Science Opportunity

The AOAC Annual Meeting & Exposition provides unparalleled professional development, networking, and collaboration in methods-based science. 

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* Take advantage of emerging methods, best practices, and trending topics like food fraud and cannabis purity and potency. 
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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.

Per-and Polyfluorinated Substances (PFAS) are diverse synthetic chemicals widely used in industry because of their thermal stability, hydrophobicity, and relative inertness. These “forever chemicals” have been detected in food and food packaging and may cause harmful health effects. With thousands of PFAS compounds identified, processing non-targeted PFAS data poses a significant challenge.
In this study, 40 paper-based food contact materials (FCMs) from shops in Montreal, Canada -- including to-go boxes, microwave popcorn bags, wrappers, paper straws, and baking liners -- were analyzed for the occurrence of PFAS using LC-QTOF MS. The highest PFAS concentrations were detected in clamshell to-go boxes, with levels reaching up to 356.6 ng/g. PFOA (up to 187.2 ng/g) and PFDA (up to 92.2 ng/g) were major contributors to the overall PFAS content. FluoroMatch Modular was used to annotate previously unidentified PFAS, confirming a homologous series  (C3 to C14) of perfluoroalkyl carboxylic acids (PFCA) and C6 and C8 perfluoroalkyl sulfonic acids (PFSA). 
The study also demonstrated that the migration of both short-chain (PFHxA and PFHpA) and long-chain (PFOA, PFDA, PFNA) PFAS into food simulants increased upon heating (65 ℃), indicating that higher temperatures facilitate the release of these substances from packaging into food. These findings raise concerns about potential for PFAS exposure through food consumption, particularly under heated conditions such as hot meals and microwave heating.
The widespread detection of PFAS in clamshell to-go boxes and other FCMs in this study highlights the need for more stringent regulations and oversight to minimize PFAS use in FCMs.

Non-Targeted Analysis of PFAS in Food Contact Materials: Unveiling Hidden Dangers with LC-QTOF MS and FluoroMatch

NMR spectroscopy is an incredibly powerful analytical tool that provides a wide array of techniques for conducting metabolomics studies. It has become an essential tool in for assessing quality, authenticity, safety, and geographical origin. However, several challenges hinder its broader application in food research, such as low sensitivity, signal overlap, and the predominance of sugar signals that obscure important signals of non-sugars. To overcome this limitation, we report the implementation of past reported sugar-suppression methods combined with these conventional standard 1D and 2D NMR methods. This approach minimizes the sugar signals and allows for clearer detection of other metabolites. Total sugar and non-sugar sixty-eight metabolites were identified, and three-fold enhanced intensities and visibility of non-sugar signals in suppressed 1D 1H NMR and 2D NMR methods compared to their respective standard  NMR methods. Combining NMR-based metabolomics with multivariate analyses like PLS-DA and heat maps helps identify compositional variations in honey samples, aiding in the discrimination of botanical origins. . Glucose, cellobiose, fructose, galactose, glucose-6-phosphate, maltose, mannitol, ribose, sucrose, trehalose, uridine diphosphate (UDP)-galactose, UDP-glucose, adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine triphosphate (ATP), acetate, asparagine, betaine, formate, gallate, glutamate, glutamine, malonate, hydroxymethylfurfural, phenylalanine, pipecolate, and [AA1] proline are main responsible primary metabolites that contributed to botanical origins discrimination. Interestingly, the applied suppressed methods improved signal intensity, enabling clearer identification of metabolites amidst complex carbohydrate backgrounds. This approach also facilitated the detection of vitamins like nicotinic acid and trigonelline, emphasizing the nutritional value of the honey samples by reducing interference from dominant carbohydrate signals.
  

Untargeted Metabolomics of Honey of Four Different Botanical Origins by Conventional and Sugar Suppressed 1D and 2D NMR Spectroscopy

Dietary fiber is recognized as an essential part of human nutrition due to its health benefits. As consumers become more aware of these benefits, there is a growing interest in products with higher dietary fiber content. This underscores the need for accurate measurement of dietary fiber in food products for labeling and other purposes. Over the past decades, our understanding of dietary fibers has evolved, with new types of fibers emerging, such as those derived from algae, insects, pectin, and hemicellulose. This evolution necessitates the optimization of existing analytical methods or the development of new ones to keep up with advancements and cover various types of dietary fiber. Consequently, numerous analytical methods have been developed to determine dietary fiber content, making accurate testing more complex. 
This contribution briefly discusses the differences between the most important analytical methods for dietary fiber determination, highlighting some recently developed solutions for novel fiber ingredients and addressing some shortcomings in current methods.

Unraveling the Fiber Frontier: Innovations and Challenges in Dietary Fiber Analysis

Supplements are known globally by various names like Dietary Supplements (DS), Health Food (HF), Food Supplements (FS), Natural Health Products (NHP), and Complementary Medicines (CM). Each category must meet local market standards for compliance, leading to differing regulations based on product classification.
This session will cover a case study on the challenges and gaps in the Nutritional Fact Panel (NFP) analysis of dietary supplements, with examples from Canada and Australia markets to illustrate issues in meeting international requirements. NFP (Nutritional Facts Panel) is an analysis that requires a combination of parameter assessment. For the dietary supplements, these details are required to report on the product label by regulation which again is classified by different terms from different regulatory bodies around the world. In Canada, this is referred to as NFT (Nutritional Fact Table) as classified by Health Canada and Canadian Food Inspection Agency (CFIA). In Australia, the authority known as Food Standards Australia New Zealand (FSANZ) refers to it as the Nutritional Information Panel (NIP). Part of this NFT disclosure, calorie estimation is a critical parameter required to indicate product/formula attributes. The standard methodology for calorie estimation, as outlined by the United States Department of Agriculture in the “Energy Value of Foods,” Agricultural Handbook, and FDA 21 CFR Part 101.9 for food versus 21 CFR 101.36 for supplements, involves analyzing carbohydrates, dietary fiber, fats, protein, cholesterol, ash, moisture, and iron in any product.
This session will conduct a structured review to identify specific challenges associated with gaps in current analytical methods. It will also address how a product development approach may result in biased data interpretation to examine the regulatory requirements that impact accurate labeling. 

A Case Study: Challenges in Analyzing Nutritional Facts Panels of Dietary Supplements for Compliance with Global Regulations

AOAC 2017.16, for the measurement of total dietary fiber, in alignment with CODEX definitions, achieved final action status in 2020, and replaced AOAC 2009.01 as a CODEX Type 1 method in 2021.  AOAC 2022.01, for the measurement of soluble, insoluble and total dietary fiber is following a similar track with AOAC final action status obtained in 2024 and it is expected to replace AOAC 2011.25 as a CODEX Type 1 method this year. As these methods are implemented across the industry, new insights into analytical challenges continue to emerge. This presentation investigates chromatography-related questions, particularly the performance of recommended columns (Waters SugarPak, AOAC 2009.01; Tosoh TSKgel, AOAC 2001.03/2017.16/2022.01) alongside alternative columns sourced globally. A comprehensive carbohydrate dataset was analysed across these columns to evaluate their performance. The comparative chromatographic performance results will be discussed herein.
Additionally, regulatory developments in pet food labelling are driving a transition from crude fiber to dietary fiber analysis, as signalled by the Association of American Feed Control Officials (AAFCO). By 2029, pet food labelling will require dietary fiber determination by approved analytical methods, necessitating a major shift in analytical approaches. To support this transition, we generated relevant data using these methods, assessing their applicability to pet food matrices, the results of which will also be described.

Recent Developments in Dietary Fiber Methodology

Last decade novel carbohydrate dietary or prebiotic fibre are being developed. Products are being sent for novel food applications (Gras, Efsa, ..). To meet acceptance criteria and enable commercialization new and highly accurate and discriminative carbohydrate quantification methods are needed. Using traditional gravimetric-LC carbohydrate, sugar and fibre methods are not capable of providing accuracy and specificity to support novel food dossiers for toxicity and stability testings as well composition testing in foods. One would need new methods to do so. Calculation of carbohydrates by 100-minus methods no longer will do.
Examples of novel carbohydrate ingredients are milk oligosaccharides, fermentation based carbohydrates, algae based carbohydrates, hemicellulose based oligosaccharides (AXOS), and pectin based oligosaccharides (POS) and foods made thereof.
Analogous to accepted proteomic and amino acid profiling approaches also glycomic or so-called carbohydrate composional profiling based methods are developed using modern UHPLC-FLD-MS methods. This presentation will show not only the suitability in scientific research and the developement of the Davis Food glycopedia or glycan nutrition encyclopedia but also the potential as validated reproducible QA methods.

New QA Test Methods for Carbohydrate Prebiotic Novel Food Ingredients

Functional mushrooms are rapidly gaining popularity in the natural products market, driving the need for improved botanical identity testing. Traditional chemistry-based methods can struggle with accurate identification due to overlapping chemical profiles and environmental variability within species and tissue type. This challenge is further compounded in blended formulations, where precise identification can become even more difficult. This talk will demonstrate how DNA-based approaches overcome these challenges to showcase how DNA testing can be a powerful option for accurate species identification despite the growing conditions, tissue type, product type, or blend formulation. Case studies on Lentinula edodes (shiitake) and Cordyceps militaris (cordyceps) will illustrate successful application of DNA testing across various product matrices, including finished goods and complex blends. By integrating DNA-based authentication into quality control, the industry can establish a strong option for mushroom verification, ensuring accuracy, transparency, and trust from cultivator to consumer.

Genetic Verification of Functional Mushrooms: A Reliable Approach to Ingredient Testing in Finished Products

The increasing complexity of botanical product formulations present significant challenges in accurate species identification and quantification. Traditional analytical methods sometimes struggle to precisely determine the composition of processed botanical mixtures, particularly in cases involving mushrooms and other botanically diverse preparations. This research introduces a refined quantitative PCR (qPCR) approach for accurately estimating the composition of mushroom blends.
Quantitative PCR has previously demonstrated utility in both absolute quantification across various biological systems and is used often when testing for microbial pathogens, cell types, and more. Our study expands this methodology to the complex domain of mushroom blend analysis, developing a molecular approach that utilizes species-specific genetic markers that can precisely identify and quantify multiple species within a blend. 
The proposed technique offers significant advantages over traditional morphological and chemical identification methods, providing high-resolution quantification with improved sensitivity and specificity. Our results demonstrate the potential of qPCR as a powerful tool for comprehensive botanical blend analysis, addressing critical needs in quality control, authenticity verification, and regulatory compliance across food, nutraceutical, and pharmaceutical industries.

Quantitative Molecular Identification of Botanical Species in Complex Blends Using qPCR

Botanical genomes contain abundant information, from which species-specific DNA regions can be pinpointed for use as markers. Molecular methods that can reliably access sufficient genomic data can serve as repeatable and definitive identification techniques. Through multiple case studies, we demonstrate how next generation sequencing (NGS) is a powerful tool allowing massive data generation and individual amplicon sequencing that can overcome conventional sequencing challenges: 1) Botanical blend products contain a variety of plant-based ingredients that require authentication. We developed primer sets for ~30 ingredients that can achieve species level identifications using one or more small genomic regions (≤150bp). Individual amplicon sequencing using NGS allowed for precise identification of target material, and detection of non-targets in some cases. NGS was exceptionally cost and resource efficient in high throughput runs (10+ samples) and was designed to be robust in identifying individual species within mixtures. 2) We further developed primer panels to identify individual mushrooms within multi-fungi ingredient matrices like Lion’s Mane, Chaga, Maitake, Shiitake, and Reishi. 3) Differentiating hybrid species from their pure parental counterparts can be difficult due to the extreme similarity of chemical and molecular markers. We developed a method that reliably delineates spearmint and peppermint specimens via targeted NGS of the multi-copy nuclear barcode region, ITS2. Observing a sequence composition that reveals a mix of two parent species effectively identifies a hybrid. In conclusion, the individual-amplicon sequencing ability of NGS allows for development of useful targeted tests that can be employed in a quality control environment.

Implementing Targeted Next Generation Sequencing Methods in Botanical Authentication Programs

Next generation sequencing platforms have been commercially available for approximately the last twenty years.  In that timeframe their use in the food safety sector has steadily increased.  While their initial use was centered around sequencing genomes of individual organisms, advances in the technology began to enable the sequencing of entire communities.  Since then metagenomics, the study of the structure and function of entire nucleotide sequences isolated from all the organisms in a sample, has created the opportunity to greatly increase detection of adulterants that compromise the safety and quality of foods and supplements.  
Here we present three case studies where amplicon sequencing or shotgun metagenomics were employed to successfully detect microbial adulterants and quality defects in food and supplement samples.  In each case, culture-based and/or existing molecular methods were initially employed and proved to be unable to resolve the issues encountered by each product.  We will see how community sequencing approaches were able to provide novel insights where other technologies (e.g., traditional culture-based methods, PCR, qPCR) could not.  We will also cover best practices for employing these approaches in diverse food matrices, as well as areas where these approaches will benefit from further innovations.

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Non-Targeted Analysis of PFAS in Food Contact Materials: Unveiling Hidden Dangers with LC-QTOF MS and FluoroMatch

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