Analyte and matrix method extension of per- and polyfluoroalkyl substances in food and feed.

The development and expansion of analytical methods for per- and polyfluoroalkyl substances (PFAS) in food are essential for the continued monitoring of the United States (US) food supply and assessments of dietary exposure. In March 2022, the European Union Reference Laboratory for Halogenated Persistent Organic Pollutants in Feed and Food (EURL POPs) released a guidance document covering priority PFAS of interest, including analytical method parameters and limits of quantification (LOQs). As a result, the Food and Drug Administration (FDA) began method extension work to incorporate ten new additional analytes to method C-010.02 including long-chain perfluorosulfonic acids, fluorotelomer sulfonates, and perfluorooctane sulfonamide. Four long-chain carboxylic acids were also validated across all foods, which were previously added to C-010.02 but only validated in seafood. In December 2022, the European Union published Commission Regulation 2022/2388, establishing maximum levels for perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS) in certain foodstuffs, primarily fish, molluscs, crustaceans, and eggs. As a result, the FDA method was evaluated for performance in reaching LOQs defined in Commission Regulation (EU) 2022/1431. The FDA method was found to be able to reach all required LOQs for analytes in matrices with established maximum levels. Currently, method detection limits (MDLs), which are used by the FDA as the lower limit for reporting PFAS in surveillance samples, were in the same range as defined indicative levels. With further method modifications, required LOQs could be met in fruits, vegetables, and milk. Reaching the lower targeted LOQs for these food matrices will require moving the method to an instrument that can provide increased signal:noise gains at the lower limits of quantification.

Plasma and Skin Per- and Polyfluoroalkyl Substance (PFAS) Levels in Dairy Cattle with Lifetime Exposures to PFAS-Contaminated Drinking Water and Feed.

Plasma and ear notch samples were removed from 164 Holstein cows and heifers, which had lifetime exposures to per- and polyfluoroalkyl substances (PFAS) through consumption of contaminated feed and water sources. A suite of nine PFAS including five perfluoroalkyl carboxylic acids (PFCA) and four perfluoroalkyl sulfonic acids (PFSA) was quantified in plasma and ear notch samples by liquid chromatography-mass spectrometry. Bioaccumulation of four- to nine-carbon PFCAs did not occur in plasma or skin, but PFSAs longer than four carbons accumulated in both plasma and skin. Exposure periods of at least 1 year were necessary for PFSAs to reach steady-state concentrations in plasma. Neither parity (P = 0.76) nor lactation status (P = 0.30) affected total PFSA concentrations in mature cow plasma. In contrast, lactation status greatly affected (P < 0.0001) total PFSA concentrations in ear notch samples. Skin samples could be used for biomonitoring purposes in instances when on-farm blood collection and plasma preparation are not practical.

Analysis of Per- and Poly(fluoroalkyl) Substances (PFASs) in Highly Consumed Seafood Products from U.S. Markets.

Seafood consumption has been identified as one of the major contributors of per- and poly(fluoroalkyl) substances (PFASs) to the human diet. To assess dietary exposure, highly consumed seafood products in the United States were selected for analysis. The analytical method previously used for processed food was extended to include four additional long-chain perflurocarboxylic acids (PFCAs), which have been reported in seafood samples. This method was single-lab-validated, and method detection limits were reported at 345 ng kg-1 for perfluorobutanoic acid (PFBA) and 207 ng kg-1 for perfluoropentanoic acid (PFPeA) and below 100 ng kg-1 for the rest of the PFAS analytes. The 81 seafood samples (clams, crab, tuna, shrimp, tilapia, cod, salmon, pollock) were analyzed for 20 PFASs using the updated analytical method. Most of the seafood packaging was also analyzed by Fourier transform infrared-attenuated total reflectance (FTIR-ATR) to identify packaging potentially coated with PFASs. None of the packaging samples in this study were identified as having PFASs. A wide range of concentrations was observed among the seafood samples, ranging from below the method detection limit to the highest concentration of 23 µg kg-1 for the sum of PFASs in one of the canned clam samples. Such a wide range is consistent with those reported in previous studies. The highest concentrations were reported in clams and crabs, followed by cod, tuna, pollock, tilapia, salmon, and shrimp. Technical perfluorooctanoic acid (PFOA) dominated the profile of the clam samples, which has been consistently found in other clam samples, especially in Asia. Long-chain PFCAs, specifically perfluoroundecanoic (PFUdA) and perfluorododecanoic (PFDoA), were the most frequently detected analytes across all seafood samples. The trends observed are comparable with those in the literature where benthic organisms tend to have the highest PFAS concentrations, followed by lean fish, fatty fish, and aquaculture. The results from this study will be used to prioritize future studies and to inform steps to reduce consumer exposure to PFASs.

Extraction and analysis of an organophosphate salt nucleating agent from irradiated polypropylene resin.

Although it is well-established that irradiation of produce can reduce food-borne pathogens and spoilage organisms, data on the effect of irradiation on polymer additives in food packaging materials are limited, particularly for those additives used in packaging leafy greens or in current food packaging materials. We investigated the effects of irradiating a nucleating agent, aluminium, hydroxybis[2,4,8,10-tetrakis(1,1-dimethylethyl)-6-hydroxy-12H-dibenzo [d,g][1,3,2]dioxaphosphocin 6-oxidato]- (CAS Reg. No. 151841-65-5), at doses of 1-20 kGy in polypropylene. That nucleating agent was then extracted using accelerated solvent extraction and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography-photodiode array detection (LC-PDA), and solid-state nuclear magnetic resonance (SSNMR) spectroscopy. We found this nucleating agent was not significantly affected by radiation treatment up to 20 kGy. Therefore, this nucleating agent could potentially be useful in food packaging materials that will be irradiated at doses of 20 kGy or less. Establishing which additives are stable under anticipated irradiation doses will help support safety evaluation of food packaging materials.

Analysis of per- and poly-fluoroalkyl substances (PFAS) in processed foods from FDA's Total Diet Study.

Additional occurrence data are needed to better understand human exposure to per- and poly-fluoroalkyl substances (PFAS) from commercially available foods in the United States. The Food and Drug Administration's (FDA) Total Diet Study (TDS) collects foods that are both nationally and regionally distributed. In 2018, 172 processed foods were collected from grocery stores around Lenexa, KS, as part of the TDS national collection. A previously developed method for the analysis of PFAS in foods as part of the TDS regional collection was modified and optimized for these samples. This method was single lab validated using 5 different matrices and method detection limits were calculated. During the analysis of these samples, challenges arose with method blanks and further investigation into statistical methods to distinguish between blank and sample concentrations were done. The confirmation of two short chain PFAS, perfluorobutanoic acid (PFBA) and perfluoropentanoic acid (PFPeA), was not possible using triple quadrupole mass spectrometry and a confirmation method was developed using high-resolution mass spectrometry. This technique was also used to investigate potential detections and interferents that fell within the retention time criteria for positive detections. In the national collection, positive detections of perfluorooctanesulfonic acid (PFOS) and perfluorononanoic acid (PFNA) were found in frozen fish sticks/patties, PFOS and perfluorodecanoic acid (PFDA) in canned tuna, and PFOS in protein powder. Concentrations were all below 150 ppt, and no other detects were confirmed above the method detection limits in any other foods.

Method Development and Validation of Per- and Polyfluoroalkyl Substances in Foods from FDA's Total Diet Study Program.

Human exposure to per- and polyfluoroalkyl substances (PFAS) through the US diet has not been well-characterized. Highly consumed foods are routinely monitored through FDA's Total Diet Study program. Portions of these samples were used to develop and validate a method for PFAS in a wide variety of foods. The extraction of 16 PFAS was performed using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method and analyzed by liquid chromatography/mass spectrometry. Method optimizations are described including investigations into the QuEChERS sorbents, matrix effects, and solid-phase extraction (SPE) cartridges. The use of a custom push-through SPE cartridge showed promising results as a rapid cleanup option for food samples. Challenges in ion confirmation are discussed, and the use of enhanced product ion (EPI) full-scan MS/MS spectra is presented as a potential option for verifying false positives. The validated method was then used for the analysis of 179 total diet study samples, and positive detects for perfluorooctanesulfonic acid (PFOS) were found in two fish and one meat sample.

Investigation into the Concentrations and Sources of Nitrates and Nitrites in Milk and Plant-Based Powders.

Milk powders in the United States (US) may contain nitrates and nitrites from several potential sources. These sources include the ingestion of nitrates and nitrites by dairy cows during grazing and drinking, nitric acid used during the sanitization of dairy equipment, and the production of nitrous oxides in directly heated spray dryers. Recently, milk powders manufactured in the US have been rejected during import to other countries because nitrite concentrations were greater than 2 mg/kg (ppm). To date, the concentrations of nitrates and nitrites in milk and plant-based powders in the US are unknown. In this study the nitrate and nitrite concentrations present in diverse milk powders were investigated including 81 milk powders from local and online retailers from 2015 to 2018. In addition, 71 commercial milk powders were obtained from blinded production facilities. Nitrate and nitrite concentrations were determined using ion chromatography with conductivity and UV detection. A subset of samples was analyzed for N-nitrosodimethylamine using gas chromatography-mass spectrometry. Carbon and nitrogen bulk isotope ratios analyzed using isotope ratio mass spectrometry were used to obtain some insights into the production method (organic vs conventional) and geographic source of the milk powder samples. Background nitrate concentrations in US-produced milk powder samples averaged 17 ± 12 mg/kg. Nitrite was detected at concentrations greater than 2 mg/kg in 5 out of 39 different brands of retail milk and plant-based powders. Of these brands, two were plant-based (soy and coconut) powders and the other three had consistently high nitrites. The analysis of milk powders using stable isotope analysis revealed further information about the cow's diet.

Assessment of the Impact of Accelerated Migration Testing for Coated Food Cans Using Food Simulants.

In this study, an accelerated migration test on food can coatings into food simulants was investigated. Food simulants covering a wide range of polarity were used to conduct migration tests at 60 °C with storage times ranging from 4 h to 30 days. Epoxy-resins, acrylic-phenolic, polyester, and vinyl coatings were exposed to water, 3% acetic acid, 50% ethanol, and Miglyol 812®. Using liquid chromatography coupled to a variety of detectors (UHPLC-Q-Orbitrap-MS, UFLC-MS/MS, and HPLC-DAD), migration of several monomers and previously identified oligomers, as well as some unidentified migrants, were determined during the experiment. The data from this study was compared to our findings from previous long-term migration studies with storage times ranging from 24 h to 540 days at 40 °C using the same can coating applications. The results illustrate that performing migration experiments for short time periods at 60 °C may mimic migration results that would be obtained at 40 °C after long-term migration tests (up to 1.5 years) from food can coatings into food simulants.

Analysis of diglycolic acid in food packaging, over the counter products, direct additive carboxymethyl cellulose, and retail foods.

Carboxymethyl cellulose (i.e. CMC or cellulose gum) is used as a direct additive for foods and drugs to change texture and act as a binder. CMC can also be a fluid absorbent used in food packaging and food contact materials. CMC and other carboxymethyl starches are synthesised by condensing glycolic acid with monochloroacetic acid. Diglycolic acid (DGA) is a byproduct produced by this condensation which cannot be completely removed. Currently, there are no analytical methods to accurately detect and quantify DGA in foods and food packaging materials. A method using a methanol/water extraction coupled with weak anion exchange solid phase extraction cleanup for more complex matrices was developed. A novel LC-MS/MS method was used to determine the DGA concentration in food contact materials, food grade direct additive CMC, and foods containing CMC. This paper will discuss the development of a new method for the preparation and cleanup of various food matrices and LC-MS/MS analysis for the presence of DGA.

Determination of endogenous concentrations of nitrites and nitrates in different types of cheese in the United States: method development and validation using ion chromatography.

Nitrites and nitrates can be present in dairy products from both endogenous and exogenous sources. In the European Union (EU), 150 mg kg-1 of nitrates are allowed to be added to the cheese milk during the manufacturing process. The CODEX General Standard for Food Additives has a maximum permitted level of 50 mg kg-1 residue in cheese, while in the United States (U.S.) nitrates are unapproved for use as food additives in cheese. In order to be able to investigate imported cheeses for nitrates intentionally added as preservatives and the endogenous concentrations of nitrates and nitrites present in cheeses in the U.S. marketplace, a method was developed and validated using ion chromatography with conductivity detection. A market sampling of cheese samples purchased in the Washington DC metro area was performed. In 64 samples of cheese, concentrations ranged from below the method detection limit (MDL) to 26 mg kg-1 for nitrates and no concentrations of nitrites were found in any of the cheese samples above the MDL of 0.1 mg kg-1. A majority of the samples (93%) had concentrations below 10 mg kg-1, which indicate the presence of endogenous nitrates. The samples with concentrations above 10 mg kg-1 were mainly processed cheese spread, which can contain additional ingredients often of plant-based origin. These ingredients are likely the cause of the elevated nitrate concentrations. The analysis of 12 additional cheese samples that are liable to the intentional addition of nitrates, 9 of which were imported, indicated that in this limited study, concentrations of nitrate in the U.S.-produced cheeses did not differ from those in imported samples.

Investigation into perfluoroalkyl substances (PFASs) in a cranberry bog: method development and sampling results.

The contamination of groundwater and surface water from previous uses of perfluoroalkyl substances (PFASs), particularly products containing the contaminants perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), has become a concern for drinking water and as a potential exposure route to the food supply. In 2016, the Food and Drug Administration (FDA) was asked to investigate a bog in Massachusetts where the surface water was believed to be contaminated with PFASs. As a result, a method was developed for the analysis of PFASs in cranberries, and water and fruit from the affected bog were evaluated. A QuEChERS method was developed and validated for PFOA, PFOS, and six additional shorter-chain PFASs. Method recoveries ranged from 60% to 115% for validation spikes performed at 10, 20 and 40 ng g-1 and method detection limits ranged from 0.2 to 5.6 ng g-1. Bog water samples were analysed using Environmental Protection Agency (EPA) method 537 for PFOA, PFOS and four additional short-chain PFASs. Surface water concentrations for PFOS ranged from 16 to 122 ng L-1 and input water concentrations were 132 ng L-1 and 206 ng L-1. Of the eight water samples, seven had water concentrations that exceeded the EPA health advisory level for PFOS of 70 ng L-1. Of the 42 cranberry samples analysed, none had detects of PFOA or PFOS above their method detection limits (0.4 and 0.5 ng g-1, respectively), nor any of the other short-chain PFASs.

Simultaneous Analysis of 3-MCPD and 1,3-DCP in Asian Style Sauces Using QuEChERS Extraction and Gas Chromatography-Triple Quadrupole Mass Spectrometry.

Acid hydrolyzed vegetable protein (aHVP) is used for flavoring a wide variety of foods and also in the production of nonfermented soy sauce. During the production of aHVP, chloropropanols including 3-monochloropropane-1,2-diol (3-MCPD) and 1,3 dichloropropane-2-ol (1,3-DCP) can be formed through the reaction of the hydrochloric acid catalyst and residual fat and the reaction of 3-MCPD with acetic acid, respectively. 3-MCPD is a carcinogen, and 1,3-DCP has been classified as a genotoxic carcinogen. The European Union (EU) has set a maximum concentration of 0.02 mg/kg of 3-MCPD in aHVP, and the Food and Drug Administration (FDA) set a guidance limit of 1 mg/kg of 3-MCPD in aHVP. 1,3-DCP is not an approved food additive, and the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) has set a limit at 0.005 mg/kg, which is close to the estimated method detection limit. Currently there are few analytical methods for the simultaneous determination of 3-MCPD and 1,3-DCP without derivatization due to differences in their physical chemical properties and reactivity. A new method was developed using QuEChERS (quick, easy, cheap, effective, rugged, and safe) with direct analysis of the extract without derivatization using gas chromatography-triple quadrupole mass spectrometry (GC-QQQ). Additionally, a market sampling of 60 soy sauce samples was performed in 2015 to determine if concentrations have changed since the FDA limit was set in 2008. The sampling results were compared between the new QuEChERS method and a method using phenylboronic acid (PBA) as a derivatizing agent for 3-MCPD analysis. The concentrations of 3-MCPD detected in soy sauce samples collected in 2015 (

The Food and Drug Administration Office of Women's Health: Impact of Science on Regulatory Policy: An Update.

The U.S. Food and Drug Administration Office of Women's Health (FDA OWH) has supported women's health research for ∼20 years, funding more than 300 studies on women's health issues, including research on diseases/conditions that disproportionately affect women in addition to the evaluation of sex differences in the performance of and response to medical products. These important women's health issues are studied from a regulatory perspective, with a focus on improving and optimizing medical product development and the evaluation of product safety and efficacy in women. These findings have influenced industry direction, labeling, product discontinuation, safety notices, and clinical practice. In addition, OWH-funded research has addressed gaps in the knowledge about diseases and medical conditions that impact women across the life span such as cardiovascular disease, pregnancy, menopause, osteoporosis, and the safe use of numerous medical products.

Method development and survey of Sudan I-IV in palm oil and chilli spices in the Washington, DC, area.

Sudan I, II, III and IV dyes are banned for use as food colorants in the United States and European Union because they are toxic and carcinogenic. These dyes have been illegally used as food additives in products such as chilli spices and palm oil to enhance their red colour. From 2003 to 2005, the European Union made a series of decisions requiring chilli spices and palm oil imported to the European Union to contain analytical reports declaring them free of Sudan I-IV. In order for the USFDA to investigate the adulteration of palm oil and chilli spices with unapproved colour additives in the United States, a method was developed for the extraction and analysis of Sudan dyes in palm oil, and previous methods were validated for Sudan dyes in chilli spices. Both LC-DAD and LC-MS/MS methods were examined for their limitations and effectiveness in identifying adulterated samples. Method validation was performed for both chilli spices and palm oil by spiking samples known to be free of Sudan dyes at concentrations close to the limit of detection. Reproducibility, matrix effects, and selectivity of the method were also investigated. Additionally, for the first time a survey of palm oil and chilli spices was performed in the United States, specifically in the Washington, DC, area. Illegal dyes, primarily Sudan IV, were detected in palm oil at concentrations from 150 to 24 000 ng ml(-1). Low concentrations (< 21 µg kg(-1)) of Sudan dyes were found in 11 out of 57 spices and are most likely a result of cross-contamination during preparation and storage and not intentional adulteration.

An investigational report into the causes of pine mouth events in US consumers.

Between July 2008 and June 2012, the US Food and Drug Administration received 501 consumer reports of prolonged taste disturbances consistent with pine mouth syndrome. Consumers consistently reported a delayed bitter or metallic taste beginning hours to days following consumption of pine nuts that recurred with intake of any food or meal. This dysgeusia lasted in some cases up to a few weeks, but would eventually resolve without serious health consequences. To evaluate these reports, a questionnaire was developed to address various characteristics of the pine nuts consumed, pertinent medical history of complainants and other dysgeusia-related factors. Pine nut samples associated with 15 complaints were collected for analysis. The investigation of reports found no clear evidence of an underlying medical cause or common trigger that could adequately explain the occurrence of dysgeusia in complainants. Rather, the results of our investigation suggest that the occurrence of "pine mouth syndrome" in US consumers is correlated with the consumption of the pine nut species Pinus armandii.

Assessments and improvements in methods for monitoring seafood safety in response to the Deepwater Horizon oil spill.

As a result of the April 2010 Deepwater Horizon oil spill, sensory testing protocols were established for reopening closed seafood harvest areas. In order to improve this method and quantitatively assess petrochemical taint, a new method using solid phase microextraction (SPME) and a 5975T transportable GC/MS was developed. This method can analyze 40 samples per instrument per day and could be an alternative to the human sensory panel. In seafood samples collected from supermarkets in the Washington D.C. area and the Gulf of Mexico, all compounds related to petrochemical taint were below the method detection limit (MDL) (0.14-2.6 ng/g). Additionally, to address consumer concerns regarding the presence of n-alkanes and iso-alkanes in seafood, these compounds were investigated in samples purchased in the Washington D.C. area and the Gulf of Mexico. Concentrations in Gulf of Mexico finfish ranged from 0.066 to 1.2 mg/kg, which is within the same background range of iso- and n-alkanes measured in seafood samples purchased in the Washington D.C. area (0.0072-1.6 µg/g). These automated methods provide a transportable option to obtain rapid results for compounds indicative of petroleum taint and iso- and n-alkanes in case of a future disaster.