ABSTRACT
BACKGROUND: Since the publication of Standard Method Performance Requirements (SMPR®) for vitamin D in infant formula (SMPR 2011.004) by AOAC INTERNATIONAL, revised vitamin D limits have been recommended by the European Food Safety Authority (EFSA) for infant formula and adopted in Commission Delegated Regulation (EU) 2019/828. The vitamin D range introduced, 2-2.5 µg/100 kcal, is significantly narrower than previous limits specified by Codex Standard 72-1981 and requires lower method reproducibility metrics to adequately assess regulatory compliance. The narrower limits for vitamin D present a significant challenge for current-generation reference analytical methods that comply with SMPR 2011.004. OBJECTIVE: We evaluate the impact of Delegated Regulation (EU) 2019/828 on the demonstrated performance of AOAC Method 2016.05/ISO 20636:2018 to assess the likelihood that vitamin D results produced by the method would be found outside the EU limits when testing infant formula that is compliant as manufactured. METHODS: AOAC Method 2016.05/ISO 20636:2018, specifically data generated during multi-laboratory study, was used as a basis for statistical evaluation of the impact of the narrower EU vitamin D limits. RESULTS: The review of AOAC Method 2016.05/ISO 20636:2018 method performance against the vitamin D regulatory range introduced in (EU) 2019/828 indicates methods capable of performing in alignment with SMPR 2011.004 are likely to produce results that fail to meet EU requirements. CONCLUSIONS: Our assessment illustrates the high probability that a well-manufactured product with vitamin D levels within the EU regulatory range would fail to meet the regulatory requirements due to analytical method variability when tested using fit-for-purpose methods. Further, required method performance cannot be expected with the future development of new methods. To avoid this, consideration should be given to aligning proposed regulatory limits with method performance metrics of current-generation compendial methods. HIGHLIGHTS: Current, state-of-the-art methods cannot consistently verify infant formula product compliance for vitamin D in accordance with (EU) 2019/828.
Subject(s)
Infant Formula , Vitamin D , Food, Formulated , Humans , Infant , Infant Formula/analysis , Reproducibility of Results , VitaminsABSTRACT
The Codex Alimentarius Commission, a central part of the joint Food and Agricultural Organization/World Health Organizations Food Standards Program, adopts internationally recognized standards, guidelines, and code of practices that help ensure safety, quality, and fairness of food trade globally. Although Codex standards are not regulations per se, regulatory authorities around the world may benchmark against these standards or introduce them into regulations within their countries. Recently, the Codex Committee on Nutrition and Foods for Special Dietary Uses (CCNFSDU) initiated a draft revision to the Codex standard for follow-up formula (FUF), a drink/product (with added nutrients) for young children, to include requirements for limiting or measuring the amount of sweet taste contributed by carbohydrates in a product. Stakeholders from multiple food and beverage manufacturers expressed concern about the subjectivity of sweetness and challenges with objective measurement for verifying regulatory compliance. It is a requirement that Codex standards include a reference to a suitable method of analysis for verifying compliance with the standard. In response, AOAC INTERNATIONAL formed the Ad Hoc Expert Panel on Sweetness in November 2020 to review human perception of sweet taste, assess the landscape of internationally recognized analytical and sensory methods for measuring sweet taste in food ingredients and products, deliver recommendations to Codex regarding verification of sweet taste requirements for FUF, and develop a scientific opinion on measuring sweet taste in food and beverage products beyond FUF. Findings showed an abundance of official analytical methods for determining quantities of carbohydrates and other sweet-tasting molecules in food products and beverages, but no analytical methods capable of determining sweet taste. Furthermore, sweet taste can be determined by standard sensory analysis methods. However, it is impossible to define a sensory intensity reference value for sweetness, making them unfit to verify regulatory compliance for the purpose of international food trade. Based on these findings and recommendations, the Codex Committee on Methods of Analysis and Sampling agreed during its 41st session in May 2021 to inform CCNFSDU that there are no known validated methods to measure sweetness of carbohydrate sources; therefore, no way to determine compliance for such a requirement for FUF.
Subject(s)
Food Ingredients , Beverages , Diet , Food, Formulated , Humans , TasteABSTRACT
A brief overview of the main analytical approaches and practices to determine food authenticity is presented, addressing, as well, food supply chain and future requirements to more effectively mitigate food fraud. Food companies are introducing procedures and mechanisms that allow them to identify vulnerabilities in their food supply chain under the umbrella of a food fraud prevention management system. A key step and first line of defense is thorough supply chain mapping and full transparency, assessing the likelihood of fraudsters to penetrate the chain at any point. More vulnerable chains, such as those where ingredients and/or raw materials are purchased through traders or auctions, may require a higher degree of sampling, testing, and surveillance. Access to analytical tools is therefore pivotal, requiring continuous development and possibly sophistication in identifying chemical markers, data acquisition, and modeling. Significant progress in portable technologies is evident already today, for instance, as in the rapid testing now available at the agricultural level. In the near future, consumers may also have the ability to scan products in stores or at home to authenticate labels and food content. For food manufacturers, targeted analytical methods complemented by untargeted approaches are end control measures at the factory gate when the material is delivered. In essence, testing for food adulterants is an integral part of routine QC, ideally tailored to the risks in the individual markets and/or geographies or supply chains. The development of analytical methods is a first step in verifying the compliance and authenticity of food materials. A next, more challenging step is the successful establishment of global consensus reference methods as exemplified by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals initiative, which can serve as an approach that could also be applied to methods for contaminants and adulterants in food. The food industry has taken these many challenges aboard, working closely with all stakeholders and continuously communicating on progress in a fully transparent manner.
Subject(s)
Food Analysis/methods , Food Contamination/analysis , Food Analysis/standards , Humans , Quality ControlABSTRACT
Methods under consideration as part of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals process are to be evaluated against a set of Standard Method Performance RequirementsSM (SMPRs) via peer review by an expert review panel (ERP). A validation protocol and a checklist have been developed to assist the ERP to evaluate experimental data and to compare multiple candidate methods for each nutrient. Method performance against validation parameters mandated in the SMPRs as well as additional criteria are to be scored, with the method selected by the ERP proceeding to multilaboratory study prior to Final Action approval. These methods are intended to be used by the infant formula industry for the purposes of dispute resolution.
Subject(s)
Dietary Supplements , Food Analysis/methods , Infant Formula , Adult , Food, Formulated/analysis , Humans , Infant , Reference Standards , Reproducibility of ResultsABSTRACT
Since 2002, it is known that the probable human carcinogen acrylamide is present in commonly consumed carbohydrate-rich foods, such as French fries and potato chips. In this review, the authors discuss the body of evidence on acrylamide carcinogenicity from both epidemiological and rodent studies, including variability, strengths and weaknesses, how both types of evidence relate, and possible reasons for discrepancies. In both rats and humans, increased incidences of various cancer types were observed. In rats, increased incidences of mammary gland, thyroid tumors and scrotal mesothelioma were observed in both studies that were performed. In humans, increased risks of ovarian and endometrial cancers, renal cell cancer, estrogen (and progesterone) receptor-positive breast cancer, and oral cavity cancer (the latter in non-smoking women) were observed. Some cancer types were found in both rats and humans, e.g., endometrial cancer (observed in one of the two rat studies), but there are also some inconsistencies. Interestingly, in humans, some indications for inverse associations were observed for lung and bladder cancers in women, and prostate and oro- and hypopharynx cancers in men. These latter observations indicate that genotoxicity may not be the only mechanism by which acrylamide causes cancer. The estimated risks based on the epidemiological studies for the sites for which a positive association was observed were considerably higher than those based on extrapolations from the rat studies. The observed pattern of increased risks in the rat and epidemiological studies and the decreased risks in the epidemiological studies suggests that acrylamide might influence hormonal systems, for which rodents may not be good models.
Subject(s)
Acrylamide/toxicity , Carcinogens/toxicity , Neoplasms/etiology , Acrylamide/pharmacokinetics , Administration, Oral , Animals , Animals, Laboratory , Carcinogenicity Tests , Carcinogens/pharmacokinetics , Diet , Diet Surveys , Disease Models, Animal , Feeding Behavior , Female , Humans , Male , Neoplasms/epidemiology , Registries , Risk Assessment , Risk FactorsABSTRACT
Acrylamide, a potential human carcinogen, has been discovered in a variety of heat-treated carbohydrate-rich food products. Previously, dietary acrylamide intake was shown to be associated with endocrine-related cancers in humans. We assessed the association between dietary acrylamide intake and risk of postmenopausal breast cancer stratified by estrogen and progesterone receptor status. This study was embedded within the Netherlands Cohort Study on diet and cancer, which was initiated in 1986 enrolling 62,573 women aged 55-69 years at baseline. After 13.3 years of follow-up, 2225 incident breast cancer cases were ascertained, with hormone receptor status information for 43%. Cox proportional hazards analysis was applied to determine hazard ratios in quintiles of dietary acrylamide intake stratifying on estrogen receptor (ER) and progesterone receptor (PR) and smoking status. No association was observed for overall breast cancer or receptor-negative breast cancer risk, irrespective of smoking status. A statistically non-significantly increased risk of ER positive, PR positive and joint receptor-positive breast cancer was found in never-smoking women. The multivariable-adjusted hazard ratios were 1.31 (95% CI: 0.87-1.97, P (trend) = 0.26) for ER+, 1.47 (0.86-2.51, P (trend) = 0.14) for PR+, and 1.43 (0.83-2.46, P (trend) = 0.16) for ER+PR+, when comparing women in the highest quintile of acrylamide intake (median 36.8 microg/day) to women in the lowest (median 9.5 microg/day). This study showed some indications of a positive association between dietary acrylamide intake and receptor-positive breast cancer risk in postmenopausal never-smoking women. Further studies are needed to confirm or refute our observations.
Subject(s)
Acrylamides/adverse effects , Breast Neoplasms/epidemiology , Carcinogens, Environmental/adverse effects , Diet , Estrogens , Neoplasms, Hormone-Dependent/epidemiology , Postmenopause , Progesterone , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis , Acrylamides/analysis , Aged , Breast Neoplasms/chemically induced , Breast Neoplasms/chemistry , Carcinogens, Environmental/analysis , Cohort Studies , Feeding Behavior , Female , Humans , Incidence , Middle Aged , Neoplasms, Hormone-Dependent/chemically induced , Neoplasms, Hormone-Dependent/chemistry , Netherlands/epidemiology , Proportional Hazards Models , Risk , Smoking/epidemiologyABSTRACT
Acrylamide exposure has been related to an increased incidence of oral and thyroid tumors in animal studies. In 1986, 120,852 persons (aged 55-69 years) were included in the Netherlands Cohort Study. Dietary acrylamide intake was assessed with a food frequency questionnaire and was based on chemical analysis of all relevant Dutch foods. Hazard ratios were adjusted for smoking and other confounders. After 16.3 years of follow-up, there were 101, 83, 180, and 66 cases of oral cavity, oro-hypopharynx, larynx, and thyroid cancer, respectively. Average daily dietary acrylamide intake was 21.8 microg (standard deviation, 12.1). Dietary acrylamide intake was not associated with increased risk of oral cavity (hazard ratio (HR) per 10-microg intake/day = 0.90, 95% confidence interval (CI): 0.73, 1.10), oro-hypopharynx (HR = 0.74, 95% CI: 0.53, 1.03), larynx (HR = 1.05, 95% CI: 0.91, 1.21), or thyroid (HR = 1.03, 95% CI: 0.82, 1.27) cancer. For nonsmokers, hazard ratios were not increased either. Dietary acrylamide was statistically significantly associated with increased risk of oral cavity cancer in female nonsmokers, but case numbers were small. Dietary acrylamide intake was not positively associated with risk of head-neck and thyroid cancer, except with oral cavity cancer risk for female nonsmokers. A negative association for males was indicated.
Subject(s)
Acrylamides/adverse effects , Food Additives/adverse effects , Head and Neck Neoplasms/etiology , Thyroid Neoplasms/etiology , Acrylamides/analysis , Aged , Case-Control Studies , Female , Food Additives/analysis , Head and Neck Neoplasms/epidemiology , Head and Neck Neoplasms/prevention & control , Humans , Male , Middle Aged , Multivariate Analysis , Netherlands/epidemiology , Proportional Hazards Models , Prospective Studies , Sex Distribution , Smoking/epidemiology , Thyroid Neoplasms/epidemiology , Thyroid Neoplasms/prevention & controlABSTRACT
BACKGROUND: Acrylamide is a probable human carcinogen, which is present in several heat-treated foods. In epidemiologic studies, positive associations with endometrial, ovarian, and renal cell cancer risk have been observed. The incidence of central nervous system tumors was increased upon acrylamide administration in drinking water to rats. In the current study, the association between dietary acrylamide intake and human brain cancer risk was investigated for the first time. METHODS: In 1986, 120,852 persons (ages 55-69 years) were included in the Netherlands Cohort Study on diet and cancer. At baseline, a random subcohort of 5,000 participants was randomly selected from the total cohort for a case-cohort approach. Acrylamide intake was assessed with a food frequency questionnaire at baseline and based on acrylamide analyses in relevant Dutch foods. Hazard ratios (HR) were calculated using Cox proportional hazards analysis. Subgroup analyses were done for microscopically verified brain cancer, astrocytic gliomas, high-grade astrocytic gliomas, and never-smokers. The acrylamide risk estimates were adjusted for possible brain cancer risk factors. RESULTS: After 16.3 years of follow-up, 216 brain cancer cases were available for analysis. The multivariable-adjusted HR per 10 microg/d increment of acrylamide intake was 1.02 (95% confidence interval, 0.89-1.16). HRs were not significantly increased either when dietary acrylamide intake was analyzed as a categorical variable. Also, there was no association in the subgroups based on histology and smoking. CONCLUSION: In this prospective cohort study, acrylamide intake was not associated with brain cancer risk.
Subject(s)
Acrylamide/toxicity , Brain Neoplasms/chemically induced , Diet , Aged , Brain Neoplasms/epidemiology , Female , Follow-Up Studies , Humans , Incidence , Male , Middle Aged , Netherlands/epidemiology , Proportional Hazards Models , Prospective Studies , Risk , Surveys and QuestionnairesABSTRACT
BACKGROUND: Acrylamide is a probable human carcinogen that is present in several heat-treated foods. In epidemiological studies, positive associations between dietary acrylamide intake and the risks of endometrial, ovarian, estrogen receptor-positive breast, and renal cell cancers have been observed. The association between dietary acrylamide intake and lung cancer risk is not known. METHODS: We conducted a case-cohort study among 58 279 men and 62 573 women (aged 55-69 years) in the Netherlands Cohort Study on Diet and Cancer. Intakes of acrylamide-containing foods and risk factors for cancer were assessed with a self-administered questionnaire at baseline in 1986 and combined with acrylamide levels in relevant Dutch foods to assess total dietary acrylamide intake. The number of person-years at risk was estimated by using a random sample of participants from the total cohort that was chosen at baseline (n = 5000). Incident lung cancer cases in the total cohort were detected by computerized record linkages to the Netherlands Cancer Registry and the Netherlands Pathology Registry. Hazard ratios and 95% confidence intervals (CIs) for the risk of lung cancer associated with acrylamide intakes were estimated using Cox proportional hazards models that controlled for smoking (status, quantity, and duration) and other lung cancer risk factors. All statistical tests were two-sided. RESULTS: After 13.3 years of follow-up (September 17, 1986 up to January 1, 2000) there were 2649 cases of primary, histologically verified lung cancer (International Classification of Diseases for Oncology-3 code: C34) when cases with prevalent cancer at baseline (other than skin cancer) were excluded. The multivariable-adjusted hazard ratio of lung cancer for a 10-microg/d increment of acrylamide intake was 1.03 (95% CI = 0.96 to 1.11) for men and 0.82 (95% CI = 0.69 to 0.96) for women. The hazard ratio of lung cancer for the highest (median intake [microg/d]: men = 37.6 and women = 36.8) vs the lowest (median intake [microg/d]: men = 10.8 and women = 9.5) quintile of acrylamide intake was 1.03 (95% CI = 0.77 to 1.39, P(trend) = .85) for men and 0.45 (95% CI = 0.27 to 0.76, P(trend) = .01) for women. The inverse association in women was strongest for adenocarcinoma (hazard ratio for highest vs lowest tertile of intake = 0.40, 95% CI = 0.21 to 0.78; P(trend) = .01). CONCLUSIONS: Acrylamide intake was not associated with lung cancer risk in men but was inversely associated in women, most strongly for adenocarcinoma. This finding suggests that acrylamide is involved in human carcinogenesis through pathways other than genotoxicity.
Subject(s)
Acrylamide/administration & dosage , Acrylamide/adverse effects , Carcinogens/administration & dosage , Feeding Behavior , Lung Neoplasms/chemically induced , Lung Neoplasms/epidemiology , Adenocarcinoma/chemically induced , Adenocarcinoma/epidemiology , Aged , Case-Control Studies , Diet Surveys , Endometrial Neoplasms/chemically induced , Endometrial Neoplasms/epidemiology , Female , Follow-Up Studies , Gonadal Steroid Hormones/blood , Humans , Lung Neoplasms/blood , Male , Medical Record Linkage , Middle Aged , Multivariate Analysis , Netherlands/epidemiology , Odds Ratio , Ovarian Neoplasms/chemically induced , Ovarian Neoplasms/epidemiology , Proportional Hazards Models , Prospective Studies , Registries , Risk Assessment , Risk Factors , Sex Factors , Smoking/adverse effectsABSTRACT
Acrylamide is a probable human carcinogen that was detected in several heat-treated foods, such as French fries and crisps, in 2002. Prospective studies are needed on acrylamide and human cancer risk. We prospectively investigated the association between acrylamide and gastrointestinal cancer risk. In 1986, 120,852 men and women (aged 55-69 y) were included in the Netherlands Cohort Study on diet and cancer. At baseline, a random subcohort of 5000 participants was selected for a case-cohort approach. Acrylamide intake was assessed with a FFQ at baseline and was based on acrylamide analyses in relevant Dutch foods. After 13.3 y of follow-up, 2190, 563, 349, and 216 cases of colorectal, gastric, pancreatic, and esophageal cancer, respectively, were available for analysis. The daily acrylamide intake of the subcohort was (mean +/- SD) 21.7 +/- 12.1 microg. A 10-microg/d increment of acrylamide intake was associated with multivariable-adjusted Cox proportional hazard rate ratios (HR) (95% CI) of 1.00 (0.96-1.06), 1.02 (0.94-1.10), 1.06 (0.96-1.17), and 0.96 (0.85-1.09) for colorectal, gastric, pancreatic, and esophageal cancer, respectively. For former or never-smokers, the corresponding HR were: 1.03 (0.94-1.12), 1.09 (0.98-1.22), 1.07 (0.93-1.24), and 0.92 (0.76-1.11). There were some significantly increased risks within subgroups stratified by obesity, nonoccupational physical activity, and age, factors that were a priori selected based on their capacity to modify cytochrome P4502E1 activity. Overall, acrylamide intake was not associated with colorectal, gastric, pancreatic, and esophageal cancer risk, but some subgroups deserve further attention.
Subject(s)
Acrylamide/toxicity , Carcinogens/toxicity , Food Contamination , Gastrointestinal Neoplasms/chemically induced , Acrylamide/analysis , Aged , Case-Control Studies , Cohort Studies , Diet , Female , Humans , Male , Middle Aged , Odds Ratio , Proportional Hazards ModelsABSTRACT
In traditional Chinese medicine several Aristolochia species are used. Aristolochia spp. contain a mixture of aristolochic acids (AAs), mainly AA I and AA II which are nephrotoxicants and carcinogens. After AA-related nephropathy (AAN) and urothelial cancer were described in female patients in Belgium following intake of AA-contaminated herbal preparations, herbs with AAs were prohibited worldwide. Confusing nomenclature can cause AA contamination of certain Chinese traditional herbal preparations (THPs). Here we report the results of investigations by the Dutch Food and Consumer Product Safety Authority (VWA) into the presence of AAs in THPs sampled on the Dutch market using a liquid-chromatography--mass spectrometry method. Between 2002 and 2006 we sampled 190 Chinese THPs using recent information on Chinese THPs potentially containing AAs. AA I was found in 25 samples up to a concentration of 1,676 mg/kg. AA II was also found in 13 of these samples up to 444 mg/kg. All 25 positive samples including Mu Tong, Fang Ji, Tian Xian Teng and Xi Xin were part of a group of 68 THPs identified as possibly containing AAs. In a worst-case scenario, use of a sample of Mu Tong with the highest AA content over a 7-day period would result in the same intake levels of AAs which significantly raised the cancer risk in the Belgian AAN cases. Our results show that contaminated THPs still can be found on the market following worldwide publicity. Therefore, it can be concluded that testing of possibly AA-contaminated THPs is still essential.
Subject(s)
Aristolochic Acids/analysis , Drugs, Chinese Herbal , Plant Preparations/analysis , Aristolochic Acids/chemistry , Chromatography, Liquid , Mass Spectrometry , Molecular Structure , Netherlands , Plant Preparations/chemistryABSTRACT
Simultaneous Determination of Vitamins.--Klejdus et al. described a simultaneous determination of 10 water- and 10 fat-soluble vitamins in pharmaceutical preparations by liquid chromatography-diode-array detection (LC-DAD). A combined isocratic and linear gradient allowed separation of vitamins in 3 distinct groups: polar, low-polar, and nonpolar. The method was applied to pharmaceutical preparations, fortified powdered drinks, and food samples, for which results were in good agreement with values claimed. Heudi et al. described a separation of 9 water-soluble vitamins by LC-UV. The method was applied for the quantification of vitamins in polyvitaminated premixes used for the fortification of infant nutrition products. The repeatability of the method was evaluated at different concentration levels and coefficients of variation were <6.5%. The concentrations of vitamins found in premixes with the method were comparable to the values declared. A disadvantage of the methods mentioned above is that sample composition has to be known in advance. According to European legislation, for example, foods might be fortified with riboflavin phosphate or thiamin phosphate, vitamers which are not included in the simultaneous separations described. Vitamin B2.--Viñas et al. elaborated an LC analysis of riboflavin vitamers in foods. Vitamin B2 can be found in nature as the free riboflavin, but in most biological materials it occurs predominantly in the form of 2 coenzymes, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD). Several methods usually involve the conversion of these coenzymes into free riboflavin before quantification of total riboflavin. According to the authors, there is growing interest to know flavin composition of foods. The described method separates the individual vitamers isocratically. Accuracy of the method is tested with 2 certified reference materials (CRMs). Vitamin B5.-Methods for the determination of vitamin B5 in foods are limited because of their low sensitivity and poor selectivity. Pakin et al. proposed a post-column derivatization of pantothenic acid as a fluorescent compound and used this principle in a specific and sensitive method for the determination of free and bound pantothenic acid in a large variety of foods. A French laboratory invited European laboratories to participate in a series of collaborative studies for this method, which will be carried out in 2005/2006. A more sophisticated method was described by Mittermayer et al. They developed an LC-mass spectrometry (LC/MS) method for the determination of vitamin B5 in a wide range of fortified food products. Application of the method to various samples showed consistent results with those obtained by microbiology. Vitamin B6.-Method 2004.07, an LC method for the analysis of vitamin B6 in reconstituted infant formula, was published by Mann et al. In contrast with this method, which quantifies vitamin B6 after converting the phosphorylated and free vitamers into pyridoxine, Viñas et al. published an LC method which determines 6 vitamin B6 related compounds, the 3 B6 vitamers, their corresponding phosphorylated esters, and a metabolite. Accuracy was determined using 2 CRMs. Results were within the certified ranges. Vitamin C.-Franke et al. described an extensive study to vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. Vitamin C was determined by measuring ascorbic acid in its reduced state by LC and coulometric detection along with UV absorbance detection at 245 nm. No attempts were made to assess levels of dehydroascorbic acid. Most recent research revealed that cell uptake of dehydroascorbic acid is unlikely to play a major role, which may explain the very low vitamin C activity of orally administered L-dehydroascorbic acid in rats. The food levels found by Franke et al. are variably lower, higher, or equal in comparison to other studies. Iwase described a method for the determination of ascorbic acid in foods using L-methionine for the pre-analysis sample stabilization. Electrochemical detection was used for the quantification. Traditionally, metaphosphoric acid was proven to be a useful dissolving agent for the determination of ascorbic acid. However, it dissolves in water very slowly, it is hygroscopic, and accurate weighing is not easy. Adjustment at pH 2-3 takes a long time. It appeared to be possible to replace metaphosphoric acid by 0.2% phosphoric acid. Methionine played an important role on the stability of ascorbic acid. The method seemed to be applicable to the routine analysis of ascorbic acid in foods. Folic Acid.-Microbiological analysis of total folate in foods is often considered as the golden standard compared to other methods based on, for example, LC. Koontz et al. showed results of total folate concentrations measured by microbiological assay in a variety of foods. Samples were submitted in a routine manner to experienced laboratories that regularly perform folate analysis fee-for-service basis in the United States. Each laboratory reported the use of a microbiological method similar to the AOAC Official Method for the determination of folic acid. Striking was, the use of 3 different pH extraction conditions by 4 laboratories. Only one laboratory reported using a tri-enzyme extraction. Results were evaluated. Results for folic acid fortified foods had considerably lower between-laboratory variation, 9-11%, versus >45% for other foods. Mean total folate ranged from 14 to 279 microg/100 g for a mixed vegetable reference material, from 5 to 70 microg/100 g for strawberries, and from 28 to 81 microg/100 g for wholemeal flour. One should realize a large variation in results, which might be caused by slight modifications in the microbiological analysis of total folate in foods or the analysis in various (unfortified) food matrixes. Furthermore, optimal combination of enzymes and reaction conditions may vary depending on the composition of the food. Padrangi and Laborde showed recently that treatment with alpha-amylase had no significant effect on measured folate in spinach, although addition of protease significantly increased the release of folate. LC/MS applications gain increasing attention because of their specificity. Rychlik used stable isotope dilution assays for the determination of the folate content of broccoli and bread. Compared to data in the literature and food data bases, amounts were significantly lower. Pawlosky et al., however, found comparable values for 5-methyltetrahydrofolic acid and folic acid by HPLC analysis with fluorescent detection and HPLC/MS. Among samples analyzed were CRMs and broccoli. Besides folic acid, other water-soluble vitamins were also determined by LC/MS/MS by Leporati et al. The method was applied to the quantitative analysis of the natural content of vitamins in typical Italian pasta samples, as well as in fortified pasta samples produced for the U.S. market. Biotin.-A paper from Staggs et al. included the assertion that existing biotin data in food composition tables are inaccurate because the majority are based on bioassays with all relevant disadvantages. Data in most cases are overestimated with consequences for recommendations for dietary biotin intake. An HPLC/avidin-binding assay was used to analyze 87 foods to support the hypothesis mentioned.