Development of an improved standard reference material for folate vitamers in human serum.

The US National Institute of Standards and Technology (NIST) developed a Standard Reference Material® (SRM®) 3949 Folate Vitamers in Frozen Human Serum to replace SRM 1955 Homocysteine and Folate in Human Serum. The presence of increased endogenous levels of folic acid and 5-methyltetrahydrofolate (5mTHF) in SRM 3949, enhanced folate stability via addition of ascorbic acid, and inclusion of values for additional minor folates are improvements over SRM 1955 that should better serve the clinical folate measurement community. The new SRM contains folates at three levels. To produce SRM 3949, pilot sera were collected from 15 individual donors, 5 of whom were given a 400-µg folic acid supplement 1 h prior to blood draw to increase serum levels of 5mTHF and folic acid for the high-level material. To stabilize the folates, 0.5% (mass concentration) ascorbic acid was added as soon as possible after preparation of serum. These pilot sera were screened for five folates plus the pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyltetrahydrofolate (MeFox) at the US Centers for Disease Control and Prevention (CDC) by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Based on these results, a blending protocol was specified to obtain the three desired folate concentrations for SRM 3949. ID-LC-MS/MS analysis at the CDC and NIST was utilized to assign values for folic acid and 5mTHF, as well as several minor folates.

Lessons from the NIST micronutrients quality assurance program for vitamin C, 1993 to 2015: sample stability, assay reproducibility, and use of controls to improve comparability.

Vitamin C is a necessary micronutrient that is involved in many biological processes. In preserved human plasma and serum, vitamin C is most meaningfully analyzed as total ascorbic acid (TAA). From 1993 through 2015, the National Institute of Standards and Technology (NIST) coordinated 40 interlaboratory studies (ILS) devoted to improving the between-participant comparability of TAA measurements. The results from these ILS demonstrate that the concentration of TAA ([TAA]) is stable for at least 20 years in serum diluted 1 + 1 (volume fraction) with 10% mass concentration aqueous metaphosphoric acid (MPA) and stored at -80 °C. The between-participant relative reproducibility precision, expressed as a coefficient of variation (CV), improved from over 16% to under 9% over the course of the studies. Normalization of test samples (i.e., ex post-facto recalibrating the as-submitted results) using participant-prepared serum-free calibration solutions did not improve reproducibility. Normalization to one control sample having a similar serum-based matrix as the test samples improved the CV to 7%; normalization to two such controls reduced the CV to 4%. Multicenter studies that require the highest degree of measurement comparability among the participants should consider calibrating with materials that have a serum-based matrix as similar as possible to that of the samples of interest.

Standard Reference Material (SRM) 2378 fatty acids in frozen human serum. Certification of a clinical SRM based on endogenous supplementation of polyunsaturated fatty acids.

Dietary fatty acids can be both beneficial and detrimental to human health depending on the degree and type of saturation. Healthcare providers and research scientists monitor the fatty acid content of human plasma and serum as an indicator of health status and diet. In addition, both the Centers for Disease Control & Prevention (CDC) and the National Institutes of Health - Office of Dietary Supplements are interested in circulating fatty acids (FAs) because they may be predictive of coronary heart disease. The National Institute of Standards and Technology (NIST) provides a wide variety of reference materials (RMs) and Standard Reference Materials® (SRM®s) including blood, serum, plasma, and urine with values assigned for analytes of clinical interest. NIST SRM 2378 Fatty Acids in Frozen Human Serum was introduced in 2015 to help validate methods used for the analysis of FAs in serum, and consists of three different pools of serum acquired from (1) healthy donors who had taken fish oil dietary supplements (at least 1000 mg per day) for at least one month (level 1 material), (2) healthy donors who had taken flaxseed oil dietary supplements (at least 1000 mg per day) for at least one month (level 2 material), and (3) healthy donors eating "normal" diets who had not taken dietary supplements containing fish or plant oils (level 3 material). The use of dietary supplements by donors provided SRMs with natural endogenous ranges of FAs at concentrations observed in human populations. Results from analyses using two methods at NIST, including one involving a novel microwave-assisted acid hydrolysis procedure, and one at the CDC are presented here. These results and their respective uncertainties were combined to yield certified values with expanded uncertainties for 12 FAs and reference values with expanded uncertainties for an additional 18 FAs.

Development of a kelp powder (Thallus laminariae) Standard Reference Material.

A Standard Reference Material (SRM) of seaweed, SRM 3232 Kelp Powder (Thallus laminariae) has been developed to support food and dietary supplement measurements in compliance with the Food Safety Modernization Act (FSMA) and the Dietary Supplement Health and Education Act of 1994 (DSHEA). The material was characterized for nutritional minerals, arsenic species, isomers of vitamin K1, proximates, and toxic elements. Kelp is a rich source of vitamins and minerals, and it is an excellent source of dietary iodine. Kelp also contains a large amount of arsenic, which is toxic as inorganic species but much less so as organic species. To capture the dietary profile of kelp, certified values were issued for As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Mo, Na, Pb, and Zn. Reference values for proximates were assigned. For the first time, a certified value for iodine, reference values for isomers of vitamin K1, and reference values for arsenic species including arsenosugars were assigned in a seaweed. SRM 3232 fills a gap in Certified Reference Materials (CRMs) needed for quality assurance and method validation in the compositional measurements of kelp and similar seaweeds used as food and as dietary supplements. Graphical Absract Arsenic species and isomers of vitamin K1 were determined in the development of SRM 3232 Kelp Powder (Thallus laminariae).

Development of an Improved Standard Reference Material for Vitamin D Metabolites in Human Serum.

The National Institute of Standards and Technology (NIST) has developed Standard Reference Material (SRM) 972a Vitamin D Metabolites in Frozen Human Serum as a replacement for SRM 972, which is no longer available. SRM 972a was developed in collaboration with the National Institutes of Health's Office of Dietary Supplements. In contrast to the previous reference material, three of the four levels of SRM 972a are composed of unmodified human serum. This SRM has certified and reference values for the following 25-hydroxyvitamin D [25(OH)D] species: 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3. The value assignment and certification process included three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). The value assignment methods employed have been modified from those utilized for the previous SRM, and all three approaches now incorporate chromatographic resolution of the stereoisomers, 25(OH)D3 and 3-epi-25(OH)D3.

Development of urine standard reference materials for metabolites of organic chemicals including polycyclic aromatic hydrocarbons, phthalates, phenols, parabens, and volatile organic compounds.

Two new Standard Reference Materials (SRMs), SRM 3672 Organic Contaminants in Smokers' Urine (Frozen) and SRM 3673 Organic Contaminants in Non-Smokers' Urine (Frozen), have been developed in support of studies for assessment of human exposure to select organic environmental contaminants. Collaborations among three organizations resulted in certified values for 11 hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and reference values for 11 phthalate metabolites, 8 environmental phenols and parabens, and 24 volatile organic compound (VOC) metabolites. Reference values are also available for creatinine and the free forms of caffeine, theobromine, ibuprofen, nicotine, cotinine, and 3-hydroxycotinine. These are the first urine Certified Reference Materials characterized for metabolites of organic environmental contaminants. Noteworthy, the mass fractions of the environmental organic contaminants in the two SRMs are within the ranges reported in population survey studies such as the National Health and Nutrition Examination Survey (NHANES) and the Canadian Health Measures Survey (CHMS). These SRMs will be useful as quality control samples for ensuring compatibility of results among population survey studies and will fill a void to assess the accuracy of analytical methods used in studies monitoring human exposure to these organic environmental contaminants.

Development of a Standard Reference Material for metabolomics research.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Characterization of NIST food-matrix Standard Reference Materials for their vitamin C content.

The vitamin C concentrations in three food-matrix Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST) have been determined by liquid chromatography (LC) with absorbance detection. These materials (SRM 1549a Whole Milk Powder, SRM 1849a Infant/Adult Nutritional Formula, and SRM 3233 Fortified Breakfast Cereal) have been characterized to support analytical measurements made by food processors that are required to provide information about their products' vitamin C content on the labels of products distributed in the United States. The SRMs are primarily intended for use in validating analytical methods for the determination of selected vitamins, elements, fatty acids, and other nutrients in these materials and in similar matrixes. They can also be used for quality assurance in the characterization of test samples or in-house control materials, and for establishing measurement traceability. Within-day precision of the LC method used to measure vitamin C in the food-matrix SRMs characterized in this study ranged from 2.7% to 6.5%.

Development of botanical and fish oil standard reference materials for fatty acids.

As part of a collaboration with the National Institutes of Health's Office of Dietary Supplements and the Food and Drug Administration's Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed Standard Reference Material (SRM) 3274 Botanical Oils Containing Omega-3 and Omega-6 Fatty Acids and SRM 3275 Omega-3 and Omega-6 Fatty Acids in Fish Oil. SRM 3274 consists of one ampoule of each of four seed oils (3274-1 Borage (Borago officinalis), 3274-2 Evening Primrose (Oenothera biennis), 3274-3 Flax (Linium usitatissimum), and 3274-4 Perilla (Perilla frutescens)), and SRM 3275 consists of two ampoules of each of three fish oils (3275-1 a concentrate high in docosahexaenoic acid, 3275-2 an anchovy oil high in docosahexaenoic acid and eicosapentaenoic acid, and 3275-3 a concentrate containing 60% long-chain omega-3 fatty acids). Each oil has certified and reference mass fraction values for up to 20 fatty acids. The fatty acid mass fraction values are based on results from analyses using gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS). These SRMs will complement other reference materials currently available with mass fractions for similar analytes and are part of a series of SRMs being developed for dietary supplements.

Milk and serum standard reference materials for monitoring organic contaminants in human samples.

Four new Standard Reference Materials (SRMs) have been developed to assist in the quality assurance of chemical contaminant measurements required for human biomonitoring studies, SRM 1953 Organic Contaminants in Non-Fortified Human Milk, SRM 1954 Organic Contaminants in Fortified Human Milk, SRM 1957 Organic Contaminants in Non-Fortified Human Serum, and SRM 1958 Organic Contaminants in Fortified Human Serum. These materials were developed as part of a collaboration between the National Institute of Standards and Technology (NIST) and the Centers for Disease Control and Prevention (CDC) with both agencies contributing data used in the certification of mass fraction values for a wide range of organic contaminants including polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, polybrominated diphenyl ether (PBDE) congeners, and polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners. The certified mass fractions of the organic contaminants in unfortified samples, SRM 1953 and SRM 1957, ranged from 12 ng/kg to 2200 ng/kg with the exception of 4,4'-DDE in SRM 1953 at 7400 ng/kg with expanded uncertainties generally <14 %. This agreement suggests that there were no significant biases existing among the multiple methods used for analysis.

Certification of elements in and use of standard reference material 3280 multivitamin/multielement tablets.

Standard Reference Material 3280 Multivitamin/ Multielement Tablets was issued by the National Institute of Standards and Technology in 2009, and has certified and reference mass fraction values for 13 vitamins, 26 elements, and two carotenoids. Elements were measured using two or more analytical methods at NIST with additional data contributed by collaborating laboratories. This reference material is expected to serve a dual purpose: to provide quality assurance in support of a database of dietary supplement products and to provide a means for analysts, dietary supplement manufacturers, and researchers to assess the appropriateness and validity of their analytical methods and the accuracy of their results.

Development and certification of a standard reference material for vitamin D metabolites in human serum.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health's Office of Dietary Supplements (NIH-ODS), has developed a Standard Reference Material (SRM) for the determination of 25-hydroxyvitamin D [25(OH)D] in serum. SRM 972 Vitamin D in Human Serum consists of four serum pools with different levels of vitamin D metabolites and has certified and reference values for 25(OH)D(2), 25(OH)D(3), and 3-epi-25(OH)D(3). Value assignment of this SRM was accomplished using a combination of three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). Chromatographic resolution of the 3-epimer of 25(OH)D(3) proved to be essential for accurate determination of the metabolites.

Preparation and value assignment of standard reference material 968e fat-soluble vitamins, carotenoids, and cholesterol in human serum.

Standard Reference Material 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for total retinol, γ- and α-tocopherol, total lutein, total zeaxanthin, total ß-cryptoxanthin, total ß-carotene, 25-hydroxyvitamin D(3), and cholesterol. Reference and information values are also reported for nine additional compounds including total α-cryptoxanthin, trans- and total lycopene, total α-carotene, trans-ß-carotene, and coenzyme Q(10). The certified values for the fat-soluble vitamins and carotenoids in SRM 968e were based on the agreement of results from the means of two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the median of results of an interlaboratory comparison exercise among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol and 25-hydroxyvitamin D(3) in the SRM are the means of results obtained using the NIST reference method based upon gas chromatography-isotope dilution mass spectrometry and liquid chromatography-isotope dilution tandem mass spectrometry, respectively. SRM 968e is currently one of two available health-related NIST reference materials with concentration values assigned for selected fat-soluble vitamins, carotenoids, and cholesterol in human serum matrix. This SRM is used extensively by laboratories worldwide primarily to validate methods for determining these analytes in human serum and plasma and for assigning values to in-house control materials. The value assignment of the analytes in this SRM will help support measurement accuracy and traceability for laboratories performing health-related measurements in the clinical and nutritional communities.

Development of Kudzu (Pueraria Montana var. lobata) Reference Materials for the Determination of Isoflavones and Toxic Elements.

BACKGROUND: In collaboration with the Office of Dietary Supplements at the National Institutes of Health, the National Institute of Standards and Technology issued a suite of botanical matrix reference materials (RMs) and Standard Reference Material® (SRM) for determination of isoflavones and toxic elements in kudzu dietary supplement ingredients. OBJECTIVE: RM 8650 Pueraria montana var. lobata (Kudzu) Rhizome, SRM 3268 Pueraria montana var. lobata (Kudzu) Extract, and RM 8652 Kudzu-Containing Solid Oral Dosage Form were issued with values assigned for isoflavones (puerarin, daidzin, and daidzein), toxic elements (arsenic, cadmium, and lead), and selenium. METHODS: Isoflavone values were assigned using liquid chromatography with UV absorbance or mass spectrometry detection. Element values were assigned using inductively coupled plasma mass spectrometry and results from an interlaboratory comparison exercise. RESULTS: Mass fractions for puerarin were 32.2 ± 3.2 mg/g, 128 ± 13 mg/g, and 68.2 ± 6.9 mg/g in RM 8650, SRM 3268, and RM 8652, respectively. Arsenic increases from 156 ± 14 ng/g to 849 ± 83 ng/g and cadmium decreases from 348 ± 14 ng/g to 82.1 ± 4.9 ng/g from rhizome to extract. CONCLUSION: The kudzu RM/SRM suite complements previously issued soy-related SRMs with values assigned for isoflavones, which have been studied for their potential health benefits, and expands the analytical resource by providing values for puerarin, an isoflavone not found in soy. HIGHLIGHTS: The three new kudzurmaterials are for use in the determination of isoflavones, toxic elements, and selenium. For the isoflavones, these new kudzu materials provide higher levels of daidzin and daidzein than existing soy-related SRMs, and they provide a value for an isoflavone not in existing SRMs (puerarin). Toxic elements in RM 8650 and SRM 3268 provide new botanical matrixes for use by dietary supplement manufacturers for the verification of the safety of their raw materials.

Isotope dilution liquid chromatography - mass spectrometry methods for fat- and water-soluble vitamins in nutritional formulations.

Vitamins are essential to human health, and dietary supplements containing vitamins are widely used by individuals hoping to ensure they have adequate intake of these important nutrients. Measurement of vitamins in nutritional formulations is necessary to monitor regulatory compliance and in studies examining the nutrient intake of specific populations. Liquid chromatographic methods, primarily with UV absorbance detection, are well established for both fat- and water-soluble measurements, but they do have limitations for certain analytes and may suffer from a lack of specificity in complex matrices. Liquid chromatography-mass spectrometry (LC-MS) provides both sensitivity and specificity for the determination of vitamins in these matrices, and simultaneous analysis of multiple vitamins in a single analysis is often possible. In this work, LC-MS methods were developed for both fat- and water-soluble vitamins and applied to the measurement of these analytes in two NIST Standard Reference Materials. When possible, stable isotope labeled internal standards were employed for quantification.

Determination of fat-soluble vitamins and carotenoids in standard reference material 3280 multivitamin/multielement tablets by liquid chromatography with absorbance detection.

The concentrations of selected fat-soluble vitamins and carotenoids in Standard Reference Material (SRM) 3280 Multivitamin/Multielement Tablets have been determined by two independent LC methods, with measurements performed by the National Institute of Standards and Technology (NIST). This SRM has been prepared as part of a collaborative effort between NIST and the National Institutes of Health Office of Dietary Supplements. The SRM is also intended to support the Dietary Supplement Ingredient Database that is being established by the U.S. Department of Agriculture. The methods used at NIST to determine the concentration levels of vitamins A and E, and beta-carotene in the SRM used RPLC with absorbance detection. The relative precision of these methods ranged from 2 to 8% for the analytes measured. SRM 3280 is primarily intended for use in validating analytical methods for the determination of selected vitamins, carotenoids, and elements in multivitamin/multielement tablets and similar matrixes.

Dietary supplement laboratory quality assurance program: the first five exercises.

The National Institute of Standards and Technology (NIST) has established a Dietary Supplement Laboratory Quality Assurance Program (DSQAP) in collaboration with the National Institutes of Health Office of Dietary Supplements. Program participants measure concentrations of active and/or marker compounds as well as nutritional and toxic elements in food and dietary supplements distributed by NIST. Data are compiled at NIST, where they are analyzed for accuracy relative to reference values and concordance among the participants. Performance reports and certificates of completion are provided to participants, which can be used to demonstrate compliance with current Good Manufacturing Practices as promulgated by the U.S. Food and Drug Administration. The DSQAP has conducted five exercises to date, with total participation including more than 75 different laboratories and many more individual analysts.

An LC-ESI/MS method for determining theanine in green tea dietary supplements.

Theanine is the major amino acid present in Camellia sinensis or green tea. A method for determining theanine in its native state using liquid chromatography with positive-mode electrospray ionization mass spectrometric detection was developed. Quantitation of theanine was achieved using theanine-[(2)H(5)] as an internal standard. This approach was utilized on different green tea matrix materials that are commonly used as dietary supplements including powdered plant leaves, a powdered plant leaf extract, and an oral dosage form that contains green tea. The theanine response was linear over several orders of magnitude, and excellent measurement precision was obtained for all three materials using the developed method.

Determination of organic acids in Vaccinium berry standard reference materials.

Nine organic acids (citric acid, galacturonic acid, glycolic acid, isocitric acid, malic acid, oxalic acid, quinic acid, shikimic acid, and tartaric acid) and two anions (phosphate and sulfate) were determined in a suite of Vaccinium berry-containing dietary supplement standard reference materials (SRMs). Following solvent extraction, three independent methods were utilized in the quantification of these compounds. The first method involved reversed-phase liquid chromatography with ultraviolet absorbance detection at 210 nm and isotope dilution mass spectrometry. The second method utilized ion chromatography with conductivity detection. Finally, gas chromatography with isotope dilution mass spectrometry detection was used following derivatization with N-methyl-N-trifluoroacetamide (MSTFA). The combined data from these methods was used for the assignment of organic acid levels in the seven candidate SRMs.

Preparation and characterization of standard reference material 1849 infant/adult nutritional formula.

Standard Reference Material (SRM) 1849 Infant/Adult Nutritional Formula has been issued by the National Institute of Standards and Technology (NIST) as a replacement for SRM 1846 Infant Formula, issued in 1996. Extraction characteristics of SRM 1846 have changed over time, as have NIST's analytical capabilities. While certified mass fraction values were provided for five constituents in SRM 1846 (four vitamins plus iodine), certified mass fraction values for 43 constituents are provided in SRM 1849 (fatty acids, elements, and vitamins) and reference mass fraction values are provided for an additional 43 constituents including amino acids and nucleotides, making it the most extensively characterized food-matrix SRM available from NIST.