Determination of fortified and endogenous folates in food-based Standard Reference Materials by liquid chromatography-tandem mass spectrometry

The National Institute of Standards and Technology (NIST) is developing a wide variety of Standard Reference Materials (SRMs) to support measurements of vitamins and other nutrients in foods. Previously, NIST has provided SRMs with values assigned for the folate vitamer, folic acid (pteroylglutamic acid), which is fortified in several foods due to its role in prevention of neural tube defects. In order to expand the number of food-based SRMs with values assigned for folic acid, as well as additional endogenous folates, NIST has developed methods that include trienzyme digestion and isotope-dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Sample preparation was optimized for each individual food type, but all samples were analyzed under the same LC-MS/MS conditions. The application of these methods resulted in folic acid values for SRM 1849a Infant/Adult Nutritional Formula and SRM 3233 Fortified Breakfast Cereal of (2.33?±?0.06) μg/g and (16.0?±?0.7) μg/g, respectively. In addition, the endogenous folate vitamer 5-methlytetrahydrofolate (5-MTHF) was detected and quantified in SRM 1849a Infant/Adult Nutritional Formula, candidate SRM 1549a Whole Milk Powder, and candidate SRM 1845a Whole Egg Powder, resulting in values of (0.0839?±?0.0071) μg/g, (0.211?±?0.014) μg/g, and (0.838?±?0.044) μg/g, respectively. SRM 1849a Infant/Adult Nutritional Formula is the first food-based NIST SRM to possess a reference value for 5-MTHF and the first certified reference material to have an assigned 5-MTHF value based on LC-MS/MS. The values obtained for folic acid and 5-MTHF by LC-MS/MS will be incorporated into the final value assignments for all these food-based SRMs.     

Analysis of carcinogenic polycyclic aromatic hydrocarbons in complex environmental mixtures by LC-APPI-MS/MS

Here we developed a highly sensitive, fast and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the detection and analysis of 16 different polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs that have been identified as carcinogens and classified according to their biological potency. Comparison to standard analysis procedures based on gas chromatography-mass spectrometry (GC-MS) instrumentation demonstrated an improved easiness of sample preparation and sensitivity of detection achieved with the new LC-MS/MS method employing an atmospheric pressure photoionization (APPI) source attached to an API 4000 mass spectrometer (LC-APPI-MS/MS). The favorable outcome could be confirmed by analyzing complex mixtures such as certain Standard Reference Materials (SRMs) obtained from the National Institute of Standards & Technology (NIST), i.e., SRM 1975 and SRM 2975, and several diesel exhaust soots provided by the German automobile industry. Certified concentrations of individual analytes provided by NIST not only could be confirmed, but additional extremely potent carcinogens such as several isomeric hexacyclic dibenzopyrenes (DBPs), 5-methylchrysene (5-MC), and others have been detected in these crude samples in a concentration range down to below 1 ng g(-1) raw material.     

Recent developments in food-matrix Reference Materials at NIST

Since 1996, the National Institute of Standards and Technology (NIST) has developed several food-matrix Standard Reference Materials (SRMs) characterized for nutrient concentrations. These include SRM 1544 Fatty Acids and Cholesterol in a Frozen Diet Composite, SRM 1546 Meat Homogenate, SRM 1548a Typical Diet, SRM 1566b Oyster Tissue, SRM 1846 Infant Formula, and SRM 2383 Baby Food Composite. Three additional materials--SRM 1946 Lake Superior Fish Tissue, SRM 2384 Baking Chocolate, and SRM 2385 Spinach--are in preparation. NIST also recently assigned values for proximate (fat, protein, etc.), individual fatty acid, and total dietary fiber concentrations in a number of existing SRMs and reference materials (RMs) that previously had values assigned for their elemental composition. NIST has used several modes for assignment of analyte concentrations in the food-matrix RMs, including the use of data provided by collaborating laboratories, alone and in combination with NIST data. The use of data provided by collaborating food industry and contract laboratories for the analysis of food-matrix RMs has enabled NIST to provide assigned values for many analytes that NIST does not have the resources or analytical expertise to measure.     

Standard reference materials for foods and dietary supplements

Well-characterized certified reference materials are needed by laboratories in the food testing, dietary supplement, and nutrition communities to facilitate compliance with labeling laws and improve the accuracy of information provided on product labels, so that consumers can make good choices. As a result of the enactment of the Nutrition Labeling and Education Act of 1990 and the Infant Formula Act of 1980, the National Institute of Standards and Technology (NIST) worked to develop a series of food-matrix standard reference materials (SRMs) characterized for nutrient concentrations. These include SRM 1544 Fatty Acids and Cholesterol in a Frozen Diet Composite, SRM 1546 Meat Homogenate, SRM 1548a Typical Diet, SRM 1566b Oyster Tissue, SRM 1846 Infant Formula, SRM 1946 Lake Superior Fish Tissue, SRM 1947 Lake Michigan Fish Tissue, SRM 2383 Baby Food Composite, SRM 2384 Baking Chocolate, SRM 2385 Slurried Spinach, and SRM 2387 Peanut Butter. With the enactment of the Dietary Supplement Health and Education Act of 1994, NIST has been working to develop suites of dietary supplement SRMs characterized for active and marker compounds and for toxic elements and pesticides, where appropriate. An updated SRM 1588b Organics in Cod Liver Oil, a suite of ephedra-containing materials (SRMs 3240–3245), a carrot extract in oil (SRM 3276), and a suite of ginkgo-containing materials (SRMs 3246–3248) are available. Several other materials are currently in preparation. Dietary supplements are sometimes provided in forms that are food-like; for these, values may also be assigned for nutrients, for example SRM 3244 Ephedra-Containing Protein Powder. Both the food-matrix and dietary supplement reference materials are intended primarily for validation of analytical methods. They may also be used as “primary control materials” in assignment of values to in-house (secondary) control materials to confirm accuracy and to establish measurement traceability to NIST.     

Determination of perfluorinated alkyl acid concentrations in biological standard reference materials

Standard reference materials (SRMs) are homogeneous, well-characterized materials used to validate measurements and improve the quality of analytical data. The National Institute of Standards and Technology (NIST) has a wide range of SRMs that have mass fraction values assigned for legacy pollutants. These SRMs can also serve as test materials for method development, method validation, and measurement for contaminants of emerging concern. Because inter-laboratory comparison studies have revealed substantial variability of measurements of perfluoroalkyl acids (PFAAs), future analytical measurements will benefit from determination of consensus values for PFAAs in SRMs to provide a means to demonstrate method-specific performance. To that end, NIST, in collaboration with other groups, has been measuring concentrations of PFAAs in a variety of SRMs. Here we report levels of PFAAs and perfluorooctane sulfonamide (PFOSA) determined in four biological SRMs: fish tissue (SRM 1946 Lake Superior Fish Tissue, SRM 1947 Lake Michigan Fish Tissue), bovine liver (SRM 1577c), and mussel tissue (SRM 2974a). We also report concentrations for three in-house quality-control materials: beluga whale liver, pygmy sperm whale liver, and white-sided dolphin liver. Measurements in SRMs show an array of PFAAs, with perfluorooctane sulfonate (PFOS) being the most frequently detected. Reference and information values are reported for PFAAs measured in these biological SRMs.

Determination of vitamins in food-matrix Standard Reference Materials

In recent years, the National Institute of Standards and Technology (NIST) has developed several food-matrix Standard Reference Materials (SRMs) characterized for vitamins and other organic nutrients. NIST uses several "modes" for assignment of analyte concentrations in SRMs, one of which includes the use of data provided by collaborating laboratories. Certification modes and liquid chromatographic methods that were used by NIST for value assignment of vitamin concentrations in recently introduced food-matrix SRMs are described in this paper. These materials and methods include vitamins D and E in coconut oil (SRM 1563) by gravimetry and multi-dimensional liquid chromatography (LC); vitamins A, E, and several B vitamins by reversed-phase LC and vitamin C by ion-exchange chromatography in infant formula (SRM 1846); and carotenoids and vitamins A and E by reversed-phase liquid chromatography in a baby food composite (SRM 2383).     

Recent developments in NIST botanical SRMs

The National Institute of Standards and Technology (NIST) (formerly the National Bureau of Standards (NBS)) issued the first botanical reference material certified for elemental content in January 1971, as Standard Reference Material (SRM) 1571, Orchard Leaves. In the following years a total of nine additional botanical certified reference materials have been issued by NIST. Each of these materials was certified for major, minor and trace elements except for SRM 2695, certified for fluorine only. Botanical SRMs issued since 1991 are significantly improved over previous materials in a number of ways. Probably the most significant change is the use of a jet-milling process to grind them to extremely fine particles. This has resulted in botanical SRMs with significantly improved homogeneity. These NIST reference materials are described with information on homogeneity, drying techniques and grit content.     

Recent SRMs for organic and inorganic nutrients in food matrices

The National Institute of Standards and Technology (NIST) has recently introduced several reference materials for organic and inorganic nutrients in food matrices to assist the food industry in complying with nutrition labeling laws; the food industry and other government agencies have collaborated with NIST in this endeavor. Two food-matrix SRMs were issued in 1996: SRM 1544, Fatty Acids and Cholesterol in Frozen Food Composite; and SRM 1846, Infant Formula. Concentration values in SRM 1544 are provided for six fatty acids, cholesterol, and proximates (fat, protein, carbohydrate, etc.). Values are assigned for proximate and caloric content as well as the concentrations of sixteen vitamins and nine minerals and trace elements in SRM 1846. In 1997, NIST expects to issue two additional food-matrix SRMs: SRM 1548a, Typical Diet, and SRM 2383, Baby Food Composite. SRM 1548a will replace SRM 1548, Total Diet, and will have values assigned for proximates, major and minor inorganic nutrients, and toxic trace elements. SRM 2383 will have values assigned for proximates, vitamins, carotenoids, and inorganic nutrients.     

A review of the DNA standard reference materials developed by the National Institute of Standards and Technology

The Standard Reference Materials Program at the US National Institute of Standards and Technology (NIST) has three human DNA standard reference materials (SRM 2390, SRM 2391a, and SRM 2392) currently available¹ (Orders and requests for information concerning these SRMs should be directed to the Standard Reference Materials Program, National Institute of Standards and Technology, 100 Bureau Drive, Stop 2321, Gaithersburg, MD 20899-2321, Telephone (301) 975-6776, FAX: (301) 948-3730.) [1, 2]. Both the DNA profiling SRM 2390 and the polymerase chain reaction (PCR)-based DNA profiling SRM 2391a are intended for use in forensic and paternity identifications, for instructional law enforcement, or for non-clinical research purposes and are not intended for clinical diagnostics. The mitochondrial DNA (mtDNA) SRM 2392 is to provide standardization and quality control when performing PCR and sequencing any segment or the entire 16,569 base pairs that comprise human mitochondrial DNA. SRM 2392 is designed for use by the forensic, medical, and toxicological communities for human identification, disease diagnosis or mutation detection.     

Determination of caffeine and caffeine-related metabolites in ephedra-containing standard reference materials using liquid chromatography with absorbance detection and tandem mass spectrometry

The concentrations of caffeine and caffeine-related compounds in 2 ephedra-containing reference materials have been determined by 3 independent methods with measurements performed by the National Institute of Standards and Technology (NIST) and a collaborating laboratory. Results from the 3 methods were used for value assignment of caffeine, theobromine, and theophylline in these Standard Reference Materials (SRMs). The methods used at NIST to determine the concentration levels of caffeine, theobromine, and theophylline in SRM 3243 Ephedra-Containing Solid Oral Dosage Form and SRM 3244 Ephedra-Containing Protein Powder used reversed-phase liquid chromatography with absorbance detection and tandem mass spectrometry. These reference materials are part of the first suite in a series of NIST SRMs that provide concentration values for multiple components in dietary supplements. These SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and similar materials.     

Certification of NIST standard reference material 2389a, amino acids in 0.1 mol/L HCl—quantification by ID LC-MS/MS

An isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) measurement procedure was developed to accurately quantify amino acid concentrations in National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2389a—amino acids in 0.1 mol/L hydrochloric acid. Seventeen amino acids were quantified using selected reaction monitoring on a triple quadrupole mass spectrometer. LC-MS/MS results were compared to gravimetric measurements from the preparation of SRM 2389a—a reference material developed at NIST and intended for use in intra-laboratory calibrations and quality control. Quantitative mass spectrometry results and gravimetric values were statistically combined into NIST-certified mass fraction values with associated uncertainty estimates. Coefficients of variation (CV) for the repeatability of the LC-MS/MS measurements among amino acids ranged from 0.33% to 2.7% with an average CV of 1.2%. Average relative expanded uncertainty of the certified values including Types A and B uncertainties was 3.5%. Mean accuracy of the LC-MS/MS measurements with gravimetric preparation values agreed to within |1.1|% for all amino acids. NIST SRM 2389a will be available for characterization of routine methods for amino acid analysis and serves as a standard for higher-order measurement traceability. This is the first time an ID LC-MS/MS methodology has been applied for quantifying amino acids in a NIST SRM material.     

Determination of polychlorinated dibenzo-p-dioxin and dibenzofuran congeners in air particulate and marine sediment standard reference materials (SRMs)

Due to the limited number of environmental matrix certified reference materials (CRMs) with assigned values for natural levels of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), an interlaboratory study was undertaken by the National Institute of Standards and Technology (NIST) and Environment Canada to establish reference concentration values for selected PCDD/Fs in two well-characterized NIST Standard Reference Materials (SRMs): SRM 1649a (Urban Dust) and SRM 1944 (New York/New Jersey Waterway Sediment). Results from 14 laboratories were used to provide reference values for the seventeen 2, 3, 7, 8-substituted PCDD/F congeners, the totals for individual tetra- through hepta-substituted PCDD/F homologues, and the total amount of tetra-through hepta-substituted PCDD/Fs. The mass fractions for the individual 2, 3, 7, 8-substituted congeners range from approximately 0.01 μg/kg to 7 μg/kg dry mass.     

Adsorption of cobalt on some natural zeolites occurring in ČSFR

Neutron activation analysis is one of many analytical techniques used at the National Institute of Standards and Technology (NIST) for the certification of NIST Standard Reference Materials (SRMs). NAA competes favorably with all other techniques because of it's unique capabilities for high accuracy even at very low concentrations for many elements. In this paper, instrumental and radiochemical NAA results are described for 25 elements in two new NIST SRMs, SRM 1515 (Apple Leaves) and SRM 1547 (Peach Leaves), and are compared to the certified values for 19 elements in these two new botanical reference materials.     

Revalidation and long-term stability of National Institute of Standards and Technology Standard Reference Materials 1566, 1567, 1568, and 1570

Multiple units of Standard Reference Materials (SRMs) 1566 Oyster Tissue, 1567 Wheat Flour, 1568 Rice Flour, and 1570 Trace Elements in Spinach, produced by the National Institute of Standards and Technology (NIST, then the National Bureau of Standards), were analyzed 17-20 years after the original certification dates and 12-15 years after the certificates became invalid. Instrumental neutron activation analysis and thermal neutron prompt gamma-ray activation analysis were used to measure mass fractions for 27 elements in these SRMs to revalidate them for use in quality assurance (QA) programs required for food analysis programs within the U.S. Food and Drug Administration. With the exception of Se in SRM 1567, all element mass fractions were in agreement with certified values and literature data. Some evidence of B loss from SRM 1568 was observed. These materials were judged to be suitable for continued use in QA programs. Findings showed that these matrixes exhibited stability of moisture, mass fraction, and weight basis for far longer (> or =15 years) than was indicated by the 5-year validity statement on the NIST Certificates of Analysis.     

Certification of methylmercury content in two fresh-frozen reference materials: SRM 1947 Lake Michigan fish tissue and SRM 1974b organics in mussel tissue (Mytilus edulis)

This paper describes the development of two independent analytical methods for the extraction and quantification of methylmercury from marine biota. The procedures involve microwave extraction, followed by derivatization and either headspace solid-phase microextraction (SPME) with a polydimethylsiloxane (PDMS)-coated silica fiber or back-extraction into iso-octane. The identification and quantification of the extracted compounds is carried out by capillary gas chromatography/mass spectrometric (GC/MS) and inductively coupled plasma mass spectrometric (GC/ICP-MS) detection. Both methods were validated for the determination of methylmercury (MeHg) concentrations in a variety of biological standard reference materials (SRMs) including fresh-frozen tissue homogenates of SRM 1946 Lake Superior fish tissue and SRM 1974a organics in mussel tissue (Mytilus edulis) and then applied to the certification effort of SRM 1947 Lake Michigan fish tissue and SRM 1974b organics in mussel tissue (Mytilus edulis). While past certifications of methylmercury in tissue SRMs have been based on two independent methods from the National Institute of Standards and Technology (NIST) and participating laboratories, the methods described within provide improved protocols and will allow future certification efforts to be based on at least two independent analytical methods within NIST.     

Assessment of stability of trace elements in two natural matrix environmental standard reference materials: NIST-SRM 1547 Peach leaves and NIST-SRM 1566a Oyster Tissue

The NIST program for environmental Standard Reference Materials (SRM) includes materials covering a range of matrices, mass fraction values and analytes. For many SRMs, mass fraction data are accumulated, incidentally, over time, as these are used routinely for quality assurance purposes. Although these are not formal stability studies, data generated may be useful in assessing stability. To evaluate the potential for assessing material stability from incidental use of SRMs, results of neutron activation analysis performed from 1992 through 2008 were compiled for SRM 1547 Peach Leaves and SRM 1566a Oyster Tissue. Results indicate that incidental use of SRMs yields useful information on SRM stability.     

Synthetic musk fragrances in environmental Standard Reference Materials

Synthetic musk fragrances have been measured in water, air, sediments, sewage sludge, and biota worldwide. As the study of the environmental fate and impacts of these compounds progresses, the need for Standard Reference Materials (SRMs) for these compounds to facilitate analytical method improvement and interlaboratory comparisons becomes increasingly important. The National Institute of Standards and Technology (NIST) issues environmental matrix SRMs with certified concentrations for a variety of persistent organic pollutants including polycyclic aromatic hydrocarbons (PAHs), chlorinated pesticides, and polychlorinated biphenyl congeners (PCBs). Until now synthetic musk fragrance concentrations have not been reported in NIST SRMs. The objective of this study was to provide reference values for several commonly detected synthetic musk fragrances in several NIST natural matrix SRMs. In this study five polycyclic musk fragrances [HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-γ-2-benzopyran), AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene), ADBI (4-acetyl-1,1-dimethyl-6-tert-butylindane), AHMI (6-acetyl-1,1,2,3,3,5-hexamethylindane), and ATII (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane] and two nitro musk fragrances [musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene) and musk ketone (4-tert-butyl-3,5-dinitro-2,6-dimethylacetophenone)] were measured in selected environmental SRMs. Gas chromatography–electron impact mass spectrometry (GC/EI-MS) was used for all analyses. HHCB was the most frequently detected synthetic musk fragrance and was detected in SRM 2585 Organic Contaminants in House Dust, SRM 2781 Domestic Sludge, SRM 1974b Organics in Mussel Tissue (Mytilus edulis), and SRM 1947 Lake Michigan Fish Tissue. It was not detected in SRM 1946 Lake Superior Fish Tissue or SRM 1945 Organics in Whale Blubber. Concentrations of HHCB in these SRMs ranged from 1.12 ng/g in SRM 1947 to 92,901 ng/g in SRM 2781. All of the polycyclic musk fragrances were detected in SRM 2781 and all of the target compounds were detected in SRM 2585. Electronic supplementary material Supplementary material is available for this article at and is accessible to authorized users.     

Filling the AOAC triangle with food-matrix standard reference materials

Well-characterized reference materials are needed by laboratories in the food testing and nutrition communities to facilitate compliance with nutritional labeling laws, to provide traceability for food exports needed for acceptance in many foreign markets, and to improve the accuracy of nutrition information that is provided to assist consumers in making sound dietary choices. As a result of the enactment of the Nutrition Labeling and Education Act of 1990 and the Infant Formula Act of 1980, the National Institute of Standards and Technology (NIST) has developed a suite of food-matrix Standard Reference Materials (SRMs) characterized for nutrient concentrations. These include SRM 1544 Fatty Acids and Cholesterol in a Frozen Diet Composite, SRM 1546 Meat Homogenate, SRM 1548a Typical Diet, SRM 1566b Oyster Tissue, SRM 1846 Infant Formula, SRM 1946 Lake Superior Fish Tissue, SRM 2383 Baby Food Composite, SRM 2384 Baking Chocolate, SRM 2385 Spinach, and SRM 2387 Peanut Butter. Many of these materials were developed at the request of the food industry to populate a nine-sectored fat-protein-carbohydrate triangle developed by AOAC International. With the completion of SRM 2387, SRMs representing each sector of the triangle are now available. These food-matrix reference materials are intended primarily for validation of analytical methods for the measurement of proximates, fatty acids, vitamins, minerals, and so on in foods of similar composition. They may also be used as "primary control materials" in the value-assignment of in-house, secondary, control materials to confirm accuracy as well as to establish traceability to NIST.