Susan S.-C. Tai

Development of a Candidate Reference Measurement Procedure for the Determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in Human Serum Using Isotope-Dilution Liquid Chromatography/Tandem Mass Spectrometry

Vitamin D exists in two major forms, vitamin D(3) and vitamin D(2). Vitamin D helps the body absorb calcium and promote optimal bone health. Both forms of vitamin D are metabolized to 25-hydroxyvitamin D in the body, and the levels of 25-hydroxyvitamin D(3) [25(OH)D(3)] and 25-hydroxyvitamin D(2) [25(OH)D(2)] in serum are considered the best indicators of vitamin D status. A candidate reference measurement procedure for serum 25(OH)D(3) and 25(OH)D(2) has been developed and critically evaluated. The deuterated compounds 25(OH)D(3)-d(3) and 25(OH)D(2)-d(3) are used as internal standards for 25(OH)D(3) and 25(OH)D(2), respectively. The 25(OH)D(3) and 25(OH)D(2) and their respective labeled internal standards are simultaneously extracted from serum using liquid-liquid extraction prior to reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a cyano (CN) column for both 25(OH)D(3) and 25(OH)D(2). Atmospheric pressure chemical ionization (APCI) in the positive ion mode and multiple reaction monitoring (MRM) were used for LC-MS/MS. The accuracy of the method was evaluated by recovery studies of measuring 25-hydroxyvitamin D [25(OH)D] in spiked samples with known 25(OH)D levels. The recoveries of the added 25(OH)D(3) and 25(OH)D(2) ranged from 99.0% to 101.0%. The absolute recoveries with this method were 97% and 92% for 25(OH)D(3) and 25(OH)D(2), respectively. Excellent precision was obtained with between-set coefficients of variation (CVs) of 0.2-0.6% for 25(OH)D levels >1 ng/g and within 2% for the level of <1 ng/g. Chromatographic separation of 25(OH)D(3) and 25(OH)D(2) from their respective isomers 3-epi-25(OH)D(3) and 3-epi-25(OH)D(2) was achieved. The limit of detection at a signal-to-noise ratio of approximately 3 was 40 pg of 25(OH)D on column (or approximately 0.15 ng/g as expressed as a concentration). This candidate reference measurement procedure for serum 25(OH)D(3) and 25(OH)D(2) demonstrates good accuracy and precision and low susceptibility to interferences. It can be used to provide an accuracy base to which clinical methods for 25(OH)D(3) and 25(OH)D(2) can be compared and that will serve as a standard of higher order for measurement traceability.

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 Healths 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.

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.

An assessment of 25-hydroxyvitamin D measurements in comparability studies conducted by the Vitamin D Metabolites Quality Assurance Program.

BACKGROUND The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health Office of Dietary Supplements, established the first accuracy-based program for improving the comparability of vitamin D metabolite measurements, the Vitamin D Metabolites Quality Assurance Program. METHODS The study samples were human serum or plasma Standard Reference Materials (SRMs) with 25-hydroxyvitamin D values that were determined at NIST. Participants evaluated the materials using immunoassay (IA), liquid chromatography (LC) with mass spectrometric detection, and LC with ultraviolet absorbance detection. NIST evaluated the results for concordance within the participant community as well as trueness relative to the NIST value. RESULTS For the study materials that contain mostly 25-hydroxyvitamin D3 (25(OH)D3),the coefficient of variation (CV) for the participant results was consistently in the range from 7% to 19%, and the median values were biased high relative to the NIST values. However, for materials that contain significant concentrations of both 25-hydroxyvitamin D2 (25(OH)D2) and 25(OH)D3, the median IA results were biased lower than both the LC and the NIST values, and the CV was as high as 28%. The first interlaboratory comparison results for SRM 972a Vitamin D Metabolites in Human Serum are also reported. CONCLUSIONS Relatively large within-lab and between-lab variability hinders conclusive assessments of bias and accuracy.

Baseline Assessment of 25-Hydroxyvitamin D Assay Performance: A Vitamin D Standardization Program (VDSP) Interlaboratory Comparison Study.

The Vitamin D Standardization Program (VDSP) coordinated an interlaboratory study to assess the comparability of measurements of total 25-hydroxyvitamin D [25(OH)D] in human serum, which is the primary marker of vitamin D status. A set of 50 individual donor samples were analyzed by 15 different laboratories representing national nutrition surveys, assay manufacturers, and clinical and/or research laboratories to provide results for total 25(OH)D using both immunoassays (IAs) and LC tandem MS (MS/MS). The results were evaluated relative to bias compared with the target values assigned based on a combination of measurements at Ghent University (Belgium) and the U.S. National Institute of Standards and Technology using reference measurement procedures for the determination of 25(OH)D2 and 25(OH)D3. CV and mean bias for each laboratory and assay platform were assessed and compared with previously established VDSP performance criteria, namely CV ≤ 10% and mean bias ≤ 5%. Nearly all LC-MS/MS results achieved VDSP criteria, whereas only 50% of IAs met the criterion for a ≤10% CV and only three of eight IAs achieved the ≤5% bias. These results establish a benchmark for the evaluation of 25(OH)D assay performance and standardization activities in the future.

NIST reference materials to support accuracy in drug testing

Substance abuse is a major problem worldwide. There is considerable emphasis placed upon testing individuals for evidence of use of controlled substances. Because the consequences of a positive test can be quite severe, laboratories conducting such tests must rigorously follow a carefully designed quality assurance program. Such a QA program should include use of reference materials to assure that the methods used to detect and quantify drugs are providing accurate results. The National Institute of Standards and Technology (NIST) supports accuracy in drugs of abuse testing by providing Standard Reference Materials (SRMs) with certified concentrations of drugs of abuse in urine- and hair-based reference materials. NIST, working in collaboration with the College of American Pathologists (CAP), has developed urine-based SRMs for marijuana metabolite, cocaine metabolite, morphine and codeine, and morphine glucuronide and CAP Reference Materials for amphetamines and phencyclidine. Certification measurements performed at NIST involve two independent methods for each analyte, one of which always uses GC/MS with the other usually being an LC method with either MS or UV detection. Work has recently been completed on a seven component drug in urine SRM. In addition NIST conducts research in the analysis of hair for drugs of abuse. To assist laboratories testing hair for that purpose, NIST has developed two drugs in hair reference materials.

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 Healths 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.

Certification of drugs of abuse in a human serum standard reference material: SRM 1959

A new standard reference material (SRM) for drugs of abuse in human serum (SRM 1959) has been developed. This SRM is intended to be used as a control material for laboratories performing analysis of drugs of abuse in blood to evaluate the accuracy of their methods. SRM 1959 is a frozen human serum material fortified with seven compounds for which analyses are performed to determine evidence of illegal drug use: benzoylecgonine (BZE), methadone (METH), methamphetamine (MAMP), morphine (MOR), nordiazepam (NOR), phencyclidine (PCP), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-9-COOH). Two independent methods involving isotope dilution (ID)-gas chromatography/mass spectrometry (GC/MS) and ID-liquid chromatography/mass spectrometry (LC/MS) were used for the value assignment. For THC-9-COOH, an additional measurement using LC/tandem mass spectrometry (LC/MS/MS) was also included. All methods used isotopically labeled compounds as internal standards and solid-phase extractions to isolate the analytes from the serum. The GC/MS methods used different clean-up procedures from those used for the LC/MS-based methods. Repeatability with within-set coefficients of variation (CVs) ranged from 0.5% to 4.3% for the GC/MS methods and from 0.2% to 1.2% for the LC/MS-based methods. Intermediate precision with between-set CVs for all the methods ranged from 0.1% to 1.1%. Agreement between the GC/MS and LC/MS methods ranged from 0.8% to 8.8%. The results from the methods were combined to obtain the certified concentrations and their expanded uncertainties.

Baseline Assessment of 25-Hydroxyvitamin D Reference Material and Proficiency Testing/External Quality Assurance Material Commutability: A Vitamin D Standardization Program Study

The Vitamin D Standardization Program (VDSP) coordinated a study in 2012 to assess the commutability of reference materials and proficiency testing/external quality assurance materials for total 25-hydroxyvitamin D [25(OH)D] in human serum, the primary indicator of vitamin D status. A set of 50 single-donor serum samples as well as 17 reference and proficiency testing/external quality assessment materials were analyzed by participating laboratories that used either immunoassay or LC-MS methods for total 25(OH)D. The commutability test materials included National Institute of Standards and Technology Standard Reference Material 972a Vitamin D Metabolites in Human Serum as well as materials from the College of American Pathologists and the Vitamin D External Quality Assessment Scheme. Study protocols and data analysis procedures were in accordance with Clinical and Laboratory Standards Institute guidelines. The majority of the test materials were found to be commutable with the methods used in this commutability study. These results provide guidance for laboratories needing to choose appropriate reference materials and select proficiency or external quality assessment programs and will serve as a foundation for additional VDSP studies.

Interlaboratory Comparison for the Determination of 24,25-Dihydroxyvitamin D3 in Human Serum Using Liquid Chromatography with Tandem Mass Spectrometry.

Six laboratories associated with the Vitamin D Standardization Program (VDSP) participated in an interlaboratory comparison of LC with tandem MS (MS/MS) methods for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] in human serum. The laboratories analyzed two different serum-based Standard Reference Materials (SRMs) intended for use in the determination of 25-hydroxyvitamin D and 30 samples from the Vitamin D External Quality Assessment Scheme (DEQAS). All laboratory methods for 24,25(OH)2D3 were based on isotope dilution LC-MS/MS; three of the methods used derivatization of the vitamin D metabolites before LC-MS/MS. Laboratory results were compared to the National Institute of Standards and Technology (NIST) results, which were obtained using their newly developed candidate reference measurement procedure for 24,25(OH)2D3. Laboratory results for the SRM samples varied in comparability to the NIST results, with one laboratory in excellent agreement (-1.6% mean bias), three laboratories at 10-15% mean bias, and the remaining laboratory at 36% mean bias. For the 30 DEQAS samples, the mean bias for the five laboratories ranged from 6 to 15%; however, the SD of the bias ranged from 8 to 29%. As a result of this intercomparison study, one laboratory discovered and corrected a method calculation error and another laboratory modified and improved their LC-MS/MS method.