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1.  Development and Comparison of Three Liquid Chromatography-Atmospheric Pressure Chemical Ionization/Mass Spectrometry Methods for Determining Vitamin D Metabolites in Human Serum 
Journal of Chromatography. a  2012;1240:132-139.
Liquid chromatographic methods with atmospheric pressure chemical ionization mass spectrometry were developed for the determination of the vitamin D metabolites 25-hydroxyvitamin D2 (25(OH)D2), 25-hydroxyvitamin D3 (25(OH)D3), and 3-epi-25-hydroxyvitamin-D3 (3-epi-25(OH)D3) in the four Levels of SRM 972, Vitamin D in Human Serum. One method utilized a C18 column, which separates 25(OH)D2 and 25(OH)D3, and one method utilized a CN column that also resolves the diastereomers 25(OH)D3 and 3-epi-25(OH)D3. Both methods utilized stable isotope labeled internal standards for quantitation of 25(OH)D2 and 25(OH)D3. These methods were subsequently used to evaluate SRM 909c Human Serum, and 25(OH)D3 was the only vitamin D metabolite detected in this material. However, SRM 909c samples contained matrix peaks that interfered with the determination of the [2H6]-25(OH)D3 peak area. The chromatographic conditions for the C18 column were modified to remove this interference, but conditions that separated the matrix peaks from [2H6]-25(OH)D3 on the CN column could not be identified. The alternate internal standard [2H3]-25(OH)D3 did not suffer from matrix interferences and was used for quantitation of 25(OH)D3 in SRM 909c. During the evaluation of SRM 909c samples, a third method was developed using a pentafluorophenylpropyl column that also separates the diastereomers 25(OH)D3 and 3-epi-25(OH)D3. The 25(OH)D3 was measured in SRM 909c using all three methods, and the results were compared.
doi:10.1016/j.chroma.2012.03.091
PMCID: PMC3348387  PMID: 22533908
LC-MS; 25-hydroxyvitamin D2; 25-hydroxyvitamin D3; 3-epi-25-hydroxyvitamin D3; Standard Reference Material; human serum
2.  Development and Certification of a Standard Reference Material for Vitamin D Metabolites in Human Seruma 
Analytical Chemistry  2011;84(2):956-962.
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)D2, 25(OH)D3, and 3-epi-25(OH)D3. 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)D3 proved to be essential for accurate determination of the metabolites.
doi:10.1021/ac202047n
PMCID: PMC3287345  PMID: 22141317
3.  Biomarkers of folate status in NHANES: a roundtable summary123456 
A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999–2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991–1994 and NHANES 1999–2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007–2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.
doi:10.3945/ajcn.111.013011
PMCID: PMC3127517  PMID: 21593502
4.  Biomarkers of vitamin B-12 status in NHANES: a roundtable summary123456 
A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12–related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES—serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)—and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12–related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.
doi:10.3945/ajcn.111.013243
PMCID: PMC3127527  PMID: 21593512
5.  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 
Analytical chemistry  2010;82(5):1942-1948.
Vitamin D exists in two major forms, vitamin D3 and vitamin D2. 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 D3 [25(OH)D3] and 25-hydroxyvitamin D2 [25(OH)D2] in serum are considered the best indicators of vitamin D status. A candidate reference measurement procedure for serum 25(OH)D3 and 25(OH)D2 has been developed and critically evaluated. The deuterated compounds 25(OH)D3-d3 and 25(OH)D2-d3 are used as internal standards for 25(OH)D3 and 25(OH)D2, respectively. The 25(OH)D3 and 25(OH)D2 and their respective labeled internal standards are simultaneously extracted from serum using liquid-liquid extraction prior to reversed-phase LC-MS/MS. Chromatographic separation was performed using a cyano (CN) column for both 25(OH)D3 and 25(OH)D2. 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)D3 and 25(OH)D2 ranged from 99.0 % to 101.0 %. The absolute recoveries with this method were 97 % and 92 % for 25(OH)D3 and 25(OH)D2, 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)D3 and 25(OH)D2 from their respective isomers 3-epi-25(OH)D3 and 3-epi-25(OH)D2 was achieved. The limit of detection at a signal-to-noise ratio of ~ 3 was 40 pg of 25(OH)D on column (or ~ 0.15 ng/g as expressed as a concentration). This candidate reference measurement procedure for serum 25(OH)D3 and 25(OH)D2 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)D3 and 25(OH)D2 can be compared and that will serve as a standard of higher order for measurement traceability.
doi:10.1021/ac9026862
PMCID: PMC2838390  PMID: 20136128
25-hydroxyvitamin D3; 25(OH)D3; 25-hydroxyvitamin D2; 25(OH)D2; Reference measurement procedure; Atmospheric pressure chemical ionization; Isotope dilution; Liquid chromatography/tandem mass spectrometry; LC-MS/MS; Liquid-liquid extraction

Results 1-5 (5)