CDC's Vitamin A Laboratory-External Quality Assessment Program Shows Little Change in Laboratory Performance for Multiple Nutritional Biomarkers over a 10-Year Period (2008-2017).

BACKGROUND: The Vitamin A Laboratory-External Quality Assessment (VITAL-EQA) program operated by the CDC provides analytical performance assessment to low-resource laboratories conducting serum vitamins A (VIA), D (VID), B-12 (B12), and folate (FOL), as well as ferritin (FER) and CRP measurements for public health studies. OBJECTIVES: We aimed to describe the long-term performance of VITAL-EQA participants from 2008 to 2017. METHODS: Participating laboratories received 3 blinded serum samples biannually for duplicate analysis over 3 d. We assessed results (n = 6) for relative difference (%) from the CDC target value and imprecision (% CV) and conducted descriptive statistics on the aggregate 10-year and round-by-round data. Performance criteria were based on biologic variation and deemed acceptable (optimal, desirable, or minimal performance) or unacceptable (less than minimal performance). RESULTS: Thirty-five countries reported VIA, VID, B12, FOL, FER, and CRP results from 2008-2017. The proportion of laboratories with acceptable performance ranged widely by round: VIA 48%-79% (for difference) and 65%-93% (for imprecision), VID 19%-63% and 33%-100%, B12 0%-92% and 73%-100%, FOL 33%-89% and 78%-100%, FER 69%-100% and 73%-100%, and CRP 57%-92% and 87%-100%. On aggregate, ≥60% of laboratories achieved acceptable differences for VIA, B12, FOL, FER, and CRP (only 44% for VID), and over 75% of laboratories achieved acceptable imprecision for all 6 analytes. Laboratories participating continuously in 4 rounds (2016-2017) showed generally similar performance compared to laboratories participating occasionally. CONCLUSIONS: Although we observed little change in laboratory performance over time, on aggregate, >50% of the participating laboratories achieved acceptable performance, with acceptable imprecision being achieved more often than acceptable difference. The VITAL-EQA program is a valuable tool for low-resource laboratories to observe the state of the field and track their own performance over time. However, the small number of samples per round and the constant changes in laboratory participants make it difficult to identify long-term improvements.

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.

National Estimates of Serum Total 25-Hydroxyvitamin D and Metabolite Concentrations Measured by Liquid Chromatography-Tandem Mass Spectrometry in the US Population during 2007-2010.

BACKGROUND: The 2007-2010 NHANES provides the first US nationally representative serum 25-hydroxyvitamin D [25(OH)D] concentrations measured by standardized liquid chromatography-tandem mass spectrometry. OBJECTIVE: We describe patterns for total 25(OH)D and individual metabolites in persons aged ≥1 y stratified by race-ethnicity and grouped by demographic, intake, physiologic, and lifestyle variables. METHODS: We measured 25-hydroxycholecalciferol [25(OH)D3], 25-hydroxyergocalciferol [25(OH)D2], and C3-epimer of 25(OH)D3 [C3-epi-25(OH)D3] in serum samples (n = 15,652) from the 2007-2010 cross-sectional NHANES [total 25(OH)D = 25(OH)D3 + 25(OH)D2]. RESULTS: Concentrations (median, detection rate) of 25(OH)D3 (63.6 nmol/L, 100%) and C3-epi-25(OH)D3 (3.40 nmol/L, 86%) were generally detectable; 25(OH)D2 was detectable in 19% of the population. Total 25(OH)D, 25(OH)D3, and C3-epi-25(OH)D3 displayed similar demographic patterns and were strongly correlated (Spearman's r > 0.70). Concentrations of 25(OH)D2 (90th percentile) were much higher in persons aged ≥60 y (17.3 nmol/L) than in younger age groups (≤4.88 nmol/L). We noted significant race-ethnicity differences in mean total 25(OH)D [non-Hispanic blacks (NHBs), Hispanics, and non-Hispanic whites (NHWs): 46.6, 57.2, and 75.2 nmol/L, respectively] and in the prevalence of total 25(OH)D <30 nmol/L overall (24% of NHBs, 6.4% of Hispanics, and 2.3% of NHWs) as well as stratified by season (winter months: 30% of NHBs, 7.5% of Hispanics, and 3.8% of NHWs; summer months: 17% of NHBs, 4.4% of Hispanics, and 1.6% of NHWs). Persons with higher vitamin D intakes (diet, supplements, or both) and those examined during May-October had significantly higher total 25(OH)D. Significant race-ethnicity interactions in a multiple linear regression model confirmed the necessity of providing race-ethnicity-specific estimates of total 25(OH)D. CONCLUSIONS: Race-ethnicity differences in the prevalence of low total 25(OH)D remained strong even after adjustment for season to account for the NHANES design imbalance between season, latitude, and race-ethnicity. The strong correlation between C3-epi-25(OH)D3 and 25(OH)D3 may be because the epimer is a metabolite of 25(OH)D3. The presence of 25(OH)D2 mainly in older persons is likely a result of high-dose prescription vitamin D2.

A candidate reference measurement procedure for quantifying serum concentrations of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 using isotope-dilution liquid chromatography-tandem mass spectrometry.

The inaccuracy of routine serum 25-hydroxyvitamin D measurements hampers the interpretation of data in patient care and public health research. We developed and validated a candidate reference measurement procedure (RMP) for highly accurate quantitation of two clinically important 25-hydroxyvitamin D metabolites in serum, 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3]. The two compounds of interest together with spiked deuterium-labeled internal standards [d 3-25(OH)D2 and d 6-25(OH)D3] were extracted from serum via liquid-liquid extraction. The featured isotope-dilution LC-MS/MS method used reversed-phase chromatography and atmospheric pressure chemical ionization in positive ion mode. A pentafluorophenylpropyl-packed UHPLC column together with isocratic elution allowed for complete baseline resolution of 25(OH)D2 and 25(OH)D3 from their structural C-3 isomers within 12 min. We evaluated method trueness, precision, potential interferences, matrix effects, limits of quantitation, and measurement uncertainty. Calibration materials were, or were traceable to, NIST Standard Reference Materials 2972. Within-day and total imprecision (CV) averaged 1.9 and 2.0% for 25(OH)D3, respectively, and 2.4 and 3.5% for 25(OH)D2, respectively. Mean trueness was 100.3% for 25(OH)D3 and 25(OH)D2. The limits of quantitation/limits of detection were 4.61/1.38 nmol/L for 25(OH)D3 and 1.46/0.13 nmol/L for 25(OH)D2. When we compared our RMP results to an established RMP using 40 serum samples, we found a nonsignificant mean bias of 0.2% for total 25(OH)D. This candidate RMP for 25(OH)D metabolites meets predefined method performance specifications (≤5% total CV and ≤1.7% bias) and provides sufficient sample throughput to meet the needs of the Centers for Disease Control and Prevention Vitamin D Standardization Certification Program. Graphical abstract Bias assessment using NIST standard reference materials. Legend CDC mean mass fractions (ng/g) ± U 95 (6 replicates per mean). NIST-certified mass fractions (ng/g) ± U 95 from the Certificates of Analysis.

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.

An HPLC Ultraviolet Method Using Low Sample Volume and Protein Precipitation for the Measurement of Retinol in Human Serum Suitable for Laboratories in Low- and Middle-Income Countries.

BACKGROUND: Assessing vitamin A status in populations remains a high public health priority for low- and middle-income countries. However, analytical difficulties with serum retinol measurements persist in international laboratories. Nearly all participants in a Centers for Disease Control and Prevention external quality assessment program use HPLC to measure serum retinol, but round-to-round results failing to meet acceptable criteria suggest the need to provide a straightforward stable HPLC ultraviolet (UV) method that can be adopted by these laboratories to improve performance. We present a protein precipitation HPLC-UV method that measures serum retinol below the deficiency cutoff value (<0.7 µmol/L or 20 µg/dL) that is suitable for low- and middle-income countries and uses commercially available materials. METHODS: Serum (25 µL) added to retinyl acetate was precipitated with acetonitrile (125 µL) to extract retinol. Solvent-based calibration solutions required no extraction. Calibration used either single-point (50 µg/dL) or multipoint solutions (0.52-100 µg/dL). C18 column (4.6 × 100 mm) and acetonitrile with 0.1% triethylamine/water (83/17, v/v) as isocratic mobile phase (1.1 mL/min), achieved baseline separation (7 minutes). RESULTS: With only 25 µL of serum, the limit of detection was 0.52 µg/dL. Single- and multipoint calibration generated equivalent results. Over several years, between-run imprecision was ≤7.1% in multiple quality-control materials. Overall mean (CV) method bias for NIST-certified reference materials (e-series) was -0.2% (5.8%). Maximally, 180 samples were processed within 24 h. CONCLUSIONS: This method was robust and stable over years and accurately measured serum retinol with low-volume samples. Thus, it may be of interest to low- and middle-income countries and to pediatric and finger stick applications.

The vitamin D status of the US population from 1988 to 2010 using standardized serum concentrations of 25-hydroxyvitamin D shows recent modest increases.

BACKGROUND: Temporal trends in the US population's vitamin D status have been uncertain because of nonstandardized serum 25-hydroxyvitamin D [25(OH)D] measurements. OBJECTIVE: To accurately assess vitamin D status trends among those aged ≥12 y, we used data from the cross-sectional NHANESs. DESIGN: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 25(OH)D (sum of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3), calibrated to standard reference materials, was used to predict LC-MS/MS-equivalent concentrations from radioimmunoassay data (1988-2006 surveys; n = 38,700) and to measure LC-MS/MS concentrations (2007-2010 surveys; n = 12,446). Weighted arithmetic means and the prevalence of 25(OH)D above or below cutoff concentrations were calculated to evaluate long-term trends. RESULTS: Overall, mean predicted 25(OH)D showed no time trend from 1988 to 2006, but during 2007-2010 the mean measured 25(OH)D was 5-6 nmol/L higher. Those groups who showed the largest 25(OH)D increases (7-11 nmol/L) were older, female, non-Hispanic white, and vitamin D supplement users. During 1988-2010, the proportions of persons with 25(OH)D <40 nmol/L were 14-18% (overall), 46-60% (non-Hispanic blacks), 21-28% (Mexican Americans), and 6-10% (non-Hispanic whites). CONCLUSIONS: An accurate method for measuring 25(OH)D showed stable mean concentrations in the US population (1988-2006) and recent modest increases (2007-2010). Although it is unclear to what extent supplement usage compared with different laboratory methods explain the increases in 25(OH)D, the use of higher vitamin D supplement dosages coincided with the increase. Marked race-ethnic differences in 25(OH)D concentrations were apparent. These data provide the first standardized information about temporal trends in the vitamin D status of the US population.

A Method-bridging Study for Serum 25-hydroxyvitamin D to Standardize Historical Radioimmunoassay Data to Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Serum concentrations of 25-hydroxyvitamin D (25OHD) were measured for the National Health and Nutrition Examination Survey (NHANES) over the 1988-2006 period using a radioimmunoassay (RIA). In 2010, the Centers for Disease Control and Prevention (CDC) reissued RIA-harmonized 25OHD for NHANES 2004 and 2006, and advised users to adjust the original RIA data from 1988-1994 by using an equation. Beginning with NHANES 2007-2008, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method measured 25OHD. METHODS: A method comparison (bridging) study was designed to convert original RIA 25OHD to LC­MS/MS-equivalents. This report compares the predictive ability of a competitor regression model (Model 2) to the equations that CDC publicly released in 2015 (Model 1). The models differ by time period variable and use of transformations. RESULTS: The two models provided similar adjusted R(2) (Model 1: 88.9%, Model 2: 90.4%) and root mean square error of prediction (plus or minus 9 to 10 nanomoles per liter [nmol/L]). Applying these models to NHANES 1988­2006 RIA 25OHD, the Pearson correlation of LC­MS/MS-equivalent concentrations was 0.99; the median difference between models was 0 nmol/L (interquartile range: ­2.8 to 1 nmol/L). In contrast to declining RIA-harmonized 25OHD, both models showed little change in LC­MS/MS-predicted 25OHD over the 1988­2006 period. For 2001­2006, both models predicted similar prevalences of 25OHD less than 30 nmol/L, which were lower than the prevalence estimates based on RIA-harmonized data. Mean weighted LC­MS/MS-equivalent concentrations based on either model were about 3 nmol/L lower for the 1988­1994 survey and about 3 nmol/L higher for the 2001­2006 surveys, effectively smoothing out temporal trends observed using the harmonized RIA data. CONCLUSIONS: Given minimal differences between models, final selection was based on public availability of the regression data. The bridging equations provide a way to use the previous RIA results to obtain LC­MS/MS-equivalent concentrations and evaluate temporal trends in vitamin D status.

Isotope dilution ultra performance liquid chromatography-tandem mass spectrometry method for simultaneous measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in human serum.

BACKGROUND: An ultra performance liquid chromatography-tandem mass spectrometry method with calibration traceable to NIST SRM was developed and validated to measure concentrations of 25-hydroxyvitamin D(2) (25OHD(2)), 25-hydroxyvitamin D(3) (25OHD(3)) and the C-3 epimer of 25OHD(3) (epi-25OHD(3)) in human serum. METHODS: Tri- and hexa-deuterated internal standards were added to serum (100 µl) to monitor recovery. Liquid-liquid extraction was used to extract the hexane-soluble materials. Calibration solutions [8-100 nmol/L 25OHD(2,) 12-150 nmol/L 25OHD(3), and 4-50 nmol/L epi-25OHD(3)] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1×100 mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4 ml/min, run time was 14 min per sample; 25OHD(3) and epi-25OHD(3) were baseline separated. Atmospheric pressure chemical ionization in the positive ion mode with selected reaction monitoring captured the following transitions: 25OHD(2), m/z 395.3>377.3 (209.1 qualifier); (epi-)25OHD(3), m/z 383.3>365.3 (105.1 qualifier); d(3)-25OHD(2), m/z 398.3>380.3; and d(6)-25OHD(3), m/z 389.3>371.3. RESULTS: Recovery averaged ≥98%. Total imprecision was ≤10% when concentrations were ≥20 nmol/l. Bias averaged <5%. Detection limits were <5 nmol/l. Median (nmol/l) 25OHD(2), 25OHD(3) and epi-25OHD(3) were quantitated in 98 blood donors (