Assessment of serum total 25-hydroxyvitamin D assays for Vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 1 liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays - impact of 3-epi-25-hydroxyvitamin D3 on assay performance.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D2, 25(OH)D3, 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3), and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) using isotope dilution liquid chromatography - tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 1 includes results from 14 laboratories using 14 custom LC-MS/MS assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 53% of the LC-MS/MS assays met the VDSP criterion of mean % bias ≤ |±5%|. For the LC-MS/MS assays not meeting the ≤ |±5%| criterion, four assays had mean % bias of between 12 and 21%. Based on multivariable regression analysis using the concentrations of the four individual vitamin D metabolites in the 50 single-donor samples, the performance of several LC-MS/MS assays was found to be influenced by the presence of 3-epi-25(OH)D3. The results of this interlaboratory study represent the most comprehensive comparison of LC-MS/MS assay performance for serum total 25(OH)D and document the significant impact of the lack of separation of 3-epi-25(OH)D3 and 25(OH)D3 on assay performance, particularly with regard to mean % bias.

Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 2 ligand binding assays - impact of 25-hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of ligand binding assays (Part 2) for the determination of serum total 25-hydroxyvitamin D [25(OH)D]. Fifty single-donor samples were assigned target values for concentrations of 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] using isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 2 includes results from 17 laboratories using 32 ligand binding assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 50% of the ligand binding assays achieved the VDSP criterion of mean % bias ≤ |± 5%|. For the 13 unique ligand binding assays evaluated in this study, only 4 assays were consistently within ± 5% mean bias and 4 assays were consistently outside ± 5% mean bias regardless of the laboratory performing the assay. Based on multivariable regression analysis using the concentrations of individual vitamin D metabolites in the 50 single-donor samples, most assays underestimate 25(OH)D2 and several assays (Abbott, bioMérieux, DiaSorin, IDS-EIA, and IDS-iSYS) may have cross-reactivity from 24R,25(OH)2D3. The results of this interlaboratory study represent the most comprehensive comparison of 25(OH)D ligand binding assays published to date and is the only study to assess the impact of 24R,25(OH)2D3 content using results from a reference measurement procedure.

Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and Vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2.

An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays.

Vitamin D Standardization Program (VDSP) intralaboratory study for the assessment of 25-hydroxyvitamin D assay variability and bias.

An intralaboratory study assessing assay variability and bias for determination of serum total 25-hydroxyvitamin D [25(OH)D] was conducted by the Vitamin D Standardization Program (VDSP). Thirteen assays for serum total 25(OH)D were evaluated in a single laboratory including 11 unique immunoassays and one liquid chromatography - tandem mass spectrometry (LC-MS/MS) assay. Fifty single-donor serum samples, including eight samples with high concentrations of 25(OH)D2 (> 30 nmol/L), were assigned target values for 25(OH)D2 and 25(OH)D3 using reference measurement procedures (RMP). Using four replicate measurements for each sample, the mean total percent coefficient of variation (%CV) and mean % bias from the target values were determined for each assay using the 50 single-donor samples and a 42-sample subset, which excluded 8 high 25(OH)D2 concentration samples, and compared with VDSP performance criteria of ≤ 10 % CV and ≤ ±5 % mean bias. All 12 assays achieved the performance criterion for % CV, and 9 of the 12 assays were within ≤ ±5 % mean bias. The Fujirebio Inc. assay exhibited the lowest %CV and highest percentage of individual measurements within ≤ ±5 % mean bias. Ten immunoassays exhibited changes in response due to the high 25(OH)D2 samples with Abbott, Biomérieux, DiaSorin, DIAsource, and IDS-iSYS assays having the largest deviations. The Fujirebio Inc. and Beckman Coulter assays were only minimally affected by the presence of the high 25(OH)D2 samples. Samples with high concentrations of 25(OH)D2 provided a critical performance test for immunoassays indicating that some assays may not have equal response or recovery for 25(OH)D2 and 25(OH)D3.

Gas chromatographic retention behavior of polycyclic aromatic hydrocarbons (PAHs) and alkyl-substituted PAHs on two stationary phases of different selectivity.

Retention indices (I) for 45 polycyclic aromatic hydrocarbons (PAHs) and 63 methyl-substituted PAHs were determined by gas chromatography - mass spectrometry (GC-MS) using two different stationary phases: a Rxi-PAH phase (a "higher phenyl-content stationary phase") and a 50% (mole fraction) liquid crystalline dimethylpolysiloxane phase. Retention data were obtained for parent PAHs from molecular mass (MM) 128 g/mol (naphthalene) to 328 g/mol (benzo[c]picene) and for 12 sets of methyl-PAHs (methylfluorenes, methylanthracenes, methylphenanthrenes, methylfluoranthenes, methylpyrenes, methylbenz[a]anthracenes, methylbenzo[c]phenanthrenes, methylchrysenes, methyltriphenylenes, methylbenzo[a]pyrenes, methylperylenes, and methylpicenes). Molecular shape descriptors such as length-to-breath ratio (L/B) and thickness (T) were determined for all the PAHs studied. Correlation between I and L/B ratio was evaluated for both stationary phases with a better correlation observed for the 50% liquid crystalline phase (correlation coefficients ranging from 0.22 to 1.00). Graphical Abstract GC separation of six methylchrysene isomers (m/z 242) on two different stationary phases: 50 % phenyl-like methylpolysiloxane phase and 50 % liquid crystalline phase. Retention indices (I) are plotted as a function of L/B for both phases. The data marker numbers identify each isomer based on methyl-substitution position.

Role of the National Institute of Standards and Technology (NIST) in Support of the Vitamin D Initiative of the National Institutes of Health, Office of Dietary Supplements.

Since 2005, the National Institute of Standards and Technology (NIST) has collaborated with the National Institutes of Health (NIH), Office of Dietary Supplements (ODS) to improve the quality of measurements related to human nutritional markers of vitamin D status. In support of the NIH-ODS Vitamin D Initiative, including the Vitamin D Standardization Program (VDSP), NIST efforts have focused on (1) development of validated analytical methods, including reference measurement procedures (RMPs); (2) development of Standard Reference Materials (SRMs); (3) value assignment of critical study samples using NIST RMPs; and (4) development and coordination of laboratory measurement QA programs. As a result of this collaboration, NIST has developed RMPs for 25-hydroxyvitamin D2 [25(OH)D2], 25(OH)D3, and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]; disseminated serum-based SRMs with values assigned for 25(OH)D2, 25(OH)D3, 3-epi-25(OH)D3, and 24R,25(OH)2D3; assigned values for critical samples for VDSP studies, including an extensive interlaboratory comparison and reference material commutability study; provided an accuracy basis for the Vitamin D External Quality Assurance Scheme; coordinated the first accuracy-based measurement QA program for the determination of 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3 in human serum/plasma; and developed methods and SRMs for the determination of vitamin D and 25(OH)D in food and supplement matrix SRMs. The details of these activities and their benefit and impact to the NIH-ODS Vitamin D Initiative are described.

Establishing an Accuracy Basis for the Vitamin D External Quality Assessment Scheme (DEQAS).

Until recently, the Vitamin D External Quality Assessment Scheme (DEQAS) assessed the performance of various assays for the determination of serum total 25-hydroxyvitamin D [25(OH)D] by using a consensus mean based on the all-laboratory trimmed mean (ALTM) of the approximately 1000 participants' results. Since October 2012, the National Institute of Standards and Technology (NIST), as part of the Vitamin D Standardization Program, has participated in DEQAS by analyzing the quarterly serum sample sets using an isotope dilution LC-tandem MS (ID LC-MS/MS) reference measurement procedure to assign an accuracy-based target value for serum total 25(OH)D. NIST has analyzed 90 DEQAS samples (18 exercises × 5 samples/exercise) to assign target values. The NIST-assigned values are compared with the ALTM and the biases assessed for various assays used by the participants, e.g., LC-MS/MS, HPLC, and several ligand-binding assays. The NIST-value assignment process and the results of the analyses of the 90 DEQAS samples are summarized. The absolute mean bias between the NIST-assigned values and the ALTM was 5.6%, with 10% of the samples having biases >10%. Benefits of the accuracy-based target values are presented, including for sample sets with high concentrations of 25(OH)D2 and 3-epi-25(OH)D3.

The Influence of the Aromatic Character in the Gas Chromatography Elution Order: The Case of Polycyclic Aromatic Hydrocarbons.

A link between the aromatic character of polycyclic aromatic hydrocarbons and gas chromatography elution order in columns with a polysiloxane backbone in the stationary phase is reported for the first time. The aromatic character was calculated using a method that combines the π-Sextet Rule and the Pauling Ring Bond Orders to allow the establishment of the location and migration of aromatic sextets in PAH structures. One GC column with a polysiloxane - like backbone (Rxi-PAH) and three GC columns with a polysiloxane backbone (DB-5, SE-52, and LC-50), were used for the analysis. According to the results of this study, within an isomer group, PAHs that contain a lower number of rings affected by the aromatic sextets tend to elute earlier than PAHs that contain a higher number of rings affected by the aromatic sextets. The PAHs that follow the calculated elution order are 88 % in the Rxi-PAH column, 88 % in the DB-5 column, 93 % in the SE-52 column, and 85% in the LC-50 column. It is expected that future analyses with other aromatic compounds in GC columns with a polysiloxane backbone in the stationary phase will follow a GC elution order that agrees with the aromatic character of the molecules.