Outi Itkonen 2,3* Influence of Vitamin D 2 Percentage on Accuracy of 4 Commercial Total 25- Hydroxyvitamin D Assays

25-Hydroxyvitamin D (25-OHD) is the most appropriate marker for monitoring vitamin D storage status. The 2 major forms of 25OH-D [25-OH-D2 (D2) and 25OH-D3 (D3)], differ slightly in molecular structures but have essentially the same physiological activity. Therefore, both D2 and D3 should be measured to assess vitamin D status. At our laboratory, 42% of patient samples submitted for vitamin D testing contain D2. Recognizing the ability of an assay to quantify both the D3 and D2 forms equally is therefore crucial. The analytical performance of several total 25-OH-D assays approved by the US Food and Drug Administration is well documented (1 ); however, information is limited on how D2 affects assay accuracies. Le Goff et al. (2 ) demonstrated the inconsistency between several assays in their crossreactivities toward D2. The size of their study was small, however, and the range of D2 percentages (D2%) was unknown. We used a larger sample size than previous studies and a D2% interval of 1.3%–91.2% [3.4 –95.1 ng/mL (8.5–238 nmol/ L)] and evaluated the influence of D2% on the accuracies of the following assays: DiaSorin Liaison, Abbott Architect, Roche Cobas, and Siemens Centaur. We developed and validated a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay as the reference method. Samples were prepared for the LC-MS/MS assay by mixing with internal standard (D3-d6/D2-d6), ZnSO4 solution, and methanol; liquid–liquid extraction (hexane); drying; and reconstitution. The liquid chromatography method was established with a Phenomenex 2.6m Kinetex C18 column (2.1 50 mm) at 35 °C and a flow rate of 0.35 mL/min. Mobile phases A and B were deionized water and LC-MS-grade methanol, respectively, both containing 1 mL/L formic acid and 2 mmol/L ammonium acetate. The linear gradient of the mobile phase was 60% to 100% B in 3.5 min, and the total length of the liquid chromatography run was 8 min. A Waters Micromass Quattro Micro mass spectrometer, operated with positive electrospray ionization, was used to acquire chromatograms with the following ion transitions: D3, m/z 401.03159.1; D2, m/z 413.03355.2; D3-d6, m/z 407.03159.1; and D2-d6, m/z 419.03355.2. The between-day CVs were between 5.1% and 12% at D2, D3, and total D concentrations of 12.9 –143 ng/mL (32.2–358 nmol/ L). The matrix effect was negligible. The linearity intervals for D3, D2, and total D were 3.0 –200 ng/mL (7.5–500 nmol/L), 1.0 –150 ng/mL (2.5–375 nmol/L), and 3.0 – 350 ng/mL (7.5– 875 nmol/L), respectively. The recoveries were 83.8%–109.2% (mean, 95.9%). The lower limits of quantification for D3 and D2 were 3.0 ng/mL (7.5 nmol/L) and 1.0 ng/mL (2.5 nmol/ L), respectively. We tested the 2011 College of American Pathologists proficiency testing set ABVD01– 05 with the assay, and all results were within 25% of target values (mean bias, 1.2%). Interference by 3-epi-25-OH-D3 was negligible, because our study included only samples from adults (21–97 years) (3 ). We randomly chose samples from 149 adult patients submitted for 25-OH-D testing by the Abbott Architect assay. All samples were tested by the LC-MS/MS assay (63 contained D2), and subsets of these samples were aliquoted for the following assays: 75 samples (31 with D2) by the Liaison assay, 64 samples (20 with D2) by the Centaur assay, and 55 samples (25 with D2) by the Cobas assay. Total 25-OH-D values obtained with each commercial assay were compared with those obtained by LC-MS/MS. Deming regression analysis and the determination of percent bias were performed under 2 scenarios: (a) samples with D3 only vs. (b) all samples. The regression equations and the coefficients of determination (r) obtained for each scenario are shown in Fig. 1A. For the Liaison assay, the 2 scenarios yielded similar coefficients of determination; however, these values were different for all of the other assays. The regression slopes and r values of the Architect and Cobas assays both de1 Nonstandard abbreviations: 25-OH-D, 25hydroxyvitamin D; D2, 25-OH-D2; D3, 25-OH-D3; D2%, D2 percentage; LC-MS/MS, liquid chromatography–tandem mass spectrometry. Letters to the Editor