Development and Validation of a Liquid Chromatography Mass Spectrometry Method for Simultaneous Measurement of 25-OH D3, epi-25-OH D3, 25-OH D2, Vitamin A, α-Tocopherol, and γ-Tocopherol

Background Circulatory fat-soluble vitamin levels are commonly measured to identify deficiencies that may lead to rickets, osteomalacia, night blindness, and reversible motor and sensory neuropathies. We developed and validated a rapid and robust LC-MS/MS method that simultaneously measures 25-OH D3, epi-25-OH D3, 25-OH D2, vitamin A, α-tocopherol, and γ-tocopherol for clinical use. Method 100 µL of serum was mixed with isotope-labeled internal standard and extracted using a 96-well supported-liquid extraction plate with 1.5 mL of hexanes/isopropanol (90/10) (v/v). Dried eluate was reconstituted with 100 µL of methanol/water (90/10) (v/v) and analyzed by LC-MS/MS with a 10-minute gradient. Accuracy was assessed using NIST Standard Reference Materials SRM972a and SRM968f, patient comparison analysis with a LC-MS/MS method at a reference lab, and spike-recovery studies using patient sera and vitamin D-depleted serum. Analytical measurement range (AMR) was determined by spiking 6 analytes into vitamin D-depleted serum to give 7 specimens at varying concentrations. The lower limit of the measuring interval (LLMI) was assessed using 6 pooled specimens with varying low concentrations of each analyte over 20 days. Precision (repeatability and reproducibility) was assessed using quality control materials. Interference studies were performed using pooled patient specimens spiked with varying concentrations of hemoglobin, bilirubin, or intralipid. Matrix effect was assessed by post-column infusion and by matrix dilution with saline. Results The method was linear covering physiological concentrations with r2 > 0.99. Repeatability and reproducibility were <15% CV at all QC levels. LLMI for 25-OH D3, epi-25-OH D3, 25-OH D2, vitamin A, α-tocopherol, and γ-tocopherol were 4 ng/mL (15% CV), 4 ng/mL (15% CV), 4 ng/mL (18% CV), 1 µg/dL (20% CV), 0.2 µg/mL (20% CV), and 0.2 µg/mL (8% CV). Recoveries for NIST Standard Reference Materials were between 92 - 111% and between 81 - 122% for spike-recovery studies. Passing-Bablok analyses for vitamin D total, vitamin A, and α-tocopherol demonstrated slopes between 1.04 and 1.11 and r2 between 0.94 and 0.96. Minimal matrix effect was observed. Conclusions We have developed and validated a comprehensive and rapid LC-MS/MS method for the simultaneous measurement of 25-OH D3, epi-25-OH D3, 25-OH D2, vitamin A, α-tocopherol, and γ-tocopherol for clinical use.