MSACL 2023 Abstract

Development and Validation of a LC-MS/MS Method for Simultaneous Measurement of 25-OH D3, epi-25-OH D3, 25-OH D2, Vitamin A, alpha-Tocopherol, and gamma-Tocopherol

Yi Xiao (1), Edward Leung (1, 2)
(1) Department of Pathology and Laboratory Medicine, Children’s Hospital of Los Angeles, Los Angeles, CA (2) Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA

Yi Xiao, Ph.D. (Presenter) Children’s Hospital Los Angeles

Presenter Bio: Yi Xiao, Ph.D., DABCC, CLS is currently an assistant professor of Clinical Pathology at Keck School of Medicine of USC, and assistant director of the core laboratory and the special chemistry/biochemical genetics laboratory at Children’s Hospital Los Angeles. He received his PhD in Biochemistry from Vanderbilt University, and completed clinical chemistry fellowship from a ComACC-accredited fellowship program from Children’s Hospital Los Angeles.

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

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 have 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 uL 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 uL 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 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 ug/dL (20% CV), 0.2 ug/mL (20% CV), and 0.2 ug/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.