25-hydroxyvitamin D2 and D3 composition and repeat test patterns in routine adult hospital liquid chromatography–tandem mass spectrometry testing
Yichi Mai, Ziyin Song, Cheng Yi, Lili Wei, Huanliang Liu, Yanhong XiaoAbstract
Introduction
Liquid chromatography–tandem mass spectrometry quantifies 25-hydroxyvitamin D2 (25[OH]D2) and D3 (25[OH]D3) separately, but clinical reporting often emphasizes total 25(OH)D. We characterized component composition and repeat test changes in routine adult hospital testing.
Methods
We retrospectively analyzed a pseudonymized dataset derived from routine laboratory records for vitamin D tests performed from June 2019 through November 2022. Cross-sectional analyses used first tests from 2808 adults; longitudinal analyses compared first and last tests in 446 retested adults. Vitamin D2–contributory composition was defined exploratorily as a 25(OH)D2 share of at least 10%, with sensitivity analyses using 5%, 20%, and a continuous measure.
Results
Median total 25(OH)D was 24.6 ng/mL (approximately 61.5 nmol/L), and 71.3% of results were below the local reference cutoff. Vitamin D2–contributory composition occurred in 16.3% and was associated with sex, season, and ordering service. Among retested adults, median changes were 4.3 ng/mL for total 25(OH)D, 4.2 ng/mL for 25(OH)D3, and −0.1 ng/mL for 25(OH)D2. Total change correlated with 25(OH)D3 change (ρ = 0.98; P < .001) but not with 25(OH)D2 change (ρ = 0.04; P = .46).
Discussion
Fractionated measurements revealed compositional variation within total 25(OH)D categories and predominantly D₃-related changes on repeat testing. The clinical implications of these patterns require further evaluation.