Candidate Reference Measurement Procedures for Serum 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 by Using Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry

摘要: BACKGROUND: 25-hydroxyvitamin D [25(OH)D] assays are characterized by poor between-assay comparability. This result emphasizes the need for reference measurement procedures (RMPs) to establish calibration traceability and assist in method validation. We aimed at developing candidate RMPs on basis of isotope- dilution liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS) separate quantification serum 25(OH)D2 25(OH)D3. METHODS: Hexa-deuterated 25(OH)D3/D2 was added serum. mixture extracted with n-hexane fractionated Sephadex LH-20 before 2-dimensional LC-MS/MS. In first dimension, both used a C4 column; however, second procedure C18 25(OH)D3 Zorbax SB-CN column. Calibration traceable NIST Standard Reference Material (SRM) 2972. Validation comprised assessment interference limit quantification/detection. Imprecision trueness were validated analysis SRM 972 against specifications (CV <5% bias <1.7%). The expanded uncertainty quadruplicate measurements estimated. RESULTS: Testing potentially interfering substances negative. Interference 3-epi-25(OH)D3 resolved sufficient chromatographic resolution. limits quantification/detection 1.1 nmol/L 0.09 pmol/L 1.2 0.05 25(OH)D2. Mean total CVs differences from target (± 1-sided 95% CI) 2.1% 1.1% ± 1.5% [25(OH)D3] 3% 1.3% 0.6% [25(OH)D2], respectively. respective uncertainties 3.4% 3.9%. CONCLUSIONS: From validation data, we conclude that achieved our objective 2 state-of-the-art