Candidate reference measurement procedures for serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 by using isotope-dilution liquid chromatography-tandem mass spectrometry.
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 the basis of isotope- dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for separate quantification of serum 25(OH)D2 and 25(OH)D3.


METHODS
Hexa-deuterated 25(OH)D3/D2 was added to serum. This mixture was extracted with n-hexane and fractionated on Sephadex LH-20 before 2-dimensional LC-MS/MS. In the first dimension, both procedures used a C4 column; however, in the second dimension, the 25(OH)D2 procedure used a C18 and the 25(OH)D3 procedure used a Zorbax SB-CN column. Calibration was traceable to the NIST Standard Reference Material (SRM) 2972. Validation comprised assessment of interference and limit of quantification/detection. Imprecision and trueness were validated by analysis of the SRM 972 against specifications (CV<5% and bias<1.7%). The expanded uncertainty for quadruplicate measurements was estimated.


RESULTS
Testing of potentially interfering substances was negative. Interference by 3-epi-25(OH)D3 was resolved by sufficient chromatographic resolution. The limits of quantification/detection were 1.1 nmol/L and 0.09 pmol/L for 25(OH)D3 and 1.2 nmol/L and 0.05 pmol/L for 25(OH)D(2). Mean total CVs and differences from the SRM 972 target (±1-sided 95% CI) were 2.1% and 1.1%±1.5% [25(OH)D3] and 3% and 1.3%±0.6% [25(OH)D2], respectively. The respective expanded uncertainties were 3.4% and 3.9%.


CONCLUSIONS
From the validation data, we conclude that we achieved our objective of 2 state-of-the-art candidate RMPs for serum 25(OH)D3 and 25(OH)D2.
Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
Hedwig C M
HC
Stepman
An
A
Vanderroost
Katleen
K
Van Uytfanghe
Linda M
LM
Thienpont
D015652:25-Hydroxyvitamin D 2 / Q000097:blood*; D001331:Automation; D002112:Calcifediol / Q000097:blood*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods* / Q000592:standards*; D003903:Deuterium; D006801:Humans; D011865:Radioisotope Dilution Technique; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards*
D015652:25-Hydroxyvitamin D 2; D003903:Deuterium; D002112:Calcifediol