Development of a Candidate Reference Measurement Procedure for the Determination of 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 in Human Serum Using Isotope-Dilution Liquid Chromatography−Tandem Mass Spectrometry

Abstract

Vitamin D exists in two major forms, vitamin D₃ and vitamin D₂. Vitamin D helps the body absorb calcium and promote optimal bone health. Both forms of vitamin D are metabolized to 25-hydroxyvitamin D in the body, and the levels of 25-hydroxyvitamin D₃ [25(OH)D₃] and 25-hydroxyvitamin D₂ [25(OH)D₂] in serum are considered the best indicators of vitamin D status. A candidate reference measurement procedure for serum 25(OH)D₃ and 25(OH)D₂ has been developed and critically evaluated. The deuterated compounds 25(OH)D₃-d₃ and 25(OH)D₂-d₃ are used as internal standards for 25(OH)D₃ and 25(OH)D₂, respectively. The 25(OH)D₃ and 25(OH)D₂ and their respective labeled internal standards are simultaneously extracted from serum using liquid−liquid extraction prior to reversed-phase liquid chromatography−tandem mass spectrometry (LC−MS/MS). Chromatographic separation was performed using a cyano (CN) column for both 25(OH)D₃ and 25(OH)D₂. Atmospheric pressure chemical ionization (APCI) in the positive ion mode and multiple reaction monitoring (MRM) were used for LC−MS/MS. The accuracy of the method was evaluated by recovery studies of measuring 25-hydroxyvitamin D [25(OH)D] in spiked samples with known 25(OH)D levels. The recoveries of the added 25(OH)D₃ and 25(OH)D₂ ranged from 99.0% to 101.0%. The absolute recoveries with this method were 97% and 92% for 25(OH)D₃ and 25(OH)D₂, respectively. Excellent precision was obtained with between-set coefficients of variation (CVs) of 0.2−0.6% for 25(OH)D levels >1 ng/g and within 2% for the level of 3 and 25(OH)D₂ from their respective isomers 3-epi-25(OH)D₃ and 3-epi-25(OH)D₂ was achieved. The limit of detection at a signal-to-noise ratio of ∼3 was 40 pg of 25(OH)D on column (or ∼0.15 ng/g as expressed as a concentration). This candidate reference measurement procedure for serum 25(OH)D₃ and 25(OH)D₂ demonstrates good accuracy and precision and low susceptibility to interferences. It can be used to provide an accuracy base to which clinical methods for 25(OH)D₃ and 25(OH)D₂ can be compared and that will serve as a standard of higher order for measurement traceability.