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

Vitamin D exists in two major forms, vitamin D-3 and vitamin D-2. 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 D-3 [25(OH)D-3] and 25-hydroxyvitamin D-2 [25(OH)D-2] in serum are considered the best indicators of vitamin D status. A candidate reference measurement procedure for serum 25(OH)D-3 and 25(OH)D-2 has been developed and critically evaluated. The deuterated compounds 25(OH)D-3-d(3) and 25(OH)D-2-d:1 are used as internal standards for 25(OH)D-3 and 25(OH)D-2, respectively. The 25(OH)D-3 kind 25(OH)D-2 kind 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 it cyano (CN) column for both 25(OH)D-3 and 25(OH)D-2. Atmospheric pressure chemical ionization (APCI) in the positive ion mode and multiple reaction monitoring (MRM) were used for I-C-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)D-3 and 25(OH)D-2 ranged from 99.0% to 101.0%. The absolute recoveries with this method were 97% and 92% for 25(OH)D-3 and 25(OH)D-2, 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 < 1 ng/g. Chromatographic separation of 25(OH)D-3 and 25(OH)D-2 from their respective isomers 3-epi-25(OH)D-3 and 3-epi-25(OH)D-2 was achieved. The limit of detection at a signal-to-noise ratio of similar to 3 wits 40 pg of 25(OH)D on column (or similar to 0.15 ng/g as expressed as a concentration). This candidate reference measurement procedure for serum 25(OH)D-3 kind 25(OH)D-2 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)D-3 and 25(OH)D-2 can be compared and that will serve as a standard of higher order for measurement traceability.