Quantification of serum 25-hydroxyvitamin D2 and D3 using HPLC-tandem mass spectrometry and examination of reference intervals for diagnosis of vitamin D deficiency

Serum levels of 25-hydroxyvitamin D are important in establishing true vitamin D levels in humans. The Purposes of this study were to develop a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of 25-hydroxyvitamin D-2 and D-3 and establish reference intervals for these analytes. Chromatographic separation of 25(OH)D-2 and 25(OH)D-3 was achieved after adding deuterated Delta(9)-tetrahydrocannabinol (A(9)-THC-D-3) and organic extraction. The 3 ions were ionized using positive electrospray ionization and detected in the multiple-reaction monitoring mode using mass (m)/charge (z) transitions of 318.15 > 196.20 (Delta(9)-THC-D-3), 401.15 > 365.2 [25(OH)D-3], and 413.15 > 355.20 [25(OH)D-2]. Reference interval study results were compared with current 25(OH)D recommendations.