Quantification of Serum 25-Hydroxyvitamin D₂ and D₃Using 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 D₂ and D₃ and establish reference intervals for these analytes. Chromatographic separation of 25(OH)D₂ and 25(OH)D₃ was achieved after adding deuterated ▵⁹-tetrahydrocannabinol (▵⁹-THC-D₃) 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 (▵⁹-THC-D₃), 401.15 > 365.2 [25(OH)D₃], and 413.15 > 355.20 [25(OH)D₂]. Reference interval study results were compared with current 25(OH)D recommendations.

Elution of 25(OH)D₂, 25(OH)D₃, and ▵⁹ -THC-D₃ was achieved after 3.0 minutes (total run time, 6.0 minutes). Within- and between-run coefficients of variation were less than 11%. Deming regression of radioimmunoassay and LC-MS/MS methods for total 25(OH)D levels yielded a slope of 0.97 (95% confidence interval, 0.88-1.05) and y-intercept of –1.74 ng/mL. Reference intervals were less than recommended levels (D₂, 0.0–12.1; D₃ , 5.5–41.4; total vitamin D, 6.0–43.5 ng/mL [0–30, 14–103, 15–109 nmol/L, respectively]) with no statistically significant differences in race, age, or sex. This LC-MS/MS method provides a rapid, accurate, sensitive, and cost-effective alternative to other methods for detection of 25(OH)D₂ and 25(OH)D₃ at nanomolar concentrations.