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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 (D(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. Elution of 25(OH)D(2), 25(OH)D(3), and Delta(9)-THC-D(3) 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 (D(2), 0.0-12.1; D(3), 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)D(2) and 25(OH)D(3) at nanomolar concentrations.

The sensitive and specific detection of nicotine, its metabolites, and the tobacco alkaloid, anabasine, is useful in evaluating the success of smoking cessation treatments and detecting tobacco use, passive exposure, and nontobacco nicotine exposure in potential transplant recipients, insurance clients, and elective surgical patients. Rapid sample preparation and extended high-performance liquid chromatographic separation of tobacco alkaloids and metabolites was interfaced with tandem mass spectrometry. By using deuterated internal standards and appropriate confirmatory ion mass transitions, direct injection of centrifugally clarified urine was possible. The method had excellent precision, limit of quantitation, and linearity. The rigorous separation method revealed an interferent of nicotine that had coeluted with anabasine in more rapid chromatography and that may result in tobacco use misclassification. The method provides more specific detection of tobacco exposure and illustrates the potential of centrifugal clarification for sample preparation in the detection of multiple analytes in urine.

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 (D(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 318.15196.20 (Delta(9)-THC-D(3)), 401.15365.2 (25(OH)D(3)), and 413.15355.20 (25(OH)D(2)). Reference interval study results were compared with current 25(OH)D recommendations. Elution of 25(OH)D(2), 25(OH)D(3), and Delta(9)-THC-D(3) 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 (D(2), 0.0-12.1; D(3), 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)D(2) and 25(OH)D(3) at nanomolar concentrations.