title: Determination of 1,25-dihydroxyvitamin D2 in rat serum using liquid chromatography with tandem mass spectrometry.
abstract: Vitamin D therapy is widely used for the treatment of hyperparathyroidism associated with chronic renal failure in renal disease patients. The vitamin D prodrug, 1α-hydroxyvitamin D(2) (1α(OH)D(2)), is used for the treatment of the end stage renal disease patients who as a result of impaired kidney function cannot convert the naturally occurring vitamin D to the active hormonal form namely 1,25-dihydroxyvitamin D(2) (1,25(OH)(2)D(2)). The systemic circulating levels of this active form are in the pg/mL range and represent a significant bioanalytical challenge for therapeutic monitoring. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered the gold standard for the selective and sensitive determination of small molecule therapeutics in biological matrices. However, the reported LC-MS/MS bioanalytical assays for 1,25(OH)(2)D(2) suffer from extensive sample preparation procedures or derivatization protocols to achieve the requisite sensitivity and selectivity. In this paper, we describe an assay that employs 96-well plate solid phase extraction sample preparation combined with highly sensitive LC-MS/MS instrumentation. The utility of ultra high pressure liquid chromatography to reduce the analytical run time was also demonstrated. Employing this assay a lower limit of quantitation of 25.0 pg/mL using 300 μL sample aliquot of rat serum was achieved with linearity obtained over the range of 25.0-1000 pg/mL. Both intra-day and inter-day coefficients of variation were <15% and accuracy across the assay range was within 100±7.24%. The application of the assay was demonstrated for the analysis of 1,25(OH)(2)D(2) rat serum samples to support pharmacokinetic studies conducted at doses down to sub-microgram per kilogram of 1α(OH)D(2).
authors.affiliation: DMPK & Pharmaceutics Department, Drug and Biomaterial R&D, Genzyme Corporation, 153 2nd Avenue, Waltham, MA 02451, USA.
authors.forename: Sirimas
authors.initials: S
authors.lastname: Sudsakorn
authors.forename: Abhishek
authors.initials: A
authors.lastname: Phatarphekar
authors.forename: Thomas
authors.initials: T
authors.lastname: O'Shea
authors.forename: Hanlan
authors.initials: H
authors.lastname: Liu
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D004872:Ergocalciferols / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D016014:Linear Models; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004872:Ergocalciferols; C003564:1,25-dihydroxyergocalciferol