A High Sensitivity Analytical Method for the Determination of Derivatized 1,25-Dihydroxyvitamin D3 and 1,25-Dihydroxyvitamin D2 by LC/MS/MS

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 257-280-Disorders of Vitamin D Metabolism & Action
Basic/Translational
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-278
Michael J . Y. Jarvis* and Adam Latawiec
AB Sciex, Concord, ON, Canada
For Research Use Only.  Not For Use In Diagnostic Procedures. Current methods for the determination of 1,25-dihydroxyvitamin D3 (DHVD3) are hampered by low sensitivity (in the case of the underivatized species) or low selectivity (in the case of immunoassays).  The accurate and sensitive determination of various dihydroxyvitamin D species continues to be an important research field in clinical chemistry. We report here the results of a recently developed workflow using a novel dieneophile Cookson type reagent to significantly improve the sensitivity of MS/MS based assays. 

Serum samples (200 µL) were diluted with deionized water and cleaned up using a combination of Celite and silica gel SPE type cartridges.  The target materials retained on the Celite cartridge were eluted on to the silica gel cartridge using di-isopropyl ether.  Successive elution from the silica gel cartridge using mixtures of isopropanol and hexane allowed for the selective removal of most interferences including 24,25-dihydroxy vitamin D.  Isolation, followed by solvent evaporation afforded a sample that could be immediately derivatized using Ampliflex™ Diene reagent.  After 1 hour, the derivatized sample was diluted with deionized water and analyzed by LC/MS/MS on an AB SCIEX QTRAP® 6500 system. Baseline resolution of the derivatized DHVD3 and DHVD2 could be obtained in less than 7 minutes using a small particle size C-18 column employing a simple Water:Acetonitrile gradient.

Using double charcoal stripped serum as the matrix, we were able to routinely obtain LODs as low as 5 pg/mL.  Linear calibration curves were generated from the same matrix from 5 to 250 pg/mL.  Precision values, based on multilevel calibrators and controls, were typically less than 10%, with accuracies ranging between 94-106% .

Disclosure: MJYJ: Employee, AB SCIEX. AL: Employee, AB SCIEX.

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm