Session: SUN 257-280-Disorders of Vitamin D Metabolism & Action
Poster Board SUN-278
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.
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