Liquid chromatography-tandem mass spectrometry for monitoring vitamin D hydroxymetabolites in human aqueous humor

A stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of 24,25-dihydroxyvitamin D-3 (24,25(OH)(2)D-3) and 25-hydroxyvitamin D-3/D-2 (25(OH)D-3/D-2) in human aqueous humor samples from the Eye-D study. Optimum sample preparation is based on simple liquid-liquid extraction (LLE) followed by derivatization with an Amplifex reagent prior to LC-MS/MS in order to enhance analyte sensitivity. The average recoveries for 24,25(OH)(2)D-3 (0.02, 0.05, 0.4 mu g L-1), 25(OH)D-3 (0.2, 0.5,3.75 mu g L-1) and 25(OH)D-2 (0.15, 0.4, 3 mu g L-1) ranged from 92 to 112 with a coefficient of variance (CV) lower than 15%. The limits of quantitation (LOQs) were from 0.02 mu g L-1 (24,25(OH)(2)D-3) to 0.2 mu g L-1 (25(OH)D-3) using a sample volume of 50 mu L. We demonstrated that the present method provides adequate sensitivity, selectivity, accuracy, and robustness to screen for vitamin D metabolites in aqueous humor samples by analyzing five samples which were withdrawn during cataract operation.