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

Abstract

A stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) and 25-hydroxyvitamin D₃/D₂ (25(OH)D₃/D₂) 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)₂D₃ (0.02, 0.05, 0.4 μg L⁻¹), 25(OH)D₃ (0.2, 0.5,3.75 μg L⁻¹) and 25(OH)D₂ (0.15, 0.4, 3 μg 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 μg L⁻¹ (24,25(OH)₂D₃) to 0.2 μg L⁻¹ (25(OH)D₃) using a sample volume of 50 μL. 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and robustness to screen for vitamin D metabolites in aqueous humor samples by analyzing five samples which were withdrawn during cataract operation.