Development and validation of a sensitive LC–MS/MS method for the determination of adefovir in human serum and urine

A sensitive and selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the determination of adefovir (PMEA) in human serum and urine. The analyte was separated on a Diamonsil C18 column (250 mm × 4.6 mm i.d., 5 μm particle size) by isocratic elution with methanol–water–formic acid (20:80:0.1, v/v/v) at a flow rate of 0.6 ml/min, and analyzed by mass spectrometry in multiple reaction-monitoring mode. The precursor-to-product ion transitions of m/z 274 → 162 and m/z 226 → 135 were used to measure and quantify the analyte and internal standard (I.S.), respectively. The weighted (1/x2) calibration curve was linear over serum concentration range 1.25–160.00 ng/ml and urine concentration range 0.05–8.00 μg/ml, with a correlation coefficient (r) of 0.9992 and 0.9978, respectively. The lower limit of quantification in human serum was 1.25 ng/ml. The inter- and intra-day precisions (R.S.D.%) in both serum and urine were lower than 8.64%, the mean method accuracies and recoveries from spiked serum samples at three concentrations ranged from 96.3 to 102.0% and 56.5 to 59.3%, respectively. The serum extract was stable when stored for 24 h. The developed method was successfully applied to determine PMEA in human serum and urine, and proved suitable to clinical pharmacokinetic study.