Sensitive bioassay of bupivacaine in human plasma by liquid-chromatography-ion trap mass spectrometry

A sensitive high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS-MS) method, using an ion trap spectrometer, was developed for quantitation of bupivacaine in human plasma. Bupivacaine and an internal standard (ropivacaine) were extracted in a single step from 100 μL of alkalinized plasma with diethyl-ether. The mobile phase consisted of acetonitrile with 0.1% formic acid (50:50, v/v), and was delivered at a flow rate of 0.3 mL/min. The effluent was detected by MS-MS in positive ion mode. Ionisation was performed, using an electrospray ion source, operating at 200 °C. The selected reaction monitoring transitions m/z 289 → m/z 140 and m/z 275 → m/z 126 were chosen for bupivacaine and ropivacaine, respectively. Calibration curves were linear over the concentration range of 3.90-500 μg/L with determination coefficients >0.996. The method is accurate (bias <10%) and reproducible (intra-assay and inter-assay precision <15%), with a quantitation limit of 3.90 μg/L, using only 100 μL of plasma. The high specificity and sensitivity, achieved by this fast method (total run-time <3 min), allowed the determination of bupivacaine plasma levels in pediatric patients, following epidural administration of bupivacaine.