Determination of procarbazine in human plasma by liquid chromatography with electrospray ionization mass spectrometry

Procarbazine is a cytotoxic chemotherapeutic agent used in the treatment of lymphomas and brain tumors. Its pharmacokinetic behavior remains poorly understood even though more than 30 years have elapsed since the drug was approved for clinical use. To characterize the pharmacokinetics of procarbazine in brain cancer patients during a phase I trial, a method for determining the drug in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) was developed and thoroughly validated. Plasma samples were prepared for analysis by precipitating proteins with trichloroacetic acid and washing the protein-free supernatant with methyl tert-butyl ether to remove excess acid. The solution was separated on a Luna C-18 analytical column using methanol-25 mM ammonium acetate buffer, pH 5.1 (22:78, v/v) as the mobile phase at 1.0 ml/min. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H](+) ions at m/z 222.2 for procarbazine and at m/z 192.1 for the internal standard (3-dimethylamino-2-methylpropiophenone). Procarbazine and the internal standard eluted as sharp, symmetrical peaks with retention times (mean+/-S.D.) of 6.3+/-0.1 and 9.9+/-0.3 min, respectively. Calibration curves of procarbazine hydrochloride in human plasma at concentrations ranging from 0.5 to 50 ng/ml exhibited excellent linearity. The mean absolute recovery of the drug from plasma was 102.9+/-1.0%. Using a sample volume of 150 microl, procarbazine was determined at the 0.5 ng/ml (1.9 nM) lower limit of quantitation with a mean accuracy of 105.2% and an interday precision of 3.60% R.S.D. on 11 different days over 5 weeks. During this same time interval, the between-day accuracy for determining quality control solutions of the drug in plasma at concentrations of 2.0, 15 and 40 ng/ml ranged from 97.5 to 98.2% (mean+/-S.D., 97.9+/-0.4%) and the precision was 3.8-6.2% (mean+/-S.D., 5.1+/-1.2%). Stability characteristics of the drug were thoroughly evaluated to establish appropriate conditions to process, store and prepare clinical specimens for chromatographic analysis without inducing significant chemical degradation. The sensitivity achieved with this assay permitted the plasma concentration-time profile of the parent drug to be accurately defined following oral administration of standard doses to brain cancer patients.