Atmospheric pressure ionization time-of-flight mass spectrometry coupled with fast liquid chromatography for quantitation and accurate mass measurement of five pharmaceutical drugs in human plasma

The quantitative determination and accurate mass measurement of five tricyc lic amine pharmaceutical drugs (doxepin, desipramine, imipramine, amitripty line and trimipramine) fortified in human plasma within a per sample run ti me of 18 s was accomplished by atmospheric pressure ionization (API) time-o f-flight (TOF) mass spectrometry using a turbolonspray liquid chromatograph y/mass spectrometry (LC/MS) interface coupled with high-performance liquid chromatography (HPLC), The relatively short HPLC separation (18 s) was achi eved using a short Cls column (15 x 2.1 mm i.d.) with a high aqueous mobile phase maintained at a flow-rate of 1.4 mi min(-1). An acquisition speed of 0.2 s per spectrum accommodates these fast separation conditions. This met hod employs a one-step liquid-liquid extraction procedure to isolate the fi ve tricyclic amines from biological matrix components The overall extractio n recovery was 75% for desipramine and >90% for the other four tricyclic am ines, The lower level of quantitation was 1-2 ng ml(-1) for each compound. The calibration curve was linear from 2 to 100 ng ml(-1) for desipramine an d from 1 to 50 ng ml(-1) for the other four tricyclic amines, A deuterated internal standard, imipramine-d(3), was used for all five tricyclic amines, Acceptable intra- and interassay precision (1.0-17.7%) and accuracy (0.2-1 4.5%) were obtained. The linear dynamic range was extended to 200 based on a software upgrade for correcting ion current detection saturation. The acc urate masses of the five tricyclic amines were determined by on-line LC/TOF MS analyses of biological extracts using two-point internal mass calibratio n. This was done by infusing a reference standard, Jeffamine D230, post-col umn into the HPLC effluent, All results showed a mass error not greater tha n 9 ppm for all the target compounds. These results were obtained from both synthetic mixtures when as Little as 100 pg were injected or extracts of s piked human plasma samples with analytical concentration as low as 5 ng ml( -1). The factors influencing accurate mass measurements are discussed. Copy right (C) 2000 John Whey & Sons, Ltd.