DEVELOPMENT AND COMPARISON OF HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHICMETHODS WITH TANDEM MASS-SPECTROMETRIC AND ULTRAVIOLET ABSORBENCY DETECTION FOR THE DETERMINATION OF CYCLOBENZAPRINE IN HUMAN PLASMA AND URINE

Sensitive assays for the determination of cyclobenzaprine (I) in human plasma and urine were developed utilizing high-performance liquid chr omatography (HPLC) with tandem mass spectrometric (MS-MS) and ultravio let (UV) absorbance detections. These two analytical techniques were e valuated for reliability and sensitivity, and applied to support pharm acokinetic studies. Both methods employed a liquid-liquid extraction o f the compound from basified biological sample. The organic extract wa s evaporated to dryness, the residue was reconstituted in the mobile p hase and injected onto the HPLC system. The HPLC assay with MS-MS dete ction was performed on a PE Sciex API III tandem mass spectrometer usi ng the heated nebulizer interface. Multiple reaction monitoring using the parent --> daughter ion combinations of mit 276 -->, 215 and 296 - -> 208 was used to quantitate I and internal standard (II), respective ly. The HPLC-MS-MS and HPLC-UV assays were validated in human plasma i n the concentration range 0.1-50 ng/ml and 0.5-50 ng/ml, respectively. In urine, both methods were validated in the concentration range 10-1 000 ng/ml. The precision of the assays, as expressed as coefficients o f variation (C.V.) was less than 10% over the entire concentration ran ge, with adequate assay specificity and accuracy. In addition to bette r sensitivity, the HPLC-MS-MS assay was more efficient and allowed ana lysis of more biological fluid samples in a single working day than th e HPLC-UV method.