DEVELOPMENT AND VALIDATION OF NITECAPONE IN HUMAN PLASMA USING TOLCAPONE AS INTERNAL STANDARD BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

The present LC-MS/MS method for estimation of Nitecapone in human plasma using Tolcapone as an internal standard is established and validated as per FDA guidelines. A good response was obtained with ZORBAX Eclipse plus C18 column (150 mm × 4.6 mm ID, 5 μm) and mobile phase with a mixture of 0.01 M Ammonium phosphate buffer with pH adjusted to 5.0 with OPA and Acetonitrile (70:30, v/v) at a flow rate of 0.8 mL/min by positive ion mode (API 4000) with an injection volume of 20 µL and a run time of 3.0 min. Detection is performed by atmospheric pressure electrospray ionization (ESI) tandem mass spectrometry in positive ion mode. The precursor to product ion transitions is m/z 266.20 to 156.20 for Nitecapone and m/z 274.20 to 183.10 for Tolcapone (Internal standard) were used for quantization. The retention time of Nitecapone and Tolcapone (Internal standard) were found to be 2.12 min and 2.58 min, respectively. Linearity established for Nitecapone in the range of 50 ng/mL to 2000 ng/mL with correlation coefficient (r = 0.9997), and the overall percentage recovery was 90.39 % for Nitecapone and 92.34 % for Tolcapone (Internal standard) respectively. The CV % values of accuracy and precision for Nitecapone were found to be ≤ 15%, which indicates the accuracy and precision of the proposed method. This method is suitable for routine therapeutic drug monitoring of Nitecapone.