Abstract

DEVELOPMENT AND VALIDATION OF LC-MS/MS METHOD FOR THE ESTIMATION OF NEBIVOLOL IN HUMAN PLASMA

Praveen Kumar Dasari* and Srinivasa Rao Dorapaneedi

ABSTRACT

In the present work, determination of Nebivolol in human plasma was developed and validated with a large calibration curve range (10 - 4000 pg/mL) by specific, sensitive, an accurate liquid chromatography-tandem mass spectrometry method. The analyte from the human plasma was extracted from Liquid-liquid extraction method. The separation was achieved using Xbridge Zorbax Eclipse XDB - C18 (150 x 4.6 mm, 5 μ) column with Acetonitrile: 20mM Ammonium formate (pH-3.0) (50: 50, v/v) as a mobile phase. A flow rate of 1.0 mL/min and run time 10.0 min was used for the chromatographic analysis of Nebivolol. Sensitivity of this method was found to be 10pg/mL. The analyte was analyzed by mass spectrometry in the multiple reaction monitoring modes. The precursor-product ion m/z was 406.2  151.0 m/z and Nebivolol-D4 was 410.2  151.0 m/z were used for quantification of an analyte and its IS. The method was validated in terms of accuracy, precision, selectivity, recovery, freeze-thaw stability, bench-top stability, stock solution stability and re-injection reproducibility. Within- and between-batch precision was obtained within the range 0.7 to 8.2. The mean recovery for drug was obtained 87.71% where as the mean recovery of IS was 84.97%. The % RSD value at higher concentration and lower concentration in all stability experiments was within 15%. This method is free from ion suppression, ion enhancement and any type of abnormal ionization.

Keywords: Nebivolol, LC-MS/MS, Human Plasma.