Abstract

DEVELOPMENT AND VALIDATION OF A SENSITIVE LC-MS/MS METHOD FOR DETERMINATION OF NEBIVOLOL: APPLICATION TO A PHARMACOKINETIC STUDY

Vasu Babu Ravi* and Venkateswarlu Ponneri

ABSTRACT

A simple, rapid and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay method has been proposed for the determination of Nebivolol in human plasma samples using Nebivolol D4 as internal standard (IS). Analyte and the IS were extracted from the 100 μL of K2 EDTA human plasma by Solid Phase extraction (SPE). The chromatographic separation was achieved on a Zodiac C18 column by using a mixture of Methanol and 0.1% formic acid buffer (85:15, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The calibration curve obtained was linear (r2  0.99) over the concentration range of 0.15 – 40.4 ng/mL. The Mass detection of Nebivolol involves m/z - 406.10 (parent) and 151.10 (product) and Nebivolol D4 involves m/z - 410.10 (parent) and 151.10 (product) as internal standard in Positive ion mode. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The intra-day and inter-day precision (%CV) and accuracy results in three validation batches across six concentration levels were well within the acceptance limits. A run time of 2.00 min for each sample made it possible to analyze more number of samples in short time, thus increasing the productivity. The proposed method successfully applied to a pharmacokinetic study of Nebivolol 20 mg tablets in healthy, adult, human male south Indian subjects under fed condition.

Keywords: Nebivolol, Solid-Phase extraction, human plasma, LC-MS/MS.