2.2. HPLC Determination of Nebivolol

Estimation of nebivolol in samples was carried out by a high-performance liquid chromatography (HPLC) system (Jasco LC–4000, Jasco Inc., Easton, MD, USA). The HPLC system used is made up of a Purospher C18 endcapped (150 × 4.6 mm, 5 µm) with a MD–4010 UV–Visible absorbance detector. Chromatographic separation of nebivolol as well as carvedilol (internal standard) was attained by a mobile phase comprising acetonitrile and 0.2% (v/v) triethylamine in water (80%:20% v/v). The temperature in the column was kept at 25 °C, and the flow rate of the mobile phase was adjusted to 1 mL/min. The injection was made with a sample size of 50 µL, and the chromatogram was recorded at wavelength of 280 nm. Linear regression analysis indicated good linearity in the nebivolol concentration of 1–80 µg/mL (r² = 0.999). A separate calibration curve was developed to quantify nebivolol in the rat plasma. Plasma was analyzed by HPLC for nebivolol concentration, after the proteins were precipitated by adding equal volume of acetonitrile and 2-propanol. Then, the samples were centrifuged at 8000 rpm (15 min), and the supernatant was filtered using 0.22 μm filter (syringe filter, Merck, India) and injected (50 μl) into the HPLC. The concentration of nebivolol in the range of 10–1000 ng/mL, showed good linearity (r² = 0.998). The limit of quantitation and limit of detection of the analytical method was found to be 20.28 ng/mL and 6.69 ng/mL, respectively. The coefficient of variation and accuracy ranged 6.40%–8.66% and −2.27 to −7.19, respectively.