Development and validation of a simple and sensitive high performance liquid chromatographic method for the simultaneous determination of anastrozole, bicalutamide, tamoxifen, and their synthetic impurities.

ABSTRACT

A simple and sensitive analytical method for simultaneous determination of anastrozole, bicalutamide, and tamoxifen as well as their synthetic impurities, anastrozole pentamethyl, bicalutamide 3-fluoro-isomer, and tamoxifen e-isomer, was developed and validated by using high performance liquid chromatography (HPLC). The separation was achieved on a Symmetry(®) C-8 column (100×4.6 mm i.d., 3.5 µm) at room temperature (±24 °C), with a mobile phase consisting of acetonitrile/water containing 0.18% N,N dimethyloctylamine and pH adjusted to 3.0 with orthophosphoric acid (46.5/53.5, v/v) at a flow rate of 1.0 mL min(-1) within 20 min. The detection was made at a wavelength of 270 nm by using ultraviolet (UV) detector. No interference peaks from excipients and relative retention time indicated the specificity of the method. The calibration curve showed correlation coefficients (r) >0.99 calculated by linear regression and analysis of variance (ANOVA). The limit of detection (LOD) and limit of quantitation (LOQ), respectively, were 2.2 and 6.7 µg mL(-1) for anastrozole, 2.61 and 8.72 µg mL(-1) for bicalutamide, 2.0 and 6.7 µg mL(-1) for tamoxifen, 0.06 and 0.22 µg mL(-1) for anastrozole pentamethyl, 0.02 and 0.07 µg mL(-1) for bicalutamide 3-fluoro-isomer, and 0.002 and 0.007 µg mL(-1) for tamoxifen e-isomer. Intraday and interday relative standard deviations (RSDs) were <2.0% (drugs) and <10% (degradation products) as well as the comparison between two different analysts, which were calculated by f test.