Validation of chiral liquid chromatography-tandem mass spectrometric method for the estimation of bicalutamide enantiomers in human plasma: application to a bioequivalence study

Abstract

Bicalutamide (BCT) is a non-steroidal antiandrogen used for the treatment of prostate cancer. Its chemical name is ( RS )-N-[4-cyano-3-(trifluoromethyl) phenyl]-3-[(4-fluorophenyl) sulfonyl]-2-hydroxy-2-methyl-propanamide. BCT as such is a racemic mixture, but most of its pharmacological activity is attributed to its R enantiomer. A single, simple and selective method for simultaneous estimation of (-)-BCT and (+)-BCT in human plasma was validated using (-)-d4-BCT and (+)-d4-BCT as internal standards. The compounds were separated on a Chiralpak AD-3R column under isocratic conditions consisting of 5mM ammonium acetate buffer and methanol (70:30, v/v), with a total run time of 6.5 minutes and detected by tandem mass spectrometry with negative ionization mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 429.0a185.0 for BCT enantiomers and m/z 433.0a185.0 for d4-BCT enantiomers. Linearity in plasma was observed over the concentration range 10.0 – 3000.0 ng/mL for (-)-BCT and 2.0 – 200.0 ng/mL for (+)-BCT. The mean recovery was 81.2 % and 78.0 % for (-)-BCT and (+)-BCT respectively. The coefficient of variation of the assay was less than 4.2 % and 6.8 % for (-)-BCT and (+)-BCT with an accuracy of 99.3 % to 104.1 % and 98.6 % to 103.4 % for (-)-BCT and (+)-BCT respectively. Stability was evaluated under different conditions including bench top, processed sample, freeze and thaw, autosampler and long term. The validated method was applied for the determination of individual BCT enantiomers in human plasma samples from a bioequivalence study of 150mg fixed dose formulation in 12 healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 10% incurred samples.