Liquid chromatography–mass spectrometry method for the quantification of tamoxifen and its metabolite 4-hydroxytamoxifen in rat plasma: Application to interaction study with biochanin A (an isoflavone)

Abstract

Tamoxifen is the agent of choice for the treatment of estrogen receptor-positive breast cancer. Tamoxifen is a substrate of P-glycoprotein (P-gp) and microsomal cytochrome P450 (CYP) 3A, and biochanin A (BCA) is an inhibitor of P-gp and CYP3A. Hence, it could be expected that BCA would affect the pharmacokinetics of tamoxifen. In the present study we have developed and validated a simple, sensitive and specific LC-ESI-MS/MS method for the simultaneous quantification of tamoxifen and its metabolite 4-hydroxytamoxifen with 100 µL rat plasma using centchroman as an internal standard (IS). Tamoxifen, 4-hydroxytamoxifen and IS were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column under isocratic condition using 0.01 M ammonium acetate (pH 4.5): acetonitrile (10:90 v/v) as a mobile phase. The mobile phase was delivered at a flow rate of 0.8 mL/min. The method was proved to be accurate and precise at linearity range of 0.78–200 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.89–8.54% and 3.97–10.26%, respectively; and intra- and inter-day assay accuracy was between 87.63–109.06% and 96–103.89%, respectively for both the analytes. The method was successfully applied to study the effect of oral co-administration of BCA (an isoflavone) on the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen in female rats. The coadministration of BCA caused no significant changes in the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen. However, the peak plasma concentration (Cmax) of 4-hydroxytamoxifen in BCA pretreated rats was significantly (P<0.05) lower than those from control group.