Simultaneous quantification of centchroman and its 7-demethylated metabolite in rat dried blood spot samples using LC-MS/MS.

An approach has been developed for the quantitative determination of concentrations of centchroman (I), a nonsteroidal once-a-week oral contraceptive, and its major metabolite (7-desmethyl centchroman, II) using dried blood spots (DBS) on paper, rather than conventional plasma samples. The assay employed simple solvent extraction of the DBS sample circle (6 mm) requiring small blood volumes (30 μL) followed by reversed-phase HPLC separation, combined with multiple reaction monitoring mass spectrometric detection. The calibration plot in matrix using d-trans-hydroxy chroman as internal standard (IS) was linear (r²  = 0.998) over ranges of 1.5-240 and 4.5-720 ng/mL for I and II, respectively. The recoveries of both I and II were always >60% with quantification limits (signal-to-noise ratio = 10) of 1.5 and 4.5 ng/mL for I and II, respectively. The intra-day and inter-day precision (%RSD) and accuracy (%bias) variations in blood spots for both I and II were better than 13%. Moreover, both I and II were stable in DBS for at least 3 months when stored at room temperature. The developed method was successfully applied to the pharmacokinetic interaction study after oral administration of centchroman with and without co-administration of carbamazepine in female Sprague-Dawley rats using serial sampling and results were comparable with the plasma concentrations reported earlier.