Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum

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A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol–10 m M aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol–10 m M aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient ( R) of more than 0.999 in the range of 1–500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.