High Performance Liquid Chromatography Method With Fluorescent Detection For Quantification Of Tamoxifen And Its Major Metabolites In Human Plasma: Application To A Clinical Study

AimTo set a high performance liquid chromatography method with fluorescent detection for quantification of tamoxifen and its major metabolites.Then apply the method to study the pharmacokinetics and relative bioavailability of citric tamoxifen dispersible tablet in Chinese healthy volunteers to validate the method we established.MethodsIn first part,a high performance liquid chromatography method with fluorescent detection for quantification of tamoxifen and its major metabolites has been established.Plasma samples were treated by n-hexane:n-butanol(98:2,v/v),and exposed to a 254-nm UV lamp offline for 10 min.Separation was carried out on the column of Agilent Extend C₁₈(150 mm×4.6 mm,5μm)at 50℃.The mobile phase consisted of methanol:1%triethylamine aqueous solutions(82:18,v/v)pumped at a flow rate of 1 mL·min^-1.The fluorescence detector was operated at an excitation and emission wavelengths 260 and 375 nm,respectively.In second part,the pharmacokinetics and relative bioavailability of tamoxifen citrate dispersible tablets in Chinese healthy volunteers was studied.In a randomized two period crossover study,20 healthy volunteers received tested and reference tablets 20 mg.The plasma concentrations of tamoxifen were determined by HPLC method established in the first part.The pharmacokinetics of tamoxifen were estimated by non-compartment model.ResultsIn first part,in the conditions described above,TAM,DMT and OHT exhibited good chromatography with baseline resolution of each compound.The method described in our paper was selective and specific. There were no foreign peaks interfered with analytes and internal standard at the retention times.The retention times for internal standard, OHT,DMT,TAM were 2.4,3.9,9.9 and 11 min,respectively.The linear range of TAM,DMT and OHT were 0.5～200 ng·mL^-1,0.5ng·mL^-1～200ng·mL^-1,0.1ng·mL^-1～10ng·mL^-1.Lower limit of quantification (LLOQ)was 0.5ng·mL^-1,0.5ng·mL^-1,0.1ng·mL^-1.All the inter-day and intra-day precisions were less than 15%.The stability of unprocessed and processed samples stored at 4℃was good.After 4 cycles of freeze and thaw processes,the plasma samples were stable. In second part,in a randomized two period crossover study,the main pharmacokinetics parameters of tested and reference tablets were as the following:t_max:(6.3±2.2),(6.7±2.4)h;C_max:(72±14),(68±16)μg/L; AUC_0-Ï„:(4608.43±2054.50),(4586.05±2026.23)ng·h·mL^-1;t_1/2:(143±24),(154±33)h,respectively.The relative bioavailability was(101.3±12.9)%.According to the data of t_max,C_max,AUG_0-Ï„between two tablets, they have the same bioequivalence.ConclusionsThe method presented here describes a specific,sensitive and reproducible human plasma assay using HPLC with fluorescence detection for the determination and quantification of TAM and its metabolites.And the method was validated by the pharmacokinetics of TAM in a clinical study.It demonstrated that the HPLC-FLU method we set can be applied in the clinical study of TAM kind of drugs.