Development And Validation Of LC-MS/MS Methods For The Determinations Of Concentrations In Body Fluids And Pharmacokinetic Parameters Of 2 Aromatase Inhibitors And 3 Phosphodiesterase-5 Inhibitors

OBJECTIVES:1.To explore the fragmentation rules of 2 aromatase inhibitors（AIs,including anastrozole and letrozole）and 3 phosphodiesterase-5 inhibitors（PDE5is,including sildenafil,N-desmethylsildenafil and tadalafil）by using high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry（ESI-Q-TOF/MS）method.Selecting monitoring ion-pairs to develop methods for the determination of 5 compounds in biological samples.2.To develop and validate liquid chromatography-tandem mass spectrometry（LC-MS/MS）methods for the simultaneous determination of 5 compounds in plasma and seminal plasma for therapeutic drug monitoring（TDM）to provide reference for drug dosage adjustment in male infertility patients.3.To develop and validate a LC-MS/MS method for the determination of letrozole in SD rat plasma to explore the effects of sildenafil on the single dose pharmacokinetic（PK）of letrozole in SD rats,and to provide new therapy ideas for male infertility.METHODS:1.High-resolution ESI-Q-TOF/MS method in a positive ion mode was used to analyze the relative molecular mass information of the quasi-molecular ions and the characteristic fragment ions of 5 compounds,and to determine their fragmentation rules.2.LC-MS/MS methods were developed for the determination of 5compounds by using bifonazole as internal standard（IS）.Sample preparations were performed by protein precipitation with acetonitrile or14%perchloric acid solution.Ultimate XB-C₁₈（4.6×50 mm,5μm）column was used for the online extraction of 5 compounds in human seminal plasma.5 compounds and IS were chromatographed on Agilent ZORBAX SB-Aq（4.6×150 mm,5μm）column and mobile phase was consisted of ammonium acetate-acetonitrile with gradient elution.Analysis was performed with electrospray ionization（ESI）source in a positive and multiple reaction monitoring（MRM）mode.The developed LC-MS/MS methods were validated for selectivity,residue,lower limit of quantitation（LLOQ）and calibration curve,accuracy and precision,matrix effect,and stability.3.Plasma and seminal plasma samples of male infertility patients receiving combination therapy of AIs and PDE5is were collected according to the designed requirements after medication,and the concentrations of 5 compounds were determined and analyzed.4.SD rats were given a single dose of letrozole（or letrozole+sildenafil）by intragastric administration to collect plasma samples before administration and 0.083,0.25,0.5,0.75,1,2,4,8,12,24,48 h after administration,and the concentration of letrozole was determined for PK analysis.RESULTS:1.The fragmentation of 5 compounds showed their own characteristics,and tadalafil split through a new pathway.2.The concentrations of 5 compounds in plasma and seminal plasma samples of male infertility patients were determined by the validated LC-MS/MS methods.It showed that there were individual variations among patients both in plasma and seminal plasma,and the concentration of letrozole（2.5 mg,qd）that combined with sildenafil or tadalafil therapy were lower than letrozole alone both in plasma and seminal plasma.3.The concentration of letrozole in SD rat plasma samples was determined by the validated LC-MS/MS method.It showed that sildenafil could reduce the apparent volume of distribution（V_d）of letrozole and shorten the half-life(t_1/2)of letrozole in SD rats,while there were no statistical differences in other PK parameters between the 2 groups.CONCLUSIONS:1.The fragmentation rules of 5 compounds can be used to select monitoring ion-pairs.2.The developed LC-MS/MS methods can be used for rapid and simultaneous determination of 5 compounds in plasma and seminal plasma of male infertility patients,which can be used for TDM to provide reference for drug dosage adjustment.3.In SD rat pharmacokinetic experiment,sildenafil can accelerate the metabolism of letrozole.The result can provide new therapy ideas for male infertility.