Determination Of Tenofovir,dolutegravir,raltegravir In Plasma By LC-MS And Establishment Of Population Pharmacokinetic Model

Objective:To establish a High performance liquid chromatography tandem mass spectrometry（LC-MS）for simultaneous determination of dolutegravir（DTG）and raltegravir（RAL）in human plasma.Meanwhile,a method for quantifying tenofovir（TFV）concentration in human plasma was established by LC-MS.The plasma concentrations of DTG,RAL and TFV in Chinese adult AIDS patients were determined by the established method,and Population Pharmaocokinetics（PPK）model was established to investigate the effects of population,liver and kidney function indexes and combined drugs use on the pharmacokinetics of the three drugs.To provide guidance for clinical individual drug administration,reduce the occurrence of toxic and side reactions,improve the therapeutic effect.Methods:Data Source:They were mainly from Chinese adult AIDS patients who were followed up in the AIDS voluntary counseling and testing Clinic of Shanghai Public Health Clinical Center from March 28,2021 to June 11,2021 and received antiviral therapy for at least one week.Among them,270 patients（6females and 264 males）took DTG.There were 165 patients（3 females,162 males）taking TFV and 2 patients taking RAL.The other part was from densely sampled AIDS patients in the inpatient ward of Shanghai Public Health Clinical Center,including 9 patients（2 females,7 males）who took DTG,and 30 patients（10females,20 males）who took TFV.Demographic data,liver and kidney function indexes,medication time and dose,blood collection time and drug combination were recorded in detail.The plasma samples were precipitated by protein.DTG and RAL samples were separated using Kinetex 2.6μm Phenyl Hexyl（50×3.0mm）column using 0.1%formic acid aqueous solution（A）-0.1%formic acid acetonitrile（B）as mobile phases.The TFV samples were separated by Kinetex 2.6μm butyl-hexyl（50×3.0 mm）column using 5 mmol/L ammonium formate solution（A）and methanol solution（B）as mobile phase.Electrospray ionization source was used to detect positive ions by multiple reaction monitoring（MRM）method.In this study,the nonlinear mixed effects modeling software Monlix was used to establish a population pharmacokinetic model,and the pharmacokinetic process of DTG,RAL and TFV in vivo was modeled.The effects of demographic factors,liver and kidney function indexes and combined drug use were investigated.Goodness of Fit（GOF）and Visual Predictive check（VPC）were adopted to evaluate the predictability and stability of the final model.Results:A total of 288 of DTG and 555 of TFV blood concentration points were included.In this study,a method for simultaneous determination of DTG and RAL plasma concentrations was established by LC-MS,and a method for determination of TFV plasma concentrations was established by LC-MS,in which DTG ion pairs were 420.3/277.1,RAL ion pairs were 445.2/109.0,TFV ion pairs were 288.0/176.1.The linear ranges of DTG and RAL in plasma were 25-5000ng/m L and 10-2000 ng/m L,respectively.The linear ranges of TFV were 1-200ng/m L.The intra-day and intra-day precision of DTG and RAL samples at low and high concentrations were within 15%,and the extraction recoveries were above 90%.The intra-day and intra-day precision of TFV low and high concentration quality control samples were all less than 15%,and the accuracy ranged from 85%to 115%.The extraction recoveries of low-concentration and high-concentration quality control samples were 114.36%and 112.96%,respectively,and matrix effect was 89.49%and 89.60%.The final DTG model adopts the one-compartment model of first-order absorption and first-order elimination,and the residual model screening results show that the proportional fitting accuracy is the best.The population typical value of DTG clearance（CL/F）was 0.82 L/h,and the variation between individuals was 0.13.Stepwise regression method was adopted to screen covariables and it was determined that body weight had a significant impact on CL/F of DTG.The formula of the final model is as follows:CL/F（L/h）=0.82·（WT/67）^0.37·（?）^ηCLTFV final model adopts two-compartment model with first-order absorption and first-order elimination,and residual model screening results show that proportional fitting accuracy is the best.The typical values of TFV population CL/F,V₁/F,Q/F,V₂/F and Ka were 130.73L/h,552.8L,524.83 L/h,2344.94L and1.7 h^-1.Stepwise regression was used to screen covariates,and it was found that creatinine clearance rate（Ccr）had a significant effect on CL/F of TFV.The formula of the final model is as follows:CL/F（L/h）=130.73·（Ccr/117.3）^0.7·（?）^ηCLThe model evaluation results of DTG and TFV show that the final model is stable and reliable,with high goodness of fit and good prediction performance.Conclusions:The LC-MS method for simultaneous quantification of DTG and RAL and the LC-MS method for quantification of TFV plasma concentrations established in this study showed high sensitivity,strong selectivity and good reproducibility.It was found that body weight had significant influence on DTG clearance rate,and creatinine clearance rate had significant influence on TFV clearance rate.The population pharmacokinetic model established in this study can predict the pharmacokinetic behaviors of DTG and TFV in Chinese HIV infected patients,providing guidance for clinical individual drug administration.