A High-Performance Liquid Chromatography-Mass Spectrometry method for simultaneous determination of dolutegravir, nevirapine, efavirenz, rifampicin and rifapentine concentrations in human plasma

Variations in clinical pharmacokinetics and genetics, and polypharmacy affect attainment of target drug concentrations in HIV-TB co-infected patients. However, scarcity of analytical methods hinders drug concentration measurement for therapeutic drug monitoring in this doubly vulnerable population in low-and-middle income countries. This study aimed to develop and validate an LC-MS method for simultaneous determination of dolutegravir, nevirapine, efavirenz, rifampicin, isoniazid and rifapentine concentrations in plasma. ThermoScientific LCQ ion trap operated by Xcalibur™ software was used. Analyte extraction from plasma was achieved by protein precipitation using 100% acetonitrile. Injection volume was 30µl for dolutegravir, efavirenz and nevirapine and 10µl for rifampicin and rifapentine. Analyte separation was achieved on Waters Atlantis dC18 column by 400µl flow rate of mobile phase under gradient elution. Transitions used were; dolutegravir (419.13→277.00), efavirenz (315.03→316.00), nevirapine (266.16→267.10), rifampicin (822.41→790.20), rifapentine (876.45→845.50), dolutegravir-d4 (423.13→280.10) and rifampicin-d4 (826.41→795.20). Calibration curves were constructed using quadratic regression with dynamic weighting of x = y, 1/x and 1/x ² and accepted at r ² ≥ 0.95 for all validation parameters. Method linearity and recovery ranged from 0.25µg/ml to 10.00µg/ml and 86.12% to 109.89% respectively for all analytes. Intra-and inter-day accuracy ranged from 88.73% to 109.67% and 93.38% to 104.30% respectively for all analytes. Both intra-and inter-day precision ranged from 2.47 RSD to 12.39 RSD and 5.34 RSD to 16.83 RSD respectively for all analytes. The method was selective and specific with no significant ghost peak identified in blank samples at expected retention times of dolutegravir, efavirenz, nevirapine, rifampicin and rifapentine. An LC-MS method for simultaneous determination of dolutegravir, efavirenz, nevirapine, rifampicin and rifapentine in human plasma was developed and validated. The method could be useful to the Uganda Ministry of Health for therapeutic drug monitoring in HIV-TB patients in health facilities in the country.