Anti-HIV Nnrtis Drug Prototype And Its Metabolites Mass Spectrometry Detection And Application

Non-nucleoside reverse transcriptase inhibitors(NNRTIs)combined with other antiretroviral drugs have become the focus of the therapy for HIV patients.Efavirenz and nevirapine as the early NNRTIs drugs are the cornerstone of the highly active antiretroviral therapy.Most of NNRTIs drugs in the body are metabolized as metabolites that are not resistant to viruses.Nevirapine is metabolized into two major metabolites,2-hydroxynevirapine and 3-hydroxynevirapine and efavirenz as7-hydroxyefavirenz,8-hydroxyefavirenz and 8,14-hydroxyefavirenz.On the other hand,drug concentration in hair is considered an important indicator of long-term adherence to highly active antiretroviral therapy.Evaluation based on the combination of drug prototypes with the metabolites will more fully and accurately reflect long-term adherence,such as nevirapine,efavirenz and their respective metabolites.However,no study has detected both NNRTIs and their metabolites in human hair.Therefore,this study aimed to develop two simple,highly sensitive and selective methods for simultaneous detection of nevirapine and efavirenz and their metabolites in hair,respectively.Then it explored whether there is high consistency between drug prototype and its metabolites in assessing the long-term adherence.In this study,the analytes were determined with high performance liquid chromatography tandem mass spectrometry(LC-MS/MS)under multi-reaction monitoring.Nevirapine and its metabolites were identified in positive ion mode and atmospheric pressure chemical ionization,and efavirenz and its metabolites in negative ion mode and electrospray ionization.Hair samples were washed with methanol twice and after being cut into pieces,incubated with methanol for 16 hrs in an electrothermal thermostatic water bath at 40℃.The mixture was shaken for 2 min,and centrifuged for 5 min at 12000 rpm.Then 800μL of supernatant were transferred and blow-dried under nitrogen gas at 50℃.Finally,50μL of mobile phase was added and re-dissolved into mass spectrometry for detection.The LC-ESI~--MS/MS method showed the limits of quantification at 3,15,9 and 6pg/mg for efavirenz,7-hydroxyefavirenz,8-hydroxyefavirenz,8,14-hydroxyefavirenz,and the intra-day and inter-day coefficients of variation less than 15%,and the recovery between 85 and 115%.The LC-APCI~+-MS/MS method showed the limits of quantification at 11,6 and 6 pg/mg for nevirapine,2-hydroxynevirapine and3-hydroxynevirapine,and the intra-day and inter-day coefficients of variation less than 15%,and the recovery between 85 and 115%.Population analysis revealed that patients with high adherence showed significantly higher concentrations of nevirapine,2-hydroxynevirapine and 3-hydroxynevirapine than those patients with low adherence.It implied that there was high consistence among nevirapine and its two metabolites in assessing long-term adherence to the highly active antiretroviral therapy.Additionally,the nevirapine’s concentration in hair was moderately correlated with those of the two metabolites,but there was high correlation between 2-hydroxynevirapine and 3-hydroxynevirapine.