Development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry method to quantify antiretroviral drug concentrations in human plasma for therapeutic monitoring

Abstract

Antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection. ART previously consisted of concomitant administration of many drugs, multiple times per day. Currently, ART generally consists of two- or three-drug regimens once daily as fixed-dose combinations. Drug monitoring may be necessary to ensure adequate concentrations are achieved in the plasma over the dosing interval and prevent further HIV resistance formation. Additionally, nonadherence remains an issue, highlighting the need to ensure sufficient ART exposure. Towards this effort, we developed and validated a highly selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of a panel of nine antiretrovirals: abacavir, bictegravir, cabotegravir, dolutegravir, doravirine, emtricitabine, lamivudine, raltegravir, and tenofovir in human plasma. Using only 50µL of plasma, a simple protein precipitation with acetonitrile with internal standards followed by reconstitution in 50uL (high) or 400 uL (low)was performed. Analyte separation was achieved using a multistep UPLC gradient mixture of (A: 0.1% formic acid in water and B: acetonitrile) and a Waters CORTECS T3 (2.1×100mm) column. The method was comprehensively validated according to the United States Food and Drug Administration Bioanalytical Guidelines over two clinically relevant ranges (1-250ng/mL and 100-5000ng/mL) with excellent linearity (R2 > 0.99 for all). The assay run time was 7.5minutes. This method achieves acceptable performance of trueness (89.7-104.1%), repeatability, and precision (CV <15%), and allows for simultaneous quantification of guideline-recommended ART regimens. This method can be utilized for the therapeutic monitoring of antiretrovirals in human plasma.