Development and validation of an LC-MS/MS method for quantifying total and unbound doravirine in human plasma.

A robust LC-MS/MS method was developed to quantify total and unbound doravirine in plasma samples from patients receiving daily doses of 100 mg doravirine, in combination with lamivudine and tenofovir disoproxil fumarate, in a phase 3 clinical trial. The trial is ongoing, and sample analysis is planned to commence once all samples have been collected. The method was validated to quantify both total and unbound doravirine using a single calibration curve. Protein precipitation was used to obtain the total doravirine from plasma and ultrafiltration was used to obtain the unbound doravirine. A Kinetex 1.7 µm EVO C18 100 Å column was used for chromatography using a gradient mobile phase (0.1 % formic acid in water and 0.1 % formic acid acetonitrile) at a flow rate of 250 µL/min during a five minute runtime. The analyte was ionized by positive electrospray ionization and detection was by multiple reaction monitoring on a Sciex API3200 triple quadrupole mass spectrometer. Using calibration standards prepared in whole plasma, the calibration curve fitted a quadratic regression (weighted by 1/x) over a calibration range of 7.00-2000 ng/mL and was applied successfully for the measurement of both total and unbound doravirine. Validation experiments, conducted according to international guidelines, proved the accuracy and precision of the method over three consecutive days. The method demonstrated robustness in the presence of matrix components, different anticoagulants, hemolyzed blood (2 %), and concomitant medications, showing the necessary sensitivity and selectivity for the quantification of both total and unbound doravirine concentrations expected in the study samples.