Simultaneous Quantification of Doravirine, Lamivudine, and Tenofovir Disoproxil Fumarate in Human Plasma by UPLC-MS/MS: Method Development and Validation

INTRODUCTION: A novel, high-throughput UPLC-MS/MS technique has been developed that uses Etravirine (ETR) as the internal standard to simultaneously quantify Doravirine (DOR), Lamivudine (LAM), and Tenofovir disoproxil fumarate (TDF) in human plasma. The procedure uses a precipitation extraction technique to extract the analytes from human plasma and analyze them.
METHODS: ETR was chosen as the internal benchmark for this investigation. Precipitation extraction is the technique used to prepare samples. An Agilent Zorbax XDB C18 analytical column (2.1 × 50 mm, 3.5 µm) is used for chromatographic separation, and its isocratic mobile phase consists of acetonitrile: buffer (80: 20, v/v) at a flow rate of 0.120 mL/min was used.
RESULTS: Parent-to-product ion transitions in the the values for the drugs: LAM: m/z 231.08 amu → 112.00 amu, TDF: m/z 288.33 amu → 176.17 amu, DOR: m/z 426.38 amu → 112.02 amu, and Internal Standard ETR: m/z 437.36 amu → 164.97 amu. Using a triple quadrupole mass spectrometer in the MRM positive ion mode, these transitions were observed. For DOR, LAM and TDF, the method was validated throughout concentration ranges of 2.5 – 1000ng/mL. From spiked plasma samples, the mean recovery values for three API’s were consistent. With a 3.0 minute total run time, the approach was shown to be reliable and quick.
DISCUSSION AND CONCLUSION: A triple quadrupole mass spectrometer running in the MRM positive ion mode was used to track these transitions. The run times were found reliable and quick. The mean recovery values were obtained for three API’s from plasma samples that had been spiked. The method was proved to produce precise results and specific to the determination of selected drugs through current study. The method is stable when exposed to different stressed conditions with less degradation. The current method was validated as per the ICH M10 guidelines and found meeting the desired acceptance criteria.