A sensitive HPLC–MS/MS method for the determination of dolutegravir in human plasma

• We validated the first LC–MS/MS assay for dolutegravir quantitation in human plasma.
• The assay is accurate, and precise, and sensitive with a broad calibration range.
• The assay was successfully applied to 350 clinical samples in a phase I/II trial.

A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of dolutegravir (DTG) in plasma samples. This work describes an assay system requiring only a 20 μL aliquot of human plasma that is subjected to a simple acetonitrile protein precipitation containing a stably labeled isotope of DTG used as an internal standard. Chromatography was performed on an XBridge C18, 2.1 mm × 50 mm, reversed phase analytical column, using a 60:40 acetonitrile/water mobile phase containing 0.1% formic acid. Detection of the analyte and internal standard was achieved by ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 420.1/136.0 and 428.1/283.1 for DTG and DTG-IS, respectively. The dynamic range of this assay extends from 5 to 10,000 ng/mL, with a mean coefficient of determination (r, mean ± SD) of 0.9996 ± 0.0003. The mean precision values for calibration standards ranged from 0.7 to 4.1%, while accuracy values were 98.3 to 102.0%. Validation results demonstrated high accuracy (≤6.5% deviation) and high precision (≤9.1% CV) for the quality control samples. This assay system provides an accurate, precise, and sensitive method for DTG quantitation and was successfully applied to clinical research samples as part of a phase I/II pediatric clinical trial.