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Abstract Introduction
Cabotegravir (CAB), an integrase inhibitor, and rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor, are used in combination as the first long acting injectable therapy for the treatment of HIV infection. Recent data suggests that variable absorption and drug distribution may warrant drug monitoring in patients with body habitus changes (eg obesity, pregnancy). Because these drugs are a co-formulated, we developed a fully validated LC-MS/MS method for simultaneous measurement of CAB and RPV in human plasma over a clinically relevant 1000-fold range for each analyte.
Methods
Calibration standards were prepared at eight concentrations in human plasma to cover the calibration range of 25.0 to 25000 ng/mL (CAB) and 5.00 to 5000 ng/mL (RPV). Quality controls (QCs) were prepared at four concentrations consisting of a lower limit of quantification (LLOQ), low, mid, and high concentrations (25.0, 75.0, 1000, and 20000 ng/mL for CAB; 5.00, 15.0, 200, and 4000 ng/mL for RPV). A QC three times higher than the upper limit of quantitation was used to evaluate the ability to dilute samples for accurate and precise concentration measures.
Human plasma (40µL) samples were extracted by protein precipitation by mixing with 200 µL of methanol containing stable, isotopically labeled internal standards (13C 2H2 15N-CAB and RPV-d6,). The samples were vortexed for approximately 2 minutes, centrifuged at room temperature for 5 minutes at 16,100 rcf, and mixed 1:5 with water before being transferred to a 96-well plate for LC-MS/MS analysis.
Chromatographic separation was achieved by reverse phase chromatography on a Waters Atlantis T3 (50x2.1 mm, 3 µm particle size) analytical column with 0.1% acetic acid in water (mobile phase A) and 0.1% acetic acid in methanol (mobile phase B) under gradient conditions. Detection of the analytes and internal standards was achieved on an AB Sciex API-5000 triple quadruple mass spectrometer under positive ion electrospray conditions with a total run time of 3.5 minutes.
Results
The method was fully validated to meet the acceptance criteria of the US Food and Drug Administration guidelines and met all acceptance criteria for precision and accuracy. Inter-assay precision (%CV) and accuracy (%Bias) was within ±15% for all four concentrations of QC samples and determined by replicates of 6 QCs in each of 3 separate runs.
Six different blank plasma lots were free of interferences in all monitored transitions. Those same six lots also were spiked at the LLOQ level and the precision and accuracy were within 20% criteria demonstrating that this assay performance is not affected by different plasma sources. Lastly, these six lots were spiked at the low, mid, and high QC concentrations and the average peak area ratios (analyte:internal standard) from the analyses (n=3) were plotted against QC concentration. The CV from the 6 slope values was <5% indicating lack of matrix effect. Analyte stability was established for 3 freeze-thaw cycles, at room temperature in human plasma for up to 24 hours, and in extracts for up to 3 days. Long-term stability in human plasma was also established for up to 691 days when stored at -80C.
Conclusions
A simple and fast LC-MS/MS assay has been validated for the quantitative analysis of cabotegravir and rilpivirine in human plasma.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
