The development and validation of an UHPLC-MS/MS method for the rapid quantification of the antiretroviral agent dapivirine in human plasma

Abstract

Background: Dapivirine is a non-nucleoside reverse transcriptase inhibitor designed to prevent HIV-1 viral replication and subsequent propagation. A sensitive method is required to quantify plasma concentrations to assess drug efficacy.

Results: Dapivirine-spiked plasma was combined with acetonitrile containing deuterated IS and was processed for analysis. The method has an analytical measuring range from 20 to 10,000 pg/ml. For the LLOQ, low, mid and high QCs, intra- and inter-assay precision (%CV) ranged from 5.58 to 13.89% and 5.23 to 13.36%, respectively, and intra- and inter-day accuracy (% deviation) ranged from -5.61 to 0.75% and -4.30 to 6.24%, respectively.

Conclusion: A robust and sensitive LC-MS/MS assay for the high-throughput quantification of the antiretroviral drug dapivirine in human plasma was developed and validated following bioanalytical validation guidelines. The assay meets criteria for the analysis of samples from large research trials.

Conflict of interest statement

Financial & competing interests disclosure

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1. Non-nucleoside reverse transcriptase inhibitor dapivirine (C20H19N5)

*Denotes positions of deuterium atoms for the deuterated 2H4-dapivirine IS.

Figure 2. Chromatogram of (A) dapivirine and (B) 2H4-dapivirine, with retention time demarcated at 1.15 min

Figure 3. Product ion spectrum of dapivirine m/z 330.3 shows the predominant dapivirine product ions at m/z 158.1 and 119.1

The former product ion at m/z 158.1 was monitored as the dapivirine-quantifier ion, while the latter product ion at m/z 119.1 was monitored for qualitative purposes only. DPV: Dapivirine.

Figure 4. Calibration curve of dapivirine from human plasma

The analytical measuring range of the assay is 20–10,000 pg/ml. Insert shows the analytical measuring range from 20 to 500 pg/ml. The curve is fit to the calibrators with a quadratic regression using 1/x2 weighting. The equation of the line is: y = 8.08 e10x2+ 0.00111x + 0.00483, with an r2 value of 0.9962.

Figure 5. Selectivity of dapivirine and 2H4-dapivirine transitions

(A) Chromatograms of drug-free human plasma illustrating the extracted ion current of the dapivirine transition (upper panel) and the 2H4-dapivirine transition (lower panel). (B) Overlay of dapivirine at the LLOQ (20 pg/ml) spiked into blank K2 EDTA plasma and blank plasma. The relative intensity is six-times the background level of the blank plasma (upper panel). Extracted ion current for the 2H4-dapivirine transition is depicted in the lower panel.