Quantification of dolutegravir, bictegravir, raltegravir and doravirine in human breastmilk with UPLC-MS/MS

Abstract

With the use of newer and more robust antiretrovirals, the risk of viral transmission through breastfeeding has sharply diminished. This has led to a change in guideline recommendations. Currently, breastfeeding is regarded as an equipoise of formula, in case of a well-controlled HIV infection. However, it is not yet fully established whether infant exposure to antiretroviral drugs through breastmilk is a reason for concern. Only sparse and heterogenous data exist on concentrations of dolutegravir, raltegravir, bictegravir and doravirine in breastmilk. So further research is required and to accurately describe infant exposure of antiretrovirals through breastmilk, reproducible bioanalytical methods are needed. Our existing UPLC-MS/MS method for several anti-HIV drugs in EDTA-plasma was modified for validation of dolutegravir, bictegravir, raltegravir and doravirine concentrations in human breastmilk. [¹³C,²H₅]-Dolutegravir, [¹³C,²H_2,¹⁵N]-bictegravir, [²H₆]-raltegravir and [¹³C₆]-doravirine were used as internal standard. The sample preparation involved protein precipitation and detection was performed with tandem mass spectrometry (MS/MS) in a total runtime of 10 min. The assay was validated over the concentration range of 0.0100-10.0 mg/L for dolutegravir and doravirine, 0.00500-10.0 mg/L for raltegravir and 0.0200-20.0 mg/L for bictegravir. Within-run and between-run accuracy were within ±10 % of the nominal concentration and precision <10 CV% for quality controls at high, medium and low concentrations, and ± 8.8 % and < 14 CV%, respectively, at the lower limit of quantification for all analytes. Extraction recovery was 81 % and 79 % for dolutegravir and its internal standard, 83 % and 83 % for bictegravir and its internal standard, 89 % and 92 % for raltegravir and its internal standard and 104 % and 101 % for doravirine and its internal standard. Processed samples of dolutegravir, bictegravir, raltegravir and doravirine in breastmilk were 108 %, 95.5 %, 105 % and 102 % after 14 days at 4 °C. The assay is currently being implemented successfully in pharmacokinetic studies.

Keywords: Bictegravir; Breastmilk; Dolutegravir; Doravirine; MS/MS detection; Raltegravir; Ultra-high performance liquid chromatography.