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

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. 13 C, 2 H 5 -Dolutegravir, 13 C, 2 H 2, 15 N-bictegravir, 2 H 6 -raltegravir and 13 C 6 -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. • Integrase-strand transfer inhibitors (INSTIs, such as dolutegravir, bictegravir, raltegravir) and doravirine are first line agents currently used in the treatment of HIV-infection. • A bioanalytical assay was developed to measure INSTIs and doravirine to support studies needed to determine drug transfer in breastmilk to ascertain safety. • A bio-analytical method using UPLC-MS/MS can be used to measure concentrations of often used antiretroviral drugs. • Protein precipitation with adapted preparation of calibration standards and quality controls in breastmilk are required for compounds that are highly protein bound and/or lipid soluble. • The proof-of-concept was shown by measuring a concentration curve after a single dose of doravirine or bictegravir and concentrations of dolutegravir in breastmilk (mg/L) after chronic use after a certain amount of time after drug intake.