MSACL 2024 Abstract

Introduction: The use of antiretroviral therapy (ART) in pregnant people living with HIV can prevent peri- and post-natal transmission of the virus. Dolutegravir (DTG) is an integrase strand transfer inhibitor that is utilized in several combinatorial ART regimens. DTG is a lipophilic drug, and may deposit in breast milk during the postpartum period. Previous work has shown transplacental and breastmilk transfer of DTG, resulting in neonatal and infant drug exposure. However, DTG pharmacokinetics (PK) in breast milk are incompletely understood. Bioanalytical tools are required to understand the multicompartment pharmacology and efficacy of DTG in preventing mother-to-child HIV transmission.

Objectives: To develop and validate a liquid chromatographic-mass spectrometric (LC-MS/MS) assay in breast milk to support clinical trials research.

Methods: Breast milk was purchased acquired from BioIVT (Hicksville, NY). DTG was acquired from Toronto Research Chemicals (Toronto, ON), and its isotopically labeled internal standard, 13C, 2H5-DTG, was acquired from Alsachim (Illkirch, FR). DTG was spiked into breast milk to prepare calibration standards and quality controls. Drug was isolated via protein precipitation, and reconstituted material was analyzed using an API 5500 mass spectrometer (SCIEX, Redwood City, CA) interfaced with a Shimadzu LC-40. Analytical separation occurred using a Waters Acquity C8 column, and the instrument was operated in selective reaction monitoring and positive ionization modes. Monitored transitions were 420.20/277.10 for DTG and 426.20/133.10 for 13C, 2H6-DTG. The assay was validated in accordance with FDA bioanalytical method validation recommendations.

Results: The analytical measuring range of the assay was 0.5-1000 ng/mL, with an analytical run time of 2.2 minutes. Inter-assay precision and accuracy ranged from 4.6% to 15.1% and -2.4% to 9.6%, respectively, for materials prepared at the lower limit of quantification (0.5 ng/mL), low (1.5 ng/mL), mid (75 ng/mL) and high (800 ng/mL) quality control levels. Matrix effects assessments showed 12% ion suppression for DTG and 13C, 2H5-DTG, and recovery efficiencies of 99%; relative matrix effects were negligible. DTG also demonstrated stability under freeze-thaw, re-injection, and room temperature conditions.

Conclusion: An LC-MS/MS method for DTG quantification in breast milk has been developed and validated. The assay met acceptable performance criteria and may be used in downstream applications to understand the multicompartment pharmacology and distribution of DTG into breast milk.