BACKGROUND
The highly active antiretroviral therapy, HAART, is the main resource to avoid the mother-to-child transmission of HIV. The physiological changes during pregnancy are factors to predispose to significant changes in plasmatic concentration suggesting potential variability and drug therapeutic failures. The quantification of HAART during pregnancy and their association with the therapeutic response is of critical importance. Therapeutic drug monitoring is especially useful in patients who are either taking multiple drugs or expressing unusual pharmacokinetics.
METHODS
The detection of antiretroviral drugs was performed using tandem mass spectrometry applying multiple reaction monitoring, MRM. We used positive electrospray mode. LMV, 230.18-112.08, ZDV, 268.22-127.10, LPV 629.55-447.35, and RTV 721.50-296.20. Chromatographic separation was carried-out with a BEHC18-Acquity column with the dimensions 2.1mmx50mm, 1.7um, with acetonitrile and 0.1% formic acid gradient. The injection volume was 3uL and run-time was 3.0min. The process for plasma samples consisted of protein precipitation using 0.1% formic acid in cold acetonitrile. For the breast milk samples, first we performed a lipids extraction using hexane, then protein precipitation. Both methods were validated in accordance with the Mexican standards and FDA criteria. Finally, ten plasma samples from eight HIV-positive women, in the third trimester of pregnancy and nine colostrum samples were assayed.
RESULTS
Methods validation were conducted over a range ng-mL between 50-3000, 75-4500, 250-15,000 and 25-500 for LMV, ZDV, LPV and RTV for plasma samples. For colostrum samples the range was between 50-2,500, 12.5-750, 100-6,000 and 5-300 ng-mL for LMV, ZDV, LPV and RTV. In plasma samples, we did not detect antiretroviral drugs in three samples. A patient had undetectable levels of HAART in two samples obtained in different time of pregnancy, suggesting non-treatment adherence. The colostrum samples revealed higher quantities of lopinavir and ritonavir than reported in literature.
CONCLUSIONS
These methods are selective, accurate and exact for simultaneous quantification of four antiretroviral drugs LMV, ZDV, RTV, and LPV in plasma and colostrum samples. These methodologies can be used in therapeutic antiretroviral drugs monitoring, characterizing the drugs excretion trough breast milk and the approach to breastfeeding the children from HIV-positive women, and in the measure of treatment adherence in pregnant women.
