TUPE061 - Poster Exhibition
Use of a liquid chromatography/mass spectrometry (LC/MS) in vitro assay to quantify nevirapine (NVP), efavirenz (EFV) and lamivudine (3-TC) on dried blood-spot (DBS) samples: a strategy to evaluate adherence to prevention-of-mother-to-child-transmission (
E.H. Koumans, A. Martin, M.R. Adler, J. Schulte, P.J. Weidle, E. Rivadeneira
Centers for Disease Control & Prevention, Atlanta, United States
Background: Infant prophylaxis during PMTCT is currently measured using programmatic data. LC-MS could also measure prophylaxis adherence, because it can detect antiretrovirals from DBS. However, LC/MS has not been evaluated on samples subjected to real-world conditions. We evaluated in vitro performance of LC-MS to detect and quantify NVP, EFV, and 3TC on DBS varying the levels of heat, humidity, and sample age.
Methods: Blood samples for NVP at 100ng/ml, 500ng/ml, and 3000ng/ml, EFV at 100ng/ml, 500ng/ml, and 1500ng/ml, and 3TC at 25ng/ml, 100ng/ml, and 1000ng/ml were spotted onto DBS paper and dried. Samples were stored at different heat (22⁰C or 45⁰C), humidity (16%-42%, 30%, or 90%), and duration (2, 4, 6, or 8 weeks) conditions and analyzed in quadruplicate using LC-MS. Quantification for all drugs was done using Analyst software.
Results: Of the 1152 samples that were analyzed, 288 were negative controls and 1124 had interpretable results; 16 3TC samples had no detectable drug. Some degradation occurred for most drug concentrations and conditions during the initial 2 weeks. By 8 weeks at 45⁰C and 30% humidity, the 100ng/ml NVP concentration was reduced by half; the 3000ng/ml concentration was reduced by 16% with 30% humidity, but not by heat. After 6 weeks, the 100ng/ml heat-exposed EFV sample was reduced by 47%; 500ng/ml samples exposed to high and low humidity were reduced by 37%. Concentrations of 3TC were detected at 10-25% of original concentrations, with 70%reduction at 45⁰C and 90% humidity.
Conclusions: LC-MS could detect administration of infant NVP prophylaxis and maternal NVP and EFV treatment through breastmilk. While high temperature, low humidity settings would not affect LC-MS detection of 3TC through breastmilk (expected concentration of 25ng/ml), high temperature, high humidity for even two weeks makes detection of 3TC unreliable. LC-MS could also be used to detect infants treated with 3TC, NVP, or EFV.
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