Development and validation of liquid chromatography mass spectrometry (L/C/MS/MS) assay for the determination of plasma 4betahydroxycholestrol and cholesterol in HIV infected children in Africa

Master Thesis


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University of Cape Town

4β-hydroxycholesterol (4β-OHC) is a metabolite of cholesterol formed by Cytochrome (CYP) 3A4/5/7 enzymes. It has recently been proposed as an endogenous biomarker forCYP3A4/5/7 activity. This may be useful in prediction of drug-drug interactions and other metabolic processes affected by regulators of CYP3A activity. The aim of this study was to develop and validate an LC/MS/MS assay for the determination of 4β-OHC in human plasma and use 4β-OHC as a biomarker of CYP3A4/5/7 metabolism in HIV-infected children with and without treatment in Africa. Determination of 4β-OHC from plasma was performed by saponification and derivatisation reaction processes followed by high performance liquid chromatography with MS/MS detection on an AB Sciex Qtrap 5500 mass spectrometer. Since 4β-OHC is an endogenous metabolite in human plasma, a stable isotope labelled (SIL) analogue, 4β-OHC-D7, was used as a surrogate analyte for the preparation of calibration standards and quality controls. A second SIL analogue, 4β-OHC-D4 was used as the internal standard. The transitions of the protonated derivatised products were monitored atm/z 613, 620 and 617 to the product ions m/z 490, 497 and 494 for 4β-OHC, 4β-OHC-D7and 4β-OHC-D4 respectively. The calibration curve fitted a quadratic (weighted by1/concentration2) regression over the range 2-500 ng/ml. Validation accuracy and precision statistics summary for three consecutive runs were between 98.9% and 103%, and 3.5%and 12% respectively of all quality controls. The assay's recovery, selectivity and analyte stability were established. The validated assay was successfully applied on clinical samples, where 4β-OHC was used as a biomarker to investigate the levels of CYP3A induction in HIV-infected children with and without treatment containing non-nucleoside reverse transcriptase inhibitors (NNRI).It was found that plasma 4β-OHC concentrations at baseline were significantly lower in children belonging to the naïve group compared to nevirapine (NVP) and efavirenz (EFV)groups. When NVP and EFV groups were compared at non-baseline treatment weeks, the median 4β-OHC concentrations were significantly higher in EFV group than the NVP group. Regarding the effect of time on treatment, a significant increase in 4β-OHC concentrations was observed from baseline to each of the non-baseline weeks in naïve group. Conversely, in the NVP group, there was a significant decrease in 4β-OHC concentrations from baseline to each of the non-baseline weeks. Time did not show any significant effect on 4β-OHC concentrations in EFV group. Furthermore, at baseline, age, sex and weight did not affect 4β-OHC concentrations in all the three groups. This study has provided a method that would be utilised to determine plasma 4β-OHC concentrations using relatively small volumes - typical of samples taken from children. The results of this study suggest that children on antiretroviral therapy (ART) are at risk of effects of CYP3A induction, as indicated by the increase of 4β-OHC concentrations in the NVP and EFV groups. Additionally, prolonged use of the ART may activate some nuclear receptors that regulate CYP3A enzyme activity thereby negatively affecting, for example, the regulation of lipid and glucose metabolism. The developed method may therefore be useful in predicting drug-drug interactions in the context of multiple therapy and may also be used in predicting other metabolic processes affected by regulators of CYP3A activity. Further prospective studies with larger sample sizes are required to confirm and build on the evidence shown in this study.

Includes bibliographical references