Browsing by Author "Kellermann, Tracy"

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    Open Access
    Determination of Rifapentine and 25-O-desacetyl Rifapentine from 100 µl human breastmilk by LC-MS/MS using protein precipitation and solid phase extraction
    (2019) Mkhize, Buyisile; Wiesner, Lubbe; Kellermann, Tracy; Norman, Jennifer
    There is currently no information available on the transfer of the second-line anti-TB drug, rifapentine and its metabolite, into breastmilk. The subsequent implications to the breastfed infant, as well as consequences of long-term exposure to potentially sub-therapeutic drug levels with regards to the development of drug resistant bacteria is therefore not known. A liquid chromatography method with detection by mass spectrometry (LC-MS/MS) is described for the quantification of rifapentine and its metabolite, 25-O-desacetyl rifapentine in human breastmilk, using rifampicin-d3 as an internal standard. An AB Sciex 4000 mass spectrometer at unit resolution in the multiple reaction monitoring (MRM) mode was used to monitor the transition of the protonated precursor ions m/z 877.5, m/z 835.4 and m/z 827.4 to the product ions m/z 151.1, m/z 453.2 and m/z 151.200 for rifapentine, 25-Odesacetyl rifapentine and rifampicin-d3, respectively. Ions were produced using Electro spray ionisation (ESI) in the positive ionisation mode. An Agilent Poroshell 120 EC-C18 (4.6 x 50 mm, 2.7 μm) column was used for chromatographic separation using an isocratic method of acetonitrile containing 0.1% formic acid and water containing 10% methanol and 0.1% formic acid (55:45, v/v), at a flow rate of 450 µl per minute. The retention times for rifapentine, 25- O-desacetyl rifapentine and rifampicin-d3 were ≈2.67, ≈1.88 and ≈1.96 minutes, respectively. The method was developed and validated according to FDA guidelines. The extraction method consisted of a combination of protein precipitation and C18 solid phase extraction. Rifapentine and 25-O-desacetyl rifapentine showed no significant carry over on the Agilent autosampler. The method was reproducible when analysed with human breastmilk from six different sources from Western Cape Maternity Breastmilk Bank. Rifapentine mean extraction yield was 84.2% (%CV = 1.7) and that of 25-O-desacetyl rifapentine was 71.1% (%CV = 10.8). Rifapentine had a mean process efficiency of 80.4% (%CV = 4.7) and that of 25-O-desacetyl rifapentine was 95.7% (%CV = 5.7). Intra- and inter day validations over 3 days were performed. The calibration curves fit a quadratic regression with 1/x weighting over a concentration range of 2 - 2000 ng/ml for both rifapentine and 25-Odesacetyl rifapentine based on the analyte/internal standard peak area ratios, the accuracy ranged from 92.9% to 105.5% for both rifapentine and 25-O-desacetyl rifapentine standards. The Quality Controls accuracy ranged from 97.4% to 106.0% for both rifapentine and 25-Odesacetyl rifapentine. Stock solutions were shown to be stable for 69 days at -80°C. v Rifapentine and 25-O-desacetyl rifapentine were stable in human breastmilk for up to 72 hours at approximately -80°C and -20°C, on benchtop for ≈4.5 hours on ice and after three freeze-thaw cycles. Rifapentine and 25-O-desacetyl rifapentine were shown to be stable on the autosampler over a period of approximately 48 hours after which the entire batch could be reinjected. Autosampler stability revealed a decrease in peak area ratios, indicating that a partial batch cannot be reinjected after 48 hours in case of instrument failure. This method will be utilized in the analysis of patient samples from a clinical study in South Africa in breastfeeding women with tuberculosis.
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    Open Access
    Determination of total, unbound, and intracellular concentrations of the antiretroviral drugs Efavirenz, Lopinavir, and Ritonavir
    (2022) Kriegler Foster, Katie; Wiesner, Lubbe; Kellermann, Tracy
    Efavirenz, lopinavir, and ritonavir are antiretroviral drugs used for the treatment of HIV in South Africa. Plasma concentrations of these drugs are routinely monitored to ensure efficacy, minimise adverse effects, and adjust dosing. However, variability exists in patient treatment response and tolerability, which cannot always be explained by the therapeutic drug monitoring results. This may be due to variability in the amount of drug reaching the target site within the HIV-infected cells. Therefore, intracellular drug concentrations could provide a more accurate depiction of drug exposure. An alternative to intracellular drug concentrations could be the quantitation of drug not bound to plasma proteins as this is the portion able to diffuse into tissues and cells to exert a therapeutic effect. A method is described for the quantification of intracellular efavirenz, lopinavir, and ritonavir from one million human peripheral blood mononuclear cells. In addition, the quantification of unbound efavirenz, lopinavir, and ritonavir from human plasma using ultracentrifugation is demonstrated, including a novel surrogate matrix. The two methods were validated according to the United States Food and Drug Administration and European Medicines Agency guidelines and proven to be accurate, precise, and reproducible. Both methods were submitted to the United States National Institute of Allergy and Infectious Diseases' Clinical Pharmacology Quality Assurance group for review and have been approved for use on clinical samples. A proof-of-concept correlation study of intracellular, unbound, and total drug concentrations is described using blood samples from six HIV-positive patients. A further patient unresponsive to lopinavir treatment, despite total plasma concentrations within the normal therapeutic range, was also evaluated. Paired plasma and cell samples indicated that the drug reached the target site within the cells, eliminating a possible cause of treatment failure. These findings show the utility and validity of these methods in a clinical setting to provide an overall view of treatment response and support their novel application in individualised patient care in South Africa.
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    Open Access