Browsing by Author "Joubert, Anton"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Open Access
    Simultaneous quantification of first-line anti-tuberculosis drugs and metabolites in human plasma
    (2019) Mazanhanga, Marian Tafadzwa; Wiesner, Lubbe; Joubert, Anton; Norman, Jennifer
    Tuberculosis (TB) currently kills more people than any other infectious disease worldwide, the highest burden being in Africa and Asia (1). Therapy recommended for drug sensitive TB consists of a cocktail of isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB), all given in a 2-month intensive phase, followed by only INH and RIF in a 4-month continuation phase. Clinical studies seeking to optimize dosing, gain more knowledge on the pharmacokinetics and pharmacodynamics of the drugs and compare current therapy to alternative regimens are required (2, 3). Therapeutic drug monitoring (TDM) is frequently carried out in cases responding poorly to therapy (3, 4). Both clinical studies and TDM require bioanalytical methods for quantifying drug concentrations in biological fluids. Several methods have been developed, mostly analysing individual drugs but a few analyse combinations. Ideally, quantification of all four drugs in one method is desirable as it is economical and allows high throughput. A method was developed and validated for the quantification of first line anti-tuberculosis drugs EMB, INH, PZA and RIF and the metabolites N-acetyl isoniazid (AcINH) and 25- desacetyl rifampicin (desRIF). Sample preparation consisted of protein precipitation, followed by high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) detection. Deuterated internal standards for each analyte (AcINH-d4, desRIF-d3, EMB-d4, INH-d4, PZA-15N,d3 and RIF-d3) were used. Mean recoveries of the analytes from plasma were as follows: AcINH 106.5%, DesRIF 123.2%, EMB 105.3%, INH 110.1%, PZA 132.0% and RIF 127.7%. Sample preparation was followed by reverse phase liquid chromatography on an Agilent 1200 series HPLC system using an Agilent Poroshell 120 EC- C18 2.7µm, 4.6 X 50mm analytical column. Separation of all analytes was achieved using a mobile phase gradient consisting of an aqueous mobile phase A (0.05% formic acid in water) and an organic mobile phase B (0.05% formic acid in a mixture of methanol and acetonitrile, 1:1). A T-junction splitter was used to reduce the mobile phase flow to the ion source by about 30%. Retention times for AcINH, desRIF, EMB, INH, PZA and RIF were 2.45, 5.40, 1.75, 2.22, 4.30 and 5.68 minutes respectively. An AB Sciex API 4000 triple quadrupole mass spectrometer at unit mass resolution in the multiple reaction monitoring (MRM) mode was used for detection, monitoring the following transitions for the six analytes: AcINH 180 → 121, desRIF 784 → 752, EMB 205 → 116, INH 138 → 79, PZA 124 → 81 and RIF 823 → 792. An electrospray ionisation (ESI) source in the positive ion mode was used to couple the mass spectrometer to the LC system. Accuracy and precision were assessed over three consecutive and independent runs. The calibration curves fit quadratic regressions for all analytes, with weighting of 1/x (where x=concentration) for all except PZA which had a weighting of 1/x2 over the calibration range. Calibration ranges in µg/ml were as follows: AcINH 0.050 – 12.5, desRIF 0.040 – 10.0, EMB 0.020 – 5.00, INH 0.100 – 25.0, PZA 0.32. – 80.0 and RIF 0.120 – 30.0, based on peak area ratios. A 1:4 dilution of the QC Dilute sample showed that concentrations of up to 20.0 µg/ml for AcINH, 16.0 µg/ml for desRIF, 8.00 µg/ml for EMB, 40.0 µg/ml for INH, 128 µg/ml for PZA and 48.0 µg/ml for RIF in plasma could be analysed reliably when diluted into the calibration range. No significant carry-over was observed for all analytes. The method was shown to be reproducible when human plasma samples from six different sources were analysed and endogenous matrix components had no significant effect on the assay. All analytes were stable in plasma for at least four hours on ice, and when subjected to three freeze-thaw cycles. Reinjection reproducibility experiments showed that all analytes except PZA could be reliably analysed by re-injecting an entire batch after about 48 hours. Quantification of AcINH, INH and RIF was not significantly affected by 2% hemolysis of sample while desRIF, EMB and PZA were significantly affected. Data was analysed using Analyst ® version 1.6.2 software. With wide calibration ranges, the assay is suitable for both routine TDM and PK studies. Concurrent analysis of metabolites allows inferences to be made on the PK of the two main TB drugs. The total run time of 6.5 minutes per sample combined with the simple sample preparation procedure, the method is more economical on both time and resources than single analyte assays.
  • Thumbnail Image
    Item
    Open Access
    The Development and Validation of a Direct LC-MS/MS Assay for the Determination of Tenofovir-diphosphate in Dried Blood Spots for the Analysis of Clinical Samples
    (2019) Oberholster, Lucas; Wiesner, Lubbe; Joubert, Anton
    Tenofovir (TFV) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors, often used in preexposure prophylaxis (PrEP) trials: where antiretroviral drugs are administered to high-risk, HIV-negative individuals to prevent HIV infection. Both drugs are safe when taken either daily or intermittently, which is ideal for PrEP regimens where adherence may not be high. The minimum number of doses estimated to confer high PrEP efficacy for a TFV/FTC regimen is four or more doses per week, resulting in a 95% lower risk of HIV acquisition. However, this is highly dependent on various host factors, of which adherence plays the largest role. The aim of the project was to develop a novel sensitive, specific, and robust direct method for the measurement of adherence, utilising tenofovir-diphosphate (TFV-DP) in dry blood spots (DBS) through LC-MS/MS analysis, to replace the current costly and laborious indirect method currently used to elucidate adherence of patients. This indirect method faces challenges, due to the polar nature of TFV and its metabolites, leading to separation and retention issues. The existing method applied a technique which separated the parent drug from the metabolite and then back-converted all metabolites to the parent drug before analysing the samples on LC-MS/MS. The developed alternative method aimed to reduce the time taken for each assay and the associated cost of consumables. TFV-DP is a highly polar compound and traditional reverse-phase chromatography has poor retention and separation capabilities when used to retain polar compounds, therefore alternative strategies were implemented. In this developed direct method, an anion exchange column was used along with a pH gradient, with the aim of improving separation and chromatography of TFV, TFV-DP, and tenofovir-monophosphate (TFV-MP). The method was optimised and validated using current U.S. Food and Drug Administration (FDA) and European Medical Agency (EMA) guidelines. The use of the anion exchange column resulted in a marked increase in retention time and allowed baseline separation of TFV, TFV-DP, and TFV-MP. Determination of TFV-DP from DBS was performed using three 3 mm DBS punches per sample, which underwent an extraction procedure followed by high-performance liquid chromatography with tandem mass spectrometry detection on an AB Sciex Qtrap 5500 mass spectrometer. The transitions of the protonated precursor ions were monitored at m/z 448.0 and 452.9 to the product ions m/z 350.0 and 354.9 for TFV-DP and the deuterated TFVDP internal standard, respectively. The method was validated over a range of 50–6400 fmol/punch for TFV-DP. The developed direct method had a lower limit of quantification (LLOQ) of 50 fmol/punch, which was higher than that of the indirect method; therefore, it had less sensitivity. The reduced sensitivity was acceptable, since the methods were meant for the measurement of adherence. The direct method had an ULOQ of 6400 fmol/punch, which was similar to that of the indirect method. The direct method also required significantly less on-bench sample processing and, therefore, was less time consuming and costly. To determine the suitability and accuracy of the direct method in comparison to the indirect method a comparative analysis was completed by analysing the same samples using both the indirect and direct method. The developed method met all the validation requirements and a strong correlation was observed between the results of the indirect and direct methods during the comparative analysis.