Masters Degrees (Clinical Pharmacology)

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Browsing Masters Degrees (Clinical Pharmacology) by Author "Louw, Vanessa"

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    LC-MS/MS methods for the quantification of sulfasalazine and sulfapyridine in various matrices: application to a pharmacokinetic study
    (Stellenbosch : Stellenbosch University, 2022-11) Louw, Vanessa; Kellermann, Tracy; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
    ENGLISH ABSTRACT: Introduction: An early phase clinical trial which took place at The Mercy Hospital for Women in Australia assessed the use of sulfasalazine as a treatment for preterm pre-eclampsia. This project consisted of the development and validation of a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method according to the Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines to simultaneously quantitate sulfasalazine and its metabolite, sulfapyridine, in placenta for pharmacokinetic analysis. Methods: A Shimadzu 8040 mass spectrometer was operated in multiple reaction monitoring (MRM) mode to monitor the mass-to-charge (m/z) transition of the protonated precursor ions m/z 398.90 and m/z 250.07 to the product ions m/z 381.05 and m/z 156.00 for sulfasalazine and sulfapyridine, respectively. Sulfasalazine-d4 and sulfapyridine-d4 were used as internal standards. 100 μL of placental tissue homogenate was extracted using acetonitrile:methanol (90:10, v/v) and the supernatant was eluted through hydrophilic-lipophilic balanced cartridges. The extraction procedure was followed by liquid chromatographic separation using a Poroshell C18 column. Gradient elution using a mobile phase combination of water + 0.1% formic acid (A) and acetonitrile:methanol (90:10, v/v) + 0.1% formic acid (B) was used. Accuracy and precision were assessed over three consecutive, independent runs. The ratios of analyte peak area to internal standard peak area were plotted against the nominal concentrations to generate a calibration curve which fits a quadratic regression (weighted by 1/x, x= concentration) over the range 30-30 000 ng/mL for both sulfasalazine and sulfapyridine. Results and Discussion: The average accuracy of calibration standards during inter-day validations ranged from 94.2-103.2% (%CV= 1.4-10.8) for sulfasalazine and 96.6-103.4% (%CV= 1.4-8.3) for sulfapyridine. The accuracy of quality controls ranged from 101.6-112.7% (%CV= 4.4-6.7) and 97.4-108.4% (%CV= 3.7-10.0) for sulfasalazine and sulfapyridine, respectively. Endogenous matrix components were shown to have no impact on the reproducibility of the method when placental tissue from six different sources were analysed. The average recovery of sulfasalazine and sulfapyridine from placental tissue homogenate was 121.5% and 119.6%, respectively. Autosampler stability experiments indicated that placental tissue homogenate extracts were stable on instrument for up to 48-hours at the method-defined temperature. Re-injection reproducibility experiments illustrated that the method remained accurate and precise for analysis of both analytes following a re-injection of a batch for up to 48 hours after the initial injection. Furthermore, sulfasalazine and sulfapyridine were found to be stable in placental tissue homogenate for 10 days when stored at -80 °C, for six hours when left on bench at room temperature, and when subjected to three-freeze thaw cycles. Upon analysis of patient samples (n= 9), the concentrations ranged from 491-4201 ng/g tissue for sulfasalazine and 637-26756 ng/g tissue for sulfapyridine, with two patient samples below the limit of quantitation (BLQ) of the assay for both analytes. Conclusion: An LC-MS/MS method for the quantification of sulfasalazine and sulfapyridine in human placenta was successfully validated and applied to a clinical study to evaluate the efficacy of sulfasalazine as an intervention for pre-eclampsia.