Browsing by Author "du Plessis, Christiena Hendriena"

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    The development and validation of an LC-MS/MS method for the quantification of metformin in human plasma
    (Stellenbosch : Stellenbosch University, 2023-08) du Plessis, Christiena Hendriena; Kellermann, Tracy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
    ENGLISH ABSTRACT: Background: The rising incidence of type 2 diabetes mellitus among people living with HIV residing in lower-and middle-income countries such as South Africa, leads to a greater proportion of people being prescribed both the first-line anti-diabetic medication, metformin, as well as the first-line antiretroviral drug, dolutegravir (DTG). DTG has been found to interact with metformin by increasing its plasma concentration. Drug interactions can result in supratherapeutic drug concentrations, and for this reason it is essential to develop and validate a selective, sensitive, rapid, and affordable bioanalytical method for the quantification of metformin in human plasma. This carries true clinical value, as it will not only enhance our understanding of the drug itself, but also inform on the magnitude of possible drug interactions. Methods: A Shimadzu 8040 instrument was used for analysis, to monitor the transition of metformin and metformin-d6 hydrochloride (ISTD), in the positive ion mode, [M+H]+ : m/z 129.9 → 60.1 and 136.2 → 60.1, respectively. An Agilent Zorbax Eclipse XDB-C8 column was used with gradient chromatography and mobile phases of 0.1% formic acid in water (A) and 100% acetonitrile (B) at a flow rate of 0.5 mL/min, and a retention time of ~2.50 min. A protein precipitation method was used where 200 μL of acetonitrile (ACN) was added to 50 μL plasma and 200 μL of the supernatant was transferred to 96-well plates, before injection for analysis. The % cross-talk, concomitant medication effects, whole blood stability (2 h), matrix effects, % recovery, process efficiency and the effect of 2% hemolysis were determined. Intra- and inter-batch validations were performed together with bench-top stability for ~4 h, freeze-thaw stability (3 cycles), autosampler stability (48 h). Results: The calibration curve had a quadratic regression with a weighting of 1/C2 and a concentration range of 15.6 ng/mL – 4 000 ng/mL in plasma. Metformin was stable in stock and working solutions at 4 °C, -20 °C, and -80 °C for 24 h and on bench at room temperature for ~4 h. The % cross-talk was negligible, and the presence of concomitant had no effect on the method’s performance. The analyte was stable in whole blood for 2 h. No matrix effects were observed after evaluating plasma from 6 different sources. The average percentage recovery was 69.9%, and process efficiency was 106.4%. Hemolysis at 2% did not have an effect on the quantification of metformin. Intra- and inter-batch validation results met the FDA (2018) and EMA (2011) acceptance criteria. The calibration standards had a % accuracy ranging from 98.1% - 102.1% (% CV of 5.1% - 10.2%). The quality controls had a % accuracy ranging from 91.6% – 101.5% (% CV of 4.9% - 10.5%). The analyte was stable in plasma through 3 freeze/thaw cycles, and on bench for ~4 h. Metformin was stable in the autosampler at 15 °C for ~48 h. This method was successfully applied to clinical samples. Conclusions: The developed LC-MS/MS method, using a simple protein precipitation extraction protocol, was successfully validated according to FDA and EMA guidelines. Subsequently, the robust method was applied to clinical samples to quantify metformin in human plasma to better inform patient care.