The development and validation of an LC-MS/MS method for the quantification of metformin in human plasma

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dc.contributor.advisorKellermann, Tracyen_ZA
dc.contributor.authordu Plessis, Christiena Hendrienaen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Dept. of Medicine. Division of Clinical Pharmacology.en_ZA
dc.date.accessioned2023-11-06T12:50:52Zen_ZA
dc.date.accessioned2024-01-08T14:10:43Zen_ZA
dc.date.available2023-11-06T12:50:52Zen_ZA
dc.date.available2024-01-08T14:10:43Zen_ZA
dc.date.issued2023-08en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2023.en_ZA
dc.description.abstractENGLISH 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.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Inleiding: Die stygende voorkoms van tiepe 2 diabetes mellitus onder mense met HIV, wat woon in laer- en middel-inkomste lande soos Suid Afrika, lei tot ‘n groter proporsie van mense wat beide die eerste lyn anti-diabetiese medikasie, metformin, sowel as die eerste lyn antiretrovirale dwelm, dolutegravir (DTG), voorgeskryf word. DTG is bevind om ‘n interaksie met metformin te hê, waardeur dit die plasma konsentrasie laat toeneem. Dwelm interaksies kan tot supraterapeutiese dwelm lonsentrasies lei, en vir hierdie rede is die ontwikkeling en geldigheids bepaling van ‘n selektiewe, sensitiewe, vinnige, en bekostigbare bioanalitiese metode vir die kwantifikasie van metformin in menslike plasma, noodsaaklik. Dit dra by tot ware kliniese waarde, nie net omdat dit ons verstaan van die dwelm verbeter nie, maar ook omdat dit ons kan inlig rakende die omvang van moontlike dwelm interaksies. Metodes: ʼn Shimadzu 8040-massaspektrometer is gebruik vir die analiese, om die oorgang van metformin en metformin-d6 hydrochloried (die interne standaard), in die geprotoneerde voorlooperione wyse, [M+H]+ : m/z 129.9 → 60.1 and 136.2 → 60.1, onderskeidelik, te moniteer. ‘n Agilent Zorbax Eclipse XDB-C8 kolom was gebruik met ‘n gradiënt-eluëring en ‘n mobiele fasekombinasie van water + 0.1% metanoësuur (A) en 100% asetonitriel (B) teen ‘n vloeitempo van 0.5 mL/min, en ‘n retensie tyd van ~2.50 min. ‘n Proteïen onttrekking metode was gebruik waar 200 μL asetonitriel bygevoeg was tot 50 μL plasma en 200 μL van die bodrywende stof oorgedra was na 96-put plate, voor die inspuiting vir analiese. Die % dwars-praat, gepaardgaande medikasie effek, volbloed stabiliteit (2 ure), matriks effekte, gemiddelde onttrekkings-persentasie, proses doeltreffendheid en die effek van 2% hemolise was geassesseer. Intra- en inter-bondel bevestigings validasies was uit gevoer, saam met werksbank stabiliteit vir ~4 ure, vries-ontdooi-stabiliteit (3 siklusse), en outomatiese monsternemer stabiliteit (48 ure). Resultate: Die kalibreringskromme het ‘n kwadratiese regressive met ‘n beswaar van 1/C2 , en ‘n konsentrasie reeks van 15.6 ng/mL – 4 000 ng/mL in plasma gehad. Metformin was stabiel in vorraad- en werkende-oplossings teen 4 °C, -20 °C en -80 °C vir 24 ure en op die werksbank teen kamer temperatuur vir ~4 ure. Die % dwars-praat was weglaabaar, en gepaardgaande medikasie het geen effek op die metode se oplewering gehad nie. Die analiet was stabiel in volbloed vir 2 ure. Geen matriks effekte was waargeneem na plasma evaluasie van 6 verskillende bronne nie. Die gemiddelde onttrekkings-persentasie was 69.8%, en die proses doeltreffendheid was 106.4%. Hemolise teen 2% het nie die kwantifikasie van metformin geaffekteer nie. Intra- en inter-bondel bevestigings-validasie resultate het voldoen aan die FDA (2018) en EMA (2011) aanvaardings vereistes. Die kalibreringstandaarde het ‘n gemiddelde akkuraatheid gehad wat gewissel het van 98.1% - 102.1% (%CV van 5.1% - 10.2%). Die gehaltebeheermaatreëls het ‘n gemiddelde akkuraatheid gehad wat gewissel het van 91.6% – 101.5% (%CV van 4.9% - 10.5%). Die analiet was stabiel in plasma na drie vries-ontdooi-siklusse, en op die werksbank vir ~4 ure. Metformin was stabiel in die outomatiese monsternemer teen 15 °C vir ~48 ure. Die metode was suksesvol toegepas op klienise monsters. Gevolgtrekking: Die ontwikkelde LC-MS/MS metode, waar ‘n eenvoudige proteïen ontrekking ekstraksieprosedure gebruik was, was suksesvol gevalideer in ooreenstemming met die FDA en EMA riglyne. Gevolglik, die robuuste metode was toegepas op kliniese monsters om metformin in menslike plasma te kwantifiseer, vir beter ingeligde patient-sorg.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxvii, 110 pages : illustrationsen_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/128873en_ZA
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshMetforminen_ZA
dc.subject.lcshHIV-positive personsen_ZA
dc.subject.lcshType 2 diabetes -- Alternative treatmenten_ZA
dc.subject.lcshPlasma cellsen_ZA
dc.subject.lcshDrug interactionsen_ZA
dc.titleThe development and validation of an LC-MS/MS method for the quantification of metformin in human plasmaen_ZA
dc.typeThesisen_ZA
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