Razvoj bioanalizne metode za določanje plazemskih koncentracij protimikrobnih učinkovin s tekočinsko kromatografijo visoke ločljivosti

Benda, Klavdija (author), Vovk, Tomaž (mentor)

Protibakterijske učinkovine (벵-laktami, glikopeptidi, aminoglikozidi, makrolidi, kinoloni in tetraciklini) sodijo med protimikrobne učinkovine, namenjene zdravljenju okužb. Kljub njihovi varnosti je za optimizacijo zdravljenja pri bolnikih s sepso smiselno uporabiti terapevtsko spremljanje koncentracije, saj se farmakokinetika protimikrobnih zdravil spreminja. Poleg tega se lahko za zmanjšanje vnetnega odziva pri bolnikih s sepso uporabljajo tudi hemoadsorbenti, ki lahko vežejo zdravila. V okviru magistrske naloge smo razvili bioanalizno metodo za sočasno določanje plazemskih koncentracij imipenema, amoksicilina, cefepima, meropenema, vankomicina in piperacilina v goveji plazmi. Plazemske vzorce smo pripravljali z obarjanjem proteinov z acetonitrilom in ekstrakcijo v diklorometanu. Vzorce smo analizirali s tekočinsko kromatografijo visoke ločljivosti, kjer smo za separacijo analitov uporabili C18 stacionarno fazo in mobilno fazo, ki je vsebovala 0,5 % H3PO4 (pH 7,0) in 0,5 % H3PO4 (pH 3,0) z dodatkom acetonitrila v razmerju 3 : 1 (v/v). Pretok mobilne faze je bil 1,3 mL/min. Analite smo detektirali z UV/VIS detektorjem. Metodo smo uspešno validirali v skladu s smernicami Evropske agencije za zdravila za validacijo bioanalizne metode. Na podlagi validacijskih parametrov (znotraj dnevna in med dnevna ponovljivost, točnost, specifičnost) lahko potrdimo, da je metoda primerna za analiziranje izbranih analitov v govejih plazemskih vzorcih. Območje linearnosti za cefepim, piperacilin, meropenem smo definirali v koncentracijskem območju 1–75 µg/mL, medtem ko je za vankomicin in amoksicilin 1–100 µg/mL, za imipenem pa 2,5–100 µg/mL. Vzorci za analizo so stabilni vsaj 12 ur pri 5 °C v avtomatskem vzorčevalniku. Validirano metodo smo uspešno uporabili za določanje koncentracij izbranih antibiotikov v in vitro poskusu vezave antibiotika na hemoadsorbent Cytosorb®. Ugotovili smo, da dodatek Cytosorba® vpliva na koncentracijo izbranih antibiotikov v goveji plazmi, kar kaže na vezavo antibiotikov na hemoadsorbent.

• Language: Slovenian
• Keywords: bioanalizna metoda, validacija, protibakterijske učinkovine
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-155741
• Publication date in RUL: 14.04.2024

Secondary language

• Language: English
• Title: Development of bioanalytical method for determining plasma concentrations of antimicrobial agents using high performance liquid chromatography

Abstract

Antibacterial agents (β-lactams, glycopeptides, aminoglycosides, macrolides, quinolones, and tetracyclines) belong to antimicrobial substances used for the treatment of infections. Despite their safety, therapeutic drug monitoring is recommended to optimize the treatment of patients with sepsis, as the pharmacokinetics of antimicrobial drugs is variable. In addition, hemoadsorbents are also used to reduce inflammation in septic patients, but they can also bind drugs. In the master's thesis we have developed a bioanalytical method for simultaneous determination of imipenem, amoxicillin, cefepime, meropenem, vancomycin and piperacillin concentration in bovine plasma. Plasma samples were prepared by protein precipitation with acetonitrile and extraction in dichloromethane. The analysis was performed using high-performance liquid chromatography with a C18 stationary phase and a mobile phase containing 0.5% H3PO4 (pH 7.0) and 0.5% H3PO4 (pH 3.0) with acetonitrile at a ratio of 3:1 (v/v). The flow rate of the mobile phase was 1.3 mL/min. Analytes were detected with a UV/VIS detector. The method was successfully validated according to the guidelines of the European Medicines Agency for the validation of bioanalytical methods. Based on validation parameters (intra-day and inter-day precision, accuracy, specificity), we confirm that the method is suitable for analyzing selected analytes in bovine plasma samples. The linear range for cefepime, piperacillin, and meropenem was defined in the concentration range of 1–75 µg/mL, while for vancomycin and amoxicillin, it was 1–100 µg/mL, and for imipenem, it was 2.5–100 µg/mL. Samples for analysis are stable for at least 12 hours at 5 °C in the autosampler. The validated method was successfully applied to determine the concentrations of selected antibiotics in an in vitro experiment involving the binding of antibiotics to the hemoadsorbent Cytosorb®. We found that the addition of Cytosorb® affects the concentration of selected antibiotics in bovine plasma, indicating the binding of antibiotics to the hemoadsorbent.

• Keywords: bioanalytical method, validation, antibacterial agents