Razvoj in validacija metode za merjenje protimikrobnih učinkovin v površinskih vodah s tekočinsko kromatografijo s tandemsko masno spektrometrijo

Bukovec, Simon (author), Trontelj, Jurij (mentor), Roškar, Robert (comentor)

Protimikrobne učinkovine zaradi stalne prisotnosti v naravnem okolju predstavljajo nevarnost za razvoj bakterijske odpornosti. Ker so nekatere zelo lipofilne, druge zelo hidrofilne in imajo značaj šibkih kislin ali šibkih baz, predstavljajo zelo heterogeno skupino. Zato je razvoj enotne ekstrakcijske in kromatografske metode zahteven, saj je težko doseči visok izkoristek in dobro koncentriranje ter ponovljivost za vse preiskovane analite, še posebej za manj stabilne. Cilj magistrske naloge je bil razviti in validirati analizno metodo za spremljanje onesnaženja površinskih vod s protimikrobnimi učinkovinami. V optimizirano metodo smo umestili devet skupin protimikrobnih učinkovin, ki so: makrolidi, sulfonamidi, kinoloni, tetraciklini, beta-laktami, nitroimidazoli, linkozamidi, amfenikoli in zaviralci dihidrofolat reduktaze. Metoda je obsegala ekstrakcijo na trdnem nosilcu z obratno-faznim polimernim adsorbentom in 200 mL volumna vodnega vzorca, ki je omogočil tisočkratno koncentriranje analitov in kromatografsko ločbo na učinkoviti Poroshell C18, 100 × 3 mm, 2,7 μm koloni in detekcijo s hibridnim tandemskim masnim spektrometrom vrste trojni kvadrupol-linearna ionska past. Validirana metoda ima za 22 učinkovin koncentracijski razpon med 0,5 in 50 ng/L, oziroma med 5 in 500 ng/L za analite s slabšim odzivom (spiramicin, enrofloksacin, norfloksacin in ampicilin) in ustrezno vizualno linearnost (podkrepljeno z R2 večjim od 0,99), selektivnost (odziv analit/slepi vzorec večji od 4) pa za 21 preiskovanih analitov. Spodnja meja določitve koncentracije na osnovi ustrezne točnosti, ponovljivosti, razmerja signal/šum in odziva analit/slepi vzorec je pri večini analitov 0,5 ng/L, pri treh 5,0 ng/L in pri ampicilinu 10 ng/L. Izkoristek pri nizkem kontrolnem vzorcu je v povprečju 72,6 %, znotrajdnevna ponovljivost 11,0 % RSD, meddnevna pa 9,9 % RSD. Znotrajdnevna točnost pri enakem kontrolnem vzorcu je v povprečju 90,9 %, meddnevna pa 88,2 %. V realnem vzorcu smo na osnovi dodatne funkcionalnosti programske opreme masnega spektrometra mejo zaznave analizne metode zanesljivo določili pri razmerju signal/šum, večjem od 10. V 4 vzorcih površinskih voda v Sloveniji smo v povprečju zaznali 11 (od 7 do 13) in sicer doksiciklin, roksitromicin, sulfametoksazol, eritromicin, trimetoprim, sulfadiazin, sulfapiridin, metronidazol, spiramicin, enrofloksacin, ofloksacin, norfloksacin, ciprofloksacin, klindamicin, tetraciklin, klaritromicin in azitromicin) in kvantificirali 7 (od 3 do 9) od skupaj 22 protimikrobnih učinkovin. Koncentracijski razpon le-teh je bil med 0,5 ng/L (klaritromicin) in 29,4 ng/L (enrofloksacin).

• Language: Slovenian
• Keywords: protimikrobne učinkovine, LC-MS/MS, SPE, površinske vode
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-144353
• Publication date in RUL: 16.02.2023

Secondary language

• Language: English
• Title: Development and validation of a method for measuring antimicrobials in surface waters by liquid chromatography with tandem mass spectrometry

Abstract

Because of their presence in the ecosystem, antimicrobials pose a threat for the development of bacterial resistence. Because of their diverse nature (hydrophilic or lypophilic and weak acid or basic character), they represent a very heterogeneous group. This is why it is difficult to develope a unified extraction and chromatographic method, because it is hard to achieve a high extraction efficiency and repeatability for all analytes, especially for unstable ones. The goal of this Master's thesis was to develop and validate an analytical method for the screening of antimicrobials in surface waters. Our optimised method includes nine groups of antimicobials: macrolides, sulfonamides, quinolones, tetracyclines, beta-lactames, nitroimidazoles, lincosamides, amphenicoles and dihydrofolate reductase inhibitors. The method consisted of a solid phase extraction with a reversed-phase adsorbent and 200 mL sample loading which allowed for a thousandfold concentration of the sample and a chromatographic separation on an efficient Poroshell C18, 100 × 3 mm, 2,7 μm column, coupled with a hybrid triple quadrupole-linear ion trap mass spectrometer. The validated method has a concentration range of 0.5 to 50 ng/L and from 5 to 500 ng/L for the lower response analytes (spiramycin, enrofloxacine, norfloxacine, ampicillin). The linearity (backed up with R2 greater than 0,99) was sufficient for all 22 analysed antimicrobials, and the selectivity (analyte/blank response greater than 4) was sufficient for 21. For most analytes, the limit of quantification was 0.5 ng/L, for 3 analytes at 5.0 ng/L and for ampicillin at 10.0 ng/L. The average recovery of the method for the low quality control sample was 72,6 %, the average intra- and interday repeatibilities were 11.0 % and 9.9 % RSD, respectively. Average intraday and interday accuracies of the same low quality control sample were 90.9 % and 88.2 %, respectively. By the use of an additional mass spectrometer software, we determined the limit of detection of the method at a signal to noise ratio greater than 10. In 4 analysed surface water samples from Slovenia, we detected on average 11 (7 to 13) antimicrobials, those being doxycycline, roxithromycin, sulfamethoxazole, erythromycin, trimethoprim, sulfadiazine, sulfapyridine, metronidazole, spiramycin, enrofloksacin, ofloxacin, norfloxacin, ciprofloxacin, clindamycin, tetracycline, clarithromycin in azithromycin and quantified on average 7 (3 to 9) antimicrobials. The measured concentrations ranged from 0.5 ng/L (clarithromycin) to 29.4 ng/L (enrofloxacin).

• Keywords: antimicrobials, LC-MS/MS, SPE, surface water