Monitoring plasma concentrations of β-lactam antibiotics is crucial, particularly in critically ill patients, where variations in concentrations can lead to treatment failure or adverse events. Standardized antimicrobial regimens may not be effective for all patients, especially in special groups with altered physiological parameters. Pharmacokinetic/pharmacodynamic (PK/PD) studies highlight the time-dependent antibacterial activity of these antibiotics, emphasizing the need for personalized dosing. Therapeutic drug monitoring (TDM) is essential, requiring rapid and accurate analytical methods for precise determination of drugs in biological material (typically plasma or serum). This study presents a novel capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) method designed for the simultaneous quantification of five penicillin antibiotics, two cephalosporins, one carbapenem, and two β-lactamase inhibitors in a single run. The method involves a simple sample pretreatment-precipitation with organic solvent-and has a run time of 20 min. Optimization of CZE separation conditions revealed that 20 mM ammonium hydrogen carbonate (NH4HCO3) serves as the optimal background electrolyte (BGE). Positive electrospray ionization (ESI) mode, with isopropyl alcohol (IP)/10 mM ammonium formate water solution (50/50, v/v) as the sheath liquid, was identified as the optimal condition for MS detection. Method validation according to the Food and Drug Administration (FDA) guideline for development of bioanalytical methods demonstrated satisfactory selectivity, linearity, recovery, robustness, and stability. The method's practicality was evaluated using the Blue Applicability Grade Index (BAGI), yielding a score of 77.5. Moreover, the greenness of the proposed method was evaluated by two commonly used metric tools-Analytical GREEnness (AGREE) and Green Analytical Procedure Index (GAPI). The developed CZE-MS/MS method offers a practical and reliable approach for quantifying a broad spectrum of β-lactam antibiotics in plasma. Its ability to simultaneously quantify multiple analytes in a single run, coupled with a straightforward sample pretreatment, positions it as a valuable and prospective tool for TDM in critically ill patients.

AGEL Lab Revolucni 2214 35 CZ 741 01 Novy Jicin Czech Republic

Department of Galenic Pharmacy Faculty of Pharmacy Comenius University in Bratislava Odbojarov 10 SK 832 32 Bratislava Slovakia

Department of Pharmaceutical Analysis and Nuclear Pharmacy Faculty of Pharmacy Comenius University in Bratislava Odbojarov 10 SK 832 32 Bratislava Slovakia

Toxicological and Antidoping Center Faculty of Pharmacy Comenius University in Bratislava Odbojarov 10 SK 832 32 Bratislava Slovakia

Zobrazit více v PubMed

Carlier M., Stove V., De Waele J.J., Verstraete A.G. Ultrafast Quantification of β-Lactam Antibiotics in Human Plasma Using UPLC–MS/MS. J. Chromatogr. B. 2015;978–979:89–94. doi: 10.1016/j.jchromb.2014.11.034. PubMed DOI

Dhaese S., Van Vooren S., Boelens J., De Waele J. Therapeutic Drug Monitoring of β-Lactam Antibiotics in the ICU. Expert. Rev. Anti Infect. Ther. 2020;18:1155–1164. doi: 10.1080/14787210.2020.1788387. PubMed DOI

Cusumano J.A., Klinker K.P., Huttner A., Luther M.K., Roberts J.A., LaPlante K.L. Towards Precision Medicine: Therapeutic Drug Monitoring-Guided Dosing of Vancomycin and β-Lactam Antibiotics to Maximize Effectiveness and Minimize Toxicity. Am. J. Health Syst. Pharm. 2020;77:1104–1112. doi: 10.1093/ajhp/zxaa128. PubMed DOI

Roberts J.A., Paul S.K., Akova M., Bassetti M., De Waele J.J., Dimopoulos G., Kaukonen K.-M., Koulenti D., Martin C., Montravers P., et al. DALI: Defining Antibiotic Levels in Intensive Care Unit Patients: Are Current β-Lactam Antibiotic Doses Sufficient for Critically Ill Patients? Clin. Infect. Dis. 2014;58:1072–1083. doi: 10.1093/cid/ciu027. PubMed DOI

Stašek J., Keller F., Kočí V., Klučka J., Klabusayová E., Wiewiorka O., Strašilová Z., Beňovská M., Škardová M., Maláska J. Update on Therapeutic Drug Monitoring of Beta-Lactam Antibiotics in Critically Ill Patients—A Narrative Review. Antibiotics. 2023;12:568. doi: 10.3390/antibiotics12030568. PubMed DOI PMC

Dilworth T.J., Schulz L.T., Micek S.T., Kollef M.H., Rose W.E. β-Lactam Therapeutic Drug Monitoring in Critically Ill Patients: Weighing the Challenges and Opportunities to Assess Clinical Value. Crit. Care Explor. 2022;4:e0726. doi: 10.1097/CCE.0000000000000726. PubMed DOI PMC

Gatti M., Cojutti P.G., Pascale R., Tonetti T., Laici C., Dell’Olio A., Siniscalchi A., Giannella M., Viale P., Pea F. Assessment of a PK/PD Target of Continuous Infusion Beta-Lactams Useful for Preventing Microbiological Failure and/or Resistance Development in Critically Ill Patients Affected by Documented Gram-Negative Infections. Antibiotics. 2021;10:1311. doi: 10.3390/antibiotics10111311. PubMed DOI PMC

Maguigan K.L., Al-Shaer M.H., Peloquin C.A. Beta-Lactams Dosing in Critically Ill Patients with Gram-Negative Bacterial Infections: A PK/PD Approach. Antibiotics. 2021;10:1154. doi: 10.3390/antibiotics10101154. PubMed DOI PMC

Martinez M.N., Papich M.G., Drusano G.L. Dosing Regimen Matters: The Importance of Early Intervention and Rapid Attainment of the Pharmacokinetic/Pharmacodynamic Target. Antimicrob. Agents Chemother. 2012;56:2795–2805. doi: 10.1128/AAC.05360-11. PubMed DOI PMC

Owens R.C., Shorr A.F. Rational Dosing of Antimicrobial Agents: Pharmacokinetic and Pharmacodynamic Strategies. Am. J. Health-Syst. Pharm. 2009;66:S23–S30. doi: 10.2146/090087d. PubMed DOI

Legrand T., Vodovar D., Tournier N., Khoudour N., Hulin A. Simultaneous Determination of Eight β-Lactam Antibiotics, Amoxicillin, Cefazolin, Cefepime, Cefotaxime, Ceftazidime, Cloxacillin, Oxacillin, and Piperacillin, in Human Plasma by Using Ultra-High-Performance Liquid Chromatography with Ultraviolet Detection. Antimicrob. Agents Chemother. 2016;60:4734–4742. doi: 10.1128/AAC.00176-16. PubMed DOI PMC

Carlier M., Noe M., De Waele J.J., Stove V., Verstraete A.G., Lipman J., Roberts J.A. Population Pharmacokinetics and Dosing Simulations of Amoxicillin/Clavulanic Acid in Critically Ill Patients. J. Antimicrob. Chemother. 2013;68:2600–2608. doi: 10.1093/jac/dkt240. PubMed DOI

Huttner A., Harbarth S., Hope W.W., Lipman J., Roberts J.A. Therapeutic Drug Monitoring of the β-Lactam Antibiotics: What Is the Evidence and Which Patients Should We Be Using It for? J. Antimicrob. Chemother. 2015;70:dkv201. doi: 10.1093/jac/dkv201. PubMed DOI

Gonçalves-Pereira J., Póvoa P. Antibiotics in Critically Ill Patients: A Systematic Review of the Pharmacokinetics of β-Lactams. Crit. Care. 2011;15:R206. doi: 10.1186/cc10441. PubMed DOI PMC

Abdulla A., Ewoldt T.M.J., Hunfeld N.G.M., Muller A.E., Rietdijk W.J.R., Polinder S., van Gelder T., Endeman H., Koch B.C.P. The Effect of Therapeutic Drug Monitoring of Beta-Lactam and Fluoroquinolones on Clinical Outcome in Critically Ill Patients: The DOLPHIN Trial Protocol of a Multi-Centre Randomised Controlled Trial. BMC Infect. Dis. 2020;20:57. doi: 10.1186/s12879-020-4781-x. PubMed DOI PMC

Blondiaux N., Wallet F., Favory R., Onimus T., Nseir S., Courcol R.J., Durocher A., Roussel-Delvallez M. Daily Serum Piperacillin Monitoring Is Advisable in Critically Ill Patients. Int. J. Antimicrob. Agents. 2010;35:500–503. doi: 10.1016/j.ijantimicag.2010.01.018. PubMed DOI

Kurihara Y., Kizu J., Hori S. Simple and Rapid Determination of Serum Carbapenem Concentrations by High-Performance Liquid Chromatography. J. Infect. Chemother. 2008;14:30–34. doi: 10.1007/s10156-007-0576-X. PubMed DOI

Denooz R., Charlier C. Simultaneous Determination of Five β-Lactam Antibiotics (Cefepim, Ceftazidim, Cefuroxim, Meropenem and Piperacillin) in Human Plasma by High-Performance Liquid Chromatography with Ultraviolet Detection. J. Chromatogr. B. 2008;864:161–167. doi: 10.1016/j.jchromb.2008.01.037. PubMed DOI

Pinder N., Brenner T., Swoboda S., Weigand M.A., Hoppe-Tichy T. Therapeutic Drug Monitoring of Beta-Lactam Antibiotics—Influence of Sample Stability on the Analysis of Piperacillin, Meropenem, Ceftazidime and Flucloxacillin by HPLC-UV. J. Pharm. Biomed. Anal. 2017;143:86–93. doi: 10.1016/j.jpba.2017.05.037. PubMed DOI

Verhoven S.M., Groszek J.J., Fissell W.H., Seegmiller A., Colby J., Patel P., Verstraete A., Shotwell M. Therapeutic Drug Monitoring of Piperacillin and Tazobactam by RP-HPLC of Residual Blood Specimens. Clin. Chim. Acta. 2018;482:60–64. doi: 10.1016/j.cca.2018.03.021. PubMed DOI PMC

Gaikwad A., Gavali S., Narendiran, Katale D., Bonde S., Bhadane R.P. An LC–MS–MS Method for the Simultaneous Quantification of Amoxicillin and Clavulanic Acid in Human Plasma and Its Pharmacokinetic Application. J. Pharm. Res. 2013;6:804–812. doi: 10.1016/j.jopr.2013.07.019. DOI

Piestansky J., Cizmarova I., Mikus P., Parrak V., Babiak P., Secnik P., Secnik P., Kovac A. An Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Determination of 4 β-Lactam Antibiotics, Tazobactam, and Linezolid in Human Plasma Samples. Ther. Drug Monit. 2022;44:784–790. doi: 10.1097/FTD.0000000000001017. PubMed DOI

Colin P., De Bock L., T’jollyn H., Boussery K., Van Bocxlaer J. Development and Validation of a Fast and Uniform Approach to Quantify β-Lactam Antibiotics in Human Plasma by Solid Phase Extraction-Liquid Chromatography–Electrospray-Tandem Mass Spectrometry. Talanta. 2013;103:285–293. doi: 10.1016/j.talanta.2012.10.046. PubMed DOI

Paal M., Zoller M., Schuster C., Vogeser M., Schütze G. Simultaneous Quantification of Cefepime, Meropenem, Ciprofloxacin, Moxifloxacin, Linezolid and Piperacillin in Human Serum Using an Isotope-Dilution HPLC–MS/MS Method. J. Pharm. Biomed. Anal. 2018;152:102–110. doi: 10.1016/j.jpba.2018.01.031. PubMed DOI

Farid N.F., Abdelwahab N.S. New ecological method for determination of different β-lactams: Application to real human plasma samples. RSC Adv. 2019;9:19539–19548. doi: 10.1039/C9RA02671A. PubMed DOI PMC

Nishi H., Fukuyama T., Matsuo M. Separation and Determination of Aspoxicillin in Human Plasma by Micellar Electrokinetic Chromatography with Direct Sample Injection. J. Chromatogr. A. 1990;515:245–255. doi: 10.1016/S0021-9673(01)89319-5. PubMed DOI

Mrestani Y., Neubert R., Schiewe J., Härtl A. Application of Capillary Zone Electrophoresis in Cephalosporin Analysis. J. Chromatogr. B Biomed. Sci. Appl. 1997;690:321–326. doi: 10.1016/S0378-4347(96)00425-2. PubMed DOI

Castaneda Penalvo G., Kelly M., Maillols H., Fabre H. Evaluation of Capillary Zone Electrophoresis and Micellar Electrokinetic Capillary Chromatography with Direct Injection of Plasma for the Determination of Cefotaxime and Its Metabolite. Anal. Chem. 1997;69:1364–1369. doi: 10.1021/ac9605049. PubMed DOI

Mrestani Y., Neubert R., Nagel F. Capillary Zone Electrophoresis Determination of Meropenem in Biological Media Using a High Sensitivity Cell. J. Pharm. Biomed. Anal. 1999;20:899–903. doi: 10.1016/S0731-7085(99)00100-4. PubMed DOI

Mayer B.X., Hollenstein U., Brunner M., Eichler H.-G., Müller M. Micellar Electrokinetic Chromatography for the Analysis of Cefpirome in Microdialysis and Plasma Samples Obtainedin Vivo from Human Volunteers. Electrophoresis. 2000;21:1558–1564. doi: 10.1002/(SICI)1522-2683(20000501)21:8<1558::AID-ELPS1558>3.0.CO;2-N. PubMed DOI

Gáspár A., Kardos S., Andrási M., Klekner Á. Capillary Electrophoresis for the Direct Determination of Cephalosporins in Clinical Samples. Chromatographia. 2002;56:S109–S114. doi: 10.1007/BF02494122. DOI

Mayer B.X., Petsch M., Tschernko E.M., Müller M. Strategies for the Determination of Cefazolin in Plasma and Microdialysis Samples by Short-End Capillary Zone Electrophoresis. Electrophoresis. 2003;24:1215–1220. doi: 10.1002/elps.200390156. PubMed DOI

Kitahashi T., Furuta I. Determination of Meropenem by Capillary Electrophoresis Using Direct Injection of Serum. J. Chromatogr. Sci. 2005;43:430–433. doi: 10.1093/chromsci/43.8.430. PubMed DOI

Chou Y.-W., Yang Y.-H., Chen J.-H., Kuo C.-C., Chen S.-H. Quantification of Meropenem in Plasma and Cerebrospinal Fluid by Micellar Electrokinetic Capillary Chromatography and Application in Bacterial Meningitis Patients. J. Chromatogr. B. 2007;856:294–301. doi: 10.1016/j.jchromb.2007.06.015. PubMed DOI

Andrási M., Gáspár A., Klekner Á. Analysis of Cephalosporins in Bronchial Secretions by Capillary Electrophoresis after Simple Pretreatment. J. Chromatogr. B. 2007;846:355–358. doi: 10.1016/j.jchromb.2006.08.025. PubMed DOI

Solangi A., Mallah A., Memon S. Determination of Ceftriaxone, Ceftizoxime, Paracetamol, and Diclofenac Sodium by Capillary Zone Electrophoresis in Pharmaceutical Formulations and in Human Blood Serum. Turk. J. Chem. 2010;34:921–933. doi: 10.3906/kim-1005-628. DOI

Tůma P., Jaček M., Fejfarová V., Polák J. Electrophoretic Stacking for Sensitive Determination of Antibiotic Ceftazidime in Human Blood and Microdialysates from Diabetic Foot. Anal. Chim. Acta. 2016;942:139–145. doi: 10.1016/j.aca.2016.09.008. PubMed DOI

Šlampová A., Kubáň P. Micro-Electromembrane Extraction through Volatile Free Liquid Membrane for the Determination of β-Lactam Antibiotics in Biological and Environmental Samples. Talanta. 2023;252:123831. doi: 10.1016/j.talanta.2022.123831. PubMed DOI

Liang H.-H., Lin Y.-C., Hung C.-C., Hou Y.-C., Lin Y.-H. Method Development for Determination of Doripenem in Human Plasma via Capillary Electrophoresis Coupled with Field-Enhanced Sample Stacking and Sweeping. Int. J. Mol. Sci. 2023;24:13751. doi: 10.3390/ijms241813751. PubMed DOI PMC

Al-Attas A., Nasr J.J., El-Enany N., Belal F. A green capillary zone electrophoresis method for the simultaneous determination of piperacillin, tazobactam and cefepime in pharmaceutical formulations and human plasma. Biomed. Chromatogr. 2015;29:1811–1818. doi: 10.1002/bmc.3500. PubMed DOI

Hancu G., Neacsu A., Papp L.A., Ciurba A. Simultaneous determination of amoxicillin and clavulanic acid in pharmaceutical preparations by capillary zone electrophoresis. Bras. J. Pharm. Sci. 2016;52:281–286. doi: 10.1590/S1984-82502016000200006. DOI

Pham T.N.M., Le T.B., Le D.D., Ha T.H., Nguyen N.S., Pham T.D., Hauser P.C., Nguyen T.A.H., Mai T.D. Determination of Carbapenem Antibiotics Using a Purpose-Made Capillary Electrophoresis Instrument with Contactless Conductivity Detection. J. Pharm. Biomed. Anal. 2020;178:112906. doi: 10.1016/j.jpba.2019.112906. PubMed DOI

Tůma P., Jaček M., Sommerová B., Dlouhý P., Jarošíková R., Husáková J., Wosková V., Fejfarová V. Monitoring of Amoxicilline and Ceftazidime in the Microdialysate of Diabetic Foot and Serum by Capillary Electrophoresis with Contactless Conductivity Detection. Electrophoresis. 2022;43:1129–1139. doi: 10.1002/elps.202100366. PubMed DOI

Nguyen T.A.H., Pham T.N.M., Le T.B., Le D.C., Tran T.T.P., Nguyen T.Q.H., Nguyen T.K.T., Hauser P.C., Mai T.D. Cost-effective capillary electrophoresis with contactless conductivity detection for quality control of beta-lactam antibiotics. J. Chromatogr. A. 2019;1605:360356. doi: 10.1016/j.chroma.2019.07.010. PubMed DOI

Kanchi S., Sagrado S., Sabela M., Bisetty K., editors. Capillary Electrophoresis: Trends and Developments in Pharmaceutical Research. Routledge; Abingdon, UK: 2017.

Lefeuvre S., Bois-Maublanc J., Hocqueloux L., Bret L., Francia T., Eleout-Da Violante C., Billaud E.M., Barbier F., Got L. A Simple Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry Assay for the Simultaneous Quantification of 15 Antibiotics in Plasma. J. Chromatogr. B. 2017;1065–1066:50–58. doi: 10.1016/j.jchromb.2017.09.014. PubMed DOI

Radovanovic M., Day R.O., Jones G.D.R., Galettis P., Norris R.L.G. LC–MS/MS Method for Simultaneous Quantification of Ten Antibiotics in Human Plasma for Routine Therapeutic Drug Monitoring. J. Mass. Spectrom. Adv. Clin. Lab. 2022;26:48–59. doi: 10.1016/j.jmsacl.2022.11.001. PubMed DOI PMC

Straub R.F., Voyksner R.D. Determination of Penicillin G, Ampicillin, Amoxicillin, Cloxacillin and Cephapirin by High-Performance Liquid Chromatography—Electrospray Mass Spectrometry. J. Chromatogr. A. 1993;647:167–181. doi: 10.1016/0021-9673(93)83336-Q. PubMed DOI

Zaikin V.G., Borisov R.S. Options of the Main Derivatization Approaches for Analytical ESI and MALDI Mass Spectrometry. Crit. Rev. Anal. Chem. 2022;52:1287–1342. doi: 10.1080/10408347.2021.1873100. PubMed DOI

Klampfl C.W., Himmelsbach M. Sheath Liquids in CE-MS: Role, Parameters, and Optimization. In: de Jong G., editor. Capillary Electrophoresis-Mass Spectrometry (CE-MS): Principles and Applications. Wiley-VCH Verlag GmbH & Co. KGaA; Weinheim, Germany: 2016. pp. 41–65.

Carlier M., Stove V., Roberts J.A., Van De Velde E., De Waele J.J., Verstraete A.G. Quantification of Seven β-Lactam Antibiotics and Two β-Lactamase Inhibitors in Human Plasma Using a Validated UPLC-MS/MS Method. Int. J. Antimicrob. Agents. 2012;40:416–422. doi: 10.1016/j.ijantimicag.2012.06.022. PubMed DOI

Mandell G.L., Bennett J.E., Dolin R. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 7th ed. Churchill Livingstone, Elsevier; Philadelphia, PA, USA: 2010.

Enna S.J., Bylund D.B. xPharm: The Comprehensive Pharmacology Reference. Elsevier; Amsterdam, The Netherlands: 2008.

Dasgupta A., editor. Handbook of Drug Monitoring Methods: Therapeutics and Drugs of Abuse. Humana Press; Totowa, NJ, USA: 2008.

Sillén H., Mitchell R., Sleigh R., Mainwaring G., Catton K., Houghton R., Glendining K. Determination of Avibactam and Ceftazidime in Human Plasma Samples by LC–MS. Bioanalysis. 2015;7:1423–1434. doi: 10.4155/bio.15.76. PubMed DOI

Popowicz N.D., O’Halloran S.J., Fitzgerald D., Lee Y.C.G., Joyce D.A. A Rapid, LC-MS/MS Assay for Quantification of Piperacillin and Tazobactam in Human Plasma and Pleural Fluid; Application to a Clinical Pharmacokinetic Study. J. Chromatogr. B. 2018;1081–1082:58–66. doi: 10.1016/j.jchromb.2018.02.027. PubMed DOI

US Department of Health and Human Services Food and Drug Administration, Center for Drug; Evaluation and Research (CDER) Center for Veterinary Medicine (CVM). Bioanalytical Method Validation, Guidance for Industry. [(accessed on 28 February 2024)];2018 Available online: https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf.

Schulz M., Schmoldt A., Andresen-Streichert H., Iwersen-Bergmann S. Revisited: Therapeutic and Toxic Blood Concentrations of More than 1100 Drugs and Other Xenobiotics. Crit Care. 2020;24:195. doi: 10.1186/s13054-020-02915-5. PubMed DOI PMC

Manousi N., Wojnowski W., Płotka-Wasylka J., Samanidou V. Blue Applicability Grade Index (BAGI) and Software: A New Tool for the Evaluation of Method Practicality. Green Chem. 2023;25:7598–7604. doi: 10.1039/D3GC02347H. DOI

Pena-Pereira F., Wojnowski W., Tobiszewski M. AGREE—Analytical GREEnness Metric Approach and Software. Anal. Chem. 2020;92:10076–10082. doi: 10.1021/acs.analchem.0c01887. PubMed DOI PMC

Plotka-Wasylka J. A new tool for the evaluation of the analytical procedure: Green Analytical Procedure Index. Talanta. 2018;181:204–209. doi: 10.1016/j.talanta.2018.01.013. PubMed DOI