Quantification of Cefepime, Meropenem, Piperacillin, and Tazobactam in Human Plasma Using a Sensitive and Robust Liquid Chromatography-Tandem Mass Spectrometry Method, Part 1: Assay Development and Validation

Abstract

The highly variable pharmacokinetics of β-lactam antibiotics and β-lactamase inhibitors poses a significant challenge to clinicians in ensuring appropriate antibiotic doses in critically ill patients. Therefore, routine monitoring of plasma concentrations is important for individualization of antimicrobial therapy. Accordingly, a simple and robust analytical method for the simultaneous measurement of multiple β-lactam antibiotics and β-lactamase inhibitors is highly desirable to ensure quick decisions on dose adjustments. In this study, a sensitive, simple, and robust method for the simultaneous quantification of cefepime, meropenem, piperacillin, and tazobactam in human plasma was developed and rigorously validated according to FDA guidance. Sample extraction was accomplished by simple protein precipitation. Chromatographic separation of analytes was achieved using stepwise gradient elution. Analytes were monitored using tandem mass spectrometry (MS/MS) with a turbo ion spray source in positive multiple-reaction-monitoring mode. The calibration curve ranged from 0.5 to 150 μg/ml for cefepime, 0.1 to 150 μg/ml for meropenem and piperacillin, and 0.25 to 150 μg/ml for tazobactam. Inter- and intraday precision and accuracy, sensitivity, selectivity, dilution integrity, matrix effect, extraction recovery, and hemolysis effect were investigated for all four analytes, and the results met the acceptance criteria. Compared to other reported methods, our method is more robust because of the combination of the following features: (i) a simple sample extraction procedure, (ii) a short sample run time, (iii) a wide dynamic range, and (iv) the small plasma sample volume needed. Since our method already covers β-lactams and a β-lactamase inhibitor with highly heterogeneous physicochemical properties, further antibiotic candidates may easily be incorporated into this multianalyte method.

Quantification of Cefepime, Meropenem, Piperacillin, and Tazobactam in Human Plasma Using a Sensitive and Robust Liquid Chromatography-Tandem Mass Spectrometry Method, Part 2: Stability Evaluation

“…Detailed MS parameters that gave the best sensitivities for detecting the analytes of interest are specified in part 1 of our work, entitled "Quantification of Cefepime, Meropenem, Piperacillin, and Tazobactam in Human Plasma by Using a Sensitive and Robust Liquid Chromatography-Tandem Mass Spectrometry Method, Part 1: Assay Development and Validation" (17).…”

“…Because the stability data for ␤-lactam antibiotics are highly useful, instead of burying these data in a general assay development and validation manuscript, we chose to highlight the stability results in the present, stand-alone article (part 2 of our work). The LC-MS/MS method development and non-stability-related assay validation work is presented in a separate article (17).…”

A validated LC-MSMS method for the simultaneous quantification of meropenem and vaborbactam in human plasma and renal replacement therapy effluent and its application to a pharmacokinetic study

“…Methodology to quantify drugs in biological fluids is important to support pre-clinical and clinical studies. Chromatographic methods have been reported for meropenem in plasma [10][11][12][13][14][15][16][17][18][19][20][21][22][23] and serum [24][25][26][27] using UV [12,16,26], diode array [15,20,28,29], and mass spectrometry [10, 11, 13, 14, 17-19, 21-25, 27, 30] detection. To the best of our knowledge, there are currently no analytical methods reporting the quantification of the combination of meropenem-vaborbactam in biological fluids and no methods for the quantification of newly developed β-lactamase inhibitor, vaborbactam.…”

A sensitive and high‐throughput quantitative liquid chromatography high‐resolution mass spectrometry method for therapeutic drug monitoring of 10 β‐lactam antibiotics, linezolid and two β‐lactamase inhibitors in human plasma

“…In parallel with this increased interest in optimizing dosing of β-lactam antibiotics through TDM and/or pharmacokinetic studies, there has been a growing effort to develop analytical methods that are suitable for this (Bailón-Pérez, García-Campaña, del Olmo-Iruela, Gámiz-Gracia, & Cruces-Blanco, 2009;Bruck, Roberts, Roberts, & Robertson, 2014; Cohen-wolkowiez, White, Bridges, Benjamin, & Kashuba, 2011;D'Cunha et al, 2018;Dubala, Sankarachari, Nagarajan, Vimal, & George, 2013;El-Najjar, Hösl, Holzmann, Jantsch, & Gessner, 2018;Fagerquist, Lightfield, & Lehotay, 2005;Ferrari et al, 2019;Huang, Gao, & Miao, 2012;Khuroo, Monif, Verma, & Gurule, 2008;Lara, del Olmo-Iruela, Cruces-Blanco, Quesada-Molina, & García-Campaña, 2012;Larmené-Beld, Vries-Koenjer, ter Horst, & Hospes, 2014;Lefeuvre et al, 2017;Mameli et al, 2019;Martenslobenhoffer & Bode-böger, 2017;Neugebauer, Wichmann, Bremer-Streck, Hagel, & Kiehntopf, 2019;Ohmori, Suzuki, Niwa, Ushikoshi, & Shirai, 2011;Parker et al, 2019;Partani, Gurule, Khuroo, Monif, & Bhardwaj, 2010;Rigo-Bonnin et al, 2017;Souza, Felton, Crass, Hanaya, & Pai, 2020;Szultka, Krzeminski, Szeliga, Jackowski, & Buszewski, 2013). However, very few analytical methods comply with the high number of stringent requirements that are indispensable for efficient TDM.…”

“…Because of its high specificity, HPLC coupled to mass spectrometry has been successfully applied for the fast quantification of antibiotics. Unfortunately, most of these methods can only analyze a limited number of antibiotics simultaneously in human plasma (Cohen-wolkowiez, White, Bridges, Benjamin, & Kashuba, 2011;D'Cunha et al, 2018;Ferrari et al, 2019;Mameli et al, 2019;Martens-lobenhoffer & Bode-böger, 2017;Mortensen, Jensen, Zhang, & Doogue, 2019;Parker et al, 2019;Partani, Gurule, Khuroo, Monif, & Bhardwaj, 2010;Souza, Felton, Crass, Hanaya, & Pai, 2020). In order to implement TDM in daily clinical laboratory practice, one method that can simultaneously analyze all relevant β-lactam antibiotics and their β-lactamase inhibitors used in critical care is highly desirable.…”