Elsevier

Journal of Chromatography B

Volume 1159, 30 November 2020, 122354
Journal of Chromatography B

Development and validation of a quantitative LC-MS/MS method for the simultaneous determination of ceftolozane and tazobactam in human plasma and urine

https://doi.org/10.1016/j.jchromb.2020.122354Get rights and content

Highlights

  • First LC-MS/MS method to simultaneously analyze ceftolozane and tazobactam.

  • Most sensitive ceftolozane and tazobactam LC-MS/MS method.

  • The method is rapid with a simple extraction procedure.

  • The method was validated per US FDA regulatory guidelines.

Abstract

The purpose of this work was to develop and validate a single sensitive, selective and rapid bioanalytical method to determine ceftolozane and tazobactam concentrations in human plasma and urine and to use this method to analyze samples from a human clinical study. Human plasma and urine samples were prepared by protein precipitation using a solution of acetonitrile, water and formic acid. Following protein precipitation, samples were analyzed by liquid chromatography tandem mass spectrometry. Chromatographic resolution was achieved on a Kinetex PFP column using a gradient elution, a flow rate of 0.4 mL/min, and a total run time of 5 min. Positive electrospray ionization was employed and analytes were quantitated using multi-reaction monitoring mode. Method validation was conducted in accordance with Unites States Food and Drug Administration’s regulatory guidelines for bioanalytical method validation. Calibration curves were determined to linear over the range of 0.1 to 40 µg/mL for ceftolozane and 0.05 to 20 µg/mL for tazobactam. The method was determined to be accurate (−6.24 to 12.53 percent relative error), precise (less than 13.28 percent standard deviation) and sensitive in both human plasma and urine. Ceftolozane and tazobactam were determined to be stable across a battery of stability studies including autosampler, benchtop, freeze/thaw and long-term stability. This validated method successfully applied to human clinical samples to determine the concentration versus time profiles of the intravenously administered combination of Zerbaxa (ceftolozane-tazobactam) in burn patients.

Introduction

The novel combination of ceftolozane (CEF) and tazobactam (TAZ) was initially approved by the United States (US) Food and Drug Administration (FDA) in 2014 as an injectable treatment for emerging multi-drug resistant pathogens under the brand name Zerbaxa. This novel antibiotic combination of CEF and TAZ formulated in a two-to-one ratio of CEF to TAZ (CEF/TAZ) has been shown to be active against several multidrug-resistant gram-negative bacilli (Fig. 1). CEF is a fifth-generation cephalosporin and TAZ is a β-lactamase inhibitor. Mechanistically, CEF is a cephalosporin β-lactam that exhibits its bactericidal properties by inhibiting biosynthesis of the bacterial cell wall, which is mediated by penicillin-binding proteins [1], [2], [3], [4]. TAZ by itself does not have any clinically significant activity against bacteria; however, it protects CEF from hydrolysis by irreversibly binding to β-lactamase enzymes produced by extended-spectrum β-lactamase-producing Enterobacteriaceae and certain anaerobes thereby enhancing the activity of CEF [5]. Combinations of CEF and TAZ have been used for treating complicated intra-abdominal infections in combination with metronidazole [6], [7]. The combination of CEF and TAZ has been proven to have superior efficacy to levofloxacin in treating complicated urinary tract infections, including pyelonephritis [8], [9]. In 2019, FDA expanded the indications of Zerbaxa for the treatment of hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia in patients 18 years and older [10]. Off-label uses for CEF and TAZ include skin and soft-tissue infections, bone and joint infections, bloodstream infections, and infections caused by multidrug-resistant and extensively drug-resistant strains of Pseudomonas aeruginosa [11], [12].

Patients with burns represent a unique special population where CEF/TAZ has been used due to the increased likelihood of multidrug-resistant gram-negative pathogens [13]; however, the optimal dosing of CEF/TAZ is unknown as the pharmacokinetics of CEF/TAZ has not been studied in burn patients. It is known that patients with significant burns can have altered physiology including reduced plasma volume, cardiac output and urine output as well as increased systemic vascular resistance (SVR) with resultant reduced peripheral blood flow [14]. Altered systemic drug concentrations due to these changes could impact the drug’s safety and/or effectiveness in this patient population. A dose adjustment of CEF/TAZ in burn patients may be necessary to achieve effective similar concentrations to previously published pharmacokinetic data in volunteers and patients without burns. A human clinical pharmacokinetic study of CEF/TAZ in burn patients is being conducted to provide this needed information to guide design of optimal dosing for CEF/TAZ. In order to understand the concentration versus time profiles of burn patients a sensitive, accurate and reproducible analytical method for determining concentrations of CEF and TAZ in human plasma and urine has been developed.

There are multiple accounts of HPLC-UV methods for the determination of CEF and TAZ in human plasma [15], [16], [17]; however, there are only limited accounts of liquid chromatography tandem mass spectrometric methods (LC-MS/MS) for analysis of CEF and TAZ [18], [19], [20], [21], [22], [23], [24]. There is only one published account of a validated LC-MS/MS method for the determination of CEF and TAZ in human plasma from Raúl Rigo-Bonnin and co-workers [24]. In this method, CEF was detected in positive polarity mode and TAZ was detected in negative polarity mode which required the same sample to be injected twice. There is currently no validated LC-MS/MS method available for simultaneous determination of CEF and TAZ in a single analysis.

The purpose of this study was to develop and validate a LC-MS/MS method for simultaneous analysis of CEF and TAZ in a single run in either human plasma or human urine matrix. The developed method was used to quantitate plasma and urine concentrations of CEF and TAZ in patients with burns to enable the study of concentration versus time profiles of these drugs in this patient population.

Section snippets

Chemicals and materials

Acetonitrile (Optima LC-MS grade), water (Optima LC-MS grade), and formic acid (Optima LC-MS grade) were purchased from Fisher Scientific (Hampton, NH). Ceftolozane (CEF, 98.6% purity) and tazobactam (TAZ, 99.3% purity) were obtained from MicroConstants (San Diego, CA). Cefepime (CP, 93.4% purity) was purchased from Sigma Aldrich (Laramie, WY). Human plasma for blank matrix was procured from BioIVT (Westbury, NY) and human urine blanks were collected in-house. All chemicals and reagents were

Method development

Method development started with the objective of developing a method for simultaneous quantitative determination of CEF and TAZ in human plasma or urine using LC-MS/MS. Mass spectrometer parameters were optimized for CEF and TAZ using 500 ng/mL solutions of the analytes in water-acetonitrile-formic acid (50:50:0.1, v/v/v) (Fig. 2). Tuning was performed in both positive and negative mode ionizations. It was noted that CEF produces stronger and more reproducible ions in positive ion mode. TAZ

Discussion

A quantitative method was required for the rapid and sensitive determination of CEF and TAZ in human plasma and urine. Method development was initiated using CEF and TAZ solutions made at 0.5 µg/mL in acetonitrile–water (50:50, v/v) with 0.1% formic acid to determine the compound dependent parameters and MRM transition pairs (precursor to product ion) for CEF and TAZ. These compounds were analyzed both in positive and negative modes. TAZ ionizes well in both positive and negative modes but CEF

CRediT authorship contribution statement

William C. Putnam: Conceptualization, Methodology, Writing - original draft, Writing - review & editing. Raja Reddy Kallem: Methodology, Writing - original draft, Writing - review & editing, Data curation, Validation. Vindhya Edpuganti: Methodology, Writing - original draft, Data curation, Validation. Indhu Subramaniyan: Methodology, Writing - original draft, Data curation, Validation. Ronald G. Hall: Conceptualization, Methodology, Writing - review & editing.

Declaration of Competing Interest

RGH receives grant funding from Merck. The authors declare no other competing financial interest.

Acknowledgements

This study was partially supported by the Merck Investigator Studies Program, Merck Research Laboratories (to R.G.H.). The Merck Investigator Studies Program, Merck Research Laboratories is also acknowledged for the ceftolozane and tazobactam analytical standards.

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