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

Abstract

An ultra-high pressure liquid chromatography high-resolution mass spectrometric (UHPLC-HRMS) method was developed for the simultaneous and sensitive quantification of 10 β-lactam antibiotics (cefepime, meropenem, amoxicillin, cefazolin, benzylpenicillin, ceftazidime, piperacillin, flucloxacillin, cefuroxime and aztreonam), linezolid and β-lactamase inhibitors tazobactam and clavulanic acid in human plasma. Validation according to the EMA guidelines showed excellent withinand between-run accuracy and precision (i.e. between 1.1 and 8.5%) and high sensitivity (i.e. lower limit of quantification between 0.25 and 1 mg/L). The UHPLC-HRMS method enables a short turnaround time and high sensitivity and needs only a small amount of plasma, allowing appropriate routine therapeutic drug monitoring.The short turnaround time is obtained by speeding up the protocol on multiple levels, i.e. fast and workload-efficient sample preparation (i.e. protein precipitation and dilution), short (4 min) instrument run time, simultaneous measurement of all relevant β-lactam antibiotics used in the intensive care unit and the use of the same instrument, column and mobile phases as for the other routine methods in our laboratory. K E Y W O R D Sβ-lactam antibiotics, HRMS, therapeutic drug monitoring | INTRODUCTIONSevere infection and associated septic shock persist as significant healthcare concerns as they are the most common cause of mortality in critically ill patients worldwide (Vincent et al., 2009). Since adequate and early administration of antibiotics remains the mainstay of therapy for the treatment of severe infections, there is a high level of consumption of antibiotics in intensive care units (ICU) (Dellinger et al., 2013). A single-day point prevalence study showed that 71% of the ICU patients received antimicrobial therapy (Vincent et al., 2009). However, in specific patient populations like critically ill patients, cystic fibrosis patients, elderly patients, neonates, etc., the pharmacokinetics of these antibiotics may be profoundly disturbed owing to pathophysiological changes in distribution and elimination (Roberts et al., 2014;Taccone et al., 2010;Theuretzbacher, 2012). Consequently, an empirical fixed dose approach, in which every patient is treated according to the same dosing scheme, appears to be inadequate and can lead to excessive drug exposure with an increased risk of toxicity or inadequate drug exposure with clinical failure and/or antimicrobial resistance (Roberts & Lipman, 2006). Optimizing antibiotic dosing may be a key intervention, not only to improve clinical outcome and minimize toxicity, but also to prolong the clinical lifespan of the currently available antibiotics by limiting the emergence of

Therapeutic Drug Monitoring of Antibiotic Drugs: The Role of the Clinical Laboratory

“…By contrast, in other cases, a short chromatographic run is connected to 2 alternatives for detection (either in the positive or in the negative ionization mode) to establish a compromise between the analytical TAT and numerous analytes. 55,81,87 Some procedures have been validated using alternative instrumental conditions with a separate analytical run for the most frequently requested analyses, along with a different run upon application of the same chromatography column and mobile phases if quantification of additional substances is needed. 58 Moreover, there are strong efforts to implement the analysis of antibiotic concentrations in a much more widely consolidated net of analytical procedures, including the measurement of other drug classes or even other clinical chemistry parameters (eg, vitamins and hormones).…”

“…88 Newer developments strive to automate sample pretreatment and use high-resolution mass spectrometry (HRMS) instead of the more traditional MS/MS detection approach. [55][56][57] HRMS affords advantages such as full-scan acquisition mode, allowing a retrospective analysis of samples to detect degradation products or metabolites and simplified method development without the need for molecule-specific tuning to explore the fragmentation spectrum. This alternative is particularly attractive if routine laboratory requests accompany research projects.…”

“…By contrast, in other cases, a short chromatographic run is connected to 2 alternatives for detection (either in the positive or in the negative ionization mode) to establish a compromise between the analytical TAT and numerous analytes. 55,81,87 Some procedures have been validated using alternative instrumental conditions with a separate analytical run for the most frequently requested analyses, along with a different run upon application of the same chromatography column and mobile phases if quantification of additional substances is needed. 58…”

Rapid Detection of Multiple Classes of β-Lactam Antibiotics in Blood Using an NDM-1 Biosensing Assay

“…Subsequently, 28 amino acids were removed from the N-terminus and an N-terminal His-6-tag was added to the NDM-1 gene using PCR, and the product was inserted into the expression vector pET-28a (Novagen). The expression and purification of recombinant NDM-1(rNDM-1) were performed as previously described except that the His-6 tag was not removed from the N-terminus of the recombinant enzyme [18]. The purity of rNDM-1 was determined by SDS-PAGE.…”

“…Unlike routine monitoring of aminoglycoside and glycopeptide antibiotics using commercial assays, the methods used to monitor β-lactam antibiotics have been largely developed in-house [16]. Among these, liquid chromatographic (LC) or mass spectrometric (MS) detection are the most widely used methods for determining β-lactam antibiotic concentrations in serum or plasma [17,18]. The advantages of these methods include good detection accuracy/stability, wide detection range and simultaneous detection of multiple β-lactam antibiotics/β-lactamase inhibitors (BLIs).…”