MSACL 2023 Abstract

Introduction:
β-Lactam are among the most widely used class of drugs for the treatment of bacterial infections in humans. Most β-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism. Bacteria often develop resistance to these antibiotics by synthesizing a β-lactamase, an enzyme that attacks the β-lactam ring common to this class of antibiotic. To overcome this resistance, β-lactam antibiotics can be given with β-lactamase inhibitors such as clavulanic acid. The antibacterial characteristics the drugs display are dependent on both the concentration of drug in relation to the minimum inhibitory concentration (MIC) and the time that this exposure is maintained. Therefore, monitoring their concentrations in plasma is of high importance.

In this study, a fast LC-MS/MS method with a simple sample preparation on the QTRAP 4500 system is described for the quantitative analysis of nine β-lactams antibiotics, amoxicillin (AMO), cloxacillin (CLO), piperacillin (PIP), cephazolin (CEP), cefotaxime (CEFO), ceftazidime (CEFT) and cefepime (CEFE), imipenem (IMI) and meropenem (MER).

Methods:
A 100 μL aliquot of each sample, calibrator or QC was spiked with 10 μL of internal standard mix at 100 μg/mL. Protein precipitation was carried out by the addition of 200 μL of methanol containing 0.1% formic acid. Following vortex mixing, the samples were centrifuged for 10 minutes before the supernatant was transferred to autosampler vials for injection. Chromatographic separation was accomplished using a Phenomenex Kinetex Biphenyl column (100 x 2.1 mm, 2.6 μm). Water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B) were used as mobile phase solvents. A 1 μL aliquot of the sample was injected into the UHPLC system. MS/MS detection was performed using the SCIEX QTRAP 4500 system equipped with Turbo V ion source using electrospray ionization, operating in positive mode. Multiple reaction monitoring (MRM) mode was employed, using two specific transitions of each analyte.

Results:
Calibration curves over the concentration ranges were between 1 and 150 μg/mL for all analytes except imipenem (0.05 to 7.5 μg/mL) and meropenem (0.1 to 15 μg/mL). Curves were generated using a 1/x weighted linear regression of the peak-area ratios of the antibiotic to corresponding internal standard. Regression coefficients of all calibration curves were greater than 0.99 with back-calculated concentrations of the calibration samples within ±15% (±20% at LLOQ) of the nominal concentrations. The recorded accuracy and precision were within European Medicines Agency guidelines. The application of this assay for nine β-lactams antibiotics was then used to analyze plasma samples from the Hospital of Mulhouse Antibiotic Therapeutic Drug Monitoring (TDM) program and compared to alternative methods for the analysis of these compounds.

Conclusions:
The QTRAP 4500 system was used for the quantitative analysis of nine β-lactams antibiotics, amoxicillin (AMO), cloxacillin (CLO), piperacillin (PIP), cephazolin (CEP), cefotaxime (CEFO), ceftazidime (CEFT) and cefepime (CEFE), imipenem (IMI) and meropenem (MER). Sensitivity was shown to be 1 μg/mL for all analytes except imipenem (0.05 μg/mL) and meropenem (0.1 μg/mL) in plasma.