Development and Validation of Bioanalytical LC–MS/MS Method for Pharmacokinetic Assessment of Amoxicillin and Clavulanate in Human Plasma

TL;DR

Researchers developed a new LC–MS/MS method to measure amoxicillin and clavulanate in human plasma, which is more efficient and environmentally friendly than previous methods.

Contribution

The novel contribution is a validated, environmentally friendly LC–MS/MS method for simultaneous quantification of amoxicillin and clavulanate in plasma.

Findings

The method showed linear calibration ranges of 10–15,000 ng/mL for amoxicillin and 20–10,000 ng/mL for clavulanate.

Intra- and inter-day precision was ≤7.08% for amoxicillin and ≤10.7% for clavulanate.

Clavulanate showed greater inter-individual variability in exposure compared to amoxicillin.

Abstract

Background/Objectives: We developed and validated a robust and simple LC–MS/MS method for the simultaneous quantification of amoxicillin and clavulanate in human plasma relative to previously reported methods. Methods: Amoxicillin; clavulanate; and an internal standard, 4-hydroxytolbutamide, in human K2-EDTA plasma, were deproteinized with acetonitrile and then subjected to back-extraction using distilled water–dichloromethane. Separation was performed on a Poroshell 120 EC-C18 column with a mobile-phase gradient comprising 0.1% aqueous formic acid and acetonitrile at a flow rate of 0.5 mL/min within 6.5 min. The negative electrospray ionization modes were utilized to monitor the transitions of m/z 363.9→223.1 (amoxicillin), m/z 198.0→135.8 (clavulanate), and m/z 285.0→185.8 (4-hydroxytolbutamide). Results/Conclusions: Calibration curves exhibited linear ranges of 10–15,000 ng/mL for amoxicillin (r ≥ 0.9945) and 20–10,000 ng/mL for clavulanate (r ≥ 0.9959). Intra- and inter-day’s coefficients of variation, indicating the precision of the assay, were ≤7.08% for amoxicillin and ≤10.7% for clavulanate, and relative errors in accuracy ranged from −1.26% to 10.9% for amoxicillin and from −4.41% to 8.73% for clavulanate. All other validation results met regulatory criteria. Partial validation in lithium–heparin, sodium–heparin, and K3-EDTA plasma confirmed applicability in multicenter or large-scale studies. This assay demonstrated itself to be environmentally friendly, as assessed by the Analytical GREEnness (AGREE) tool, and was successfully applied to a clinical pharmacokinetic study of an Augmentin® IR tablet (250/125 mg). The inter-individual variabilities in clavulanate exposures (AUCt and Cmax) were significantly greater than in amoxicillin, and they may inform the clinical design of future drug–drug interaction.

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