Development and Validation of Bioanalytical LC–MS/MS Method for Pharmacokinetic Assessment of Amoxicillin and Clavulanate in Human Plasma
<b>Background/Objectives</b>: 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. <b>Methods</b>: Amoxicillin; clavulanate; and an internal s...
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| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-07-01
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| Series: | Pharmaceuticals |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1424-8247/18/7/998 |
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| Summary: | <b>Background/Objectives</b>: 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. <b>Methods</b>: Amoxicillin; clavulanate; and an internal standard, 4-hydroxytolbutamide, in human K<sub>2</sub>-EDTA plasma, were deproteinized with acetonitrile and then subjected to back-extraction using distilled water–dichloromethane. Separation was performed on a Poroshell 120 EC-C<sub>18</sub> 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 <i>m</i>/<i>z</i> 363.9→223.1 (amoxicillin), <i>m</i>/<i>z</i> 198.0→135.8 (clavulanate), and <i>m</i>/<i>z</i> 285.0→185.8 (4-hydroxytolbutamide). <b>Results/Conclusions</b>: Calibration curves exhibited linear ranges of 10–15,000 ng/mL for amoxicillin (<i>r</i> ≥ 0.9945) and 20–10,000 ng/mL for clavulanate (<i>r</i> ≥ 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 K<sub>3</sub>-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<sup>®</sup> IR tablet (250/125 mg). The inter-individual variabilities in clavulanate exposures (AUC<sub>t</sub> and C<sub>max</sub>) were significantly greater than in amoxicillin, and they may inform the clinical design of future drug–drug interaction. |
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| ISSN: | 1424-8247 |