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. 2025 Jul 2;18(7):998.
doi: 10.3390/ph18070998.

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

Sangyoung Lee  1 , Da Hyun Kim  1 , Sabin Shin  1 , Jee Sun Min  1 , Duk Yeon Kim  1 , Seong Jun Jo  1   2 , Ui Min Jerng  3 , Soo Kyung Bae  1
Affiliations

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

Sangyoung Lee et al. Pharmaceuticals (Basel). .

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.

Keywords: LC–MS/MS; amoxicillin; clavulanate; human K2-EDTA plasma; pharmacokinetics; validation.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Chemical structures and product ion mass spectra of (a) amoxicillin, (b) clavulanate, and (c) 4-hydorxytolbutamide (the IS). The arrows indicate the selected product ion values (m/z) for each analyte.
Figure 2
Figure 2
Typical LC–MS/MS chromatograms of (i) amoxicillin, (ii) clavulanate, and (iii) the IS: (a) blank plasma, (b) blank plasma spiked with both analytes at LLOQs (10/20 ng/mL for amoxicillin/clavulanate) and IS, and (c) a plasma sample obtained from a volunteer 2 h after a single oral dose of Augmentin® (250/125 mg) IR tablet.
Figure 3
Figure 3
Mean plasma concentration—time profiles of (a) amoxicillin (●) and (b) clavulanate (○) after a single oral administration of Augmentin® IR tablet (250/125 mg) in healthy Korean subjects (n = 17). Vertical bars represent standard deviation.
Figure 4
Figure 4
The AGREE pictogram for this bioanalytical method greenness representation.
See this image and copyright information in PMC

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