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

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 K(2)-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(18) 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 K(3)-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 (AUC(t) and C(max)) were significantly greater than in amoxicillin, and they may inform the clinical design of future drug-drug interaction.