Validation and application of a reversed-phase Hplc method for the determination of amoxicillin tri hydrate in human plasma

A specific rapid and sensitive high performance liquid chromatographic (HPLC) method with UV detection was developed and validated for the determination of amoxicillin in human plasma and cefadroxil was used as the internal standard. The separation was carried out using waters symmetry C18, (5mm, 150×3.9 mm) column with a mobile phase consisting of potassium phosphate buffer and acetonitirile. The analytical method was validated and the relationship between analyte concentration and peak area ratio was linear within the range 1 - 80μg/ml. The LOD and LLOQ for amoxicillin in plasma were 0.080μg/ml and 0.16 μg/ml respectively. Intra-day accuracy of amoxicillin in plasma ranged from 103.00 ± 8.70% to 109.82 ±1.39% while inter-day accuracy was between 102.00± 0.98% and 110.30 ±0.36%. Amoxicillin and cefadroxil were found to be stable in human plasma samples for 24 hours at room temperature (short-term stability) and for 14 days at -20°C (long-term stability). The developed method of analysis provided a sensitive and specific assay for amoxicillin in human plasma and thus, it was applied to pharmacokinetic studies of amoxicillin hydrochloride tablets in healthy volunteers in Yemen.