Validation of the HPLC method for the quantitative determination of sodium ampicillin for routine laboratory use

This study aims to validate a method for the quantification of sodium ampicillin by high-performance liquid chromatography (HPLC) for routine use in quality control laboratories. Validation was performed according to the recommendations of the International Conference on Harmonization (ICH Q2(R1)). The evaluated criteria included specificity, linearity, accuracy, and repeatability. Analyses were carried out using an Agilent Infinity 1260 HPLC system equipped with a DAD detector and a Microsorb-MV 100-5 C8 column (250 mm × 4.6 mm, 5 µm). The mobile phase consisted of a mixture of ethanol and ultrapure water (40:60, v/v) in isocratic mode, with a flow rate of 0.6 mL/min, an injection volume of 10 µL, and detection at 210 nm. The concentration range studied was from 60% to 140% (0.12 to 0.28 mg/mL) for both the standard and the reconstituted pharmaceutical form.
The method was specific; no interfering peaks were observed at the retention time of ampicillin (4.3 min). Linearity was excellent (R² = 0.9991 for the standard). The mean recovery percentages were 100.07% (standard) and 100.11% (reconstituted form), both within the acceptance range (80–120%). Repeatability yielded coefficients of variation (CV) of 0.13% for the standard and 0.28% for the reconstituted form at the target concentration (0.2 mg/mL), well below the 5% limit.
The developed HPLC method is specific, linear, accurate, and reliable. It can be used as a routine method for the assay of sodium ampicillin in injectable pharmaceutical forms in laboratory settings.