Validation of a method for determination of ampicillin in human plasma using LC-DAD

Abstract

We describe the validation data of a simple but selective chromatographic method for determination of ampicillin in human plasma using liquid chromatography-diode array detector. Blank plasma free of drugs was transferred to eppendorf's tubes and spiked with ampicillin stock solution to obtain quality control samples at 1.00, 2.50, 5.00, and 10.00 μg/mL. Extraction of ampicillin and cephalexin (internal standard) from plasma samples (250 μL) was investigated using three different methods: precipitation with perchloric acid, ultra-filtration and solid-phase extraction. Chromatographic separation was achieved using a Shimpak C18 column (300 mm × 4.6 mm i.d.; 5 μm), and detection was done at 215 nm with a diode array UV-Vis detector. The mobile phase consisted of dihydrogen phosphate (pH 3.5)-acetonitrile (87.5:12.5, v/v) delivered at a flow rate of 1.00 mL/min. Selectivity was evaluated with different pools of human plasma. Perchloric acid precipitation showed an excellent selectivity for normal plasma. The precipitation method presented recoveries above 84.0 ± 3.3% and 82.0 ± 1.6%, (n = 3) for ampicillin and cephalexin, respectively. The method has a limit of detection of 0.15 μg/mL and is linear in the range of 0.30 to 100.00 μg/mL. Standardized residue analysis demonstrated normality and homocedasticity. Inter-day precision was 4.5%, and accuracy was 11.1% (n = 9). Stability studies demonstrated instability of β-lactamics in human plasma at 20 and 2°C after 6 and 360 h of storage, respectively.