Assay of amikacin in the skin by high-performance liquid chromatography

Amikacin is used in the systemic treatment of serious infections, but also locally for the treatment of skin infections. The aim of this work was to develop and validate a simple procedure for amikacin determination inside the epidermal tissue: this implies a simple method for an efficient drug extraction from the skin and a clean and easy HPLC analysis. Amikacin was extracted from epidermis samples with 500 μl of a mixture methanol–water–0.05 M NaOH (5:5:2 v/v/v) at 60 °C for 1 h. After filtration, the obtained solution was derivatized (1-fluoro-2,4-dinitrobenzene at 90 °C for 10 min) and analyzed by HPLC, on a C18 μBondapack 300 mm × 4.6 mm column thermostatted at 45 °C. The mobile phase was a mixture of acetonitrile–water–acetic acid (47:53:0.1 v/v/v) at a flow rate of 1.5 ml/min and the UV detector was set at 365 nm. The derivatization and HPLC analysis were validated in the concentration interval 1.64–49.21 μg/ml. The linearity resulted very good (R = 0.9995); the R.S.D.% varied between 0.20% and 3.89% depending on the concentration and the ER% was included between 5.4 and 0.9. The extraction method used demonstrated to be specific and the recovery resulted about 93%. The extraction, derivatization and HPLC assay has good reproducibility, sensitivity and specificity resulting in a reliable method for biopharmaceutical studies of AK distribution in the epidermis.