Determination of etimicin in rat plasma using 9-fluorenylmethyl chloroformate precolumn derivatization by HPLC with fluorescence detection

A new and sensitive method was developed for the determination of the etimicin (ETM) using precolumn derivatization (PCD) with 9-fluorenylmethyl chloroformate (FMOC-Cl) by a reversed phase separation and subsequent fluorescence detection. The PCD conditions were fully optimized towards the lowest limit of detection. The chromatographic separation was carried out on an Agilent XDB-C8 column at 25 ºC using a constant flow rate of 1.0 mL min-1 and mobile phase of acetonitrile/water (87:13, v/v). The etimicin-FMOC-Cl derivative was monitored by fluorescent detection at λexcitation 265 nm and λemission 315 nm. Neomycin (NMC), a similar base structure compound with ETM, was used as an internal standard. The statistical evaluation of the method was examined and the method was found to be precise and accurate with a linearity range of 0.038-9.69 µg mL-1 (r > 0.9994). The intra- and inter-day precision studies showed good reproducibility with relative standard deviation (R.S.D.) less than 5%, and the relative recovery was 97.80-100.09 % (n = 3). The limit of detection (LOD) and lower limit of quantification (LLOQ) in plasma were 0.01 and 0.02 µg mL-1, respectively. A volume of 50 µL of plasma was sufficient for the determination of etimicin. The established method provides a reliable bioanalytical method to carry out etimicin pharmacokinetics in rat plasma.