A multiresidue method for the simultaneous determination of ten macrolide antibiotics in human urine based on gradient elution liquid chromatography coupled to coulometric detection (HPLC–ECD)]

A new generation of coulometric detector coupled to a high performance liquid chromatograph (HPLC) was used in this work with the aim of developing a multiresidue method allowing for the first time the separation, in a single run, of ten commercially available macrolide antibiotics, erythromycin (ERY), dirithromycin (DIR), tylosin (TYL), tilmicosin (TILM), spiramycin (SPI), josamycin (JOS), kitasamycin (KIT), rosamicin (ROS), roxithromycin (ROX) and oleandomycin (OLE). The procedure was therefore fully optimised for the separation of standard mixtures of the ten macrolides using a gradient elution on a C8 reversed phase HPLC column, and very reasonable resolution was achieved among the thirteen signals, arising from these ten. In the following step, the quantification of all the macrolides was achieved in standard mixtures, using ROX as an internal standard (I.S.), with detection limits, on column, below 2.5 ng for each. The method was then applied to the determination of macrolides in human urine, after a simple and fast sample preparation procedure involving liquid–liquid extraction. Calibration curves were carried out using the method of matrix-matching. The detection limits for each drug, in the human urine, were below 3 ng injected (equivalent to an initial concentration in the urine below 0.06 mg l^-1). Overall recoveries of the entire method were estimated for each drug in human urine fortified at different levels of concentration within the linear range and averaged values were between 49.9 and 81.2% for low concentration levels and between 53.2 and 92.0% for high concentration levels for all macrolides.