Chromatographic determinations of a β-lactam antibiotic, cefaclor by means of fluorescence, chemiluminescence and mass spectrometry

We report analytical informations on the quantification of cefaclor (CCL), a β-lactam antibiotic by three detection methods. The methods were based on the chemical derivatization of the drug with 4-(2′-cyanoisoindolyl)phenylisothiocyanate (CIPIC) under the reaction conditions with heating at 80 °C for 7 min in the presence of pyridine. The CIPIC reagent could react with the primary amino group of the drug to form CIPIC-conjugated CCL. The derivative emitted not only fluorescence (FL) at maximum emission wavelength of 410 nm with the irradiation at 310 nm excitation, but also chemiluminescence (CL) in the presence of H2O2, borate buffer (pH 9.5) and acetonitrile. After reversed-phase liquid chromatographic separation of the CIPIC-conjugated CCL in blood, the derivative could be monitored with a FL detector, indicating the detection limit (S/N=3) of 10 pmol/injection. The CIPIC-conjugated CCL was further monitored most sensitively by a CL detector after simply mixing H2O2 and borate buffer with the column eluate. The CL detection limit was 1.0 pmol/injection. In addition, we attempted to detect the CIPIC-conjugated CCL by liquid chromatographic-mass spectrometry (LC/MS). The MS method permitted the specific detection of the CIPIC derivative of the drug, though the sensitivity was 104-times lower than that of the CL detection.