Planar chromatography mediated screening of tetracycline and fluoroquinolone antibiotics in milk by fluorescence and mass selective detection

A rapid and efficient method for preliminary screening of four tetracyclines (tetracycline, chlortetracycline, oxytetracycline, doxycline) and three fluoroquinolones (enrofloxacin, ciprofloxacin, marbofloxacin), mostly detected in milk, by high-performance thin-layer chromatography–fluorescence detection and electrospray ionization mass spectrometry (HPTLC–FLD–ESI/MS) is highlighted. The optimized separation of the target antibiotics on ethylenediamine tetraacetic acid modified silica gel plates showed marked benefits for screening purposes. Besides, selective and sensitive densitometry in fluorescence mode was established with excitation at 366 nm for the tetracyclines, 300 nm for enrofloxacin and ciprofloxacin, and 280 nm for marbofloxacin. Limits of detection (LOD) and quantitation (LOQ) with 95% confidence were in the range of 12–25 and 45–95 μg/kg, respectively, in milk samples. Recoveries of target antibiotics from milk samples spiked at three critical levels (50, 100 and 150 μg/kg) ranged from 76% to 105%. More importantly, a mass selective detection (MSD) was established as additional tool for confirmatory purposes. Using the elution-head based TLC–MS interface, the optimized elution flow consisting of acetonitrile/ammonium formate buffer (9/1, v/v) at a rate of 0.3 mL/min enabled time-dependent resolution of analytes from the major interfering compounds, thus circumventing serious ion suppression effects. The established MSD assay also offered high sensitivity (25 μg/kg) for confirmation, meeting Commission Regulation (EU) No. 37/2010.