Browsing by Subject "Antibiotics"

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Rapid screening of antibiotics in foods by HPTLC-FLD/EDA/MS
(2015) Chen, Yisheng; Schwack, Wolfgang
Nowadays, the usage and partly abuse of veterinary antibiotics resulted in a very pressing need to control residues in foods of animal origin. Particularly, the increasingly demanding MRL issues and the huge number of samples to be monitored raised great challenges in this field. Microbial growth inhibition assays are traditionally employed for screening purposes, while sophisticated HPLC-MS methods are alternatively used or only used for confirmation purposes. To substitute the time consuming growth inhibition assays, HPTLC as a platform hyphenated to multi detection modes was employed in this study for the development of a high throughput, sensitive and cost-efficient screening-oriented methodology for antibiotics residues. The first step was focused on tetracyclines and fluoroquinolones, which are the most problematic antibiotics in the European Union and account for the most of the used veterinary antibiotics. To prevent strong tailing effects, the separation was optimized on normal-phase silica gel plates modified with ethylenediamine tetraacetic acid (EDTA). Besides, selective and sensitive fluorescence densitometry was optimized to achieve best signal/noise ratios. Under these conditions, limits of detection (LODs) and quantitation (LOQs) were in the range 12–25 and 45–95 µg/kg, respectively. Recoveries from milk samples, spiked at 50, 100 and 150 µg/kg and extracted by a modified QuEChERS procedure, ranged from 76 to 105%. To circumvent the ion suppressions due to EDTA, HPTLC-mass spectrometry (HPTLC-MS) was optimized, allowing the selective confirmation of positive findings, also offering high sensitivity of 25 µg/kg, and meeting Commission Regulation (EU) No. 37/2010. In the second step, sulfonamides were targeted, which are the secondly most administered veterinary antibiotics in the European Union. Separation of twelve most important sulfonamides was achieved on HPTLC silica gel plates, followed by fluram derivatization and sensitive and selective quantitation by fluorescent densitometry. LODs and LOQs were determined to 15–40 and 35–70 µg/kg, respectively. Samples of bovine milk, porcine liver and kidney were extracted according to the “QuEChERS” strategy. Additionally, a confirmative detection by HPTLC-MS was optimized, offering straightforward identification of target zones. The method was validated to meet the enforced Commission Regulation (EU) No. 37/2010. Finally, a more universal screening method based on HPTLC-bioautography was developed for most of the first-line veterinary antibiotics. A comprehensive HPTLC plate test revealed that the bio-compatibility of different plate layer materials to the applied bioluminescent bacteria (A. fischeri DSM No. 7151) was surprisingly different. It was then discovered that both bright bioluminescent background and significant inhibition zones of antibiotics can only be achieved on HPTLC amino F254S plates. In this case, HPTLC was not used for the chromatographic separation of individual antibiotics extracted with acetonitrile, but in terms of planar solid phase extraction to separate bioactive matrix compounds and to focus the analytes within two distinct target zones of different polarity. Together with HPTLC-MS for identification and confirmation purposes, the developed procedure enabled the rapid, sensitive and efficient multi-class screening of antibiotic residues (16 species of 5 groups, except sulfonamides and penicillins, which only affect Gram positive bacteria). The multi-sample plate images provided the results within a few hours. Thanks to the high sensitivity and the great matrix tolerance, the established method was successfully applied to bovine milk and porcine kidney samples, each spiked at the EU MLRs.