Antibiotic Identification by High Voltage Electrophoresis Bioautography

Abstract

The high voltage electrophoresis bioautography method is applicable to meat, milk, and animal feeds. Meat is freeze-dried, powdered, and extracted with acetonitrile-water (9 + 1), and the extract is concentrated by evaporation at room temperature. Milk is examined directly or following acetonitrile-water extraction. Feed is extracted with acetonitrile- water. Samples or extracts are applied to preliminary assay plates of antibiotic medium No. 1 at pH 6 and 8, seeded with Micrococcus luteus (ATCC 9341), M. luteus DHSR (ATCC 9341A), Bacillus cereus (ATCC 11778), or B. cereus K250 TR (NCIB 11183), and nutrient agar at pH 7 seeded with B. subtilis BGA. Inactivation of penicillinase indicates beta-Iactam antibiotics. Addition of trimethoprim increases sensitivity to sulfonamides. After 18-24 h incubation at 30°C, plates yielding clear inhibition zones guide selection of conditions for subsequent electrophoresis bioautography. Extracts are applied (5-100 |xL) to 10 mm diameter wells on electrophoresis plates 60 cm long and 40 cm wide, with a gel depth of 1.6 mm. The support medium is 1% agar and 1 % agarose in Tris/succinic acid buffers pH 6 and pH 8. A potential of 1500 V is applied for 1.5 h at 15°C. Following electrophoresis, the migrated antibiotics are visualized by over-layering with antibiotic medium No. 1, pH 6 or 8, seeded with M. luteus or B. cereus spore suspension; plates are incubated for 18-24 h at 30°C. Identification is based on results of preliminary screening together with electrophoretic migration distances and inhibition zone appearances compared with standards

In-house validation method for quantification of amoxicillin in medicated feedingstuffs with the use of HPLC-DAD technique

“…of ampicillin from amoxicillin ( 2 ). Micellar electrokinetic capillary chromatography ( 18 ) or capillary electrophoresis ( 8 , 20 , 25 ) are techniques used for the determination of penicillins in feeds. Many chromatographic methods with fluorescence detection (FLD) or UV detection ( 1 , 4 , 11 , 12 , 16 , 17 , 19 , 21 , 22 ), liquid chromatography–mass spectrometry (LC-MS) ( 5 , 6 , 9 , 14 , 19 , 23 ) or ultra-high performance liquid chromatography–mass spectrometry ( 5 , 7 , 33 ) are also used for the determination of penicillins in food of animal origin.…”

Trace analysis of oxytetracycline and tetracycline in milk by high-performance liquid chromatography

“…Several physicochemical methods including thin-layer chromatography (Ryan and Dupont, 1974), thin-layer chromatography and bioautography (Bossuyt et al, 1976;Hamann et al, 1979), electrophoresis and bioautography (Staudhouders et al, 1981;Lott et al, 1985; Pietrangeli et al, 1986), column chromatography and bioassay (Hamann, 1983), and gas-liquid chromatography (Hamann et al, 1979) have already been reported for the determination of TCs in milk. Most of them provide increased specificity and sensitivity and can be useful in classifying unknown residues in milk, but cannot be considered sufficiently sensitive or precise.…”

Προσδιορισμος Πενικιλλινων Στο Γαλα Αγελαδας Με Υγροχρωματογραφια Υψηλης Αποδοσης

“…Μέχρι σήμερα, όμως, δεν έχει αναφερθεί η δυ νατότητα ασφαλούς ποσοτικού προσδιορισμού των ουσιών αυτών σε τέτοια δείγματα δεδομένου ότι ορισμένες πενικιλλίνες, όπως η PEN G και η PEN V, που εμφανίζουν παρόμοια ηλεκτροφορητική κινητικότητα και παραπλήσια αντιμικροβιακή δράση, είναι αδύνατον να διαχωριστούν Vaughan 1978, Bates et al 1983). Έτσι οι ηλεκτροφορητικές μέθοδοι χρη σιμοποιούνται σήμερα σε ημιποσοτικές, κυρίως, αναλύσεις πενικιλλινών (Lott et al 1985).…”