MATERIALS & METHODS

Collection of Samples: A total of 100 samples honey samples were collected, 40 branded and 60 non-branded.

Chemical & Reagents used: Standard tetracycline, penicillin, streptomycin and gentamycin, methanol, acetonitrile (ACN), O-phosphoric acid and ethyl acetate. All solvents used were of TLC and HPLC grade, including the deionized water. They were filtered through a 0.45μm filter membrane and degassed by an ultrasonic cleaner for 20 minutes.

Extraction procedure for TLC: 5g of sample were extracted with a mixture of ethyl acetate:water (80:20) by centrifugation at 3,000rpm for 10 minutes and the supernatant was used for spotting on the TLC plate.

TLC Analysis of Antibiotic: The spot of each sample were loaded standard on TLC plate with the help of micro syringes by an automatic TLC spotter. Then placed in the mobile phase of methanol:water (95:5) and the detection was carried out by comparing the Rf value of sample with that of standard.

Extraction procedure for HPLC: The samples for HPLC were subjected to a deproteinizing chemical procedure using ACN. A 2 g of honey sample was placed into a 10mL test tube and shaken intensively with 3mL ACN for 1 min. The mixture was centrifuged for 15 min at 5000 rpm. The supernatant was collected and dried under nitrogen stream at 40°C. The residue was redissolved in methanol, filtered through 0.45μm filter membrane and 10 μl was injected to the HPLC system.

HPLC Analysis of Antibiotics Residues: A Hitachi (D-2000 Elite system manager) with a dual pump (L-2130), auto sampler L-2200 and UV-Visible detector L-2420 was used for the quantification of targeted antibiotic residue, in which the separation was achieved using Column oven L-2300 and column Intersil ODS-3 C18 (GL Sciences Inc. Tokyo Japan 5μm, 250mm×4.6 mm). All solvents were filtered through 0.45 μm sartolon polyamide membrane by filtration assembly of (Rocker-300 Model Taiwan) and degassed by ultrasonic cleaner Ceia (Model CP-104 Italy). The determination of these compounds were performed using different mixture of an aqueous mobile phase (A) Acidified water and organic mobile phase (B) methanol/ACN, both with a flow rate of 1 ml/min. The compounds were detected at 210-240 nm. The quantification was achieved by comparison of the peak area of the sample with that of the external standard. The identical chromatogram was quantified by the peak area of sample with that of standard in same retention time.