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Comparative Efficacy of Four Different Solid Phase Extraction Cartridges Used to Determine Tetracycline in Foods of Animal Origin

M. J. Islam
Institute of Food and Radiation Biology, AERE, Savar Dhaka-1349, Bangladesh

S. U. Talukder
Institute of Food and Radiation Biology, AERE, Savar Dhaka-1349, Bangladesh

A. Y. K. M. M. Rana
Institute of Food and Radiation Biology, AERE, Savar Dhaka-1349, Bangladesh

A. S. M. Saifullah
Institute of Food and Radiation Biology, AERE, Savar Dhaka-1349, Bangladesh

Abstract/ Executive Summary:

Four different types of Solid Phase Extraction (SPE) cartridges namely R-Biopharm (RB), Chromabond (CB), Isolute (IS) and Megabond (MB) were used in this study. Control and spiked samples of beef, chicken and shrimp were also used. Optical Density (OD) values of control and spiked samples were measured with the help of Enzyme Linked Immunosorbent Assay (ELISA) reader. Percent binding values for each SPE cartridges were calculated using OD values of respective control and spiked samples. Based on % binding values a comparative study of 4 different Solid Phase Extraction (SPE) cartridges was carried out. Analysis of variance indicated no significant differences among the % binding values considering different samples irrespective of SPE cartridges (P=0.821266, F = 0.201279) or different SPE cartridges irrespective of samples (P = 0.168119, F = 2.180932). From this study, it can be recommended that any of the four SPE cartridges available in the working area can be used for the analysis of tetracycline from foods of animal origin.

Keywords: Solid phase extraction, ELISA, Optical density, % binding

Location: Institute of Food and Radiation Biology, AERE, Savar Dhaka

Start Date: 00-00-2015

End Date: 00-00-2015

Program Area: Food Safety and Security

Commodity: Diseases

Objectives:

1. To compare the efficiency of several SPE cartridges for tetracycline determination from animal tissue by ELISA and to mark out optimal experimental conditions that can later be applied for screening and the quantification of the veterinary drug residue analysis.

Methodology:

Sample Type - Control samples of muscle tissue of beef, chicken and shrimp were obtained from International Atomic Energy Agency (IAEA), Vienna, Austria. Tetracycline hydrochloride (21.6 mg) was taken in a 20ml volumetric flask and methanol was added up to the mark to prepare stock solution (1mg/ml). The stock solution was then diluted with distilled water to the desired concentration. Citric acid monohydrate (12.9g), Na₂HPO₄ (10.9g), EDTA sodium salt (37.2g) was taken in a 1000ml volumetric flask and distilled water was added up to the mark. The pH was adjusted with saturated citric acid. NaCl (9.0g), Na₂HPO₄ x 2H₂O (2.85g), NaH₂PO₄ x H₂O (0.55g), Tween 20 (1ml) was taken in a 1000ml volumetric flask and distilled water was added up to the mark. The pH was adjusted with sodium base. Methanol (100%) was added with 20mM oxalic acid (1.8 g/l) or oxalic acid dehydrate (2.522 g/l). ELISA test kit and microplate reader - Ridascreen tetracycline ELISA test kit was purchased from R-Biopharm AG (Darmstadt, Germany), kit lot no. 14420, Art. No. R3503. Test kit contained microplate, tetracycline standard solution, tetracycline-conjugate, anti-tetracycline-antibody, red chromogen, stop solution, buffer and washing buffer. The microplate reader machine was Thermo Scientific Multiskan FC. Solid Phase Extraction (SPE) cartridges - Four different types of SPE cartridges namely R-Biopharm (RB) (100mg), Machery-Nagel Chromabond C18ec (CB) (3ml/200mg), Biotage Isolute C18ec (IS) (6ml/1g) and Mega Bond Elute C18 (MB) (6ml/1g) were used. Preparation of control and spiked samples - Eight control samples each from beef, chicken and shrimp were taken. The samples were homogenized with a rod homogenizer (Ultra turrax) and 5g each were taken into 50ml separate centrifuge tubes. Four of each from beef, chicken and shrimp were used as control. Five hundred μl oxytetracycline dihydrate solution (100μg/kg) was added in rest 4 tubes from beef, chicken and shrimp that were used as spiked samples. Sample processing - Twenty five ml Mcllvaine buffer was added in each tubes, vortexed well and used for refrigerated centrifugation (4000g/15^oC/10min.). The supernatant was filtered into 50ml separate test tube using folded filter paper and the extraction procedure was repeated using same buffer for one more time. Again the supernatant was filtered into the respective tubes. Solid Phase Extraction (SPE) - Supernatant of one control and one spiked from beef, chicken and shrimp were used for four different SPE cartridges. They were placed on to suction chamber connected to a pump and the pressure raised to 7 inches Hg. They were conditioned with 2ml methanol (100%), 2ml distilled water was added and allowed to pass. Five ml supernatant was put on the respective cartridges, 3ml distilled water was added and allowed to pass. Vacuum was reduced to 4 inches Hg with vessel controller. Two ml elution solution was added, eluents were collected in separate glass tubes and vortexed. Hundred μl eluent was taken into glass tube (12x75mm) and 400μl kit buffer was added. Fifty μl of this solution was taken for ELISA. It was done for all control and spiked samples. ELISA test procedure - Fifty μl from each tubes were pipetted into corresponding wells. Fifty μl tetracycline antibody solution was added to each well. The plate was covered, mixed gently and incubated at room temperature for 1 hour without shaking. The microplate was washed with washer (Well wash, Thermo Scientific) 3 times with 250μl washing buffer. Hundred μl enzyme conjugate solution was added to each well. The plate was covered, mixed gently and incubated at room temperature for 15 minutes. Again the plate was washed as before. Substrate/chromogen solution of 100μl was added to each well. The plate was covered, mixed gently and incubated at room temperature for 15 minutes. Hundred μl stop solution was added to each well and mixed gently. Finally the measurement was made photometrically at 450nm.

Source of Information: Bangl. J. Vet. Med. (2015). 13 (1): 89-91

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

It could be concluded that whatever SPE cartridges used, there will be no significant differences among the results. It is also recommended that the cartridges (especially among these four types) which are available in the working area would be used for the analysis of tetracycline in foods of animal origin.

Knowledge Management: Journal