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CHAPTER THREE: MATERIALS AND METHODS

3.1 Data Collection Procedure

In order to achieve the results of the first objective of the study which was to investigate the common antibiotics used in Nakawa division, the researcher used a questionnaire design to collect data from 50 employees of the 5 selected poultry farms in Nakawa division where each farm had 10 employees purposely selected.

Sample of Questionnaire

Dear Respondents,

I am MUGARURA GASTON, a student of Kyambogo University pursuing A master’s degree in chemistry. Am conducting a study on “on determination of antibiotics residues in chicken meat”. The research study is a partial requirement leading to the award of a master degree. Therefore, the information provided therein is for only academic purposes and will be highly treated with utmost confidentiality.   

Part I: BIO DATA

Please tick your appropriate option

What is the name of this farm…………………………………………………………………?

PART II: THE COMMON ANTIBIOTICS USED AT THIS FARM

In a bid to achieve the results of the second objective of the study which is to investigate the common antibiotics used in Uganda, the researcher poses different questions with the alternatives to the poultry farmers in Nakawa division and the respondents are expected to tick their appropriate option

Which of the following antibiotics do you use here?

Please tick your appropriate option

If any other antibiotic used, specify………………………………………

Who administers these antibiotics?

Where do you get these antibiotics?

Any other source, specify………………………………………………

Why do you use these antibiotics?

3.2: Sample Collection

The fresh samples of Breast Muscle, Liver and Gizzard of broilers fifty (50) in number respectively were randomly collected from difference markets of Nakawa, Bugolobi, Luzira, Ntinda and Banda in Nakawa Division, located in Kampala City Council Authority. The coordinates of the division are 0° 20’00.0″N, 32° 37’00.0″E (Latitude: 0.333333; Longitude: 32.616667 (UBOS, 2014).

The collected samples, were packed in properly labelled sterile polyethylene bags at room temperature and transported to the Directorate of Government Analytical Laboratory (DGAL) in Wandegeya, Kampala and kept under refrigeration at 4^oC.

3.3: Equipment

Laboratory blender (Thomas Scientific, USA), Vortex mixer-VM18 (Schiltern Scientific, Beds, UK), Centrifuge (Hettich D-78532, Germany), Centrifuge tubes, Pasteur pipettes and Rubber bulbs, Nitrogen evaporator – 6 Position N-Evap (Thomas Scientific, USA), Mas Hunter work station software LC/MS Data Acquisition for 6400 series triple quadrupole, version B.08.00 and Quantitative Analysis version B.07.01/Build 7.1.524.0 for QQQ.

3.4: Sample Preparation

The 150 whole tissue samples of liver, breast muscle and gizzard of broilers were blended using a laboratory blender (Thomas Scientific, USA) each sample separately to make minced samples that could easily be analysed. 4 g of the minced samples were measured and properly stored at -18 ^OC in preparation for samples analysis.

3.5: Chemicals

The purity of all standard chemicals and reagents were at least 99% HPLC grade, methanol (Merck-Germany), Trichloroacetic acid (TCA), diethyl ether and acetone were used.

3.6: Preparation of standards

Enrofloxacin (ENR), Oxytetracycline (OTC), Tetracycline (TC), Penicillin G and Chloramphenicol (CAP) are USP reference standard (USP pharmacopeial convention), USA. The standards for the selected antibiotics were prepared by dissolving 0.1 gm of powder in 4 mL solution of methanol and standard solutions were stored in – 4°C.

3.7: Sample analysis

The minced samples that were analysed were stored in a refrigerator to temperatures of -4 ⁰ C for an hour.  However, where the analysis could not be performed within 2 days, the samples were stored to a temperature of -18 ⁰ C and to further ensure that the samples remained in the same state after two days, 50 µL of work solution of internal standards (multresidue) were added to 4 g of minced samples in their storage facilities.

Sample extraction was performed according to Poppelka (2005) where to the 4 g of each minced samples, 10 mL Phosphate Buffer Saline (pH-6.5) was then added and mixed by vortexing using a Centrifuge (Hettich D-78532, Germany) @ 4000 rpm for 20 min after mixing with 2 mL 30% TCA. The supernatant was removed using Pasteur pipette and filtered by Whatman filter paper of 50 µm in thickness. The Filtrate was collected in another falcon tube and 2 mL of diethyl ether was added and left for 10 min in room temperature. The bottom layer was collected and extraction was repeated twice using diethyl ether. Final volume of the filtrate was pooled carefully into screw cap vial and kept into refrigerator for future analysis.

The tube containing the filtrate was put into a water bath at 40^oC under nitrogen flow and ether was evaporated to dryness.

3.8: Sample analysis

The remains of the filtrate were reconstituted in 1 ml water/methanol/acetonitrile (50/25/25) +/- 0.05% acetic acid and vortexed.

The filtrate was transferred in a LC-MS vail and capped with a capper. Five (5µL) of the filtrate was injected in heated LC-MS system where the mobile phase entered the column from the left, passed through the particle bed, and exited at the right.  The flow occurred in the column at time zero (the moment of injection), when the sample extracts entered the column and begun to form a band, a mixture of dyes, appeared at the inlet of the column as a single black band where a detector was used to monitor the separated compounds as they eluted. After a few minutes during which mobile phase had flown continuously and steadily past the packing material particles, the individual dyes moved in separate bands at different speeds. Since each dye band moved at different speed, separation of residues chromatographically was achieved and the data collected by the computer was used to analyse the levels of antibiotics in the samples.

The analysis of the antibiotic residues Oxytetracycline (OTC), Tetracycline, Enrofloxacin, Penicillin G and Chloramphenicol were done using analytical column C18 Eclipse Plus, 95, 1.8µm, 21 x 100 mm, manufactured by Agilent Technologies, Column Description: ZORBAX RRHD Eclipse C18, Pore Size: 95, Particle Size: 1.8µ, Inner Diameter: 2.1mm, Length: 100mm, Carbon Load: 9%, USP Number: L1, Category: Reversed Phase (RP) part number 959758-902.

The separation of antibiotics was accomplished at 30 °C, the flow rate and injection volume were 0.5 mL/min and 10 μL, respectively. The mobile phases used were (A) water and (B) acetonitrile. The gradient elution program started with 10% B for 1 min, increased to 65% for 6 min, then increased to 95% for 1 min and returned to the initial conditions in 1 min. The final run time of the method was 12 min. The separation of sulfamides, tetracyclines, and quinolones was accomplished at 40 °C. The flow rate and injection volume were 0.3 mL/min and 10 μL, respectively. The mobile phases used were (A) TFA (0.1%) in water and (B) acetonitrile. The gradient elution program was as follow: A (90%) (3min), A (25%) (5 min), and A (90%) (1min); the final run time of the method was 15 min.

3.9 Data analysis

Data analysis on the concentration of antibiotics was analysed using SPSS statistical program (SPSS Inc., Chicago, IL, USA). Analysis of variance, ANOVA, was performed to compare levels of antibiotic residues in chicken breast muscle, liver and gizzard.