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CHAPTER THREE: MATERIALS AND METHODS

3.1 Data Collection Procedure

To achieve the first objective of the study—identifying the common antibiotics used in poultry farms within Nakawa Division—a structured questionnaire was employed. Data was collected from 50 respondents, comprising employees from five purposively selected poultry farms, with 10 participants from each farm.

Sample Questionnaire Excerpt

Dear Respondents,

I am MUGARURA GASTON, a postgraduate student at Kyambogo University, pursuing a Master’s degree in Chemistry. I am conducting research titled “Determination of Antibiotic Residues in Chicken Meat.” This study is a partial requirement for the award of the Master’s degree. All information provided will be treated with strict confidentiality and is solely intended for academic purposes.

Part I: Bio Data
(Please tick the appropriate option)

• Gender: Male / Female

• Age Group: 20–29 / 30–39 / 40–49 / 50–59 years

• Marital Status: Single / Married / Widowed

• Education Level: Certificate / Diploma / Degree / Masters

• Duration of Employment at This Farm: <1 year / 2–4 years / >5 years

• Name of the Farm: ___________________________

Part II: Common Antibiotics Used
Respondents were asked to indicate which antibiotics are commonly used on their farms and answer related questions.

1. Which of the following antibiotics are used on this farm?

2. Who administers these antibiotics?

3. Source of Antibiotics

4. Reasons for Using Antibiotics

3.2 Sample Collection

Fresh tissue samples (breast muscle, liver, and gizzard) from a total of 50 broilers were randomly obtained from various markets within Nakawa Division—namely Nakawa, Bugolobi, Luzira, Ntinda, and Banda. Nakawa Division is located in Kampala Capital City Authority with coordinates 0°20’00.0″N, 32°37’00.0″E (UBOS, 2014).

Samples were sealed in sterile, labeled polyethylene bags at ambient temperature, then transported to the Directorate of Government Analytical Laboratory (DGAL) in Wandegeya, Kampala. Upon arrival, samples were refrigerated at 4°C.

3.3 Equipment Used

The laboratory equipment used included:

• Laboratory Blender (Thomas Scientific, USA)

• Vortex Mixer VM18 (Schiltern Scientific, UK)

• Centrifuge (Hettich D-78532, Germany)

• Nitrogen Evaporator – 6 Position N-Evap (Thomas Scientific, USA)

• LC-MS software: MassHunter Workstation (Version B.08.00) and Quantitative Analysis (Version B.07.01)

3.4 Sample Preparation

A total of 150 tissue samples (liver, breast, and gizzard) were blended individually using the laboratory blender to create finely minced samples. Each minced sample (4 g) was stored at -18°C until further analysis.

3.5 Chemicals

All reagents and chemicals used were of at least 99% HPLC-grade purity. These included:

• Methanol (Merck, Germany)

• Trichloroacetic acid (TCA)

• Diethyl ether

• Acetone

3.6 Preparation of Standards

USP-certified reference standards were used, including:

• Enrofloxacin (ENR)

• Oxytetracycline (OTC)

• Tetracycline (TC)

• Penicillin G

• Chloramphenicol (CAP)

Each standard was prepared by dissolving 0.1 g of the powdered compound in 4 mL of methanol and stored at -4°C.

3.7 Sample Extraction

Minced samples were kept at -4°C for one hour. If not analyzed within two days, samples were preserved at -18°C. To ensure sample integrity, 50 µL of multiresidue internal standards were added to 4 g of each sample.

Following Poppelka (2005), extraction was conducted as follows:

• 4 g of minced sample + 10 mL phosphate buffer saline (pH 6.5) + 2 mL 30% TCA

• Vortexed and centrifuged at 4000 rpm for 20 min

• Supernatant filtered using 50 µm Whatman filter paper

• 2 mL diethyl ether added; repeated twice

• Final filtrate collected in screw-cap vials and refrigerated

Evaporation was done at 40°C in a water bath under nitrogen flow to dryness.

3.8 Sample Analysis

The dry residue was reconstituted in a 1 mL solution of water/methanol/acetonitrile (50:25:25) with 0.05% acetic acid and vortexed. Filtrate was placed in LC-MS vials and analyzed using an LC-MS system.

Analysis was performed using an Eclipse Plus C18 column (Agilent Technologies):

• Column dimensions: 2.1 mm ID × 100 mm length

• Particle size: 1.8 µm, Pore size: 95

• Temperature: 30°C

• Flow rate: 0.5 mL/min; Injection volume: 10 µL

• Mobile phases: (A) water, (B) acetonitrile

• Gradient: 10% B → 65% B → 95% B → back to 10% B in 12 minutes

For other residues:

• Temp: 40°C, Flow rate: 0.3 mL/min

• Mobile phases: (A) 0.1% TFA in water, (B) acetonitrile

• Gradient run time: 15 minutes (A: 90% → 25% → 90%)

Chromatographic separation and detection allowed quantification of antibiotics present in the samples.

3.9 Data Analysis

Data on antibiotic concentrations were analyzed using SPSS (Statistical Package for the Social Sciences). Analysis of Variance (ANOVA) was applied to compare the concentration of antibiotic residues across the different tissues (breast, liver, and gizzard).