Sample collection
Two hundred chicken eggs and 540 chicken tissues (comprising 180 pieces each of liver, kidney and muscle) were taken from slaughtered, matured and market-ready birds that were randomly selected from farms in three different districts (Offinso, Kwabre and Kumasi) in the Ashanti region of Ghana in 2013. The samples cover all the available chicken poultry products that would have been offered for sale at the retail market. All samples were stored on ice and transported to the laboratory. Eggs were kept in the refrigerator while chicken tissues were frozen at −20 °C until analysis. Since the emphasis of sampling was on obtaining samples that reflect the broad range of chicken poultry products consumed in the Ghanaian home, the exact age of birds, the dose of veterinary drugs used and the frequency of drug use were not controlled in this study. All birds were matured and market-ready and all eggs were freshly laid. All muscles were comprised of breast tissue.
Reagents
Acetonitrile (MeCN), methanol (MeOH) and hexane were of HPLC grade (Fisher Scientific, UK). Potassium dihydrogen phosphate and sodium dihydrogen phosphate were of reagent grade (Fisher Scientific, UK). Standards of veterinary drugs provided in ampules include sulphathiazole, sulphamethoxazole, tiamulin, oxytetracycline, albendazole, piperazine, chloramphenicol and levamisole (Sigma-Aldrich, St. Louis, USA).
Standard solutions
Stock solutions (100 mg/mL) of each of the eight drug standards were prepared by pipetting the appropriate aliquot into a 25 mL volumetric flask and then dissolving and diluting to the mark with MeCN:MeOH (30:70) solution. Stock solutions were stored refrigerated and prepared fresh every 3 months. Working solutions of standards (100 μg/mL and 10 μg/mL) were freshly prepared through dilution of an appropriate aliquot of the stock solutions with MeCN:MeOH (30:70) solution. They were stored refrigerated and prepared fresh monthly. The blank standard was taken to be the dilution solution of MeCN:MeOH (30:70) with no added drug. Distilled water was used to prepare all aqueous solutions. All solutions prepared for HPLC were filtered through a 0.45 μm nylon filter before use.
Sample extraction
Chicken tissue
Tissues samples were minced and ground to homogeneity using a food processor. Homogenized tissues were then kept frozen at −20 °C until use. To 5.0 g of the homogenized tissue was added 50 mL MeCN and the mixture shaken vigorously for 3 min in a 100 mL centrifuge tube. The tube was centrifuged and the supernatant filtered into fresh containers. The residue was extracted two additional times, each time with 50 mL of MeCN and the filtrate pooled. The combined filtrate was transferred into a separatory funnel containing 30 mL of MeCN-saturated n-hexane and shaken for 5 min. The MeCN layer was collected into a flask and the MeCN evaporated to dryness under vacuum on a rotavap (Buchi, USA).
Eggs
Whole eggs were homogenized in an Ultra-Turrax T25 basic homogenizer (Staufen, Germany) for 1 min at 7000 rpm. A 2 mL aliquot of homogenized egg sample and 20 mL of MeCN were blended together at high speed in the homogenizer for 5 min and the supernatant filtered through a Whatman 0.45 μm filter paper. Two additional homogenization with MeCN and filtration were performed. The combined filtrate was transferred into a separatory funnel containing 30 mL portion of MeCN-saturated n-hexane and the mixture shaken for 5 min. The MeCN layer was collected into a concentration bottle and reduced to dryness under vacuum on a rotavap (Buchi, USA).
Sample clean-up
Extracts were cleaned up using C18 sorbent columns (Supelclean, 1 g × 6 mL, ENVI-18 SPE cartridge) from Sigma-Aldrich, USA. The absorbent was pre-conditioned by sequential washing with 10 mL of methanol and 10 mL of 0.05 M sodium dihydrogen phosphate solution. The semi-dry residues emanating from tissue and egg extraction were re-dissolved in 10 mL of sodium dihydrogen phosphate solution and applied onto the ENVI-18 SPE cartridge. The concentration bottle was washed with 5 mL sodium dihydrogen phosphate solution and 5 mL MeOH and applied to the SPE column. The eluate was collected into a 15 mL polypropylene centrifuge tubes and evaporated to dryness at 40 °C in a rotary evaporator. The dry matter was reconstituted with 1 mL of MeCN/H2O (3/7, v/v) and spiked 0.5 mL of MeCN saturated n-hexane. The resulting solution was then centrifuged at 3000 rpm for 5 min. The acetonitrile layer was collected and filtered through a 0.45 μm nylon membrane prior to HPLC analysis (Kao et al. 2001).
Instrument
HPLC analysis was carried out on an Agilent 1260 automated HPLC series with an SPD-M6A photodiode array detector operating at 270 nm wavelength with a 50 nm bandwidth. A 20 μL aliquot of each extracted sample were injected into the RP-18 Mightysil HPLC column (150 mL, 4.6 mm, 15 μm) maintained at 40 °C with a column heater. All HPLC analysis (tissues and eggs) utilized the same mobile phase that was a 70:30 (v/v) solution of 0.01 M potassium dihydrogen phosphate (KH2PO4): MeCN. Peaks were well resolved within 30 min run (Fig. 1).
HPLC analysis
Five calibration standards were prepared from their working stock solutions by transferring the appropriate aliquot and bringing the total volume to 1 mL using MeCN/KH2PO4 buffer. A new set of calibration standards was run before each set of samples were assayed. Calibration standard solutions, reagent blanks and reagent blank spikes for each veterinary drug were HPLC-analyzed in the same way as the sample. Samples were run in eight analytical batches, with each analytical batch consisting of five standards, three reagent blanks and two reagent blank spikes and triplicates of ten samples. In all cases, the concentrations of drug residues in samples were determined by interpolation from a five-point calibration curve generated via measurement of the HPLC peak area.
Limit of detection (LOD)
The LOD for the solution was initially estimated for each analytical batch as three times the standard deviation of the three reagent blanks. The solution LODs (mg/mL) are as follows: albendazole (0.001), chloramphenicol (0.001), levamisole (0.001), oxytetracycline (0.002), piperazine (0.001) and sulphamethoxazole (0.001), sulphathiazole (0.001) and tiamulin (0.001). Sample LODs were then calculated for each drug by multiplying the solution limit of detection by the dilution volume and dividing by the weight of the actual sample. The sample LOD (mg/kg) are as follows: albendazole (0.025), chloramphenicol (0.025), levamisole (0.025), oxytetracycline (0.025), piperazine (0.015) and sulphamethoxazole (0.025), sulphathiazole (0.025) and tiamulin (0.002).
Quality assurance and quality control measures
To ensure the validity of results, the following measures were taken. Prior to sample analysis, standards for all 8 drugs were analyzed to verify adequate system performance. Agreement of HPLC data with analyzed standards prior to sample analyses and in between ten sample runs were satisfactory. Each batch of sample analysis was prepared to include reagent blank in triplicate to control for background contamination and three spiked samples in triplicate to confirm satisfactory drug recovery greater than 70 %. In addition, the correlation coefficient for the calibration curve was required to be greater than 0.995. All samples were analyzed in triplicates.
Recovery of spiked samples
Recovery test was performed in triplicate by spiking standards at three different levels of the veterinary drugs. Five gram of homogenized samples were spiked with 5 μL of internal working standard solution (10 μg/mL) and vortexed for 1 min to mix thoroughly. Spiked and unknown samples are treated in the same way. Average recoveries registered were in the range of 76.0–98.8 %. For both tissues and eggs, sulphamethoxazole and tiamulin showed the least recovery with mean values between 76.0–78.0 % respectively, while albendazole obtained the highest mean recoveries at 98.0–98.8 %.
Statistical analysis
All data were reported as the mean ± standard deviation of the set of triplicates determinations. A one way analysis of variance (ANOVA) was conducted using GraphPad prism 5 in the assessment of variation in drug concentrations within same tissues and between different tissues. Tests were considered statistically significant at p < 0.05.
Dietary exposure assessment
Dietary exposure was quantitatively estimated using as input, the food consumption data and the total concentrations of residues found in chicken tissues and in eggs (FAO/WHO 2011). Dietary exposure (acute and chronic) was computed using expression (1) assuming an average adult weight of 60 kg (FAO/WHO 2004; FAO/WHO 2005).
$$ Dietary\; expoure\kern0.36em =\frac{{\displaystyle \sum Residue\; concentration}\;x\; Acceptable\; daily\; intake}{Body\; weight} $$
(1)
Global estimated acute dietary exposure (GEADE) was calculated based on the 97.5th percentile food consumption amount using equation (2) as:
$$ GEADE=\frac{97.{5}^{th}\; percentile\; food\; consumption\ x\; high\ residue\ tissue}{Body\; weight} $$
(2)
The 97.5th percentile food consumption amount was used because of its statistical robustness and is more representative than the maximum food consumption amount (FAO/WHO 2005; FAO/WHO 2011). Global estimated chronic dietary exposure was calculated as the product of the highest exposure animal product and total mean exposure (JECFA 2014).