Solid-phase extraction and high performance liquid chromatography with diode array detection method for the determination of antibiotic residues in poultry tissues

This article presents information on high performance liquid chromatography with diode array detection (HPLC-DAD) method for the simultaneous determination of six antibiotic residues (enroﬂoxacin, sulfadimethoxine, sulfamerazine, sulfamethoxazole, sulfamox- ole, and tylosin) in three poultry tissues. The target antibiotic residues were extracted from raw poultry samples following concentration, clean-up through solid phase extraction. The data describe the extraction, determination and screening procedures of these common antibiotic residues in 111 samples of fresh and frozen poultry meats. The limit of detection (LOD) ranged from 5.37–55.4 ng/g, while the quantiﬁcation limit (LOQ) was in the range of 17.9–184 ng/g, respectively, with minimal matrix effect. The calibration curves obtained exhibited a good linear response with the coeﬃcient of determination, R 2 > 0.996. Some concentrations exceeded their maximum residue limits in most samples. These ﬁndings indicated elevated levels of antibiotic residues in tissues of locally produced and illegally imported poultry meat samples.


How data were acquired
Agilent 1220 High Performance Liquid Chromatography with Diode Array Detection HPLC-DAD (Agilent Technologies, Waldbronn, Germany) system consisting of a binary high-pressure pump, autosampler, a thermostat column compartment, a fluorescence detector, and refractive index detector. Data was acquired and processed using ChemStation (version 1.9.0) software (Agilent Technologies, Waldbronn, Germany). Analytical column was an XTerra MS C18 column (Agilent Technologies, Waldbronn, Germany) was used (4.6 × 100 mm, 3.5 μm particle size). Mobile phase solvent A (Ultra-pure water) and B (acetonitrile) contained 0.1% formic acid. Column temperature was maintained at 40 °C, and separation done under gradient elution with the organic phase increasing linearly from 5 to 30% in 6 min, and further increasing to 70% within 12 min. The mobile phase was pumped at a flow rate of 1.2 mL/min and the detection wavelength was 275 nm, with a post-run time of 1 min before the next injection to equilibrate the column.

Data format
Raw, Analysed Parameters for data collection 111 samples of live and frozen poultry meats were collected. Antibiotic residues analysed were enrofloxacin, sulfadimethoxine, sulfamerazine, sulfamethoxazole, sulfamoxole, and tylosin.

Description of data collection
Poultry tissues were extracted using SPE method with water/methanol (4:1, v/v) mixture, and the compounds eluted with 2 mL of 10% ammonium hydroxide/methanol (1:19, v/v). The eluate collected was dried under N 2 with heating at 40 °C and reconstituted in 1 mL of phosphate buffer that was filtered with a 0.45 μm Acrodisc® syringe filters before injection into the HPLC system. Analyses of extracts for antibiotic residues were carried out using HPLC-DAD.

Data source location
Ogun

Rationale
Veterinary antibiotics are chemically synthesized antimicrobial drugs consistently used over the years in animal production for the prevention and treatment of infectious diseases. However, in recent years, the use of antibiotics in controlled quantities has been extended from animal therapeutic purposes to growth promoters and prophylactics in feed additives [2][3][4][5] . Over the past three decades, poultry production has increased as part of human's quest for alternate sources of protein for consumption. Thus, veterinary pharmaceuticals such as amphenicols, fluoroquinolones, beta lactams, tetracyclines, aminoglycosides, macrolides, among others. are widely used for growth promotion, prophylactic and therapeutic purposes [2,6,7] .
Antibiotic residues have been reported in trace concentrations in edible parts of poultry meats [2,8] . These residues may consist of the parent compounds as well as their conjugates, and possibly all could be present together resulting in direct toxic effects on consumers and allergic reactions in hypersensitive individuals, and above all antimicrobial resistance. The World Health Organisation (WHO) has warned about an imminent antimicrobial resistance crisis, thus putting the antibiotic era at risk if urgent remedial actions are not taken to reduce antibiotic usage in human and veterinary medicine [9] . To safeguard public health and ensure some level of consumer protection, there is, therefore, a need to establish surveillance systems that allow for the collection of reliable data on antibiotic usage and residues.
Antibiotic residues in foods could pose serious indirect and direct long-term health hazards, and therefore present a significant research interest due to their extensive use as well as their persistence and prevalence in animal tissues, because they portend undesirable consequences such as treatment failure and disease severity to the consumer [2,6,10] . Consequently, it is necessary to provide reliable analytical data on multiresidues of different classes of antibiotics in poultry products and, in particular, those imported into Nigeria to ensure that there is no risk of secondary exposure to the consumer.

Collection of samples
Smuggled frozen poultry products and fresh chicken (poultry meat) consisting of layers, broilers and cockerels were purchased directly from major farms and markets in the study area between May and September 2017.

Sample preparation and extraction
Using the modified method of Zhao et al. [11] , the drugs residues were extracted from 2 g of blended tissues of poultry by weighing and transferring into a 50 mL previously washed stainless centrifuge tube. 10 mL of phosphate buffer solution (0.01 M adjusted to pH 7.0) was added to the samples. Each sample was allowed to stand for 15 mins at room temperature, and then vortex mixed for about 20 s before centrifuging for 5 mins at 3500 rpm. The supernatant was transferred to Table 1 Distribution and concentration (ng/g) of antibiotic residues in frozen turkey muscle tissues ( n = 14). -:Below limit of detection (LOD); MRL: Maximum residue limit.

Table 2
Distribution and concentration (ng/g) of antibiotic residues in frozen turkey gizzards ( n = 8). another flask, and the extraction process repeated two more times. 10 mL of the combined extracts were passed through an SPE (Supelclean TM ) column previously conditioned with 2 mL of methanol and HPLC grade water, respectively. Later, it was washed with 3 mL water/methanol (4:1, v/v) mixture, and the compounds eluted with 2 mL of 10% ammonium hydroxide/methanol (1:19, v/v). The eluate collected was dried under N 2 with heating at 40 °C and reconstituted in 1 mL of phosphate buffer that was filtered with a 0.45 μm Acrodisc® syringe filters before injection into the HPLC system. To enhance instrument response, sample extracts were spiked with 50 μg/mL mixed standards of the drugs.

HPLC-DAD analysis
Sample extracts were analysed using an Agilent 1220 High Performance Liquid Chromatography coupled with diodearray detector (HPLC-DAD) (Agilent Technologies, Waldbronn, Germany) system comprising a binary high-pressure pump, autosampler, a fluorescence detector, a thermostat column compartment, and refractive index detector. The analytical column used was an XTerra MS C18 column (4.6 × 100 mm, 3.5 μm particle size) (Agilent Technologies, Waldbronn, Germany). Mobile phase solvent A (ultra-pure water) and B (acetonitrile) contained 0.1% formic acid. Column temperature was maintained at 40 °C, and separation done under gradient elution with the organic phase increasing linearly from 5 to 30% in 6 min, and further increasing to 70% within 12 min. The mobile phase injection flow rate was 1.2 mL/min, and the detection λ was 275 nm, with a post-run time of 1 min before the next injection to equilibrate the column. Data acquisition and processing was carried out using ChemStation (version 1.9.0) software (Agilent Technologies, Waldbronn, Germany).
The antibiotics were eluted singly from the column after optimising the chromatographic parameters and their retention time obtained. Standards mix of the different antibiotics were prepared with a concentration range of 0-10 0 0 ng/mL. The six antibiotics including sulfamozole, sulfamerazine, sulfadimethoxine, enrofloxacin, tylosin and sulfamethoxazole in that order were eluted as shown in the chromatogram. A 15-point calibration curve was prepared using the standard's retention time and the integrated peak area to obtain related linear equations. To enhance analyte signal and prominent peaks, sample extracts were spiked with 50 ng/mL standard mix. Analytes were quantified using their peak areas from linear equations and the spiked value subsequently subtracted from the concentration value upon evaluation.

Table 4
Distribution and concentration (ng/g) of antibiotic residues in layer muscle tissues ( n = 8).

Table 5
Distribution and concentration (ng/g) of antibiotic residues in layer liver tissues ( n = 13).  antibiotic residues concentration varied according to each sample tissue analysed. The distribution and concentration of antibiotic residues in muscle, liver and gizzard tissues obtained from laying chickens are presented in Tables 4 , 5 and 6 , respectively. Also presented are the respective levels of enrofloxacin, sulfadimethoxine, sulfamerazine, sulfamethoxazole, sulfamoxole, and tylosin in muscle ( Table 7 ), liver ( Table 8 ), and gizzard ( Table 9 ) tissue samples collected from broilers ( Gallus gallus domesticus ) raised mainly for meat consumption. Tables 10 , 11 , and 12 show the distribution and concentrations of antibiotic residues in muscle, liver, and gizzard samples of cockerels. Antibiotic residues were not detected in some tissue samples analysed whereas concentrations above EU Commission maximum residue limits (MRL) were observed in most samples. Fig. 1 shows the representative HPLC-DAD chromatogram of samples analysed in this study. The human exposure risks assessment was calculated using Estimated Daily Exposure Dose and the Hazard index, and the data are presented in Table 13 .

Sample ID
The dataset provides an insight into the distribution of six (6) antibiotics including a fluoroquinolone, macrolide and four sulfonamides that could serve as primary data for drug residues in food chain originating from poultry meat. These dataset are useful for further toxicological and safety investigations into antibiotics levels in investigated foodstuffs and human health risk assessment associated with exposure to antibiotic residues. Details of extraction, analysis and characterisation  -: Below limit of detection (LOD); MRL: Maximum residue limit.

Table 7
Distribution and concentration (ng/g) of antibiotic residues in broiler muscle tissues ( n = 6).  provide information for further evaluation of antibiotic residues in poultry meat and human exposure assessment. The data yields information on the potential safety concerns associated with poultry products illegally imported into Nigeria and those produced locally with respect to antibiotic residues.

Human health risk exposure assessment
The estimated daily exposure dose of antibiotics from the different poultry products for adults (male and female) and children based on their average daily consumption was calculated using the modified formula [11] : Where, E d = estimated daily exposure dose, μg/kg/day; C L = antibiotic content in poultry produce, μg/kg; M L = daily adult consumption of poultry meat, g/day; M B = average body weight, kg. The estimated poultry meat consumption in Nigeria as at 2014 stood at 1.41 metric tonnes [12] and this was projected to increase by 2% annually resulting from rapid population and economic growth [13] giving a current estimate of 1.56 million metric tonnes for 2019. Daily consumption Table 9 Distribution and concentration (ng/g) of antibiotic residues in broiler gizzards ( n = 6). Enrofloxacin  Sulfadimethoxine  Sulfamerazine  Sulfamethoxazole  Sulfamozole  Tylosin   BCG-1  ------BCG- -: Below limit of detection (LOD); MRL: Maximum residue limit.

Table 10
Distribution and concentration (ng/g) of antibiotic residues in cockerel muscle tissues ( n = 8).

Table 12
Distribution and concentration (ng/g) of antibiotic residues in cockerel gizzards ( n = 7). estimated from this data for the present study is 21.92 g/day for a population of approximately 195 million [14] . The average body weight for adults and children (age range 6-18 years), in Nigeria, was 70 and 48 kg, respectively [15] . The hazard index (HI) was computed using [16] :