Ran Wang,1,2 Ruicheng Wei,2 Ming Chen,2 Tian Wang1
1College of Animal Science, Nanjing Agricultural University, China
2Institute of Food Safety, Jiangsu Academy of Agricultural Sciences, Nanjing, China
Corresponding author: Dr. Ran Wang, Jiangsu Key Laboratory for Animal-derived Food Safety, Institute of Food Safety, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu Province, 210014 PR China. Tel./Fax: +86.25.84391617. E-mail: email@example.com
Key words: Analytical method, Chlortetracycline, High-performance liquid chromatography (HPLC), Pig solid manure.
Acknowledgments: this study was supported by the Jiangsu Provincial Natural Science Foundation Grant (No: BK2006163) and Environmental Protection Special Project funded by Department of Environmental Protection (No: 200809092A), China.
This work is licensed under a Creative Commons Attribution 3.0 License (by-nc 3.0).
©Copyright R. Wang et al., 2010
Licensee PAGEPress, Italy
Italian Journal of Animal Science 2010; 9:e37
Chlortetracycline (CTC) is the most frequently used antibacterial in today’s swine production for disease treatment and growth promotion. It is not absorbed completely and a considerable amount is excreted into manure when CTC is administrated to animal. For this reason there is high concern on potential risks that manure-derived CTC enters into the surface water and selects for resistant microorganism. In order to support the monitoring of CTC and study its fate in manure and environment, a simple and rapid analytical procedure was developed to determine chlortetracycline (CTC) in pig solid manure. The extraction solution, composed of acetone-4mol.L-1 hydrochloric acid-water (13:1:6, v/v/v), was mixed into the manure sample, and the solution was pH adjusted at 2.0-2.2 before extraction for 20 min. Chromatographic separation was carried out on a C18 column at 375 nm with UV detector, using isocratic elution with oxalic acid-methanol-acetonitrile (72:14:14), v/v/v) as mobile phase at room temperature. Recoveries of CTC spiked samples at the levels of 1.0 mg.kg-1, 2.0 mg.kg-1 and 10.0 mg.kg-1 were 92.2%-100.73% with relative standard deviation (RSD) of 2.81-7.11% intra-day and 1.34-3.25% inter-day, respectively. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.2 mg.kg-1 and 0.8 mg.kg-1, respectively. The procedure developed in this study was tested to be simple, sensitive and suitable for the determination and screening of CTC in pig solid manure samples.
Chlortetracycline (CTC), the first member of the tetracycline (TC) family, is a compound of clinically important natural products and semi-synthetic derivative characterized by a broad spectrum of activity against pathogenic microorganisms, including gram-positive and gram-negative bacteria and protozoa (Corcoran et al., 1975). It is widely used in animal industry for growth promoters, prophylaxis and treatment of respiratory and alimentary tract infections in pigs, poultry and other farm animals.
In China, more than 9000 tons of antibiotics are administered annually for veterinary purposes (including antibiotics used in animal feeds). Half of them are tetracycline-group drugs, and especially CTC is frequently added to feed for the treatment or prevention of bacterial diseases in pig-fattening. Recently, more and more Chinese farmers tend to scrape the solid manure out from swine farm instead of flushing, because of high disposal cost of liquid manure and much stricter regulations. The manure, after scraped out, is often stockpiled outside on the field, or spread on land as fertilizer. Thus, there is a potential for manure-derived antibiotics to ultimately reach surface and ground water by runoff or leaching and lead to more tetracycline-resistant microbes, which have become a serious public health concern (Koike et al., 2007). New investigations show that up to 46 mg.kg-1 CTC was found in pig manure in Austria (Martinez-Carballo et al., 2007) and 100 µg.kg-1 in liquid manure (Hamscher et al., 2002). Therefore it is important and necessary to establish a simple, sensitive and specific analytical method for CTC residue detection that could support its monitoring and study its fate and potential ecological risks in the environment.
Although CTC can be successfully determined using high performance liquid chromatography coupled with UV, fluorescence detection, or mass spectrometry in various matrices (Cinquina et al., 2003; Samanidou et al., 2007; Bogialli et al., 2007), few methods were appropriate for manure samples. Most of the methods involved a complicated extraction step followed by solid-phase extraction (SPE). However, these procedures can be laborious and time-consuming or the use of some type disposable cartridges can lead to poor and inconsistent recoveries (Blanchflower et al., 1997). Sunderland et al. (2003) developed a HPLC method to detect CTC in pig faeces that also involved the SPE clean-up step, but the limit of quantitation (LOQ) of 3.5 mg.kg-1 was too high for monitoring the lower level in pig manure. Martinez-Carballo et al. (2007) developed a liquid chromatography-quadrupole mass spectrometer (LC-MS/MS) method for determination of TC antibiotics including CTC in liquid manure. Mass spectrometry requires costly instruments, and liquid manure contains 95% water and less interfering peaks from endogenous compounds than solid manure.
The aim of this study was to develop a simple and rapid high-performance liquid chromatography (HPLC) method for the determination of CTC in pig manure. The precision, specificity and detection capacity of the whole procedure were validated.
Materials and methods
Chemicals and reagents
Chlortetracycline hydrochloride (purity 99%) was purchased from Dr. Ehrenstorfer (Augsburg, Germany). Acetonitrile and methanol (HPLC grade) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Acetone and hydrochloric acid were of analytical reagent grade. Oxalic acid (purity 99.6%) was purchased from Shanghai Chemical Industries Company (Shanghai, China). Deionized water was obtained from a Milli-Q system from Millipore (Millipore, Milford, MA, USA). Stock solutions (1 mg.mL-1) of CTC were prepared by dissolving 10.0 mg of the CTC standard in 10 mL of methanol and stored in the dark at -20°C. Working standard solutions were prepared daily by dilution of stock solutions in the mobile phase. The extraction solutions consisted of a mixture of acetone, 4 mol.L-1 hydrochloric acid and water (13:1:6, v/v/v) and were weekly prepared.
Twelve manure samples analyzed was collected from 12 pig farms around Jiangsu Province (China) and kept at -20°C before analysis. Blank pig manure samples were obtained from the experimental pig farm in Jiangsu Academy of Agricultural Sciences, where 100 pigs were not fed with CTC for 2 months. No CTC was detected in blank manure. All manure samples collected were tested and the pH, moisture and C/N ratio were 7.9-8.1, 78-81% and 12.5-14.2, respectively.
Analysis was carried out on an Agilent 1100 Series LC equipped with UV detector (Agilent Technologies, Palo Alto, CA, USA), column thermostat and a 20 μL injection loop. The chromatographic separation was performed with elution on a ZORBAX SB-C18 analytical column (250 mm×4.6 mm, 5 µm) at 375 nm. The mobile phase consisted 0.01 mol.L-1 oxalic acid-methanol-acetonitrile (72:14:14, v/v/v).
An accurately weighed 3.0 g amount of manure sample was placed into a 50 mL centrifuge tube, 12 mL of extraction solutions were added and shaken 30 s in a vortex mixer at high speed. The solution was pH adjusted at 2.0-2.2 with 4 mol.L-1 hydrochloric acid, before extraction for 20 min with an oscillator by the maximum speed. After centrifugation at 13,000 rmp for 10 min (at 4˚C), supernatants were transferred and filtered through 0.22 µm membrane filters Millipore (Sao Paulo, Brazil), and 20 µL was injected into HPLC for analysis.
The specificity was evaluated by the analysis of 10 blank samples taken from the pig manure and observation of the interfering peak around CTC. The wavelength of 375 nm and 362 nm was compared for the separation under the selected chromatographic separation condition.
The recoveries of CTC from manure were evaluated by analyzing four replicates of quality control samples at three fortified levels of CTC: 1.0 mg.kg-1, 2.0 mg.kg-1 and 10.0 mg.kg-1. The spiked samples were kept in the dark at 4˚C for 30 min before analysis. Four replicates of samples at each level were analyzed in one day to evaluate the intra-day recoveries and variations. As well, four replicates of samples at each level were analyzed on different days (n=3) under the same conditions to evaluate the inter-day recoveries and variations of this procedure. The recoveries were calculated by means of the standard calibration curves with the peak area.
Six-point matrix-matched calibration curves were prepared by spiking blank manure extracts with CTC in concentrations of 0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 µg.mL-1 for linearity evaluation. Four replicates for each concentration were conducted. The LOQ was obtained by calculation as a peak height at least three times and greater than the baseline noise and a % CV of <10 for four replicates. The limits of detection (LOD) was obtained from the calculation as a peak height greater than three times the baseline noise but with a % CV of >10 for four replicates.
In addition, in this study four extraction solutions were compared: i) 5% perchloric acid; ii) 0.1 mol.L-1 Na2EDTA-McIlvaine buffer solution (pH 4.0); iii) the McIlvaine buffer solution (pH 4.0); and iv) acetone-4 mol.L-1 hydrochloric acid-water (13:1:6) for extraction followed by adjusting the pH at 2.0 to 2.2 with 4 mol.L-1 hydrochloric acid. 2.0 mg.kg-1 of CTC was spiked into blank manure and stood for 30 min at 4°C. The analysis was conducted following the analytical procedures already described with four replicates. Recoveries were obtained from the standard calibration curves.
Results and discussion
Specificity and LC separation
Figure 1 shows the chromatograms obtained from the analysis of the blank pig manure and the standard solutions with different mobile phase. The retention time of CTC was between 15.12 min and 15.86 min under the selected chromatographic conditions (Figure 1C). No interfering peaks from endogenous compounds were found in the retention time of CTC at 375 nm with the selected mobile phase composed of 0.01 mol.L-1 oxalic acid-methanol-acetonitrile (72:14:14, v/v/v), which indicated that the assay was selective.
CTC is a well-known compound forming chelates with divalent and trivalent cations (Jin et al., 2007). In order to avoid forming chelates, in this study, reversed phase column chromatography, mobile phases with low pH and oxalic acid were used to improve the peak shape. The different combination ratio of mobile phase was compared, and the optimal separations were achieved with methanol-acetontrile and 0.01 mol.L-1 oxalic acid in 14:14:72 (v/v/v) mixtures for CTC (Figure 1).
Recovery and precision
Table 1 summarizes the recoveries and intra- and inter-day relative standard deviation (RSD) of spiked manure of CTC; recoveries of CTC ranged from 90.21% to 101.95% at the three spiked levels. Intra- and inter-day RSD were less than 7.11% and 3.25%, respectively (Table 1). The results indicate that the procedure is reliable, reproducible and accurate.
Linearity and sensitivity
The linearity for pig manure was good over the range 0-20 mg.kg-1. The linear regression equation and correlation coefficients were Y=19.064X-0.9721 and 0.9994, respectively. The correlation coefficient was satisfactory and indicated that this procedure is reliable for quantitative detection. The LOQ and LOD of this method were 0.8 mg.kg-1 and 0.2 mg.kg-1, respectively.
Extraction of CTC
The recoveries from four extraction solutions are shown in Figure 2. Obviously, the recoveries from the first three extraction solutions, lower than 70%, were markedly lower than the fourth extraction. The first three extraction solutions were successfully used to extract CTC residue from cheese, kidney and plasma (Posyniak et al., 2005; Bogialli et al., 2007; Cherlet et al., 2006). However, pig manure is a very heterogeneous and complex matrix in which the composition of ions, organic carbon and nitrogen and phosphorus varies significantly depending on the animal tissue. More optimistic results were obtained when solid manure was treated with acetone-4 mol.L-1 hydrochloric acid-water (13:1:6) and the pH of mixed solution was adjusted into 2.0 to 2.2 with 4 mol.L-1 hydrochloric acid. CTC might form chelate complex with metal ions and bind proteins of solid manure samples. The extraction solution pH of 2.0-2.2 could keep CTC presence in ionic form and expose to extraction solution, and thus, extraction efficiency were significantly improved.
Applicability of the method
Twelve pig manures, collected from 12 farms, were processed according to the analytical procedure described in material and methods. The presence of CTC residues was found in 8 samples at levels from 1.1 mg.kg-1 to 25.6 mg.kg-1 (Table 2).
The described method is sufficiently good, rapid and low-cost; it can therefore be used for quantification and confirmation of CTC in pig manure. It can be especially applied for monitoring the contaminant in pig manure when large number of samples has to be analysed. Another advantage of this method is that no SPE column or complicated extraction solutions are needed.
|Figure 1. HPLC chromatogram for (1) CTC standard solution and (2) blank pig manure sample in mobile phase composed of 0.01 mol.L–1 oxalic acid-methanol-acetonitrile with volume ratio of (A) 64:18:18; (B) 68:16:16; (C) 72:14:14.|
|Figure 2. Effect of four extraction solutions on recoveries of CTC.|
|Table 1. Recoveries and precision of CTC determined in spiked pig manure samples (n=4).|
|Table 2. CTC concentration (mg/kg, mean ± SD) determined in 12 pig manures (n=4).|
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