Analysis of tetracyclines in chicken tissues and dung using LC–MS coupled with ultrasound-assisted enzymatic hydrolysis
Introduction
Antibiotics are normally used agriculturally for therapeutic, prophylactic and growth-promoting purposes. The annual consumption of antibiotics in the United States (US) was estimated at 10,000 metric tons in 2001 (Austin, Kristinsson, & Anderson, 1999), and the consumption has continued growing exponentially in recent years. Tetracycline antibiotics (TCs) are usually added to animal feeds for disease prevention and treatment, as well as for the promotion of growth. The most commonly used TCs include oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC) (Fig. 1) (Leng, Zhao, Wang, & Li, 2013). On one hand, part of the added TCs will remain in the animal bodies, which presents a risk that the residual TCs are taken in by humans due to the intake of animal meats. To ensure food safety for the consumers, the EU has set the maximum residue limits (MRLs) for TCs as 0.3 mg kg−1 in liver, 0.6 mg kg−1 in kidney, 0.2 mg kg−1 in egg, and 0.1 mg kg−1 in milk or muscle tissue (European Economic Community, 1990). The USA Food and Drug Administration (FDA) has set the MRLs for the sum of TCs residues as 2 mg kg−1 in muscle, 6 mg kg−1 in liver, 12 mg kg−1 in fat and kidney, and 0.4 mg kg−1 in milk (Code of Federal Regulations, 2003, chap. 1). At this point, it is necessary to develop a fast and exact analysis method of TCs for screening all types of foodstuffs of animals, such as muscle and liver. On the other hand, most of the added TCs may be excreted by animals as parent compounds or metabolites and, thus, enter the environment through manure or ground water as a carrier vector. Due to the application of animal excrement as a fertilizer, the disposal of TCs will greatly increase their exposure to the ecological environment. Therefore, any TC residues in manure must be subjected to strict control.
Because of the recent awareness of the potentially dangerous consequences of the presence of TCs in the environment, significant attention has been paid to the analytical methodology used in the determination of TCs in complex matrices. Most of the reported analytical methods for TCs analysis were based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (Li et al., 2008), diode array detection (DAD) (Viñas, Balsalobre, López-Erroz, & Hernández-Córdoba, 2004) or fluorescence detection (Gustavo, Susanne, & Felix, 2010). In recent decades, liquid chromatography–mass spectrometry (LC–MS) has experienced impressive progress in terms of both technological development and application (Choi et al., 2007, Consuelo et al., 2011; Lindsey, Meyer, & Thurman, 2001). In principle, LC–MS/MS is a good technique to assay polar pharmaceuticals and their metabolites and is especially suitable for environmental analysis because of its selectivity. However, in some animal matrices (e.g., chicken muscle, liver or manure), antibiotics at residue levels are often masked due to matrix interferences. Thus, almost all analysis requires a sample pretreatment step to enrich the TCs and remove the interferences. Solid-phase extraction (SPE) is commonly used as a proper pre-concentration and clean-up step (Patrícia, Ernani, Susanne, & Felix, 2009). Fedeniuk, Ramamurthi, and McCurdy (1996) analyzed OTC in different samples using HPLC combined with SPE on a Bond-Elute C18 cartridge, and found that the recovery of OTC was above 90% in water samples, but it was lower than 70% in porcine tissue. The loss of OTC in porcine tissue may be due to the specific interactions between OTC and protein, which is the main component of tissue. This behavior also strongly suggests that SPE could not completely eliminate the influence of the protein-containing matrix and, therefore, is unable to meet the analytical requirements for TCs in animal-related matrices.
In general, the analysis of protein-containing samples requires a pretreatment for deproteination, which is usually accomplished by treating the samples with acid (trichloroacetic acid, TCA), organic solvents (methanol and acetonitrile (ACN)) or heat. However, these treatments are seemingly not very efficient at removing the interference. Granelli and Branzell (2007) developed an LC–MS/MS approach for the multi-residue screening of five classes of antibiotics in muscle and kidney samples from pig, cattle, sheep, horse, deer and reindeer by extracting the antibiotics from the animal tissues with 15 mL of 70% methanol and 10 min of shaking. They found that the recoveries of sulfonamides, quinolones, β-lactams and macrolides were suitable, but the ones for TCs were rather low: 47%–62% in muscle, and 26%–39% in kidney. Cherlet, Schelkens, Croubels, and Backer (2003) analyzed TCs and their 4-epimers in pig tissues using HPLC combined with positive-ion electrospray ionization mass spectrometry and found that the TCA deproteination pretreatment produced very low recoveries of TCs (<25%) with a minimum of only ∼7% for DC in liver samples. The authors had no explanation for the particularly low recoveries. In our opinion, all of the low recoveries of the TCs are possibly due to the interactions of the TCs with the proteins in the complex matrices based on our preliminary experiments. Conventional deproteination methods led to high losses of TCs because the TCs were strongly bound on protein aggregates. We noted that Hu, Pan, Hu, Huo, and Li (2008) reported a method for trace analysis of TCs in chicken feed, chicken muscle and milk with satisfactory recoveries of TCs between 71.6% and 93.7%. To achieve satisfactory recoveries of TCs, however, they found that it was necessary to use different extraction methods for different samples: microwave assisted extraction for feed, ethyl acetate buffer extraction for muscle, and hydrochloric acid extraction for milk. Therefore, there is certainly a need for develop a method that is able to not only eliminate the effects of proteins in complex biological backgrounds but is also suitable for different matrices.
In the present work, our goals were to develop a novel approach that combines ultrasound and enzymatic hydrolysis to eliminate the co-protein effects and an LC–MS/MS method for the quantitative analysis of TCs. Through comparison with acid deproteination and normal enzymatic hydrolysis, the merits of the new enzymatic approach were clearly confirmed: it not only significantly improved the recoveries of TCs but also greatly shortened the pretreatment time. After the satisfactory recoveries of TCs were confirmed by analyzing spiked animal samples of chicken muscle, liver and manure, the method was applied to the simultaneous multi-residue analysis of TCs in manures, giving satisfactory results.
Section snippets
Chemicals and materials
Hydrochloride salts of OTC, CTC and DC were obtained from Sigma–Aldrich (St. Louis, Missouri, USA). TC hydrochloride was purchased from Dr. Ehrenstorfer GmbH (Bgm Schlosser-Str., Augsburg, Germany). Trypsin and BSA were purchased from Regal Biological Company (Shanghai, China). Methanol and ACN were used as the mobile phase and were of HPLC grade from Tedia Company Inc. (Fairfield, OH, USA). All of the other chemicals were analytical grade reagents from Sinopharm Chemical Reagent Co., Ltd
Deproteination with TCA or methanol as a pretreatment
It is well-known that TCs have a marked interaction with BSA, as evidenced by the fact that the intrinsic fluorescence of BSA could be significantly quenched by adding TCs (Bi et al., 2005). Therefore, deproteination was needed to remove proteins from the samples. Fig. 2 shows the recoveries of TCs in the presence of different amount of BSA with a pretreatment of TCA and methanol denaturation at 4 °C. For each of the TCs, the recovery was significantly decreased with increasing the molar ratio
Conclusion
The experimental results confirmed that the protein in animal matrices could be removed via chemical denaturation but led to a loss of TCs as high as 60%–70%, due to the co-precipitation and adsorption within protein aggregates. This interference was efficiently eliminated by using ultrasound-assisted enzymatic hydrolysis as a pretreatment approach, which is capable of disrupting the folded, native structures of proteins. Coupled with SPE and LC–MS/MS, this method was successfully used for
Acknowledgment
This work was partially supports by the National High Technology Research and Development Program of China (No. 2012AA06A304).
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