Development and validation of HPLC methods for the determination of tetracyclineantiobiotics residues in various food products of animal origin

Tetracycline antibiotics (TCs) are broad-spectrum agents, exhibiting activity against infections caused by both Gram-positive and Gram-negative bacteria, as well as chlamydia, mycoplasmas, rickettsiae and protozoan parasites. TCs are the most widespread group of antibiotics used in veterinary medicine and animal husbandry, not only to prevent and treat certain diseases, but also to fraudulently promote growth. However the abundant and in some cases improper use of TCs may result in the presence of residues in edible animal tissues, which can be toxic and dangerous for human health and potentially cause allergic reactions. Moreover the long-term presence of TCs as residues even in low doses may generate the evolution of micro-organisms provoking resistance to antibiotics. In the present study high-performance liquid chromatographic methods were developed and validated for the determination of seven tetracyclines: minocycline (MNC), tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), doxycycline (DC), demeclocycline (DMC) and methacycline (MTC), in various food products of animal origin. The separation of the examined TCs was achieved on a Kromasil 250 x 4 mm, C18 5 μm analytical column, operated at ambient temperature, with the following elution system: C2H2O4 (0,01 M, pH=2)-10-4Μ Na2ΕDTA/CH3CN according to the gradient program: 0 min 88:12v/v, 2 min 80:20v/v, 4 min 73:27v/v, 10 min 70:30v/v, flow rate 0 min 1.65 mL/min, 3 min 1.65 mL/min, 5 min 1.2 mL/min. Detection and quantitation of the seven TCs was performed on a photodiode array detector at 355 nm. The total analysis time for the separation of TCs with the described chromatographic conditions was less than 10 min and the order of elution was: MNC at 2.994 min, OTC at 4.809 min, TC at 5.569 min, DMC at 6.639 min CTC at 8.001 min, MTC at 8.453 min and DC at 9.025 min. The quantitative determination of the TCs was based on the internal standard technique. For this purpose colchicine was chosen as the most suitable compound at a concentration level of 5 ng/μL. Under experimantal conditions the examined TCs were well separated according to the values of resolution factors. Linearity of the proposed method in standard solutions of TCs is considered satisfactory since r values vary from 0.9941 to 0.9980. Limits of detection and limits of quantitation achieved, were at the level of 12 and 20 ng/μL and 37 to 62 ng/μL respectively. In order to apply the developed method to the determination of TCs in a number of selected food products of animal origin which were chosen (bovine, porcine and chicken tissues, milk, egg yolk, liver and kidney tissues), various protocols were tested for the preparation of the spiked samples of those products. Sample preparation of each matrix includes extraction of TCs from the samples with a buffer and cleaning-up the extracts with SPE. Oxalate buffer (pH 4) is considered as the most suitable buffer for the extraction of TCs from all kind of matrices. For milk, egg yolk, liver, kidney and chicken tissues, proteins were first precipitated. Three different cartridges (Nexus, Discovery, Lichrolut) were chosen to be used during the optimization of the SPE procedure for the purification of extracts taken from each matrix. Application of the most efficient for each sample-matrix preparation protocol, lead to the absence of unknown peaks due to endogenous compounds which co-elute with TCs preventing their detection or quantitation. The procedure for each sample-matrix was validated according to the European Union regulation 2002/657/EC determining selectivity, decision limit, detection capability, accuracy, precision and stability. Limits of quantitation of all seven TCs determined in each matrix were lower than from the Maximum Residue Level (MRL) enacted by E.U. in each case. Both mean recoveries of TCs from the matrices and CV values calculated during accuracy and precision tests are very satisfactory after comparison with the limits set by 2002/657/EC decision. Decision limits (CCα) and detection capability (CCβ) values were also calculated for each TC in every matrix at the MRL. Finally during stability study the examined TCs showed a uniform behaviour, since they were stable at all spiked samples for 4 weeks. Stability was also assessed after four freezing-defrosting cycles. TCs concentrations were stable for two cycles of freezing and de-frosting the tissue samples, while a mean decrease of 30% in concentration was observed after the third cycle. In conclusion it should be noticed that the characteristics of the developed method such as quantitation limits lower than MRL, good sensitivity, satisfactory recoveries, simple sample pretreatment method, relatively short analysis time, demonstrate that the method can be easily and reliably applied to the detection and determination of TC antibiotic residues in a number of food products of animal origin