Introduction

Fluoroquinolones group of antibacterial agents have broad-spectrum antibacterial activity and so have been in use in veterinary medicine. They are mainly used against urinary, pulmonary and digestive infections (Bauditz, 1990). These antimicrobial substances are used for preventive and therapeutic purposes in farm animals (Salehzadeh et al., 2007). In fact they have been used successfully to treat infection caused by Gram positive and Gram negative bacteria. These compounds act by inhibiting the bacterial DNA-gyrase (Gilles et al., 1991). Chemical structure of fluroquinolones when changed, an increase in the spectrum of activity and potency of the drugs is seen (Spoo et al., 1995).Enrofloxacin is one of the antibacterial agent which is widely used in veterinary medicine (Nouws et al., 1988). But indiscriminate use of this agent possesses a considerable risk due to the presence of residues in meat meant for human consumption. Enrofloxacin residues are reported in animal tissues (Schneider, 2001; Cornejo et al., 2011). The FAO /WHO, 2002 have recommended Maximum Residue Limits (MRL) for Enrofloxacin in animal tissues (MRL of kidney: 0.3 µg/g; liver: 0.2 µg/g and muscle: 0.1 µg/g). The north-eastern states of India is characterized by a high proportion of tribal people for whom pig rearing is integral to their way of life and pig meat is considered as an important food item. Enrofloxacin may be found as residues in meat of slaughtered pig. Thus, the present study was undertaken to detect residues of Enrofloxacin in marketed pork of north-eastern states of Assam, Meghalaya and Mizoram using Ultra High Performance Liquid Chromatography (UHPLC) System.

Materials and Method

The analysis was done as per the method of Salehzadeh et al., 2007.

Chemical and Reagents

Enrofloxacin standard (Dr. Ehrenstofer, Germany); HPLC grade Acetonitrile (Fisher), Methanol (Fisher); other chemicals and solvents of analytical grade and HPLC grade water (Fisher) were used for the study.

Sample Collection

A total of 586 numbers of swine tissues samples comprising of muscle, kidney and liver were collected from different markets of Assam Meghalaya and Mizoram (Table 1).

Table 1: Collection of Pork Samples

The samples were collected in sterile containers from the market and after collection were preserved under freezing temperature of -20 °C.

Chromatographic Condition

The levels of Enrofloxacin were determined using a UHPLC system of make- Dionex equipped with an autosampler and Diode Array Detector (DAD) operated at 277 nm. The samples were separated on a RP-C₁₈ column and were eluted with a mobile phase of mixture of water and acetonitrile in the ratio of 67:33 v/v. The isocratic mode was run at a flow rate of 1.0 ml.min^-1.

Preparation of Sample

The fascia and fat of pork were removed and then cut into small pieces. 10 g of the sample was taken in a blender and to it added equal volume of distilled water. Ten grams of each blended sample was transferred to centrifuge tube. After few minutes 10 ml of acetonitrile was added. The sample was ultrasonicated and left undisturbed for 10 min. The samples were centrifuged and the collected supernatant was filtered. The filtrate then was passed through C₁₈ polymeric cartridge after which it was further filtered using 0.22µm filter paper.

Statistical Analysis

Statistical analysis was performed to determine the Mean ± S.E. using Microsoft Excel, 2010.

Results and Discussion

Linear calibration curve of Enrofloxacin having correlation coefficient (R²) of 0.99 was achieved. Recoveries of enrofloxacin in pork ranged from 86-97%. This was similar to that reported by Cinquina et al., 2003. Enrofloxacin was eluted under isocratic conditions. The excitation/emission wavelengths used for the UV detection were 277 nm for Enrofloxacin. The separation of the analytes was achieved in less than 5 mins. Acetonitrile was effective in the deproteinization of pork samples and in the isolation of analytes from spiked samples. This method allows the determination of residues of Enrofloxacin in different matrices with higher sensitivity.

Over all, six districts of Assam, four districts of Meghalaya and four districts of Mizoram have been selected and a total of 526 samples of pork comprising of muscle, liver and kidney were collected. A total of 35 pork samples were detected to be positive of Enrofloxacin residue. As listed in Table 2, only 13 kidney, 10 liver and 13 muscle samples showed detectable Enrofloxacin residues using UHPLC. All the samples were below the permissible limit. Residue level of Enrofloxacin detected in muscle, kidney and liver were 0.005-0.078µg.g^-1, 0.015-0.257µg.g^-1 and 0.008-0.123 µg.g^-1 respectively (Table 3).

Table 2: Enrofloxacin Residues Detected in Pork Sample

ND-Not detected

Table 3: Enrofloxacin Residue level in Swine Tissues

Conclusion

A total of 586 samples of pork were collected from different pork markets of Assam, Meghalaya and Mizoram. Out of the screened samples, 36 samples were detected to be positive for trace residues of Enrofloxacin using UHPLC which were all below the MRL. Thus, it can be concluded from the present study that the pork of North East States are safe for consumption.

Acknowledgement

The authors acknowledge all help and assistance received from ICAR and AAU, Jorhat.

References

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