|Year : 2014 | Volume
| Issue : 1 | Page : 72-74
High-performance liquid chromatography method validation for determination of tetracycline residues in poultry meat
Vikas Gupta, Sushma Ahlawat, Bhavesh Patel, Om Shankar, Kuldeep Kumar, Sanjeev K Shukla, Shubhra Shukla, Navneet Kaur, Manisha Dubey, Lakshya Veer Singh
Department of Biochemistry and Bioprocess Technology, Sam Higginbottom Institute of Agriculture, Technology and Science, Allahabad, Uttar Pradesh, India
|Date of Web Publication||25-Mar-2014|
Sam Higginbottom Institute of Agriculture, Technology and Science, Allahabad - 211 007, Uttar Pradesh
Source of Support: This work was supported by the Sam Higginbottom Institute of Agriculture, Technology & Science, Allahabad, India., Conflict of Interest: None
| Abstract|| |
Background: In this study, a method for determination of tetracycline (TC) residues in poultry with the help of high-performance liquid chromatography technique was validated. Materials and Methods: The principle step involved in ultrasonic-assisted extraction of TCs from poultry samples by 2 ml of 20% trichloroacetic acid and phosphate buffer (pH 4), which gave a clearer supernatant and high recovery, followed by centrifugation and purification by using 0.22 μm filter paper. Results: Validity study of the method revealed that all obtained calibration curves showed good linearity (r2 > 0.999) over the range of 40-4500 ng. Sensitivity was found to be 1.54 and 1.80 ng for oxytetracycline (OTC) and TC. Accuracy was in the range of 87.94-96.20% and 72.40-79.84% for meat. Precision was lower than 10% in all cases indicating that the method can be used as a validated method. Limit of detection was found to be 4.8 and 5.10 ng for OTC and TC, respectively. The corresponding values of limit of quantitation were 11 and 12 ng. Conclusion: The method reliably identifies and quantifies the selected TC and OTC in the reconstituted poultry meat in the low and sub-nanogram range and can be applied in any laboratory.
Keywords: Extraction, high-performance liquid chromatography, poultry, residues, separation, tetracycline
|How to cite this article:|
Gupta V, Ahlawat S, Patel B, Shankar O, Kumar K, Shukla SK, Shukla S, Kaur N, Dubey M, Singh LV. High-performance liquid chromatography method validation for determination of tetracycline residues in poultry meat. Chron Young Sci 2014;5:72-4
|How to cite this URL:|
Gupta V, Ahlawat S, Patel B, Shankar O, Kumar K, Shukla SK, Shukla S, Kaur N, Dubey M, Singh LV. High-performance liquid chromatography method validation for determination of tetracycline residues in poultry meat. Chron Young Sci [serial online] 2014 [cited 2019 Dec 8];5:72-4. Available from: http://www.cysonline.org/text.asp?2014/5/1/72/129344
| Introduction|| |
Tetracyclines (TCs) are an important group of antibiotics used in livestock and poultry production. TCs are given to animals destined for human consumption not only to prevent and treat certain diseases, but also to fraudulently promote growth.  Improper use of TCs may result as residues in edible animal tissues, which can be toxic and dangerous for human health and potentially cause allergic reactions. The long-term presence of TCs residues may generate the evolution of micro-organisms provoking resistance to antibiotics.  For human health, the maximum residue level values of TCs residues in animal products have been established by Codex Alimentarius Commission is 200 ng/g in muscle, 600 ng/g in liver, 1200 ng/g in fat and kidney (The Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives, 2004). Therefore, the study was planned to validate this method, which is needed to detect the level of TCs in animal products.
Sample preparation includes an extraction step with a suitable solvent system along with clean up procedure. It is difficult to extract the TCs from biological materials through organic solvent,  due to less log P value. So extraction technique involves acidic phosphate buffer at pH 4.0 by protein precipitation with 20% trichloroacetic acid (TCA) as well as with perchloroacetic acid. But TCA gives good recovery compare to perchloroacetic acid. Though an additional extract clean up to isolate TCs from interference of a complex sample matrix using solid phase extract and/or liquid-liquid extraction technique is required prior to the chromatographic analysis. Separation of TC residues isolated from biological matrixes was performed on a variety of high-performance liquid chromatography (HPLC) columns using different mobile phases by isocratic and gradient elution. Reversed-phase HPLC is the preferred mode although ion-exchange can also been used. The most widely used reversed-phase column type was C18 column. A great variety of mobile phases has been used such as phosphoric acid or phosphate buffers at acid pH, citrate buffers and phosphate buffer  and organic modifiers include acetonitrile,  methanol  and acetonitrile-methanol.  The detection of TCs residue is, generally done by a suitable detector like as ultraviolet (UV) and florescence detectors. Therefore, the present work is an attempt to develop an accurate, precise and sensitive HPLC-diode array detector (DAD) method.
| Materials and Methods|| |
Stock standard solution of each TC compound was prepared by dissolving 10 mg of the compound in 10 ml of methanol to obtain a final concentration of 1 mg/ml. Stock standard solutions were put in amber glass to prevent the photo-degradation and stored at −20°C. The solution was stable for at least 4 weeks. Stock solutions were diluted with methanol water (50:50) to give a series of working standard solutions that were prepared weekly.
Fortification of samples
Fortified by 0.9% saline solution, chicken samples were spiked with TCs standard mixture at two levels of 100 ng and 200 ng of each compound per gram meat. Fortified samples were allowed to stand at 4°C for 1 h before analysis.
Take 5 g sample and put it in 50 ml capacity polypropylene tube and then add saline solution and acetonitrile, homogenate with homogenizer in ice bath using TCA as an extracting solvent then centrifuge with cooling centrifuge.
Clean up techniques
The cleanup of the aqueous based extracts was performed on 0.22 μm Millipore filter.
The HPLC system of a HP 1100 chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with an auto-sampler set at 150l, quaternary pump and a DAD was used. The analytical column was reversed-phase (Phenomenex 100 C18, 25 cm × 4.6 mm I.D., 5 m, Germany). The oven temperature was set at 20°C. Refrigerator centrifuge, ultrasonic bath (Buhler, Hechingen, Germany) and homogenizer (Mechanika Precyzyjna, Model type ST-2) were used for sample preparation.
Chromatographic separation of TCs
Various mobile phases were evaluated for separating TCs on a Phenomenex 100 C18 column coupled with a guard column. The mobile phases tested were as follows:
- The mixture of acetonitrile/50 mM potassium dihydrogen phosphate (40:60, v/v) was used as an isocratic elution at flow rate of 1 ml/min.
- The mixture of methanol/acetonitrile/50 mM potassium dihydrogen phosphate (10:30:60, v/v/v) was used as an isocratic elution at flow rate of 1 ml.
- The gradient elution using methanol (a) acetonitrile (b) 50 mM phosphate buffer (c) was applied starting, by volume, with 5:25:70 (a:b:c) which remained isocratic for 7 min, changing to 10:25:65 in 5 min, then to 17.5:22.5:60 after 4 min and going back to the initial conditions in 3 min.
- The gradient elution using methanol (a) acetonitrile (b) and 50 mM phosphate buffer (c) was applied starting, by volume, with 0:8:92 (a:b:c) which changed to 0:18:82 in 2 min and to 5:20:75 in 0.1 min which remained isocratic for 4.9 min, then changed to 10:25:65 in 3 min, then to 15:20:65 in 1 min and to 15:25:60 in 3 min, which remained isocratic for 2 min and going back to the initial conditions in 3 min.
Detection and quantitation
The separated TCs were detected with DAD and the quantitation was integrated by Chemstation chromatographic software interfaced to a personal computer.
| Results and Discussion|| |
Selection of the optimum detection wavelength
In multi-residue analysis of TC in food, HPLC with UV detectors were the most commonly used one because they are more readily available and convenient to use in labs particularly in the developing countries. DAD is getting popularity, TC is typically read at the range of 350-365 nm in an acidic solution.  In the present study, TCs standard solution were separated on the Nucleosil 100 C18 column and two wavelengths, i.e., 351 and 365 nm, were tested to monitor the separated TCs by measuring the response factor of the DAD. It was found that the detector response factor at 365 nm was 52.97 mAU for TC. The corresponding values obtained at 351 nm were 136.14 and hence, 351 nm selected as optimum detection wavelength for TCs determination and used throughout this study. Therefore, selection of mobile phase to separate TCs on the Phenomenex 100 C18 column is needed. In the present work, both isocratic and gradient elution were evaluated for their efficiency to separate TCs. The separation and peak shapes of TCs standards were better than those obtained with the other tested mobile phases. As well as, good resolution (Rs) between the examined chromatogram peaks was assured by the obtained values of Rs with regard to the previous eluted peak, using the equation: Rs = 2 (retention times [tR2 − tR1])/(wh1 + wh2), were 2.7, for oxytetracycline (OTC)-TC. Chromatographic parameters, i.e., tR, capacity factor (k) and peak symmetry of the separated TCs peaks were calculated [Table 1] and [Figure 1].
In order to obtain optimal extraction efficiency, various widely acceptable extraction solutions like McIlvaine buffer (procedure B) as aqueous based extraction and acetonitrile (procedure C) as organic based extraction were tested to extract TCs from the samples fortified with a mixture of the two TCs at level of 100 and 200 ng/g meat.
Linearity and sensitivity
The study was extended to assay the validity of the suggested method. Linearity was evaluated by calibration in the range of 40-4500 ng for each compound at five points with triplicate analysis. The obtained results are given in [Table 2]. It is clear that the response of the DAD was linear and highly correlated with the amounts of TC injected, where the calculated coefficient (r2 ) ranged from 0.9991 to 0.9998 and each TCs had own linear equation. The sensitivity (the change in analytical signal units per nanogram analyte) of the method is usually represented by the slope of the analytical calibration curve.  The sensitivity of the suggested method was found to be 1.54 and 1.80 ng/g for OTC and TC.
Limit of detection (LOD) and limit of quantitation (LOQ)
The LOD is considered to be the quantity yielding a detector response approximately equal to thrice the background noise.  The LOQ is the lowest amount that can be analyzed within acceptable precision and accuracy at signal to noise ratio of 10.  Accuracy was in the range of 87.94-96.20 and 72.40-79.84. Both LOD and LOQ were measured and the obtained data were given in [Table 2]. It could be observed that LOD of the suggested method was 4.8 and 5.10 ng for OTC, TC. The corresponding values of LOQ were 11 and 12 ng.
| Acknowledgment|| |
This work was supported by the Sam Higginbottom Institute of Agriculture, Technology and Science, Allahabad, India.
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[Table 1], [Table 2]