KMITL Sci. J. Vol.8 No.

2 (Section B) July December, 2008

DETERMINATION OF TETRACYCLINE ANTIBIOTIC RESIDUES IN HONEY SAMPLES COLLECTED FROM NORTHERN PART OF THAILAND BY HPLC
Narin Taokaenchan1 and Supaporn Sangsrichan1
1

Department of Chemistry, Faculty of Science, Maejo University, Chiang Mai, 50290, Thailand

ABSTRACT
A high performance liquid chromatography method utilizing fluorescence detection was optimized and validated to determine tetracycline residues in honey. The separation of four tetracycline residues; oxytetracycline, tetracycline, chlortetracycline and doxycycline was observed on a reversephase C 8 column with a gradient elution. A mobile phase consisted of 50% (v/v) methanol and 25 mM sodium acetate buffer (containing disodium ethylenediaminetetraacetate and calcium chloride, pH 8.10). Fluorescence detection was observed at 518 nm (excitation wavelength at 393 nm) with analysis time of 20 min. The extraction with disodium ethylenediaminetetraacetate (Na 2EDTA)-McIlvaine buffer pH 4 was used followed by HLB cartridge clean up step. The good recovery for tetracycline and chlortetracycline were found above 89%, low recoveries for oxytetracycline and doxycycline which will be optimized to achieve higher percentage recovery.

KEYWORDS: tetracyclines, honey, fluorescence detection, high- performance liquid chromatography

1. INTRODUCTION
Antibiotics such as tetracyclines have been widely used for treating diseases in animal including bees [1]. The occurrence of antibiotic residues in human food, arising from its veterinary use, is a cause of concern to consumers worldwide, because of possible toxic or allergic reactions and the possibility that pathogenic organisms could become resistant to these drugs [2]. The application of the law in relation to these antibiotics is not harmonized across all member states of the European Union. The Commission of the European Union laid down the procedure for establishing maximum residue limits (MRLs) of veterinary medical products in foodstuffs of animal origin [3]. However, no MRLs have been fixed for using with bee products; nevertheless, some countries, such as Switzerland, have set MRLs for the TCs in honey at 20 gkg1. In Japan, base on microbiological research, a value of 0.1 mgkg -1 was introduced as the allowed residual quantity of tetracycline in honey [4]. Although the Thai law related to quality of honey affirms that it is illegal any type of additive or substance apart from honey at any level, but, there is no law in about the control of these drugs in honey, yet. Screening of antibiotics in honey is carried out by the Charm test [5, 6] or enzyme-linked immunosorbent assay (ELISA) [7, 8] prior to quantify the residues of the positively tested samples mostly by HPLC. Tetracyclines can be successfully determined in various biological matrices, HPLC in a reversephase mode, with different detection modes, such as UV [9-15], fluorescence [2, 16-20], chemiluminescence methods [21-23] and mass spectrometry [1, 24-28] have also been reported. The UV detection has lower sensitivity than mass spectrometry.

Corresponding Author. Tel. +66-53-873544-5 Fax. +66-53-873648 E-mail: supaporn-s@mju.ac.th

KMITL Sci. J. Vol.8 No.2 (Section B) July December, 2008

Oxytetracycline (OTC) : R1=H, R2=R3=OH, pKa1 = 3.2 Tetracycline (TC) : R1=R3=H, R2=OH, pKa1 = 3.3 Chlotetracycline (CTC) : R1=Cl, R2=OH, R3=H, pKa1 = 3.3 Doxycycline (DC) : R1=R2=H, R3=OH, pKa1 = 3.0

Figure 1 Structure of tetracyclines. (MS) [28], while still require costly instruments. In general, fluorescence detection is highly sensitive and selective with relatively lower cost than mass spectrometry. In this research, the extraction and chromatographic detection of four tetracyclines (see Fig. 1) were optimized and validated for the determination of tetracyclines in honey at LODs below 10 gkg-1. The separation of four tetracycline residues; oxytetracycline, tetracycline, chlortetracycline and doxycycline was observed on a reversed phase C8 column with a gradient elution. The tetracycline residues was extracted with buffer and clean up by using solid phase extraction (SPE), detected on a high performance liquid chromatography method utilizing fluorescence detection.

2. MATERIALS AND METHODS

2.1 Apparatus
The HPLC system consisted of Agilent model HP 1100 system (Agilent, USA). The column used was a reversed phase Zorbax C8 (150 x 4.6 mm I.D., 5m, Agilent, USA). Fluorescence detector was set an excitation wavelength of 380 nm and an emission wavelength of 518 nm.

2.2 Reagent and Material

Tetracycline, oxytetracycline and chlortetracycline standards were purchased from Merck, Germany and doxycycline standard was purchased from Fluka, USA. Disodium ethylenediaminetetraacetate, sodium acetate, calcium chloride and methanol were analytical-reagent grade (Merck, Germany). The Oasis HLB extraction cartridges, 6 cm3 (200 mg) were purchased from Water, USA. Individual stock standard solutions were prepared in 100 mL of methanol containing 100 mg of neat standard in a volumetric flask and were stored at -20 C in amber glass vials for a maximum period of 1 month. The working standards were mixtures of four compounds prepared by a serial dilution of the stocks in the 50% (v/v) methanol and 25 mM sodium acetate buffer (containing disodium ethylenediaminetetraacetate, pH 8.1). McIlvaine-EDTA buffer was prepared as described by Pena et al. [2]. A mobile phase system was consisted of 50% (v/v) methanol and 25 mM sodium acetate buffer (containing disodium ethylenediaminetetraacetate and calcium chloride, pH 8.1) at flow rate of 0.5 mLmin-1.

2.3 Calibration procedure

The calibration graphs were constructed with standard solution concentrations range from 10-1000 ngmL -1. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. The detection limit was defined as the signals three times the noise levels; 3S/N.

2.4 Honey Samples.

Honey samples, commercially available in Chiang Mai markets, were purchased from different beekeepers during 2008. Samples were stored at 4C in darkness until processing.

2.5 Extraction and Cleanup Optimized Procedure.

Spiked honey samples were extracted with 20 mL of McIlvaine-Na 2EDTA buffer and filtered. The filtrate was loaded onto Oasis HLB cartridge previously conditioned with 3 mL of methanol, 2 mL of water and 3 mL of McIlvaine-Na2EDTA buffer, respectively. Contaminants removing and elution efficiency were

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KMITL Sci. J. Vol.8 No.2 (Section B) July December, 2008 compared by using several solvents. The variables (Table 1); pH of McIlvaine-Na 2EDTA buffer, washing solvents and elution solvents, were optimized by means of multivariate method using full factorial design. The experimental design and the statistical study were carried out by using The Unscrambler 9.7 (Camo, Norway) software. The optimal conditions obtained for solid phase extraction have been applied for the analysis of fortified samples at 100 gkg-1. Table 1 Variable and levels considered for optimization of the SPE procedure for tetracyclines . Parameters pH of buffer Wash solvent Elution solvent Studied values 3, 6 5 % MeOH, 5 % ACN MeOH, DMF: IPA (80:20) Reference [2] 4 H2O MeOH

3. RESULTS AND DISCUSSION

3.1 HPLC conditions
The four tetracycline standards were separated by gradient elution programs: solvent B was linearly increased from 50 to 100% in 5 min, kept at 100% for 15 min and returned to the initial conditions. The flow rate was 0.5 mLmin-1 and the fluorescent detection had an excitation wavelength of 393 nm and emission wavelength of 518 nm.

3.2 Calibration
Under the optimized experimental condition, tetracyclines were found good linearity between their concentrations and peak area responses. Their detection limits, defined as 3 times signals to noise levels, were also determined. Method validation results were satisfaction; linear ranges of 10-1000 ngmL -1, with correlation coefficient more than 0.997. Detection limits were found to be 3.84, 1.89, 0.11 and 8.62 ngmL -1 for oxytetracycline, doxycycline, tetracycline and chlortetracycline, respectively.

3.3 Extraction and clean up

The Unscrambler software is the complete multivariate analysis and experimental design software. Optimization via multivariate analysis allows main effect and interaction between variables analysis with lower number of experiments than the one variable at the time (OVAT) method. For SPE optimization, the analysis of variables and % recovery of each extraction are shown in Table 2. By using The Unscrambler software, analyzing the main effect plots and the analysis of variance are shown in Fig. 2 and Table 3, respectively. From Fig. 2, it can be concluded that the most significant variables for the solid phase extraction process were pH adjustment of the sample, washing and elution solvents. It can be seen that the lower pH of the sample, the higher the percentage recovery were obtained. From Table 3, pH was the variable that affected the recovery of only oxytetracycline. Wash solution were significantly affected on the percentage recovery for all tetracyclines. Using water or 5 % methanol-water as wash solution, gave higher recovery than using 5 % acetronitrile-water. The elution solution power of methanol and 20 % DMFisopropanol are significantly different (p<0.01). Eluting with 100 % methanol gave the higher recovery for all tetracyclines. The significant interaction effect was found between pH and wash solution on TCT only (p<0.01).

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KMITL Sci. J. Vol.8 No.2 (Section B) July December, 2008

Table 2 The 3 factor 2 level factorial design proposed by The Unscrambler 9.7 (Camo, Norway) software and mean percentage recovery of tetracyclines (n=2, a n=3).
Parameter Experiment 1 2 3 4 5 6 7 8 9 10 11 12
a

% Recovery Elution solvents MeOH MeOH MeOH DMF:IPA (80:20) DMF:IPA (80:20) DMF:IPA (80:20) MeOH MeOH MeOH MeOH DMF:IPA (80:20) DMF:IPA (80:20) MeOH OTC 147.40 119.54 140.17 69.17 106.58 85.44 134.46 121.11 128.59 32.01 21.75 30.10 110.38 DC 145.28 124.06 136.32 142.71 126.34 102.57 124.23 128.03 131.19 72.83 45.99 77.98 107.71 TC 112.37 88.23 104.86 109.64 95.53 78.66 113.62 111.87 118.68 39.74 25.23 36.19 96.69 CTC 118.15 83.34 106.67 120.13 94.71 79.30 80.33 73.79 65.49 14.07 12.07 40.42 75.81

pH of buffer 3 3 3 3 3 3 6 6 6 6 6 6 4

Washing solvents H2O 5% MeOH 5% ACN H2O 5% MeOH 5% ACN H2O 5% MeOH 5% ACN 5% ACN H2O H2O H2O

Reference

Table 3 The result of the analysis of variance (ANOVA).

Variables pH (A) Wash (B) Elution (C) AB AC BC ABC p-Values Oxytetracycline 0.0008 0.0000 0.0010 0.0064 0.6027 0.0198 0.4836 Doxycycline 0.3658 0.0097 0.0038 0.5450 0.6881 0.1898 0.0800 Tetracycline 0.0532 0.0009 0.0066 0.2908 0.5778 0.0103 0.0282 Chlortetracycline 0.1642 0.0125 0.0000 0.2855 0.8642 0.0430 0.0237

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KMITL Sci. J. Vol.8 No.2 (Section B) July December, 2008

OTC

DC

TC CTC

(A)

OTC

DC

TC CTC

(B)

OTC

DC

TC

CTC

(C)

Figure 2 Main affect plots of variables on the average relative recovery of tetracyclines: (A) pH (3,6; ,), (B) wash solution (water, 5%MeOH-water and 5% ACN-water; ,,), (C) elution solution (methanol and 20%DMF-IPA; ,).

Table 4 Recovery extraction of spiked honey sample* at 100 gkg-1 (n = 2).

Tetracyclines Spiked level ( gkg-1) OTC DC TC CTC 100 100 100 100

Recovery mean (%) 61.46 0 89.85 93.84

* TCs free honey sample

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KMITL Sci. J. Vol.8 No.2 (Section B) July December, 2008

TC OTC DC CTC

(A)

(B)

OTC

TC CTC

(C)

Figure 3 Chromatogram of standard solution of tetracyclines 1 gmL-1 (A, honey blank sample (B) and spike honey 100 gkg-1 (C).

A good chromatographic separation of standard mixtures was obtained with less than 15 min analysis time (Fig. 3A). There was high back ground matrix found in TCs free honey sample as shown in Fig. 3B. When optimal conditions for solid phase extraction were applied for the analysis of fortified samples at 100 gkg-1, the extraction recoveries (Table 4) from spiked honey sample for tetracycline and chlortetracycline were satisfactory at high recovery 89 and 94 % but lower recoveries was obtained for oxytetracycline (61%). Recovery of doxycycline was not shown because of its insufficient separation (Fig. 3C) even though many gradient programs were carried out. Sample clean up with HLB cartridges has still not sufficient in removing the interferences from honey sample. This can be explained by the fact that the honey samples can vary in terms of complexity or content in natural products. As it was the case for other authors [2], our findings also show that a more effective clean up procedure that can separate tetracyclines from impurities in all types of honeys is essential.

4. CONCLUSIONS
The method can be applied to the analysis of tetracycline and chlortetracycline in honey sample with a HPLC-Fluorescence detector without post column requirement as reported by Pena [2]. However, a single clean up step is not always sufficient for the residue analysis of oxytetracycline and doxycycline in honey sample. Further investigation will be proceed to a second clean up step by using cationic exchange cartridge to remove the interferences from honey sample.

5. ACKNOWLEDGEMENTS
The authors wish to thank the TRF-Master Research Grants (MRG WII 505S057) and Maejo University for financial support.

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KMITL Sci. J. Vol.8 No.2 (Section B) July December, 2008

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