005 Determination of Teteracycline1

7/29/2019 005 Determination of Teteracycline1

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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 Sangsrichan11

1Department of Chemistry, Faculty of Science, Maejo University, Chiang Mai, 50290, Thailand

ABSTRACT

A high performance liquid chromatography method utilizing fluorescence detection was optimized andvalidated to determine tetracycline residues in honey. The separation of four tetracycline residues;oxytetracycline, tetracycline, chlortetracycline and doxycycline was observed on a reversephase C8column with a gradient elution. A mobile phase consisted of 50% (v/v) methanol and 25 mM sodiumacetate 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 20min. The extraction with disodium ethylenediaminetetraacetate (Na 2EDTA)-McIlvaine buffer pH 4 wasused followed by HLB cartridge clean up step. The good recovery for tetracycline and chlortetracyclinewere found above 89%, low recoveries for oxytetracycline and doxycycline which will be optimized toachieve 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 concernto consumers worldwide, because of possible toxic or allergic reactions and the possibility that pathogenicorganisms could become resistant to these drugs [2]. The application of the law in relation to theseantibiotics is not harmonized across all member states of the European Union. The Commission of theEuropean Union laid down the procedure for establishing maximum residue limits (MRLs) of veterinarymedical 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 20gkg1. In Japan, base on microbiological research, a value of 0.1 mgkg -1 was introduced as the allowedresidual quantity of tetracycline in honey [4]. Although the Thai law related to quality of honey affirms thatit is illegal any type of additive or substance apart from honey at any level, but, there is no law in about thecontrol 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 mostlyby HPLC. Tetracyclines can be successfully determined in various biological matrices, HPLC in a reverse-phase 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 UVdetection has lower sensitivity than mass spectrometry.

1

Corresponding Author. Tel. +66-53-873544-5 Fax. +66-53-873648E-mail: [email protected]


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Figure 1 Structure of tetracyclines.

(MS) [28], while still require costly instruments. In general, fluorescence detection is highly sensitive andselective with relatively lower cost than mass spectrometry.

In this research, the extraction and chromatographic detection of four tetracyclines (see Fig. 1) wereoptimized and validated for the determination of tetracyclines in honey at LODs below 10 gkg-1. Theseparation of four tetracycline residues; oxytetracycline, tetracycline, chlortetracycline and doxycyclinewas observed on a reversed phase C8 column with a gradient elution. The tetracycline residues wasextracted with buffer and clean up by using solid phase extraction (SPE), detected on a high performanceliquid chromatography method utilizing fluorescence detection.

2. MATERIALS AND METHODS

2.1 ApparatusThe 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 anexcitation wavelength of 380 nm and an emission wavelength of 518 nm.

2.2 Reagent and MaterialTetracycline, oxytetracycline and chlortetracycline standards were purchased from Merck, Germany anddoxycycline standard was purchased from Fluka, USA. Disodium ethylenediaminetetraacetate, sodiumacetate, calcium chloride and methanol were analytical-reagent grade (Merck, Germany). The Oasis HLBextraction cartridges, 6 cm3 (200 mg) were purchased from Water, USA.

Individual stock standard solutions were prepared in 100 mL of methanol containing 100 mg ofneat standard in a volumetric flask and were stored at -20 C in amber glass vials for a maximum period of1 month. The working standards were mixtures of four compounds prepared by a serial dilution of thestocks in the 50% (v/v) methanol and 25 mM sodium acetate buffer (containing disodiumethylenediaminetetraacetate, 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 (containingdisodium ethylenediaminetetraacetate and calcium chloride, pH 8.1) at flow rate of 0.5 mLmin-1.

2.3 Calibration procedureThe 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 squareregression 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 beekeepersduring 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-Na2EDTA buffer and filtered. The filtratewas loaded onto Oasis HLB cartridge previously conditioned with 3 mL of methanol, 2 mL of water and 3mL of McIlvaine-Na2EDTA buffer, respectively. Contaminants removing and elution efficiency were

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


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compared by using several solvents. The variables (Table 1); pH of McIlvaine-Na2EDTA buffer, washingsolvents 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 theanalysis of fortified samples at 100 gkg-1.

Table 1 Variable and levels considered for optimization of the SPE procedure for tetracyclines.

Parameters Studied values Reference [2]

pH of buffer 3, 6 4

Wash solvent 5 % MeOH, 5 % ACN H2O

Elution solvent MeOH, DMF: IPA (80:20) MeOH

3. RESULTS AND DISCUSSION

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

3.2 CalibrationUnder the optimized experimental condition, tetracyclines were found good linearity between theirconcentrations 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, withcorrelation 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 upThe Unscrambler software is the complete multivariate analysis and experimental design software.Optimization via multivariate analysis allows main effect and interaction between variables analysis withlower number of experiments than the one variable at the time (OVAT) method. For SPE optimization, theanalysis of variables and % recovery of each extraction are shown in Table 2. By using The Unscramblersoftware, 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 phaseextraction process were pH adjustment of the sample, washing and elution solvents. It can be seen that thelower pH of the sample, the higher the percentage recovery were obtained. From Table 3, pH was thevariable 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 higherrecovery than using 5 % acetronitrile-water. The elution solution power of methanol and 20 % DMF-isopropanol are significantly different (p


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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, an=3).

Experiment

Parameter % Recovery

pH ofbuffer

Washingsolvents

Elutionsolvents

OTC DC TC CTC

1 3 H2O MeOH 147.40 145.28 112.37 118.15

2 3 5% MeOH MeOH 119.54 124.06 88.23 83.34

3 3 5% ACN MeOH 140.17 136.32 104.86 106.67

4 3 H2ODMF:IPA(80:20)

69.17 142.71 109.64 120.13

5 3 5% MeOHDMF:IPA(80:20)

106.58 126.34 95.53 94.71

6 3 5% ACNDMF:IPA(80:20)

85.44 102.57 78.66 79.30

7 6 H2O MeOH 134.46 124.23 113.62 80.33

8 6 5% MeOH MeOH 121.11 128.03 111.87 73.79

9 6 5% ACN MeOH 128.59 131.19 118.68 65.49

10 6 5% ACN MeOH 32.01 72.83 39.74 14.07

11 6 H2ODMF:IPA(80:20)

21.75 45.99 25.23 12.07

12 6 H2ODMF:IPA(80:20)

30.10 77.98 36.19 40.42

a Reference 4 H2O MeOH 110.38 107.71 96.69 75.81

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

Variablesp-Values

Oxytetracycline Doxycycline Tetracycline Chlortetracycline

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

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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) Recovery mean(%)

OTCDCTCCTC

100100100100

61.46089.8593.84

* TCs free honey sample

(B)

(C)

OTC DC TC

CTC

OTC

OTC

DC

DC

TC

TC

(A)CTC

CTC

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

A good chromatographic separation of standard mixtures was obtained with less than 15 min analysistime (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 chlortetracyclinewere 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 thoughmany gradient programs were carried out. Sample clean up with HLB cartridges has still not sufficient inremoving the interferences from honey sample. This can be explained by the fact that the honey samplescan vary in terms of complexity or content in natural products. As it was the case for other authors [2], ourfindings also show that a more effective clean up procedure that can separate tetracyclines from impuritiesin all types of honeys is essential.

4. CONCLUSIONS

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

5. ACKNOWLEDGEMENTS

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

OTC DC

TC

CTC

(A)

OTCTC

CTC

(B)

(C)

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005 Determination of Teteracycline1