Tetracyclines In Honey Research Articles

Development of an indirect competitive assay-based aptasensor for highly sensitive detection of tetracycline residue in honey

Tetracycline (TC) is widely used for prevention and control of animal diseases for its broad spectrum antimicrobial activity and low cost, but its abuse can seriously affect human health and may result in trade loss. Thus there is an imperative need to develop high-performing analytical technique for TC detection. In this study, we developed a biosensor based on an indirect competitive enzyme-linked aptamer assay (ic-ELAA). A 76mer single-stranded DNA (ssDNA) aptamer, selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), was applied for the recognition and detection of TC in honey. The limit of detection was 9.6×10−3ng/mL with a linear working range from 0.01 to 100ng/mL toward TC in honey, and a mean recovery rate of 93.23% in TC-spiked honey was obtained. This aptasensor can be applied to detect TC residue in food with high sensitivity and simplicity, and it is prospective to develop useful ELAA Kits for TC determination in food.

Determinative and confirmatory method for residues of tetracyclines in honey by LC-MS/MS

A limited number of substances are authorised for the treatment of bees. Maximum residue limits (MRLs) are set for tetracyclines in several matrices, but not for honey. Nevertheless, tetracycline antibiotics may be used in order to prevent bacterial diseases and the loss of honey bee populations. In this study, a sensitive multi-residue LC-MS/MS method was developed and optimised for the quantitative and qualitative determination of tetracycline residues in honey. Homogenisation of samples under acidic conditions was performed and solid-phase extraction was carried out. The eluate was evaporated under nitrogen and dissolved in an aqueous methanol solution prior to filtration. A mobile phase composed of acetic acid–water and acetic acid–acetonitrile was used. Separation of tetracycline, oxytetracycline, chlortetracycline and doxytetracycline was achieved by using gradient elution on a C18 chromatography column. The analytical method was validated according to Commission Decision 2002/657/EC by the analysis of spiked samples around the recommended concentration of 20 μg kg−1 by EURL Guidance Paper, December 2007. A matrix effect was observed, so quantification was based on an external matrix calibration curve. Calculated decision limits (CCα) were lower than 10 μg kg–1 for all tetracyclines. Good linearity, repeatability and within-laboratory reproducibility were achieved.

Validation of a commercial receptor kit for tetracycline residues in honey according to the European guideline for screening methods

A rapid receptor test dedicated to the screening of tetracycline (TTC) residues, the Tetrasensor® kit in different matrices, has been evaluated and validated according to the European regulation to achieve the French control plan in honey. The detection capabilities (CCβs) were included between 5 and 15 µg kg−1 for chlortetracycline (CTC), oxytetracycline (OTC), TTC and doxycycline (DOXY). The cross-reactions of both kits towards other families of antibiotics were negligible and there was no masking effect of these antibiotics on the detection of tetracyclines. The false positive rate was equal to 9.4%. This kit was applicable to a wide variety of honeys [monofloral and multifloral, different floral origins, different geographical origins and different consistencies (liquid or solid), different colours]. Finally, the Tetrasensor® test is a robust method, quick, easy to perform and easy to read which is now applied to implement the French control plan for the detection of tetracyclines in honey.

Analysis of Tetracyclines (TCs) Residues in Honey with HPLC - UV

Simultaneous determination of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), and doxycycline (DC) residues in honey was performed using a high-performance liquid chromatography with ultraviolet spectrophotometer ant this method was validated by analysis of precision and accuracy. Honey was digested using perchloric acid of 2% mass concentration. Matrix matched calibration curves showed a linearity exceeding 0.99. The recovery ratios lay between 75.9% and 92.5% and the coefficient variation was lower than 16.6%. Limits of detection and quantitation were below 0.03µg mL-1 and 0.09µg mL-1, respectively, for OTC and TC, and below 0.07µg mL-1 and 0.20µg mL-1, respectively, for CTC and DC. The experiments demonstrated that it is a simple method suitable for detection of residual TCs in honey.

A HPLC with fluorescence detection method for the determination of tetracyclines residues and evaluation of their stability in honey

A high-performance liquid chromatography with fluorescence detection method for the simultaneous determination of oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) residues in honey was developed and validated. Sample preparation was done in a single solid phase extraction step. The chromatographic separation was performed on a C 8 column. Matrix matched calibration curves showed linearity higher than 0.99. The accuracy values lay between 86% and 111% and the precision was lower than 11%. Limits of detection and quantitation were 8 μg kg −1 and 25 μg kg −1, respectively. The method was employed to evaluate the stability of the tetracyclines in honey during 60 days of storage. The residue levels of OTC, TC and CTC were reduced 97%, 76% and 71%, respectively. Honey samples ( n = 22) collected from the retail market were analyzed. No tetracyclines residues were detected.

Practical Method for the Detection of Tetracyclines in Honey by HPLC and Derivative UV-Vis Spectra

A technique to detect residual tetracyclines (TCs) in honey by HPLC and derivative UV-Vis spectra is examined in this study. Chromatograms of honey products often experience overlapped peaks and sloping background due to similar retention properties of components or to the presence of impurities. The derivatives provide resolution of overlapped peaks in an UV-Vis spectrum and eventually bring chromatograms that show pure elution behavior of TCs. Calibration models for oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) are built out of the derivative spectra of standard solutions of honey and their prediction abilities are examined. The results show that the calibration models for OTC, TC and CTC can yield sufficient levels for the determination. The technique reported here may be effectively used for quantitative determination of TCs.

Quantitative detection of tetracycline residues in honey by a simple sensitive immunoassay

Tetracyclines (TCs) are widely used for prevention and control of infectious diseases and have a great activity against variety of Gram-positive and Gram-negative bacteria. Due to the widespread use of TCs in animal husbandry, it can lead to an increase the risk of TCs remaining in human food. To protect consumers, many countries have set acceptable tolerance levels for these drugs. Therefore, it is necessary to establish a suitable analytical technique with specificity, sensitivity and simplicity. The biotin–avidin mediated ELISA method was performed to determine TC residues in honey quantitatively. By using PBS-EDTA assay buffer at pH 7.2, a honey solution of TC standard was prepared and diluted. And no additional pre-treatment of sample was required in this method. The limit of detection and limit of quantitation of the optimized method were 3.98 × 10 −10 M (0.19 μg L −1) and 7.94 × 10 −10 M (0.38 μg L −1), respectively, and the dynamic range was from 1.52 μg L −1 to 152 μg L −1 of TC in honey. No cross-reactivity was observed with the structurally similar compounds, and mean percent recoveries of TC spiked in honey ranged from 95% to 101%. Compared to other methods, this method was superior in terms of detection limit, dynamic range, and % recovery with simple sample-preparation.

Validation of the Tetrasensor Honey Test Kit for the Screening of Tetracyclines in Honey

Regarding anti-infectious agents, no maximum residue limits are fixed for honey in the European legislation. Discussions are being conducted in order to set working limits at the European level; for example, for tetracyclines, 20 microg/kg was proposed. The Tetrasensor Honey test kit is a receptor-based assay using dipsticks for a rapid screening (30 min) of honey on the presence of tetracyclines. The test was validated according to Commission Decision 2002/657/EC. The test detects tetracycline, oxytetracycline, chlortetracycline, and doxycycline in honey in a specific and sensitive way. Depending on the type of tetracycline, detection capabilities (CCbeta) between 6 and 12 microg/kg were obtained (4-7 microg/kg for dried dipsticks). The test is rugged and participation with the test in an international ring trial gave compliant results. It can be concluded that the Tetrasensor Honey test kit is a simple and reliable test that can even be used at the production site.

Determination of Tetracyclines in Honey Using Liquid Chromatography with Ultraviolet Absorbance Detection and Residue Confirmation by Mass Spectrometry

AbstractA determination method has been optimized and validated for the simultaneous analysis of tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC) and doxycycline (DC) in honey. Tetracyclines (TCs) were removed from honey samples by chelation with metal ions bound to small Chelating Sepharose Fast Flow columns and eluted with Na2EDTA‐Mcllvaine pH 4.0 buffers. Extracts were further cleaned up by Oasis HLB solid‐phase extraction (SPE), while other solid‐phase extraction cartridges were compared. Chromatographic separation was achieved using a polar end‐capped C18 column with an isocratic mobile phase consisting of oxalic acid, acetonitrile, and methanol. LC with ultraviolet absorbance at 355 nm resulted in the quantitation of all four tetracycline residues from honey samples fortified at 15, 50, and 100 ng/g, with liner ranges for tetracyclines of 0.05 to 2 µg/mL. Mean recoveries for tetracyclines were greater than 50% with R.S.D. values less than 10% (n=18). Detection limits of 5, 5, 10, 10 ng/g for oxytetracycline, tetracycline, chlortetracycline and doxycycline, respectively and quantitation limits of 15 ng/g for all the four tetracyclines were determined. Direct confirmation of the four residues in honey(2–50 ng/g) was realized by liquid chromatography‐tandem mass spectrometry (LC/MS/MS). The linear ranges of tetracyclines determined by LC/MS/MS were between 5 to 300 ng/mL, with the linear correlation coefficient r>0.995. The limits of detection of 1 to 2 ng/g were obtained for the analysis of the TCs in honey.

OPTIMIZATION OF HPLC METHOD FOR QUANTIFYING TETRACYCLINE RESIDUE IN HONEY

Tetracyclines are used for the treatment or prevention of American and European fouldbrood in bee colonies which are caused especially by two species of bacteria: Paenibacillus larvae larvae and Melissococcus pluton, respectively. In the present study a method for determination of residual tetracyclines in honey has been optimized and validated. It was used the reversed-phase high performance liquid chromatography (HPLC) with UV detection. Tetracyclines were extracted from honey with 0.01M sodium succinate buffer. For the clean up step, solid phase extraction using metal chelate resin was applied. Tetracyclines were eluted with buffer containing EDTA. Linearity, precision, recovery and sensitivity were satisfactory. The limit of detection was established at 5 µg/kg

Analysis of tetracyclines in honey by high‐performance liquid chromatography/tandem mass spectrometry

A confirmatory method coupling liquid chromatography to tandem mass spectrometry (LC/MS/MS) is described for the determination of tetracycline, oxytetracycline, doxycycline and chlortetracycline in honey. Demeclocycline, another tetracycline molecule not reported for its usage in honey, was used as internal standard to quantify the four analytes. The sample preparation entails a clean-up on an Oasis HLB solid-phase extraction cartridge and analyses were realised by LC/MS/MS in selected reaction monitoring mode. The stability of tetracyclines was checked under various storage conditions at -20, +4 and +20 degrees C (both under dark and light exposures). Indeed, tetracyclines are not stable molecules and the epimerisation phenomenon was evaluated in this work. Appropriate correction factors of the MS/MS responses of each epimer were studied for each of the four tetracyclines to accurately quantify them. Moreover, the matrix effects encountered during the LC/MS/MS analyses were also studied in spiked experiments from blank honey samples of various geographical origins and different flower types.

Simple and rapid screening and confirmation of tetracyclines in honey and egg by a dipstick test and LC–MS/MS

The illegal use of antibiotics such as tetracyclines in honey production has recently been focused. The need for simple test methods has resulted in the development of a dipstick-test based on receptor binding, Tetrasensor ®, Unisensor, Belgium. In this paper, two types of the test, the kit for analysis of tetracyclines in honey and the kit for analysis of tetracyclines in egg, fish and tissues from several animal species, have been evaluated and the results have been confirmed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Different batches of Swedish honey and egg were analysed blank or spiked with oxytetracycline, tetracycline and chlortetracycline at 25 (honey) and 200 μg kg −1 (egg). The confirmation by LC–MS/MS was performed directly on diluted test extract without further purification (honey) or after a simple filtration (egg). This combination of a simple dipstick-test and LC–MS/MS proved to be a suitable and rapid way for the surveillance of tetracyclines in honey and egg.

Sensitivity Improvement of the Oxidation of Tetracycline at Acetylene Black Electrode in the Presence of Sodium Dodecyl Sulfate

AbstractThe oxidation of tetracycline (TC) was investigated at an acetylene black electrode in the presence of sodium dodecyl sulfate (SDS). TC exhibited a sensitive oxidation peak in the medium system, which was attributed to the enhanced adsorption of protonated TC at the negatively charged SDS film on the electrode surface. The mechanism of the electrode process was discussed, and some experimental conditions were optimized. The oxidation peak current was proportional to TC concentration in the range of 1.2×10−7–6.0×10−5 M, and based on three times the background signal, a detection limit of 1.2×10−8 M was obtained for 150 s accumulations. The method has been used to determine TC in honey and pharmaceutical, and the results compared favorably.

Influence of antibiotics on the offspring production of the Wolbachia-infected parthenogenetic parasitoid Encarsia formosa.

Three different concentrations of the antibiotic tetracycline in honey were tested for their influence on the offspring production and longevity of the parasitoid wasp Encarsia formosa. Several earlier publications did not provide a conclusive answer on the effect that the Wolbachia have on these wasps. The results of our experiments show that at high tetracycline hydrochloride concentrations in honey (50mg/ml) the antibiotic is toxic to the females, all females died within three days after the antibiotic treatment. The concentration 5mg/ml was less toxic although the treated females also lived shorter and produced less offspring than the control females. At the lowest tested concentration of 1mg/ml there was no significant difference either in offspring production or in longevity between the control and the treated females. The antibiotic treatment at both 5 and 1mg/ml resulted in exclusively male progeny after the first two days of oviposition. These results are consistent with the theory that in species in which all individuals are infected the Wolbachia should not impose a large fitness cost.

Improvement of chemical analysis of antibiotics. 22. Identification of residual tetracyclines in honey by frit FAB/LC/MS using a volatile mobile phase

A reliable identification method for residual tetracycline antibiotics (TCs), oxytetracycline, tetracycline, chlortetracycline, and doxycycline in honey, has been established using frit FAB/LC/MS with a volatile mobile phase. The TCs were recovered from a C 18 solid phase extraction cartridge and separated on an end-capped phenyl-bonded silica gel column synthesized from 99.99% pure silica gel (5 μm, 250×2.1 mm i.d.) using a mobile phase consisting of methanol-acetonitrile0.005 M trifluoroacetic acid (2:2:11) containing 1.0% thioglycerol. The column effluent was introduced into the frit FAB ion source after splitting at a ratio of 1:20

Identification of Residual Tetracyclines in Honey by TLC/FABMS

In order to reliably identify residual tetracycline antibiotics (TCs), oxytetracycline, tetracycline, chlortetracycline, and doxycycline, in honey, a confirmation method using thin-layer chromatography/mass spectrometry (TLC/FABMS) with the following characteristics has been established. Use of thioglycerol as a matrix enabled us to measure the FAB mass spectra of TCs without interfering ions due to the matrix and honey. Reversed-phase C8 TLC conditions gave reliable separation with good reproducibility, and the location of TCs extracted from honey could be readily estimated on the basis of the location of neighboring visualized spots of TCs. The condensation technique improved the detection limits 50-fold with good reproducibility. The clean-up procedure using a combination of C18 and COOH (weak cation exchange) cartridges proved to be effective for the identification of residual TCs by TLC/FABMS. The method can reliably identify TCs fortified at concentrations of 0.1ppm in honey, and has been successfully applied to the identification of residual chlortetracycline in a commercial honey previously found positive by high performance liquid chromatography.

Simplified Detection Method for Residual Tetracyclines and Sulfa Drugs in Honey by Microbiological Assay

A simplified method was developed for the detection of residual tetracyclines and sulfa drugs in honey by microbiological assay. A honey sample was homogenized in 0.01M di-Na EDTA McIlvaine buffer (pH 4.0) and centrifuged. The supernatant was mixed well with chloroform and centrifuged. The chloroform layer was evaporated to dryness and the residue was dissolved in phosphate buffer (pH 8.0) to prepare fraction A. The aqueous layer was passed through a Sep-pak C18 cartridge column. The Sep-pak C18 cartridge column was washed with water and absorbed antibacterial agents were eluted with methanol. The eluate was evaporated to dryness, and the residue was dissolved in phosphate buffer (pH 4.5) to prepare fraction B.Fractions A and B contained sulfa drugs and tetracyclines, respectively. The pulp disk method with Bacillus subtilis ATCC 6633 and Bacillus cereus var. mycoides ATCC 11778 as test organisms was employed for the assay of sulfa drugs and tetracyclines. As little as 0.05μg/g (oxytetracycline), 0.01μg/g (chlortetracycline) or 0.1μg/g (sulfa drugs) was detectable.Thin layer chromatographic identification was performed for positive test solutions. The test solution A was spotted on an aluminum oxide plate (Merck 5550) and a silica gel plate (Merck 5553), then developed in chloroform-methanol (7:3) and ethyl ether-chloroform-n-butanol (5:1:1), respectively. The test solution B was spotted on a cellulose plate (Merck 5552), which was developed in n-butanol saturated with water. Rf values were determined by bioautography with B. subtilis and B. cereus as the test organisms. Microbioautography was suitable for separation and identification of sulfa drugs and tetracyclines in honey. We used this method to identify sulfa drugs and tetracyclines detected in 22 samples out of 297 commercial honeys. It was found that 8 specimens contained oxytetracycline, 12 specimens chlortetracycline, 1 specimen sulfadimethoxine, and 1 specimen sulfisomidine.

Fluorometric Determination of Tetracyclines in Honey by High Performance Liquid Chromatography

A sensitive and selective method for determination of oxytetracycline (OTC) and chlortetracycline (CTC) in honey by high performance liquid chromatography with a fluorescence detector was developed.A honey sample was dissolved in 0.1N hydrochloric acid and passed through a Sep-Pak C18 cartridge. After washing of the cartridge with water, OTC and CTC were eluted with methanol and purified by counter-ion extraction. The isolated OTC was treated with magnesium acetate-sodium barbital solution and CTC was treated with 0.05M KOH to obtain a fluorescent derivative. OTC and CTC were separated on an Asahipak Gel ODP-50 column and quantitated by fluorometry. The mobile phase for OTC was 0.1M glycine buffer (pH 10)-methanol (20:1) containing 20mM sodium 1-heptane-sulfonate (final concentration) and that for CTC was 0.01M glycine buffer (pH 10)-methanol (5:1).The recoveries of OTC and CTC added to honey at levels of 0.5ppm and 1ppm were 68.5 and 73.2% for OTC, and 53.1 and 68.1% for CTC, respectively. The limits of detection were 0.02ppm for OTC and 0.005ppm for CTC.

Rapid Determination of Sulfathiazole, Oxytetracycline and Tetracycline in Honey by High-Performance Liquid Chromatography

Abstract A simple, rapid and precise analytical method for sulfathiazole, oxytetracycline and tetracycline residues in honey has been established using micellar reversed-phase high-performance liquid chromatography with photometric detection. The mean recoveries of sulfathiazole, oxytetracycline and tetracycline were 97.7, 95.3 and 100.4%, respectively. Detection limits in honey were 0.9 μg/g for sulfathiazole, 1.2 μg/g for oxytetracycline and 3.0 μg/g for tetracycline.

Improvement of chemical analysis of antibiotics : XII. Simultaneous analysis of seven tetracyclines in honey

An analytical system for the simultaneous determination of residual oxytetracycline, tetracycline, chlortetracycline, doxycycline, methacycline, demethylchlortetracycline and minocycline in honey has been established by a combination of simple thin-layer chromatographic (TLC) and precise high-performance liquid chromatographic (HPLC) methods. In this system, screening by TLC can detect tetracyclines (TCs) at a level of 0.1 ppm in honey without the need for special equipment, and the quantitative method by HPLC can determine TCs with good recovery (83.7–99.6%) and coefficient variation (0.9–4.3%).