Microwave method synthesis of magnetic ionic liquid/gold nanoparticles as ultrasensitive SERS substrates for trace clopidol detection.

Clopidol is one of the most widely used anti-coccidiosis drugs. Its residues in poultry products and the environment pose a serious threat to human health. In this work, microwave-assisted synthesis of magnetic ionic liquid/gold nanoparticles (MIL-Au NPs) as the SERS substrates were first designed for sensitive and reliable determination of clopidol residue in egg samples. The experiment shows that MIL(1-methyl-3-hexyl imidazole ferric tetrachloride ([C6mim]FeCl4)) and microwave play a key role in the dispersion and morphology of Au NPs. Under the optimal conditions, the as-prepared MIL-Au NPs were applied to the SERS detection of clopidol in methanol and egg solution and its detection limits can be as low as to 0.5 µg/kg (equal to 0.5 ppb) in both solutions. The standard curves with regression coefficients of 0.9298 and 0.93496 were constructed in the linear range of 100-1000 ppb and 0.5-50 ppb for clopidol in egg solutions. Moreover, satisfactory recoveries (97.5-103.2%) were obtained for egg samples. The developed SERS method provides a way for quantitation of clopidol and can be applied for the convenient, reliable, and highly sensitive detection of antibiotic residues in food and environment, which has great potential in food safety and biological monitoring. Graphical abstract.

Development of an enzyme-linked immunosorbent assay for detection of clopidol residues in chicken tissues.

BACKGROUND: Clopidol is mainly used for the prevention and treatment of coccidiosis, which poses a serious potential hazard to public health, in veterinary medicine. The aim of this study was to prepare monoclonal antibodies (mAbs) against clopidol (CLOP) and develop an immunoassay for detecting CLOP residues in chicken tissues. After derivation, CLOP hapten was conjugated to carrier proteins to synthesize the artificial antigen, and immunized Balb/C mice were employed to screen mAbs. RESULTS: A sensitive hybridoma named C1G3 was screened out and two indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curves were established. For the traditional two-step assay the linear range was from 0.06 to 98 ng mL(-1) , with half-maximal inhibitory concentration (IC50 ) and limit of detection (LOD) values of 2.76 ng mL(-1) and 0.03 ng mL(-1) respectively, while the rapid one-step icELISA had a working range from 0.08 to 102 ng mL(-1) , with IC50 and LOD values of 3.52 ng mL(-1) and 0.03 ng mL(-1) respectively. It was also indicated that a 10-fold dilution in chicken muscles gave an inhibition curve almost the same as that obtained in phosphate-buffered saline. When applied to spiking tests in chicken samples, the correlation coefficient (R(2) ) between concentrations added and measured was 0.9534. CONCLUSION: The results of this study suggest that the immunoassay described is a promising alternative for screening CLOP residues in biological matrices and is suitable for routine diagnostics.

[Simultaneous determination of clopidol, sulfonamide and quinolone residues in chickens by ultra performance liquid chromatography-tandem mass spectrometry].

An analytical method for the simultaneous determination of clopidol, sulfadiazine, sulfamethazine, sulfametoxydiazine, sulfamethoxypyridazine, norfloxacin, ofloxacin, ciprofloxacin, enrofloxacin in chickens by ultra performance liquid chromatography coupled with tandem quadrupole mass spectrometry (UPLC-MS/MS) in positive ion mode with multiple reaction monitoring (MRM) has been developed and validated. The samples were homogenized and extracted with acetonitrile. After defatted with high speed frozen centrifugation, the supernatant solution was evaporated and the residue was dissolved with the mobile phase and defatted with n-hexane. It was then analyzed with UPLC-MS/MS. The limit of detection of this method was 0.1 microg/kg, and the limit of quantification was 0.5 microg/kg. The average recoveries (spiked at the levels of 0.5, 1.0, 2.0 microg/kg) ranged from 81.5% to 97.6%, with the relative standard deviations between 2.1% and 8.9%. The results demonstrated that the method is simple, accurate and suitable for the identification and quantification of these drug residues in chickens.

Determination of residual clopidol in chicken muscle by capillary gas chromatography-negative chemical ionization-mass spectrometry.

A selective, sensitive gas chromatography-mass spectrometry (GC-MS) method with negative chemical ionization (NCI) was developed for the detection and quantification of clopidol in chicken muscle. Chicken muscle samples were extracted with acetonitrile and concentrated to dryness; the residue was redissolved in ethyl acetate and applied to an Alumina B cartridge for cleanup. The residue was derivatized with Sylon BFT and analyzed by GC-NCI-MS. The selected-ion monitoring mode was performed at m/z values of 156, 158, 191, and 193. The differences in the ratios for the standards and spikes in chicken muscle were within the acceptability criteria. All recoveries of the drug from chicken muscle spiked at 0.5, 5.0 and 50.0 microg/kg were 74.5-95.6% intra-day, and 71.6-94.8% inter-day, respectively, with relative standard deviations being lower than 15%. The limits of detection and quantitation were 0.1 and 0.5 microg/kg, respectively. The NCI mode had better selectivity and sensitivity than the electron impact (EI) mode for clopidol.

[Determination of clopidol residue in poultry products by liquid chromatography-electrospray ion trap tandem mass spectrometry].

A method is presented for the determination of clopidol residue in poultry products by liquid chromatography-electrospray ion trap tandem mass spectrometry (LC-ESI-MS/MS). Clopidol residue was extracted with methanol and cleaned-up by n-hexane from different poultry products. The extract was cleaned-up by an LC-18 column and an ion exchange solid phase extraction (SPE) column. Quantification was achieved by matrix calibration. The recoveries of clopidol in poultry products were in the range from 55.38% to 132.44% at the four spiked levels, 2, 5, 10, and 20 microg/kg. The intra-day relative standard deviation (RSD) and inter-day RSD were less than 9.54% and 15.27%, respectively. The linearity of the method was good from 1 microg/kg to 40 microg/kg. The limit of detection (LOD) was 0.5 microg/kg, and the limit of quantification (LOQ) was 2.0 microg/kg. The method is selective without interference and suitable for the determination and confirmation of clopidol residue in poultry products.

[Determination of clopidol residues in chicken muscle by gas chromatography-mass spectrometry].

A confirmative method to determine clopidol residues in chicken muscle by gas chromatography-mass spectrometry (GC-MS) was developed. The analyte was extracted with ace tonitrile, and then purified with an Alumina-B cartridge column. The drug was derived at 80 degrees 3 for 60 min with Sylon BFT, and more toluene was added and then applied to GC-MS. The mass spectral characteristics of trimethylsilyl derivative of clopidol were interpreted, and selected ion monitoring (SIM) mode was performed at m/z 212, 214, 248 and 263. The clopidol was qualitatively identified by the ratio of relative abundance of the selected ions and determined quantitatively by SIM mode at m/z 248. In the meantime, the matrix effect was evaluated. The range of linearity was 5.0 - 500 microg/L with the correlation coefficients better than 0.998, and the detection limit was 0.5 microg/kg (S/N = 3) for clopidol. The average recoveries from chicken muscle fortified at 5, 10 and 20 microg/kg were 77.0%, 84.5% and 89.4%, respectively, and the relative standard deviations (RSD) were less than 6.9%. The established method is simple, sensitive and reproducible for the identification and quantification of clopidol residues in chicken muscle tissue.

Determination of residual clopidol in chicken muscle by capillary gas chromatography/mass spectrometry.

Clopidol was extracted from chicken with acetonitrile, and the extract was evaporated to about 5 mL. The concentrated solution was centrifuged and applied to a glass column filled with basic alumina. The column was eluted with methanol and the methanol was evaporated to dryness. The residue was derivatized with Sylon BFT and analyzed by gas chromatography/mass spectrometry (GC/MS). Mass characteristics of trimethylsilyl derivative of clopidol were interpreted manually. The selected-ion monitoring mode was performed at m/z 191, 248, 263, and 265. A sensitive, reproducible GC/MS method was developed for monitoring residues of clopidol in chickens. All recoveries of the drug from chicken muscle fortified at 1, 10, and 100 ng/g were > 65%, and relative standard deviations were < 15%. The detection limit was about 0.5 ng/g, and the quantitation limit was the lower limit of the standard curve, which was about 1 ng/g.

Determination of clopidol residues in chicken tissues by liquid chromatography: collaborative study.

Eighteen laboratories participated in a collaborative study on the determination of clopidol residues in chicken muscle tissues by liquid chromatography. Of these, results from 16 laboratories which rigorously followed the method were subjected to statistical analysis. The method performance was assessed by all participants using 14 samples of chicken muscle fortified at concentrations ranging from 0.1 to 5.0 mg/kg. In addition, 9 participants each reported results for 6 clopidol-incurred samples in chicken muscle. Test portions were extracted with acetonitrile, and the extracts were purified with alumina and anion exchange resin solid-phase extraction cartridges in sequence. Clopidol was separated by reversed-phase liquid chromatography and quantified at 270 nm. Average recoveries ranged from 81.8 to 85.4%, reproducibility relative standard deviation (RSDR) ranged from 11.9 to 22.6%, and repeatability relative standard deviation (RSDr) ranged from 9.9 to 15.1%. For clopidol-incurred samples at concentrations of 0.100-0.687 mg/kg, the mean determination value range was 0.099-0.659 mg/kg; RSDR was 12.6-19.8%, RSDr was 3.1-8.5%; and HORRAT values were 0.7-1.1. The accuracy and precision of the method are in conformity with the requirements specified by AOAC INTERNATIONAL. The method was adopted Official First Action in April 2003.

Determination of clopidol residues in chicken tissues by liquid chromatography: part I. Optimization of analytical conditions and comparison with AOAC gas chromatography method.

A simple and specific liquid chromatographic method was developed for determination of clopidol in chicken tissues. Samples were extracted with acetonitrile. The extracts were cleaned up on an alumina column and an anion exchange column. The clopidol was separated on a column (30 cm x 3.9 mm) of microBondapak C18 (10 microm) by using acetonitrile-water (20 + 80, v/v) as mobile phase, and determined quantitatively at 270 nm. Recoveries were 86.0-97.6%, with relative standard deviations of 2.14-9.42% at 0.010-2.0 mg/kg from 4 spiked matrixes of chicken muscle, egg, liver, and kidney. The limit of detection was 0.005 mg/kg. Compared with the modified AOAC gas chromatographic method, the present method is simple and fast to operate. Its results are accurate and reliable, making it favorable for environmental protection and meeting requirements for human safety. Thus, it is suitable for routine analysis of large quantities of samples.

Determination of clopidol residues in chicken tissues by liquid chromatography: part II. Distribution and depletion of clopidol in chicken tissues.

A study was made of the distribution and depletion of clopidol residues at different tissue locations in chickens fed with feeds incurred with clopidol. Experiments showed that the residue levels were not identical at 5 different tissue locations in each chicken. The sequence of residue levels from high to low was livers, kidneys, upper breast, lower breast, and leg meat. The maximum residue values after suspension of the drug for 8 h were (mg/kg): livers, 4.600; kidneys, 3.619; upper breast, 1.742; lower breast, 1.641; leg meat, 1.525. The averages were taken after values for 10 chickens were determined. After suspension of the drug for 3 days, >80% residue clopidol was depleted, and the depletion was nearly completed within 7 days. The speed of depletion varied at different tissue locations in each chicken, with the sequence from fast to slow being equivalent to that of the residue levels. Analytical results of 350 samples during 7 days showed that the proposed method is specific for determination of clopidol in chickens.

Determination of clopidol residues in chicken tissues by liquid chromatography: part III. Quality control analysis of export chickens.

A summary is presented of the liquid chromatographic (LC) determination of clopidol residues in export chickens from 1992 to 1999. Over the past 8 years, we analyzed >7000 chicken samples. A total of 0.66% of samples contained clopidol residues, ranging from 0.005 to 0.79 mg/kg; however, none exceeded 3 mg/kg, the Chinese maximum residue limit. In comparison with 1992, the number of samples multiplied 70-fold, and the frequency of clopidol found in samples decreased 148-fold in 1999. The chickens inspected were exported to foreign countries; all were cleared by the buyers with no disputes. This report also introduces the critical control points of the proposed LC method as well as identification of false-positive procedures by both experience and LC/mass spectrometric confirmation. Our experiences over the past 8 years have demonstrated that the ruggedness of the proposed LC method is quite good.

Determination of clopidol residues in chicken tissues by high-performance liquid chromatography-mass spectrometry.

A high-performance liquid chromatographic-mass spectrometric (HPLC-MS) method has been developed for determination of clopidol residues in chicken tissues. Samples are extracted with acetonitrile. The extracts are cleaned up on an alumina column followed by an anion-exchange column. The clopidol is separated on a column (150 cmx4.6 mm) of Intertsil by using acetonitrile-water (20:80) as mobile phase. The clopidol was qualitatively identified by molecule mass and determined quantitatively by selected ion monitoring mode at 190 m/z. The recoveries with RSDs ranged from 91.6+/-10.1 to 97.3+/-5.7 at 0.010 to 10.0 mg/kg by spiking three matrices (chicken muscle, liver, and kidney). The limit of detection was 0.005 mg/kg, and the limit of quantification was 0.010 mg/kg.

Determination of nicarbazin and clopidol in poultry feeds by liquid chromatography.

A rapid and very effective analytical procedure for the simultaneous liquid chromatographic determination of two coccidiostats, clopidol (CLOP) and nicarbazin (NICA), in poultry feeds was developed and tested. The ground feed samples were extracted using aqueous dimethylformamide after moistening with water. Co-extracted feed constituents were removed with a solid-phase extraction alumina-basic column and the eluates were directly analyzed on an ODS column (250x4.6 mm, 5 microm) with acetonitrile-0.01 M acetate buffer (pH 4.6) as eluent. UV detection of CLOP and the 4,4'-dinitrocarbanilide portion of NICA was carried out at 265 and 345 nm, respectively. The mean recovery from NICA spiked samples was 95% with a RSD of 4% in a concentration range of 2-150 mg/kg while for CLOP it was 98% with a RSD of 5% in a concentration range of 5-150 mg/kg. The limits of detection of NICA and CLOP in feed, based on a detector signal-to-noise ratio of 3, were estimated to be 1 mg/kg and 2.5 mg/kg, respectively, and the lowest levels tested in feeds by this procedure were 2 mg/kg and 5 mg/kg, respectively.

An automated HPLC determination of meticlorpindol in eggs with UV absorbance detection, using on-line dialysis and pre-concentration as sample clean-up; occurrence in and carry over to eggs.

A fully automated HPLC determination of the coccidiostat meticlorpindol in whole egg, egg white and yolk is described. The sample homogenate is dialysed on-line against water. The dialysate is concentrated on-line on a short reversed-phase (RP) column. The contents of this column are transferred to the reversed-phase analytical column by means of the mobile phase. Meticlorpindol is detected using an absorbance detector at 270 nm. Linear calibration graphs are obtained in the range 40-900 ng/g in whole egg and egg white (detection limit 10 ng/g) and 80-1800 ng/g in yolk (detection limit 20 ng/g). Out of 111 commercially obtained egg samples 12 contained meticlorpindol with levels varying from 10 to 433 ng/g. A group of laying hens, kept in cages, received 10 mg/kg of Lerbek (meticlorpindol and methylbenzoquate; Dow Chemical) in the feed for 10 days. Meticlorpindol residues in the eggs rose to a level of 622 ng/g. Meticlorpindol was found in the eggs until 6 days after withdrawal of the medicated feed. Another group received 110 mg/kg in the feed. Meticlorpindol residues rose to levels of 4480 ng/g in the eggs, 5880 ng/g in the egg white and 2660 ng/g in the yolk. Meticlorpindol was found in the eggs and the egg white until 14 days and in the yolk until 8 days after withdrawal of the medicated feed.

[Method for the simultaneous determination of residues of chemotherapeutic agents, antiparasitic agents and growth promoters in food of animal origin. 3. Determination of chloramphenicol and meticlorpindol]. / Multimethode zur Bestimmung der Rückstände von Chemotherapeutica, Antiparasitica und Wachstumsförderern in Lebensmitteln tierischer Herkunft. 3. Mitteilung: Bestimmung von Chloramphenicol und Meticlorpindol.

Chloramphenicol and meticlorpindol may be determined by HPLC with UV-detection with a limit of 0.02-0.03 mg/kg. Residues of 0.001 mg/kg Chloramphenicol are determined by Capillary-GC with an ECD after an additional clean up step using chromatography on a miniaturized silica column, followed by a silylation step. HMDS/TMCS/Pyridine forms the disilylated Chloramphenicol. In samples of eggs, milk and meat the limit of detection is roughly 0.0002 mg/kg and is adequate to check the FDR tolerance levels for these foods. The proposed method was checked with residues of chloramphenicol in the range of 0.0005 to 0.005 mg/kg in milk from animals dosed with chloramphenicol. Results obtained by this method and results of a RIA-method correlated very well. The analytical procedure (applicable for all substances) was abbreviated compared to the method reported in the first communication on this topic.

Gas chromatographic determination of clopidol in chicken tissues.

A method has been developed for the determination of clopidol residues in chicken tissues. After extraction and cleanup, clopidol is esterified in a 2-phase system to clopidol propionate, which is determined by gas chromatography. The 2-phase system includes, in addition to the clopidol dissolved in methanol, aqueous borax solution, hexane, propionic anhydride, and pyridine. Use of these reagents precludes the use of explosive or carcinogenic chemicals in the derivatization step, and the method is therefore suitable for routine laboratory analysis. Levels of 0.5 ppb clopidol in tissue can be determined.

Rapid liquid chromatographic determination of clopidol in chicken muscles.

A simple, rapid, and sensitive liquid chromatographic method is described for the quantitative determination of trace amounts of clopidol (3,5-dichloro-2,6-dimethyl-4-pyridinol) in chicken muscles. Clopidol was extracted from tissues with acetonitrile and the extract was cleaned up on an alumina column and separated on an anion exchange column, using 0.5% acetic acid in methanol as eluant. The solvent in the eluate was evaporated and the residue was dissolved in methanol containing benzamide as internal standard. Clopidol was separated on a Zorbax ODS column (4.6 X 250 mm) by using acetonitrile-0. 05M phosphate buffer, pH 7.0 (15 + 85 v/v%), and detected at 270 nm with 0.005 AUFS . Recoveries of clopidol added to chicken tissues at levels of 0.02, 0.05, and 0.1 microgram/g were 94.8, 94.8, and 97.0%, respectively. This method is applicable to levels as low as 0.01 ppm clopidol in chicken muscle.

Improved method for gas-liquid chromatographic determination of clopidol in chicken tissues.

A sensitive and specific gas-liquid chromatographic (GLC) method has been developed for determining low levels of clopidol in chicken tissues. Clopidol is extracted from the tissues with methanol, and cleaned up on an alumina column and an anion exchange resin column with 0.1% acetic acid--methanol as eluate. Clopidol is methylated with diazomethane, and then determined by GLC. 2,4-Dinitro-1-chlorobenzene is used as an internal standard. The method is applicable to levels as low as 2 ppb in chicken tissues. Recoveries of 2--20 ppb clopidol added to tissues averaged 87% for muscle, 84% for liver, 80% for kidney, and 76% for fat.

Comparison of electron-capture and electrolytic conductivity detection for the gas-liquid chromatographic analysis of some perfluoro derivatives of four agricultural chemicals.

The gas chromatographic response of four perfluoro derivatives, of four agricultural chemical [diethylstilbestrol] (DES) clopidol, linuron and carbofuran] was examined. The derivatives varied in fluorine content from 3 to 30 atoms per molecule. The sensitivities of the derivatives were found to be ca. 10-100 times greater by electron-capture detection than by electrolytic conductivity detection (halogen mode). The sensitivity also was found to increase with increasing fluorine content for all derivatives by electrolytic conductivity, whereas by electron capture, DES and clopidol exhibited similar responses with either the penta-, hepta-, or pentadeca-fluoro derivatives. The sensitivity of linuron and carbofuran derivatives by electron capture varied with increasing fluorine content. For detection by electron capture, the hepta-fluorobutyryl derivatives were preferred for DES and clopidol, and either the hepta-fluorobutyryl or the pentadecafluorooctanoyl derivatives for linuron and carbofuran. For detection by electrolytic conductivity, the pentadecafluorooctanoyl derivatives were superior for all four compounds.