Chloramphenicol Residues Research Articles

A gel-based visual immunoassay for non-instrumental detection of chloramphenicol in food samples

A gel-based non-instrumental immuno-affinity assay was developed for the rapid screening of chloramphenicol (CAP) in food samples with the limit of detection (LOD) of 1μgL−1. The immuno-affinity test column (IATC) consisted of a test layer containing anti-CAP antibody coupled gel, and a control layer with anti-HRP antibody coupled gel. Based on the direct competitive immuno-reaction and the horseradish peroxidase enzymatic reaction, the test results could be evaluated visually. Basically, blue color development represented the negative results, while the absence of color development represented the positive results. In this study, CAP spiked samples of raw milk, pasteurized milk, UHT milk, skimmed milk powder, acacia honey, date honey, fish and shrimp were tested. Little or none sample pretreatment was required for this assay. The whole procedure was completed within 10min. In conclusion, the gel-based immuno-affinity test is a simple, rapid, and promising on-site screening method for CAP residues in food samples, with no instrumental requirement.

Removal of Chloramphenicol by Macroporous Adsorption Resins in Honey: A Novel Approach on Reutilization of Antibiotics Contaminated Honey

The effects of different steps in honey production on chloramphenicol (CAP) levels and CAP removal from honey using macroporous adsorption resins (MARs) were investigated in this study. CAP residues in honey were quantified by enzyme-linked immunosorbent assay after each processing step including preheating, filtration, vacuum concentration and pasteurization. Vacuum concentration contributes the most reduction of CAP level (9.9%). Meanwhile, 5 types of MARs (including LSI-1, LSI-2, LSI-3, LS-803, and LS-903) were used in CAP adsorption. The results showed that LS-803 resin had higher adsorption rate of 86% than other resins in removing CAP from honey, and its optimal adsorption time and temperature were 40 min and 55 °C, respectively. The treated honey could be used as feed additive or biomass energy. Therefore, it would be a novel approach to reutilization of antibiotics contaminated honey.

Detection of Ultratrace Chloramphenicol Residues in Milk and Chicken Muscle Samples Using a Chemiluminescent ELISA

A competitive indirect chemiluminescent enzyme-linked immunoassay (CL-ELISA) for chloramphenicol (CAP) residues in milk and chicken muscle has been developed. Due to the unique characteristic of the polyclonal antibody, special reaction system and modified extract method, then after optimization (concentration of Tween-20, concentration of PB and pH, incubation time, and temperature), the method gave a detection limit of 0.92 ng/L and a detection range of 3.16–3035 ng/L, with the IC50 of 17.29 ng/L in optimum condition and real sample matrix. When CAP was spiked in milk and chicken muscle at levels of 5–100 ng/L, recoveries ranged from 104.9%–114.8% and 101.0%–118.8%, with coefficients of variation of 3.0%–14.6% and 9.5%–14.4%, respectively. In an actual chicken muscle residue study, although the extract of samples diluted 10-fold, or even 100-fold, which represents extremely lower concentration of CAP, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The developed method is therefore suitable for screening of ultratrace CAP residues in milk and chicken muscle samples.

Chloramphenicol Residues in Muscle of Rainbow Trout Following Two Different Dose Treatments

Depletion of chloramphenicol (CAP) in muscle of rainbow trout was evaluated following 4days of oral administration with two dosages (42 and 84mg/kg/day). Sampling was conducted during treatment and for 35days following the end of treatment. Analysis was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Concentrations observed during treatment were more than 300μg/kg. A significant elimination occurred within 9days after the cessation of treatment in both groups. Higher CAP levels were measured in the group treated with higher dose. CAP was not detectable after 13 and 15 after the end of treatment in both groups.

Simultaneous determination of chloramphenicol and florfenicol in liquid milk, milk powder and bovine muscle by LC–MS/MS

A validated method based on European and Brazilian legislation is reported. It is applicable to the simultaneous determination of chloramphenicol (CAP) and florfenicol (FF) by LC-MS/MS in liquid milk, milk powder and bovine muscle. The chromatographic analysis is completed in 6 min and the extraction procedure is very simple, involving only one step liquid-extraction with ethyl acetate. Where it proved necessary to include clean-up, an efficient and rapid step using C18-dispersive solid was added. Initially, a complete validation was performed with liquid milk matrix; later the scope was extended to the other matrices through extending the inter-day precision (within laboratory reproducibility) RSD values. An internal standard (d5-CAP) was employed for quantitative purposes. The method was shown to have good accuracy and precision for determining CAP residues at the level of 0.3–0.6 µg kg−1 and FF residues at the level of 5–15 µg kg−1.

Chloramphenicol Use and Prevalence of its Residues in Broiler Chickens and Eggs in Ibadan, Nigeria

Antibiotic use in poultry could result in deposition of residues in edible products of which chloramphenicol is of particular concern because its toxicity and non-dose dependent fatal aplastic anemia in humans. This study assessed the use of antibiotic in poultry production, determined the prevalence and concentration of chloramphenicol residues in meat and eggs from poultry farms and commercial outlets in Ibadan. Semi-structured questionnaires were administered to 75 poultry farmers to assess the use of antibiotics in poultry and the presence of chloramphenicol in chicken eggs and meat randomly obtained from broiler farms and commercial egg outlets were screened for chloramphenicol residues using ELISA technique. The results showed that 50% of the farmers reported administering chloramphenicol to their flocks. Also the survey of five veterinary drugs retail outlets revealed four brands of antibiotics sold for poultry use containing chloramphenicol as an active ingredient. The residue was detected in 51.1% of poultry organs and 25.4% of eggs with mean concentration ranged from 61.8 to 74.6 ng kg -1 in meat and 283.5 to 298.9 ng kg -1 in eggs. The prevalence of chloramphenicol residue was significantly higher in organs than in eggs, but the concentrations were significantly higher in eggs than in meat ( p <0.05). This study documented widespread sales and use of chloramphenicol in poultry in Ibadan with high concentrations of chloramphenicol residues posing public health risks and international trade barrier. We recommend prudent antibiotic use in food animals, alternative treatment regimens and enforcement of appropriate regulatory efforts to prohibit the use of chloramphenicol in food animals. Keywords : Antibiotics, Chloramphenicol Residues, Poultry

Preparation and purification of monoclonal antibodies against chloramphenicol.

Monoclonal antibodies (McAbs) against chloramphenicol (CAP) were produced to detect CAP residues, which could be toxic and possesses a potential threat to human health. The CAP-BSA conjugate was obtained by bovine serum albumin (BSA) coupled with CAP, and used to immunize the mice. The splenocytes from the immunized mice were fused with mouse myeloma cells SP2/0 to form hybridoma, which may secrete McAbs against CAP. Hybridomas 1D(1) and 3G(12) secreting McAbs against CAP were obtained by screening. Ascites containing McAbs were prepared by injecting 1 x 10(6) cells of hybridoma 1D(1) and 3G(12) into the abdomen of mice. Protein A affinity chromatography was used to purify McAbs against CAP in a single chromatographic step with recovery yield above 80% and purity above 95% and full recovery of antibody activity. Experiments showed that McAb 3G(12) was highly specific for CAP and had no cross-reactivity with analogues which have a structure similar to CAP. The IC(50) value was 50.8 ng/mL.

ELISA screening and liquid chromatography-tandem mass spectrometry confirmation of chloramphenicol residues in chicken muscle, and the validation of a confirmatory method by liquid chromatography-tandem mass spectrometry

In the period December 2008 to August 2009, 180 chicken meat samples, including 90 thigh and 90 breast meats in Bursa province, Turkey, were collected. The determination of chloramphenicol (CAP) residues in the samples was screened by ELISA, and a confirmatory method based on liquid chromatography coupled with tandem mass spectrometry was described and validated. The ELISA screening of the samples was performed after extraction with ethyl acetate and defatting with n-hexane. The results showed that 15 (8.3%) of the chicken meat samples were positive for CAP residues from 12.64 to 226.22 ng/kg, with a mean of 45.32 ng/kg. Confirmatory analysis of the results from ELISA was practiced after an extraction with ethyl acetate. Chromatographic seperation was carried out by using a Synergy MAX-RP 80A column and the mixture of acetic acid-water as a mobile phase. The mass spectral acquisition was done in the negative-ion mode applying selective reaction monitoring with the following ions (mass-to-charge ratio, m/z): m/z 321 → 152 and m/z 321 → 194 for CAP. By liquid chromatography-tandem mass spectrometry, CAP was confirmed in 2 of 15 ELISA positive samples and 1 of 45 negative samples, with concentration levels that varied between 150 and 361 ng/kg. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear with a typical r(2) value of 0.9966. The recovery values ranged from 97.3 to 104.0% and within-laboratory repeatability was lower than 5%. The decision limit was 0.10 µg/kg and detection capability was 0.11 µg/kg. To evaluate the presence of CAP residues, this method was successfully implemented in chicken meat samples.

Immunomagnetic reduction assay on chloramphenicol extracted from shrimp

The application of the assay methodology, called immunomagnetic reduction, using bio-functionalized magnetic nanoparticles as labeling markers for chloramphenicol was investigated. The reduction in the alternative-current (ac) magnetic susceptibility χ ac of magnetic nanoparticles caused by the association between magnetic nanoparticles and chloramphenicol was detected as a function of the concentration of chloramphenicol. In this study, the characterizations used to detect chloramphenicol, such as low-detection limit and interference, were also conducted. Furthermore, the extracting processes for chloramphenicol from shrimp were explored. Thus, the platform for detecting chloramphenicol residue in shrimp via immunomagnetic reduction was demonstrated. Such platform showed features of a 0.1-ppb low-detection limit, low interference from other kinds of antibiotics, and easy operation.

Elimination of Chloramphenicol in Rainbow Trout Receiving Medicated Feed

Chloramphenicol muscle residue levels in rainbow trout were determined after oral administration of 84 μg kg-1d-1 of chloramphenicol for four days. Samples were taken one day before treatment and for 43 days after the treatment was over. Chloramphenicol was analysed using an in-house enzyme linked immunoassay (ELISA) validated against the criteria of the Commission Decision 2002/657/EC. Validation parameters confirmed that the method was appropriate for the detection of chloramphenicol at levels below the minimum required performance limit (MRPL) of 0.3 μg kg-1. The highest chloramphenicol levels were observed on the first day after the treatment had ended (144.3 μg kg-1). Elimination was significant over the first seven days; significant differences were detected between days 1 and 3 (p<0.001), 3 and 5 (p<0.001), and 5 and 7 (p<0.05). Chloramphenicol levels dropped below MRPL to 0.17 μg kg-1 on day 9 after the end of treatment. From day 11 to 43, chloramphenicol residues were detectable in a range from 0.091 μg kg-1 (highest) to 0.011 μg kg-1 (lowest). Our results indicate that trout muscle tissue could be compliant with health requirements for consumption 10 days after withdrawal from chloramphenicol treatment.

Validation of the Determination of Chloramphenicol Residues in Animal Feed by Liquid Chromatography with an Ion Trap Detector Based on European Decision 2002/657/EC

A new liquid chromatographic method with an ion trap detector for the determination of chloramphenicol residues in animal feed is described. The developed method consists of an organic extraction and a two-step cleanup. For the cleanup, solid phase and liquid–liquid extractions were applied to minimize the ion suppression effects. One precursor and three product ions were monitored obtaining 5.5 identification points, which provided the method with a very high selectivity/specificity and gave a great confidence to the results. The described method was fully validated according to the requirements of the 2002/657/EC European Decision and the estimated decision limit and detection capability were 6 and 8 μg Kg−1, respectively.

Discrimination of eight chloramphenicol isomers by liquid chromatography tandem mass spectrometry in order to investigate the natural occurrence of chloramphenicol

This paper describes the discrimination of eight different isomers of chloramphenicol (CAP), an antibiotic banned for use in food producing animals, by reversed phase and chiral liquid chromatography in combination with tandem mass spectrometric detection. Previously, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) the presence of CAP was confirmed in some grass and herb samples collected on Mongolian pastures up to concentrations of 450 μg kg −1. It was not possible to establish the cause of CAP residues which has initiated research on the natural occurrence of this drug. CAP occurs in the para-configuration and in the meta-configuration and contains two chiral centers thus eight different isomeric configurations exist, namely four (RR, SS, RS, SR) meta-stereoisomers and four para-stereoisomers. It is known that only RR- p-CAP has antimicrobial properties. To find out if the CAP detected in the plant material samples is the active configuration, a high resolution reversed phase LC-MS/MS system was tested for its ability to separate the different isomers. This system proved to be able to discriminate between some isomers, but not between RR- p-CAP and SS- p-CAP, also called dextramycin. Despite a detailed elucidation of the product ions and the fragmentation patterns of all isomers, MS/MS did not add sufficient specificity for full discrimination of the isomers. Therefore a chiral liquid chromatographic separation with MS/MS detection that is able to distinguish all isomers was developed and finally the isomeric ratio of non-compliant plant material samples and some CAP formulations was determined using this system. This showed that Mongolian grass and herb samples only contain the biological active isomer of CAP as do the obtained formulations. Therefore the CAP present in the plant material might origin from the production by soil organisms or from a manufactured source.

Determination of Chloramphenicol Residues in Foods by ELISA and LC-MS/MS Coupled with Molecularly Imprinted Solid Phase Extraction

The combination of molecularly imprinted solid-phase extraction (MISPE) with ELISA and LC-MS/MS was developed for the detection of chloramphenicol (CAP) in honey samples. Significant recoveries of 99.1 ± 7.1 and 98.8 ± 8.2% were obtained for intra- and inter-assay determination by ELISA determination, respectively. The limit of detection of CAP was 0.034 μg kg−1 and the limit of quantification was 0.046 μg kg−1. Determination and validation of CAP by using LC-MS/MS were performed following the same extraction and purification process as for the ELISA. The results demonstrated that the CAP samples purified by using MISPE were simultaneously applicable to analysis by ELISA and LC-MS/MS.

Multi-walled carbon nanotubes as solid-phase extraction adsorbent for the ultra-fast determination of chloramphenicol in egg, honey, and milk by fused-core C18-based high-performance liquid chromatography–tandem mass spectrometry

In the present work, a high-performance liquid chromatography-tandem mass spectrometry method has been developed for the residue analysis of chloramphenicol (CAP) in several food matrices. Following the addition of D(5)-CAP as internal standard, egg, honey, and milk were extracted and cleaned by means of solid-phase extraction, utilizing multi-walled carbon nanotubes as sorbent. The extracts were separated on a Halo fused-core C18 column (50 mm × 2.1 mm, 2.7 μm) and quantified by a 4000 Q-trap mass spectrometer equipped with a TurboIonSpray™ interface using electrospray ionization and multiple-reaction monitoring mode. The method validation was performed according to the criteria of Commission Decision 2002/657/EC. The decision limit (CCα) and detection capability (CCβ) of CAP in milk were calculated for m/z 320.8 > 151.9. Due to the existence of slight signal suppression, quantification was performed by matrix-matched calibration curves, ranging from 0.1 to 100 ng mL(-1), with regression coefficients of 0.9993, 0.9998, and 0.9997 for egg, honey, and milk, respectively. Mean recoveries of the CAP ranged from 95.8% to 102.3%, with the corresponding intra- and inter-day variation (relative standard deviation) less than 7.13% and 8.89%, respectively. The limit of detection and limit of quantification of the method were also reported. This method successfully applied to several food matrixes (egg, honey, and milk) and can serve as a monitoring tool to avoid unacceptable levels of residues of CAP entering the food chain.

Chloramphenicol residues in chicken liver, kidney and muscle: A comparison among the antibacterial residues monitoring methods of Four Plate Test, ELISA and HPLC

There are many concerns about safety of food contaminated with antibacterial residues. This study was designed to investigate the occurrence of chloramphenicol (CAP) residue in broiler chickens tissues, namely liver, kidney and muscle. One hundred and sixty broiler chickens carcasses were collected from three provinces of Iran. Four Plate Test (FTP), ELISA and HPLC were used to qualify and quantify the contamination of the samples with CAP. The results of FPT revealed that up to 17.5% of the samples were contaminated with the antibiotic. The ELISA assay showed that out of 28 positive samples in FPT, 22 liver, 21 kidney and 14 muscle samples were positive for CAP. ELISA analyses demonstrated that the minimum and maximum levels of 0.54 and 155.2 ng/g were detected in the kidney and liver, respectively. HPLC analyses confirmed the ELISA findings although the level of contamination was lower than that of ELISA. These data showed that despite the prohibition of CAP application in food animals including poultry, the CAP residue was detectable indicating an illegal use of this antibiotic. Our findings also demonstrated the application of sensitive and more specific analytical assays in screening and quantitation of CAP residues in food products.

DEVELOPING METHOD OF DIFFERENTIAL PULSE POLAROGRAPHIC FOR ANALYSIS OF CHLORAMPHENICOL RESIDUE IN MILK

A differential pulse polarographic method for a quantitative analysis of chloramphenicol residue in milk had been developed. Result showed that the method using dropping mercury electrode as working electrode, Ag/AgCl electrode as reference electrode, and platinum electrode as auxiliary electrode with an acetic buffer solution of pH 4.7 as supporting electrolyte had a recovery for chloramphenicol of (96.88 ± 3.17)% with a detection limit of 0,027 µg/mL, a quantitation limit of 0,089 µg/mL, and a determination limit of 0,010 µg/mL. Keywords: differential pulse polarography, residue, chloramphenicol, milk

Determination of Chloramphenicol Residues in Milk by Enzyme-Linked Immunosorbent Assay: Improvement by Biotin−Streptavidin-Amplified System

A sensitive biotin-streptavidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed for the determination of chloramphenicol residues in milk. The biotin-streptavidin system was applied to enhance the sensitivity. After optimization, the detection limit of the method was found to be 0.042 +/- 0.006 ng mL(-1), which is 8-fold more sensitive than the traditional competitive ELISA using the same antibody and coating antigen. The amplification mechanism of the biotin-streptavidin system and the major factors affecting the sensitivity of detection are discussed. This method was successfully applied to determine the chloramphenicol residues in milk samples with a simple and rapid extraction procedure, and good recoveries (85.66-109.67%) were obtained. The result indicated that the biotin-streptavidin system may be a valuable tool to improve the specific detection of trace veterinary drug residues and could be widely used for routine monitoring of food samples.

Screening of Chloramphenicol Residues in Broiler Chickens Slaughtered in an Industrial Poultry Abattoir in Mashhad, Iran

The use of chloramphenicol is prohibited in food producing animals due to its harmful and even potentially fatal side effects in human. In order to screen broiler carcasses for the drug residues, 31 broiler chickens from different farms were sampled. The samples from kidneys were homogenized, extracted using ethyl acetate and dried under N2 flow. The samples were then assayed using enzyme-linked immunosorbent assay (ELISA). In the next phase, the concentration of chloramphenicol in the kidney, liver and thigh muscle of 13 positive chickens was compared following extraction and ELISA as mentioned. More than half of the samples (54.8%) showed detectable concentrations of chloramphenicol. The highest concentrations of the drug were in the kidney and liver. According to the current research, there seems be a public health threat due to the illegal use of chloramphenicol in broiler farms and that kidney samples can be used for screening tests.

Otimização e validação intralaboratorial de método para análise de resíduos de cloranfenicol em leite caprino por cromatografia gasosa com detecção por captura de elétrons (CG/DCE)

The presence of chloramphenicol residues in goat milk can cause toxic effects in the population. The present work consists of the optimization and validation of analytical methodology for determination of chloramphenicol residues in goat milk by GC/ECD. The extraction was made with ethyl acetate and the clean-up with SPE-C18. The identification was made by comparison of retention time and GC/MS, and the quantification by external standard. The method was selective, linearity (0.998), precise (5.8-13.4%), exact (69.87-73.71%) and robust. The LOD and LOQ of method were 0.030 and 0.10 μg/kg, respectively. The method was efficiently for analysis of chloramphenicol in goat milk.

Characterization of Anti-Chloramphenicol Antibodies by Enzyme-Linked Immunosorbent Assay

Polyclonal antibodies against conjugates of chloramphenicol succinate and chloramphenicol base with proteins were obtained and characterized in direct ELISA. Antiserum against a conjugate of chloramphenicol (CAP) base with BSA (direct coupling) was very specific and showed cross-reactivity only with CAP succinate (11.3%) and CAP base (4.6%); whereas, antisera against a conjugate of CAP succinate with a protein recognized CAP succinate strongly as an initial compound. In direct ELISA, antisera against a conjugate of CAP succinate with KLH (homologous assay) and CAP base with BSA (heterologous assay) showed similar sensitivity: IC50 were 1.3 and 1.5 ng mL−1, respectively. Applicability of the immunoreagents obtained was shown in the analysis of CAP residues in milk (3.5% fat content). Detection limit of 0.3 ng mL−1 was obtained for milk diluted 5 times.