Chloramphenicol Residues Research Articles

Determination of chloramphenicol residues in crustaceans: Preparation and evaluation of a proficiency test in Germany

Chloramphenicol (CAP) is banned for use in food-producing animals and is, thus, controlled on the basis of the National Residue Control Plans in the European Union. Due to current problems with residues of CAP in shrimp, crayfish and prawns, a sensitive GC/NCI/MS method was optimised and in-house validated. The validation study resulted in a decision limit (CCα) of 0.07 µg kg−1, a recovery of 95% and a within-laboratory reproducibility of 9%. The method was used for preparing a proficiency test to assess the quality of residue control in Germany. The proficiency test involved analysis of five samples and the results were very satisfactory. The reproducibility standard deviation for five samples ranged from 17 to 24%, and the median concentrations lay between 0.43 and 0.51 µg kg−1 CAP. These values are clearly below the corresponding Horwitz standard deviation of about 50%. From the study, it can be concluded that there are, irrespective of the method applied, well-established and proper working analytical procedures for the control of CAP around the minimum required performance limit (MRPL) of 0.3 µg kg−1.

A linear modulation-based stochastic resonance algorithm applied to the detection of weak chromatographic peaks

A simple stochastic resonance algorithm based on linear modulation was developed to amplify and detect weak chromatographic peaks. The output chromatographic peak is often distorted when using the traditional stochastic resonance algorithm due to the presence of high levels of noise. In the new algorithm, a linear modulated double-well potential is introduced to correct for the distortion of the output peak. Method parameter selection is convenient and intuitive for linear modulation. In order to achieve a better signal-to-noise ratio for the output signal, the performance of two-layer stochastic resonance was evaluated by comparing it with wavelet-based stochastic resonance. The proposed algorithm was applied to the quantitative analysis of dimethyl sulfide and the determination of chloramphenicol residues in milk, and the good linearity of the method demonstrated that it is an effective tool for detecting weak chromatographic peaks.

Pharmacokinetics of chloramphenicol in carp (Cyprinus carpio L.) after drug administration

Chloramphenicol (CAP) is a banned substance in fish farming; yet, residues are sometimes detected in farmed fish products. Therefore, it is important that tissue distribution and elimination rates of CAP are measured to allow monitoring of fish farms. The tissue distribution and residue of CAP in carp (Cyprinus carpio L.) after muscle injection was investigated. Chloramphenicol concentrations were determined using high-performance liquid chromatography. Pharmacokinetics were used for the studies of CAP residue and the results showed that the CAP was easily assimilated in the liver, serum and gill, with the absorption rates of 6383, 3.02 and 1.39 h−1, respectively, following a single-dose injection of 80 mg kg bw−1. The elimination half-lives (t1/2) were 22.28, 15.47, 14.87, 9.28 and 5.32 h for the liver, serum, gill, muscle and kidney respectively. The t1/2 after 5 days of multi-doses of repeated CAP injection with 40 mg kg bw−1 was 1.09, 0.887, 0.872, 0.476 and 0.617 days for the five tissues respectively. The results validated that CAP in the liver, serum and gill could be markers for CAP residue analysis.

Comparison of the determination of chloramphenicol residues in aquaculture tissues by time-resolved fluoroimmunoassay and with liquid chromatography and tandem mass spectrometry

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of chloramphenicol (CAP) residues in aquaculture tissues based on monoclonal antibodies. The limits of detection (LOD) were determined to be 0.04 ng g−1 and the limits of quantification (LOQ) were less than 0.15 ng g−1. The intra-assay variations were below 10% and the interassay variations ranged between 9.7 and 13.3%. The mean recoveries established at six concentration levels varied from 83.7–109.6% and the coefficient of variation was from 8.3–11.5%. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC-MS-MS). This proposed technique could be applied to routine residue analysis.

Determination of chloramphenicol residues in meat, seafood, egg, honey, milk, plasma and urine with liquid chromatography–tandem mass spectrometry, and the validation of the method based on 2002/657/EC

A simple and rapid method for the determination and confirmation of chloramphenicol in several food matrices with LC–MS/MS was developed. Following addition of d 5-chloramphenicol as internal standard, meat, seafood, egg, honey and milk samples were extracted with acetonitrile. Chloroform was then added to remove water. After evaporation, the residues were reconstituted in methanol/water (3 + 4) before injection. The urine and plasma samples were after addition of internal standard applied to a Chem Elut extraction cartridge, eluted with ethyl acetate, and hexane washed. Also these samples were reconstituted in methanol/water (3 + 4) after evaporation. By using an MRM acquisition method in negative ionization mode, the transitions 321 → 152, 321 → 194 and 326 → 157 were used for quantification, confirmation and internal standard, respectively. Quantification of chloramphenicol positive samples regardless of matrix could be achieved with a common water based calibration curve. The validation of the method was based on EU-decision 2002/657 and different ways of calculating CCα and CCβ were evaluated. The common CCα and CCβ for all matrices were 0.02 and 0.04 μg/kg for the 321 → 152 ion transition, and 0.02 and 0.03 μg/kg for the 321 → 194 ion transition. At fortification level 0.1 μg/kg the within-laboratory reproducibility is below 25%.

A surface plasmon resonance biosensor assay for the simultaneous determination of thiamphenicol, florefenicol, florefenicol amine and chloramphenicol residues in shrimps

A rapid and sensitive screening qualitative method using a surface plasmon resonance (SPR) biosensor was developed which can detect of all fenicol antibiotic residues in shrimps from a single sample extract. This method requires ethyl acetate extraction followed by a single wash with isooctane/chloroform. Each sample extract is injected over the surfaces of two biosensor chip flow cells, one surface having the capability to detect florefenicol amine (FF amine), florefenicol (FF), and thiamphenicol (TAP) and the second surface for chloramphenicol (CAP) detection. The estimated detection capabilities (CCβ) were 0.1, 0.2, 250, and 0.5 ppb for CAP, FF, FF amine, and TAP, respectively. This quick, simple test allowed the detection of CAP residues in shrimps at the minimum required performance limit (MRPL) of 0.1 μg kg −1 for this compound and of FF, FF amine, and TAP below their maximum residue limits (MRLs).

Performance characteristics of an analytical procedure for determining chloramphenicol residues in muscle tissue by gas chromatography–electron capture detection

Validation of an analytical method for determining chloramphenicol residues in muscle tissue by gas chromatography-electron capture detection (GC-ECD) was performed according to the latest European Union criteria for the analysis of veterinary drugs in food, laid down by Commission Decision 2002/657/EC. The method using the meta isomer of chloramphenicol as an internal standard proved to be very selective, specific to other related phenicols and accurate to within +3.6% at a concentration level of 8.9 microg/kg, as present in the certified reference material available. The correlation coefficient of the calibration curve was 0.9991. At all three fortification levels studied (0.3, 0.45 and 0.6 microg/kg), repeatability and intra-laboratory reproducibility were <8 and < or =9%, respectively. The decision limit (CCalpha) and detection capability (CCbeta) were 0.07 and 0.12 microg/kg, respectively. The validation results and the results of participation in an international inter-laboratory proficiency test indicate that the method presented is completely suited for regulatory control to screen and quantify chloramphenicol residues in various muscle tissues on a routine basis.

Determination of Chloramphenicol Residue in Chicken Tissues by Immunoaffinity Chromatography Cleanup and Gas Chromatography with aMicrocell Electron Capture Detector

A rapid and sensitive gas chromatography (GC) method was developed to detect chloramphenicol in chicken tissues. The extracted samples were cleaned up using the immunoaffinity column prepared by coupling antichloramphenicol monoclonal antibody with cyanogen bromide-activated Sepharose 4B. The dynamic column capacity of chloramphenicol was 3265 ng/mL gel. The eluate was evaporated to dryness, and residues were derivatized and determined by GC with a microcell electron capture detector. Average recoveries were 86.6 to 96.9% for chicken muscle and 74.3 to 96.1% for chicken liver. The limit of quantitation of the method was 0.05 ng/g for chicken muscle and 0.1 ng/g for chicken liver.

Chemiluminescence enzyme immunoassay (CLEIA) for the determination of chloramphenicol residues in aquatic tissues

A competitive indirect chemiluminescence enzyme immunoassay (ic-CLEIA) for chloramphenicol (CAP) residues in shrimp has been developed. After optimization (incubation time, concentration of Tween-20, concentration of PBS and pH), the method gave a limit of detection of 0.01 ng/mL and a detection range of 0.03-23.7 ng/mL, with an ED(50) of 0.47 ng/mL. The method has been validated on spiked shrimp samples in terms of precision (intra- and interassay coefficient variations of less than 10% and 15%, respectively) and accuracy (mean recovery 95-123%). The assay performance is better than the ELISA method which is widely used to detect chloramphenicol and indicates that the CLEIA method can be used to test aquatic samples instead of ELISA.

ＬＣ／ＭＳ／ＭＳによるハチミツおよびローヤルゼリー中のクロラムフェニコールの分析

A sensitive and selective method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was developed for the determination of chloramphenicol (CAP) in honey and royal jelly. Mass spectral acquisition was performed in the negative mode by applying multiple reaction monitoring. In LC separation, Mightyl RP-18GP and 10 mmol/L ammonium acetate-acetonitrile were used as the column and mobile phase, respectively. CAP in honey samples was diluted with water, while CAP in royal jelly was extracted with 1% metaphosphoric acid-methanol (4 : 6). The solutions were cleaned up with an Oasis HLB cartridge. The quantification limits of CAP in honey and royal jelly were 0.3 ng/g and 1.5 ng/g, respectively. The recoveries of CAP from both honey and royal jelly at the quantification limits were over 92%. Twenty honey products and seven royal jelly products were analyzed by the developed method. CAP was detected in one honey product at 0.6 ng/g and in six royal jelly products at the level of 1.5-17.8 ng/g. These results show that the developed method has satisfactory sensitivity selectivity and is useful for the determination of CAP residues in honey and royal jelly.

Quantitative analysis of chloramphenicol residues in shrimp muscle tissues by Chemiluminescent enzyme immunoassay

A competitive indirect chemiluminescent enzyme immunoassay (ic-CLEIA) has been developed for the determination of chloramphenicol (CAP) residues in shrimp. After the optimisation of four physico-chemical parameters, i.e. incubation time, concentration of Tween-20, concentration of PBS and its pH, the method developed gave a limit of detection of 0.01 ng/ml and a detection range from 0.03 ng/ml to 23.7 ng/ml, with an ED&lt;sub&gt;50&lt;/sub&gt; of 0.47 ng/ml. The developed method has been validated on spiked shrimp samples in terms of precision (intra- and interassay coefficient variations of less than 10% and 15%, respectively), and of accuracy (mean recovery from 95% to 123%). All these parameters being better than those of the ELISA method which is widely used to detect chloramphenicol, it may be suggested that the CLEIA method can be used to detect aquatic samples instead of ELISA. &amp;nbsp;

The Tissue Kinetics and Residues of Chloramphenicol in Sokoto Red Goats

The Tissue Kinetics and Residues of Chloramphenicol in Sokoto Red Goats

Determination of chloramphenicol residues in animal feeds by liquid chromatography with photo-diode array detection

A reversed-phase liquid chromatographic method with photo-diode array detection is presented for use in the determination of chloramphenicol. A new amide-based stationary phase prevents the interaction of the analyte with the residual silanol groups, thus avoiding the appearance of tailed peaks. Specificity was demonstrated by the retention characteristics, UV spectra and peak purity index. Linearity, precision, recovery and sensitivity were satisfactory. The procedure was applied to the analysis of residual chloramphenicol in different types of animal feed. Samples were extracted by using liquid–liquid extraction steps with organic solvents and, for the clean-up, solid-phase extraction using cartridges was applied. Detection limit in the samples using the proposed procedure was 0.7 μg kg −1.

In‐house validation of a liquid chromatography/electrospray tandem mass spectrometry method for confirmation of chloramphenicol residues in muscle according to Decision 2002/657/EC

In this work we present an in-house validation study for the confirmatory analysis of chloramphenicol (CAP) in muscle according to the Commission Decision 2002/657/EC requirements. CAP is extracted in acetonitrile and after liquid-liquid partitioning with n-hexane is identified and quantitatively determined by ion trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) analysis in the negative ion mode. CAP was identified using the precursor ion and at least two product ions, meeting the qualitative and quantitative criteria set by the European Commission in the Decision 2002/657/EC for confirmation of prohibited veterinary drug residues. We calculated mean drug recoveries, CCalpha and CCbeta of the method, and reported data on specificity, ruggedness and within-laboratory reproducibility. Finally, we point out and discuss some problems and questions arising from controversy about the application of Decision 2002/657/EC.

Development and Validation of a Thin-Layer Chromatographic Method for Determination of Chloramphenicol Residues on Pharmaceutical Equipment Surfaces

A thin-layer chromatographic (TLC) method with densitometric quantitation using the absorption reflectance mode at 280 nm was developed and validated for the determination of chloramphenicol residues in controlling pharmaceutical equipment cleanliness. Simulated samples at residue levels 0.5, 1, and 1.2 mg/m2 were prepared by spreading the calculated amount of chloramphenicol solution on a 10 dm2 stainless steel surface. After evaporation of the solvent, the residue was removed by 2 methanol-wetted cotton swabs, which were then extracted with methanol. The extract was applied on a high-performance TLC (HPTLC) silica gel F254 plate together with standards ranging from 10 to 60 ng. Plates were developed in a horizontal developing chamber from both sides (36 applications per plate) by using n-hexane-ethyl acetate (35 + 65, v/v) as developing solvent. The mean recovery (n=6) at 1 mg/m2 was 95.8%, and the coefficient of variation was 5.8%. The absolute detection limit was 3 ng, and the quantitation limit 10 ng. The method detection limit was 0.3 mg/m2 by swabbing 2.5 dm2 and 0.075 mg/m2 by swabbing 10 dm2. Chloramphenicol was stable on the plate 2 h before and 24 h after development. Additionally, it was stable during 7 days storage on the cotton swabs in the solvent at room temperature and in diluted standard solution stored in darkness at 4 degrees C. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from the stainless steel surface areas of 2.5 to 10 dm2, and an acceptable residue limit of 1 mg/m2.

Chemiluminescence immunoassay for chloramphenicol

In recent years, various chemiluminescent clinical immunoassay kits have been widely applied to the detection of hormones. However, a kit for chloramphenicol (CAP) is often absent from most commercial product lists, even though it is important to control the levels of CAP residues in foodstuffs too. Therefore, we describe a simple, solid-phase chemiluminescence immunoassay (CLIA) for the measurement of CAP in foodstuffs. A rabbit anti-CAP IgG is passively adsorbed onto the walls of polypropylene plates. The labeled antigen is horseradish peroxidase (HRP) conjugate of CAP. Luminol solution is used as the substrate of HRP. The light yield is inversely proportional to the concentration of CAP. The method has a similar sensitivity (0.05 ng/ml), specificity, precision, and accuracy to a conventional enzyme immunoassay (EIA). The intra-assay and inter-assay CVs of ten samples were <8% and <20%, respectively, and the analytical recovery of the method was 87-100%. The experimental correlation coefficient of dilution was found to be 0.999 using milk supernatant as buffer. The detection limit for the method was 0.1-10 ng/ml, and it displayed good linearity.

Stability of chloramphenicol residues in shrimp subjected to heat processing treatments

The stability of chloramphenicol residues in white shrimp ( Penaeus indicus) subjected to cooking (100 °C) for 10, 20 and 30 min (C1, C2 and C3) as well as retorting (121 °C) for 10 and 15 min (R1 and R2) was studied by a microbial assay method using Photobacterium leiognathi as the test organism. The microbial assay method was found to have a good sensitivity of 1 μg/ml the loss of chloramphenicol in shrimp subjected to cooking for 10, 20 and 30 min was 6%, 12% and 29%, respectively. Similarly, the loss was 9% and 16% from the shrimp subjected to retorting for 10 and 15 min, respectively. The loss of chloramphenicol was found to increase with increase in temperature and duration of heating. This study showed that chloramphenicol is an unstable aquaculture drug that is destroyed or degrades during heat processing treatments.

Determination of Chloramphenicol Residues in Milk, Eggs, and Tissues by Liquid Chromatography/Mass Spectrometry

A new liquid chromatography/mass spectrometry (LC/MS) method is presented for the determination of chloramphenicol (CAP) residues in milk, eggs, chicken muscle and liver, and beef muscle and kidney. CAP is extracted from the samples with acetonitrile and defatted with hexane. The acetonitrile extracts are then evaporated, and residues are reconstituted in 10mM ammonium acetate--acetonitrile mobile phase and injected into the LC system. CAP is determined by reversed-phase chromatography using an Inertsil ODS-2 column and MS detection with negative ion electrospray ionization. Calibration curves were linear between 0.5-5.0 ng/g for all matrixes studied. The relative standard deviations for measurements by this method were generally <12%, and average recoveries ranged from 80 to 120%, depending on the matrix involved. The method detection limits of CAP ranged from 0.2 to 0.6 ng/g, which are comparable to previously reported results. The proposed method is rapid, simple, and specific, allowing a single analyst to easily prepare over 40 samples in a regular working day.

Screening, determination and confirmation of chloramphenicol in seafood, meat and honey using ELISA, HPLC–UVD, GC–ECD, GC–MS–EI–SIM and GCMS–NCI–SIM methods

Studies on screening, determination and confirmation of chloramphenicol (CAP) in 10 kinds of matrices, including seafood, meat, honey, etc. by enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC) with UV detector (HPLC–UVD) and gas chromatography in combination with electronic capture detector (GC–ECD) and mass spectrometry detector (GC–MS) in both electronic ionization mode (EI) and the negative ion chemical ionization mode (NCI) with selected-ion monitoring (SIM) acquisition method (GC–MS–EI–SIM and GCMS–NCI–SIM) have been carried out. Methods have been developed for both qualitative and quantitative detection of chloramphenicol (CAP). Extraction, clean-up, derivatization and analysis procedure have been optimized. The ELISA was carried out for screening, HPLC, GC and GC–MS were applied to determine and confirm CAP residue concentration in suspect samples. The ELISA procedure was carried out on an aqueous extract of the samples. Determination and confirmation of suspect samples were performed after extraction with phosphoric buffer solution (PBS, pH = 6.88)/ethyl acetate, defatting with hexane, analyzed by HPLC or GC–ECD, GC–MS–EI, GC–MS–NCI method. Samples for GC analysis, were further clean-up with solid-phase extraction using LC-Si and LC-C18 cartridges and derivatized to form volatile derivatives by derivatization agent. These techniques are able to detect chloramphenicol residues at the level of 0.1–10 μg/kg. Overall recoveries were 75–120% with R.S.D. values at 5.4–8.1%.

Development of a liquid chromatography/electrospray tandem mass spectrometry method for confirmation of chloramphenicol residues in milk after alfa‐1‐acid glycoprotein affinity chromatography

In this work we present a method for confirmatory analysis of chloramphenicol (CAP) in bovine and buffalo raw milk. CAP is extracted in acetonitrile and purified by affinity chromatography on an alpha-1-acid glycoprotein (AAG) column, then is identified and determined by ion-trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) analysis in the negative ion mode. CAP was identified at the minimum required performance limit (MRPL) of 0.30 ppb, by monitoring the [M-H]- ion and at least two product ions, meeting the qualitative and quantitative criteria set by the European Commission in Decision 2002/657/EC for confirmation of prohibited veterinary drugs. The trueness and within-day and between-day repeatability data are also reported. Moreover, the loading capacity of affinity columns towards CAP was tested. This method, based on the molecular recognition between drug and AAG during the purification step to improve sample cleanup, represents a quantitative and repeatable procedure for confirmatory analysis, and fits the requirements for the routine official control of CAP residues in raw milk.