Chloramphenicol Residues Research Articles

A LC-MS/MS confirmatory method for determination of chloramphenicol in real samples screened by competitive immunoassay

A new liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed to confirm chloramphenicol (CAP) residues in foods of animal origin and in urine samples, which were earlier found positive under the screening analysis, performed by competitive enzyme-linked immunoassay (ELISA) technique. The developed LC-MS/MS method was applied to four non-compliant samples from 2008 to 2012; giving concentrations of CAP residues from 1.18 to 3.68 μg kg−1. All samples, qualified positive by ELISA, were confirmed with the LC-MS/MS technique and found to be non-compliant. The effectiveness of the confirmatory method was proven by participating in a successful proficiency test in year 2010. Both LC-MS/MS and ELISA methods were validated according to the European Union 2002/657/EC decision. The decision limit of the confirmatory method was determined as 0.02 μg kg−1 for CAP in each validated matrix, while the detection capability of the screening test was 0.15 μg kg−1.

Gold nanocatalyst-based immunosensing strategy accompanying catalytic reduction of 4-nitrophenol for sensitive monitoring of chloramphenicol residue

A new competitive-type immunosensing system based on gold nanoparticles toward catalytic reduction of 4-nitrophenol (4-NP) was developed for sensitive monitoring of antibiotic residue (chloramphenicol, CAP, used in this case) by using ultraviolet–visible (UV–vis) spectrometry. Gold nanoparticle (AuNP) with 16nm in diameter was initially synthesized and functionalized with CAP–bovine serum albumin (CAP–BSA) conjugate, which were used as the competitor on monoclonal anti-CAP antibody-coated polystyrene microtiter plate (MTP). In the presence of target CAP, the labeled CAP–BSA on the AuNP competed with target CAP for the immobilized antibody on the MTP. The conjugated amount of CAP–BSA–AuNP on the MTP decreased with the increase of target CAP in the sample. Upon addition of 4-NP and NaBH4 into the MTP, the carried AuNP could catalytically reduce 4-NP to 4-aminophenol (4-AP), and the as-produced 4-AP could be monitored by using UV–vis absorption spectroscopy. Experimental results indicated that the absorbance at 403nm increased with the increment of target CAP concentration in the sample, and exhibited a dynamic range from 0.1 to 100ngmL−1 with a detection limit (LOD) of 0.03ngmL−1 at the 3sblank level. Intra- and inter-assay coefficients of variation were lower than 5.5% and 8.0%, respectively. In addition, the methodology was evaluated for CAP spiked honey and milk samples, respectively. The recovery was 92–112%.

Detection of chloramphenicol residue in bovine meat using Liquid Chromatography Mass Spectrometry

Chloramphenicol (CAP) is a broad spectrum antibiotic that has been banned in many countries due to its serius side effect to human. Detection of CAP residue in food has been determined to a minimum required performance limit (MRPL) of 0.3 ng/g. The purpose of this research was to conduct the analysis of CAP residue in bovine meat by using LCMS and to study the presence of CAP residue in marketed bovine meat samples. LC separation was done on a Shimpack column C18 with ammonium acetate 10 mM/water as mobile phase, and ESI-MS analysis in negative ion mode. The coefficient of determination, R2 = 0.9981 at concentration of 0.125, 0.25, 0.63, 1,00 and 2.00 ng/g. Recovery at three fortification levels (0.25, 0.50 and 1.00 ng/g) was in the range 77.5, 97.3 and 83.4%. The decision limit and the detection capability were 0.15 ng/g and 0.17 ng/g respectively. Analysis results of 52 marketed samples showed that CAP residue were detected in 9 samples in the concentration range of 0.14 to 2.70 ng/g and 6 among those positive samples were above the MRPL value. Therefore, it is important to increase the awareness and also to monitor regularly CAP residues in food originated from animal to provide safe food for the consumers. Key Words : Chloramphenicol, Residue, LCMS, Bovine Meat

Simultaneous determination of chloramphenicol and clenbuterol in milk with hybrid chemiluminescence immunoassays

Two novel chemiluminescence immunoassays using the horseradish peroxidase (HRP)–luminol chemiluminescence system and two different enzymatic systems—HRP and alkaline phosphatase (ALP) chemiluminescence systems—were established for simultaneous detection of chloramphenicol (CAP) and clenbuterol (CLE) residues in milk. Milk samples were detected utilizing the two hybrid chemiluminescence immunoassays, with limits of detection (LODs) of 0.006 μg L−1 for CAP and 0.02 μg L−1 for CLE (Scheme 1A), 0.008 μg L−1 for CAP and 0.023 μg L−1 for CLE (Scheme 1B), respectively. The recoveries for CAP and CLE in individual and co-spiked samples were found to be between 80.0% and 96.7%. The applicability of the proposed method has been validated by determining CAP and CLE in field milk samples with satisfactory results. Thereafter, the developed hybrid chemiluminescence immunoassays can be used as reliable, rapid, and cost effective screening techniques for the simultaneous determination of CAP and CLE residues in milk.

Evaluation of matrix effect in isotope dilution mass spectrometry based on quantitative analysis of chloramphenicol residues in milk powder

In the present study, we developed a comprehensive strategy to evaluate matrix effect (ME) and its impact on the results of isotope dilution mass spectrometry (IDMS) in analysis of chloramphenicol (CAP) residues in milk powder. Stable isotope-labeled internal standards do not always compensate ME, which brings the variation of the ratio (the peak area of analyte/the peak area of isotope). In our investigation, impact factors of this variation were studied in the extraction solution of milk powder using three mass spectrometers coupled with different ion source designs, and deuterium-labeled chloramphenicol (D5-CAP) was used as the internal standard. ME from mobile phases, sample solvents, pre-treatment methods, sample origins and instruments was evaluated, and its impact on the results of IDMS was assessed using the IDMS correction factor (θ). Our data showed that the impact of ME of mobile phase on the correction factor was significantly greater than that of sample solvent. Significant ion suppression and enhancement effects were observed in different pre-treated sample solutions. The IDMS correction factor in liquid–liquid extraction (LLE) and molecular imprinted polymer (MIP) extract with different instruments was greater or less 1.0, and the IDMS correction factor in hydrophilic lipophilic balance (HLB) and mix-mode cation exchange (MCX) extract with different instruments was all close to 1.0. To the instrument coupled with different ion source design, the impact of ME on IDMS quantitative results was significantly different, exhibiting a large deviation of 11.5%. Taken together, appropriate chromatographic conditions, pre-treatment methods and instruments were crucial to overcome ME and obtain reliable results, when IDMS methods were used in the quantitative analysis of trace target in complex sample matrix.

A rabbit monoclonal antibody-based sensitive competitive indirect enzyme-linked immunoassay for rapid detection of chloramphenicol residue

A sensitive competitive indirect enzyme-linked immunoassay (ciELISA) based on a rabbit monoclonal antibody (RabMAb) against chloramphenicol (CAP) has been developed and validated in this study. After optimisation of several key physicochemical factors, such as Tween-20 percentage, pH value and ionic strength, the immunoassay showed excellent performance within the linear range of 0.18–6.37 ng mL−1, with the 50% inhibition concentration (IC50) of 1.06 ng mL−1 and the limit of detection (LOD) of 0.1 ng mL−1. In addition, the cross-reactivities of RabMAb towards chloramphenicol succinate, florfenicol and thiamphenicol were 2.09, 12.45 and 18.10%, respectively. Finally, the developed method was applied in spiked swine urine, milk and honey samples, with recoveries ranging from 71.03 to 109.62%. The result demonstrated that the developed immunoassay is a valuable method for screening and quantitation of CAP residues in real samples.

Screening Procedures for Chloramphenicol Residue Determination in Serum, Milk, and Feedstuff Using Immunochromatographic Assay

The technology developed from this study is based on an immunochromatographic procedure that utilizes antigen-antibody properties using membrane carriers with immobilized immunoreactants and provides rapid and out-of-laboratory techniques. An evaluation of a colloidal gold-based immunochromatographic assay for chloramphenicol (CAP) residue detection in serum, milk and feed is described. Polyclonal antibodies against CAP were produced using CAP-IPA-MAA-ABA-bovine serum albumin (CAP-ed-BSA) conjugate as the immunogen, which exhibited no cross-reactivities with applied competitors in the studied concentration range. The test strip assay could be completed within 7 min, with a visual detection limit of 0.3 μg kg−1 CAP in serum and milk and 0.5 μg kg−1 CAP in feed. The established strip assay performed similarly as a commercial ELISA assay: the CAP test strip revealed a sensitivity of 90% (89–92%) and a specificity of 94% (91–98%), which showed appreciable accuracy and precision. The described strip is rapid, simple and cost-effective as well as sensitive and specific enough for reliable and accurate on-site screening.

Determination of Chloramphenicol Residual in Freshwater Fish by HPLC

HPLC (High Performance Liquid Chromatography) method for chloramphenicol residues in freshwater fish was developed. The samples were extracted with ethyl acetate. The LC was performed on a C18 column, mobile phase consisted of methanol-water (45:55 v/v) and eluted at 0.2mL/min at 20°C, inject volume was 20μl. The linear rang for chloramphenicol residual is within 0.5-20μg/mL, the limit of detection is 0.01ng/g. The average recovery is 80.06%-92.71%, and the RSD is 1.46%-5.61%. This method is employed to analyze freshwater fishes, such as catfish, chub and carp.

A highly sensitive europium nanoparticle-based lateral flow immunoassay for detection of chloramphenicol residue

A europium nanoparticle-based lateral flow immunoassay for highly sensitive detection of chloramphenicol residue was developed. The detection result could be either qualitatively resolved with naked eye or quantitatively analyzed with the assistance of a digital camera. In the qualitative mode, the limit of detection (LOD) was found to be 0.25ng/mL. In the quantitative mode, the half-maximal inhibition concentration (IC50) was determined to be 0.45ng/mL and the LOD can reach an ultralow level of 0.03ng/mL, which is ~100 times lower than that of the conventional colloidal gold-based lateral flow immunoassay. Potential application of the established method was demonstrated by analyzing representative cow milk samples.

Substitution of antibody with molecularly imprinted 96-well plate in chemiluminescence enzyme immunoassay for the determination of chloramphenicol residues

A direct competitive chemiluminescence enzyme immunoassay was developed by using the molecularly imprinted 96-well plate as an artificial antibody to detect chloramphenicol (CAP). The artificial antibody was synthesised on the well surface of MaxiSorp polystyrene 96-well plate and CAP was conjugated with the horseradish peroxidase. The results showed that the imprinted plate exhibited antibody-like binding ability. The plate showed fast adsorption rate, 66% adsorption was finished within 20 min. The cross-reactivity for CAP, florfenicol and thiamphenicol were 100%, 1.25% and 2.08%, respectively. And the imprinted plate could be reused for many times without loss of sensitivity. The IC50 and the detection limit values under optimum conditions were 30±2 µg·L−1 and 0.9±0.01 µg·L−1, respectively. The plate was used to detect CAP in sea cucumber, which showed excellent recoveries ranging from 89% to 98.7%. And the result correlated well with that obtained by the CAP enzyme-linked immunosorbent assay (ELISA) kit.

Development of a new fluorescence immunochromatography strip for detection of chloramphenicol residues in chicken muscles

Chloramphenicol (CAP), an antimicrobial drug that is widely used in animal feed, would have a negative effect on human health due to its low elimination rate and relatively high residue in animal food. It is important to develop a rapid and economic method to determine CAP in animal food to ensure that human health is not affected. A new fluorescence immunochromatography strip was developed and established for the detection of CAP residue in chicken muscles for the first time. A CAP-bovine serum albumin conjugate, monoclonal antibody and polyclonal antibody against CAP were applied to constitute a fluorescence immunochromatography strip. The fluorescence intensity was detected by a charge-coupled device scanner and transformed to a digital value. The CAP linearity working range was from 0.1 ng mL(-1) to 20 ng mL(-1) with a limit of detection of 0.1 ng mL(-1) within 10 min. The performance of the strip assay was compared with a commercial ELISA kit and the correlation coefficient was 0.99, which indicated that the new strip assay had a good quantification ability for CAP. The fluorescence immunochromatography strip was successfully applied to the detection of CAP residues in chicken samples. To our knowledge, it is the first report regarding the development of a fluorescence immunochromatography method for screening CAP in animal samples.

Determination of Chloramphenicol Residues in Aquatic Products Using Immunoaffinity Column Cleanup and High Performance Liquid Chromatography with Ultraviolet Detection

A method based on HPLC with UV detection was developed for the quantitative determination of chloramphenicol (CAP) residues in aquatic products. The samples were extracted with ethyl acetate-ammonium hydroxide (98 + 2, v/v), followed by a cleanup step using an immunoaffinity column. The analytes were determined by HPLC-UV. Optimal conditions for the extraction and cleanup procedures are described. The linear regression equation was y = 91.47x - 8.60 with R = 0.9998 (y = peak area and x = CAP concentration) and showed a good reproducibility. The LOQ was 0.25 microg/kg for determining CAP spiked in the aquatic products. The mean recoveries of CAP from fish and shrimp samples fortified at 0.25-1.0 microg/kg were 88.7-93.1 and 92.0-97.3%, respectively; the repeatability RSDs were less than 8.1%. It was concluded that the method is simple, highly sensitive, and low cost for quantitatively measuring CAP residues in aquatic products. Analyte identification was confirmed by HPLC/MSIMS analysis.

Rapid Determination of Chloramphenicol in Honey by Laser Diode Thermal Desorption Using Atmospheric Pressure Chemical Ionization-Tandem Mass Spectrometry

Abstract A high-throughput, rapid, and reliable method based on laser diode thermal desorption (LDTD) and atmospheric pressure chemical ionization coupled to tandem MS (APCI-MS/MS) was used to identify and quantify chloramphenicol (CAP) residues in honey. Sample pretreatment consisted of a liquid–liquid extraction of diluted honey in water with ethyl acetate containing stearic acid. After extraction, a 2 μL aliquot of the organic phase was deposited into a 96-well plate prior to detection by LDTD-APCI-MS/MS. The total analysis time was less than 6 s compared to several minutes for traditional chromatographic methods used for CAP detection, since no chromatographic separation was necessary. The extraction and analysis were made with honey samples of different color grading ranging from extra white to dark amber. The method showed good linearity (R2 = 0.99995) within a concentration range of 0.1 to 500 ng/g for CAP with D5-CAP as an internal standard. The RSD varied between 8 and 24% over the calibration range (n = 4). The method LOD for CAP in honey was 0.19 ng/g. This work demonstrates that LDTD-APCI-MS/MS could be used for fast and effective quantification of CAP in honey samples.

Occurrence of Chloramphenicol in Crops through Natural Production by Bacteria in Soil

Due to the unexpected findings of the banned antibiotic chloramphenicol in products of animal origin, feed, and straw, the hypothesis was studied that the drug is naturally present in soil, through production by soil bacteria, and subsequently can be transferred to crops. First, the stability of chloramphenicol in soil was studied. The fate of chloramphenicol highly depends on soil type and showed a half-life of approximately one day in nonsterile topsoil. It was found to be more stable in subsoil and sterile soils. Second, the production of chloramphenicol in soil was studied, and it was confirmed that Streptomyces venezuelae can produce chloramphenicol at appreciable amounts in nonsterile soil. Third, a transfer study was carried out using wheat and maize grown on three different soils that were weekly exposed to aqueous chloramphenicol solutions at different levels. Chloramphenicol was taken up by crops as determined by chiral liquid chromatography coupled to tandem mass spectrometric analysis, and the levels in crops were found to be bioavailability related. It was concluded that chloramphenicol residues can occur naturally in crops as a result of the production of chloramphenicol by soil bacteria in their natural environment and subsequent uptake by crops.

Occurrence of Chloramphenicol-Resistance Genes as Environmental Pollutants from Swine Feedlots

Chloramphenicol-resistance genes could be propagated to the surrounding environment via agricultural application of swine waste. This study investigated the potential risks of chloramphenicol-resistance genes from swine feedlots and their surrounding environment. We applied a culture-independent method to investigate levels of chloramphenicol-resistance genes in the wastewater from swine feedlots and the correspondingly impacted agricultural fields in Beijing. The cmlA, floR, fexA, cfr, and fexB genes were present in all samples, with the highest absolute concentrations of 1.50 × 10(6) copies/g in soil and 6.69 × 10(6) copies/mL in wastewater. The concentration of chloramphenicol residue was determined by ultra performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS), with the highest concentrations of 0.83 ng/g in soil and 11.5 ng/mL in wastewater. Significant correlations were found between chloramphenicol-resistance genes and chloramphenicol residues (r = 0.79, p = 0.0008) as well as between chloramphenicol-resistance genes in swine feedlots and corresponding agricultural soils (r = 0.84, p = 0.02). Consequently, swine feedlot wastewater could become a source of chloramphenicol-resistance genes, which could then lead to the spread of antibiotic resistance and eventually pose a risk to public health. To our knowledge, this is the first study to examine the occurrence of floR, fexA, cfr, and fexB genes in the environment using a culture-independent method.

An ultrasensitive chemiluminescent ELISA for determination of chloramphenicol in milk, milk powder, honey, eggs and chicken muscle

A competitive, direct, chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for chloramphenicol (CAP) residues in milk, milk powder, honey, eggs and chicken muscle has been developed. The method gave a detection limit of 0.7 ng L−1 and a linear range of 2.1–92.4 ng L−1, with the IC50 of 13.6 ng L−1 under optimal conditions, dramatically better than any previously reported ELISA method for CAP detection. Spiked at levels of 5–60 ng L−1 in different food samples, recoveries were in the range of 72.1–116.0%, with coefficient of variations of 4.2–20.2%. In a study of incurred residues, the chicken muscle samples diluted 5-, 10- and 20-fold, results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector and traditional ELISA. The developed CL-ELISA method is, therefore, suitable for rapid screening trace CAP residues in food samples.

Simultaneous Determination of Chloramphenicol, Melamine, Metronidazole, Ronidazole in Infant Milk Powder by LC-MS/MS

建立了婴幼儿配方乳粉中三聚氰胺、氯霉素、甲硝唑和洛硝达唑残留的高效液相色谱-串联四极杆质谱联用测定方法。样品经碱性乙腈提取,以MGⅢ-C18色谱柱（150 mm×2.1 mm,5!m）分离,流动相为60 mmol/L乙酸铵溶液（含0.4‰甲酸）和甲醇,梯度洗脱,流速为0.25 mL/min。采用多反应监测正离子或负离子模式,可以同时对婴幼儿配方乳粉中的三聚氰胺、氯霉素、甲硝唑和洛硝达唑进行快速定性和定量测定。在优化条件下,4种化合物检出限为0.3～50.0!g/kg,方法回收率为61.4%～96.2%,相对标准偏差（RSD）为1.8%～9.8%。采用本方法测定了多种市售婴幼儿配方奶粉。本方法操作简单、测定结果准确,可用于婴幼儿配方奶粉中三聚氰胺、氯霉素、甲硝唑和洛硝达唑残留的同时快速测定。

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.