Chloramphenicol Residues Research Articles

Determination of chloramphenicol residue in fish and shrimp tissues by gas chromatography with a microcell electron capture detector.

A gas chromatography method with microcell electron capture detection was developed for the determination of chloramphenicol residue in fish and shrimp muscle tissues. The tissue samples were extracted with ethyl acetate, defatted with hexane, and derivatized with Sylon BFT [N,O-bis (trimethylsilyl) trifluoroacetamide-trimethylchlorosilane (99 + 1)]. The limit of detection was 0.04 ng/g and the limit of quantitation 0.1 ng/g. Average recoveries were 70.8-90.8% for fish and 69.9-86.3% for shrimp, respectively. The method was validated for the determination of practical samples.

Quantitative determination of chloramphenicol in milk powders by isotope dilution liquid chromatography coupled to tandem mass spectrometry

A method is described for the determination of residues of the illegal antibiotic chloramphenicol (CAP) in milk powders. The analyte is quantified by liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-ESI-MS-MS) operating in negative ion multiple reaction monitoring mode (MRM) after a liquid-liquid extraction followed by a clean-up step on solid phase extraction (SPE) cartridge. Because of the presence of two chlorine atoms in the CAP molecule, four specific transition reactions of CAP were monitored by MS-MS in selecting m/z 321 --> 257, 321 --> 152 (35Cl2) and m/z 323 --> 257, 323 --> 152 (37Cl35Cl). Two calibration curves were constructed by plotting the area ratio of m/z 321 --> 152 versus 326 --> 157 and m/z 321 --> 257 versus 326 --> 262 against their corresponding amount ratio. Indeed, even if m/z 321 --> 152 was found to give a higher MS-MS response (calibration curve used by default), an interfering chemical substance was sometimes observed for some milk extracts and not for the transition m/z 321 --> 257. The quantitation method was validated according to the European Union (EU) criteria for the analysis of veterinary drug residues at 0.1, 0.2 and 0.5 microg/kg concentration levels using d5-CAP as internal standard. The decision limit (CCalpha) and detection capability (CCbeta) of CAP in milk were calculated for m/z 321 --> 152 at 0.02 microg/kg and 0.03 microg/kg, respectively, and for m/z 321 --> 257 at 0.02 microg/kg and 0.04 microg/kg, respectively. At the lowest fortification level (i.e. 0.1 microg/kg), repeatability and within-laboratory reproducibility were calculated for m/z 321 --> 257 both at 0.02 microg/kg and for m/z 321 --> 152 at 0.03 and 0.05 microg/kg, respectively. Moreover, the measurement of uncertainty of the analytical method was calculated at the same spiking levels and falls within the precision values of the within-laboratory reproducibility. This method can be applied to several types of milk powders (e.g. full cream, skim) and can serve as a monitoring tool to avoid that unacceptable levels of residues of CAP enter the food chain.

Quantitative determination of chloramphenicol in milk powders by isotope dilution liquid chromatography coupled to tandem mass spectrometry

A method is described for the determination of residues of the illegal antibiotic chloramphenicol (CAP) in milk powders. The analyte is quantified by liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC–ESI-MS–MS) operating in negative ion multiple reaction monitoring mode (MRM) after a liquid–liquid extraction followed by a clean-up step on solid phase extraction (SPE) cartridge. Because of the presence of two chlorine atoms in the CAP molecule, four specific transition reactions of CAP were monitored by MS–MS in selecting m/ z 321 → 257, 321 → 152 ( 35Cl 2) and m/ z 323 → 257, 323 → 152 ( 37Cl 35Cl). Two calibration curves were constructed by plotting the area ratio of m/ z 321 → 152 versus 326 → 157 and m/ z 321 → 257 versus 326 → 262 against their corresponding amount ratio. Indeed, even if m/ z 321 → 152 was found to give a higher MS–MS response (calibration curve used by default), an interfering chemical substance was sometimes observed for some milk extracts and not for the transition m/ z 321 → 257. The quantitation method was validated according to the European Union (EU) criteria for the analysis of veterinary drug residues at 0.1, 0.2 and 0.5 μg/kg concentration levels using d 5-CAP as internal standard. The decision limit (CCα) and detection capability (CCβ) of CAP in milk were calculated for m/ z 321 → 152 at 0.02 μg/kg and 0.03 μg/kg, respectively, and for m/ z 321 → 257 at 0.02 μg/kg and 0.04 μg/kg, respectively. At the lowest fortification level (i.e. 0.1 μg/kg), repeatability and within-laboratory reproducibility were calculated for m/ z 321 → 257 both at 0.02 μg/kg and for m/ z 321 → 152 at 0.03 and 0.05 μg/kg, respectively. Moreover, the measurement of uncertainty of the analytical method was calculated at the same spiking levels and falls within the precision values of the within-laboratory reproducibility. This method can be applied to several types of milk powders (e.g. full cream, skim) and can serve as a monitoring tool to avoid that unacceptable levels of residues of CAP enter the food chain.

Determination of chloramphenicol residues in rainbow trouts by gas chromatography–mass spectometry and liquid chromatography–tandem mass spectometry

A methodology for the identification and quantification of chloramphenicol (CAP) residues was developed and validated. The method is based on gas chromatography–mass spectrometry (GC–MS) for screening, in electron impact (EI) mode, after a solid phase extraction (SPE) step using C18 columns, and data acquired in selective ion monitoring (SIM) mode with the following ions: m/ z 225, m/z 208 and m/ z 242. Confirmatory method consists on liquid chromatography–tandem mass spectrometry (LC–MS/MS), in ionspray-negative mode, after the same extraction and clean-up procedure. The m/ z 321 was selected as a parent ion and m/ z 152 and m/ z 194 as daughter ions. The data were acquired in the negative multiple reaction monitoring (NMRM) mode, by monitoring the transitions 321 > 152 and 321 > 194. The method gave a decision limit (CCα) and a detection capability (CCβ) of 0.267 and 0.454 μg kg −1, respectively. The described methodology was applied on 40 samples of rainbow trout, collected in supermarkets of the Centre Region of Portugal, in order to evaluate the presence of CAP residues. Positive results were confirmed in 9 of the 15 suspected samples, which correspond to 22.5% of the whole samples collected.

Distribution of chloramphenicol residues in lactating cows following an external application

The aim of the present investigation was to demonstrate the presence of eventual chloramphenicol residues in blood plasma, urine and milk of dairy cows after topical application of a product, which contains 5% chloramphenicol in an alcohol solution and was commercially used as a spray registered for animals not intended for human consumption. The experiment was performed on two (A + B) Holstein–Frisian cows in the early lactation period. The indication for treatment with the described antibiotic solution was in the first case (cow A) a teat lesion and in the second case (cow B) skin lesions on the shoulders and the claws. Four consecutive dermal administrations inside first 41 h of the experiment were performed for both animals at the same time points. Milk, blood and urine samples were taken immediately before first treatment and in regular intervals inside applications and up to 12 days after the last application. All investigated matrices were analyzed using capillary GC-ECD after chloramphenicol derivatization. The outcome of our study confirmed our hypothesis that illegal and violative treatment (food-producing animals) of wounds with a spray containing chloramphenicol results in considerable residues of the active substance in milk, blood plasma and urine of the injured animals.

Development of a rapid and sensitive SPE-LC-ESI MS/MS method for the determination of chloramphenicol in seafood.

A method based on liquid chromatography-tandem mass spectrometry was developed and validated for the qualitative and quantitative detection of chloramphenicol (CAP) in seafood samples. The analysis of CAP residues in seafood is important because CAP can cause serious acute reactions in humans, including aplastic anemia and leukemia. The proposed methodology includes a cleanup solid-phase extraction procedure with high recovery efficiency (>90%). Chromatographic separation of CAP and the internal standard (IS) was carried out on a C(18) column, followed by mass spectrometric detection using electrospray ionization in the negative-ion mode. The precursor/product ion transitions 321-->257 (CAP) and 354-->290 (IS) were monitored. Statistical evaluation of this multiple reaction monitoring mass spectrometric procedure reveals good linearity, accuracy, and inter- and intraday precisions. The limit of detection was 0.1 ng/mL, and the limit of quantification for CAP in seafood samples is 0.02 microg/kg. Application in seafood samples allowed the detection of CAP in low parts per billion levels.

Analysis of Chloramphenicol Residues in Honey by Liquid Chromatography–Tandem Mass Spectrometry

Antibiotics are often used in bee-keeping to control European and American foulbrood. The broad spectrum antibiotic chloramphenicol (CAP) was used for curative purposes in veterinary medicine, but is now forbidden in numerous countries, although still used in south-east Asia. A liquid-chromatographic method with tandem mass spectrometric detection (LC–MS–MS) has been developed for analysis of sub-μg kg−1 residues of chloramphenicol in honey. Results from full validation of the procedure and analysis of 75 honey samples obtained commercially in Switzerland are presented. These show the method is satisfactory and useful for monitoring chloramphenicol residues in honey.

Multilaboratory Validation of a Method To Confirm Chloramphenicol in Shrimp and Crabmeat by Liquid Chromatography-Tandem Mass Spectrometry

An existing method for chloramphenicol (CAP) determination in shrimp using a gas chromatograph with electron capture detector was adapted for confirmation of CAP with a liquid chromatograph interfaced to a triple quadrupole mass spectrometer. CAP residues are extracted from tissue with ethyl acetate, isolated via liquid-liquid extraction, and concentrated by evaporation. Extracts are chromatographed by using a reversed-phased column and analyzed by electrospray negative mode tandem mass spectrometry. Four product ions (m/z 152, 176, 194, and 257) of precursor m/z 321 were monitored. Moving from gas chromatography to liquid chromatography-tandem mass spectrometry improved the sensitivity of the method greatly, enabling reliable confirmation of CAP residues at 0.3 microg/kg (ppb). The method meets confirmation criteria recommended by the U.S. Food and Drug Administration and 4-point identification criteria established by the European Union. With slight modifications to accommodate different equipment, the method was validated in 3 laboratories.

Inter-laboratory studies for the evaluation of ELISA kits for the detection of chloramphenicol residues in milk and muscle

The use of chloramphenicol in veterinary medicine was banned in the EU in 1994. As the Community Reference Laboratory for antibiotic residues in food of animal origin, one of our functions is to organize inter-laboratory studies. A first inter-laboratory study for the analysis of chloramphenicol (CAP) in milk by ELISA kits was organized in 2001 and a second one for the detection of CAP in pig muscle by ELISA kits in 2002. These studies were intended to allow participants to control their CAP ELISA methods when used routinely and also to compare the performances of various ELISA kits for the detection of chloramphenicol in milk (commercial or in-house kits). In 2001, 15 participants received ten randomly coded frozen milk samples (four blank samples and six spiked milk samples from 0.5 to 5.0 μg/l). In 2002, 20 participants received eight randomly coded frozen muscle samples (two blank samples and six incurred muscle samples from 2.1 to 6.5 μg/kg). They were asked to analyse each sample in triplicate with the ELISA kit of their choice. Different kits from different suppliers were used and compared in the two studies. The results of the two inter-laboratory studies on ELISA kits were satisfactory regarding qualitative results. The global rates of false compliant results of 2.2% for milk and 0.0% for pig muscle samples were lower than 5% whichever kit was used. The global rates of false non-compliant results (16.7% and 10% for milk and muscle respectively) were also satisfactory. The distribution of false non-compliant results depends on the kind of kit used and on the detection limit for milk as well as for muscle. Moreover the sample preparation was very important to avoid false non-compliant results. Finally, this study demonstrates that ELISA kits for chloramphenicol in milk and muscle globally show good repeatability and accuracy. So these kits could be considered as suitable tests for screening purposes.

Chloramphenicol Residues in Food Samples: Their Analysis and Stability During Storage

In this study, two methods for the determination of chloramphenicol (CAP) residues in chicken muscle and milk, are proposed. Chromatographic determination was carried out by reversed phase HPLC with a UV photodiode array detector. Confirmation of CAP was performed by comparison of UV spectra. Limit of detection was 0.4 ng g−1 in milk and 2 ng g−1 in muscle samples. The methods were validated and the mean recovery was 78.9% with CV of 9.36% for milk samples, and 86.8% with CV of 7.87% for chicken muscle. Furthermore, the effects of different storage temperatures on the stability of CAP were tested. The results showed a high stability in muscle during refrigeration and freezing storage conditions, but milk freezing produced significant losses of CAP.

Determination of chloramphenicol residues in fish meats by liquid chromatography–atmospheric pressure photoionization mass spectrometry

A liquid chromatography–atmospheric pressure photoionization (APPI) mass spectrometry method was developed for the determination of chloramphenicol (CAP) in fish meats (young yellowtail and flatfish). For the optimization of APPI, several APPI ion source parameters and mobile phases were investigated. CAP with APPI using the optimized parameters gave simple mass spectra and a strong signal corresponding to [M–H] − was observed. Further, APPI was compared with atmospheric pressure chemical ionization (APCI) and APPI gave similar results in terms of structural information and a better signal-to-noise ratio. The samples were extracted with acetonitrile and evaporated to dryness followed by a clean-up step using the liquid–liquid distribution between acetonitrile and n-hexane. The mean recoveries of chloramphenicol from a young yellowtail meat and a flatfish meat spiked at 0.1–2 ng/g were 89.3–102.5 and 87.4–94.8%, respectively. The limit of detection (signal-to-noise ratio=3) of the young yellowtail meat and the flatfish meat were 0.1 and 0.27 ng/g

Simultaneous Determination of Residues of Chloramphenicol, Thiamphenicol, Florfenicol, and Florfenicol Amine in Farmed Aquatic Species by Liquid Chromatography/Mass Spectrometry

A liquid chromatographic (LC)/mass spectrometric (MS) method was developed for determining the residues of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in a number of aquatic species. The phenicols are extracted with acetone, the extracts are partitioned with dichloromethane, the aqueous layer is removed, and the organic layer is evaporated to dryness. The residue is dissolved in dilute acid and defatted with hexane, and the aqueous layer is prepared for analysis by LC. The phenicols are determined by reversed-phase LC by using a Hypersil C18-BD column with a water-acetonitrile gradient and MS detection using selected-ion recording. Calibration curves were linear for all analytes between 0.015 and 0.425 ng injected. The relative standard deviations for measurements by the proposed method were < 10% for all of the analytes studied, with recoveries ranging from 71% for florfenicol amine to 107% for florfenicol in salmon tissue spiked at the 2 ng/g level. Detection limits of 0.1 ng/g for florfenicol and chloramphenicol, 0.3 ng/g for thiamphenicol, and 1.0 ng/g for florfenicol amine are easily obtainable. The operational errors, interferences, and recoveries for spiked samples compare favorably with those obtained by established LC methodology. The proposed method is simple, rapid, and specific for monitoring residues of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in a number of aquatic species.

Analysis of chloramphenicol in honeys of different geographical origin by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

A sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to detect trace amounts of the antibiotic chloramphenicol (CAP) in honey. The methodology entailed a solid-phase extraction of aqueous honey solutions followed by liquid-liquid partitioning, filtration and direct injection onto the LC-MS/MS system. Honey extracts were spiked with an isotopically labelled internal standard (d5-CAP) to compensate for analyte loss and potential ion suppression during the MS stage. Detection of the analyte was achieved by negative ionization electrospray in the selected reaction monitoring (SAM) mode. For confirmation, four characteristic mass transitions were monitored each for the analyte and the surrogate standard. The method was validated according to the latest European Union criteria for the analyses of veterinary drug residues in food. At all three fortification levels studied (0.1, 0.2, 0.5 μg kg-1) the method was accurate to within 15%. The repeatability and within-laboratory reproducibilities were <12 and 18%, respectively. The decision limit (CCα) and detection capability (CCβ) were both <0.1 μg kg-1. The procedure provides a sensitive and reliable method for the determination of residues of chloramphenicol in honey. Numerous raw honeys of various geographical origins were analysed, showing extensive contamination particularly those of Chinese origin.

Determination of chloramphenicol residues in shrimps by liquid chromatography–mass spectrometry

A liquid chromatographic method with mass spectrometric detection and identification (LC–MS) is presented for the determination of chloramphenicol (CAP) in shrimp tissues. Homogenized shrimp samples were extracted with phosphate buffer (pH 7.0). Clean-up was carried out on a C 18 SPE cartridge. Chloramphenicol was determined by LC–MS-ESI in negative mode. The column used was a Symmetry Shield with a mixture of acetonitrile–water (25:75) as mobile phase. Shrimp samples were fortified at CAP levels between 0.2 and 50 ng g −1 with 5D-CAP as internal standard. At these levels, accuracies lay between 101 and 110% and between-day reproducibilities were lower than 7.1%, expressed as the variation coefficient of the mean. Limit of decision (CCα) was 0.02 ng g −1. Limit of quantitation (LOQ) was 0.2 ng g −1.

Matrix Solid-Phase Dispersion Extraction and Gas Chromatographic Determination of Chloramphenicol in Muscle Tissue

A method based on matrix solid-phase dispersion (MSPD) was developed for the gas chromatographic (GC) determination of chloramphenicol (CAP) residues in animal muscle tissue. Muscle tissue was blended with octadecylsilyl-derivatized silica (C(18)). A column made from the C(18)/muscle tissue matrix was washed with n-hexane and acetonitrile/water (5 + 95), after which CAP was eluted with acetonitrile/water (50 + 50) and partitioned into ethyl acetate. The final extract was evaporated, and a trimethylsilyl derivative of CAP was prepared with Sylon HTP and detected by GC with an electron capture detector (ECD) and a mass spectrometer. For quantitation, the internal standard used was the meta isomer of CAP (m-CAP) for GC-ECD. Muscle tissue samples were fortified at three concentration levels. At 5, 10, and 15 microg/kg levels the respective mean recoveries were 93, 96, and 98%, and the repeatabilities were 13, 11, and 3%. The detection and quantitation limits with ECD were 1.6 and 4.0 microg/kg, respectively. No statistically significant difference was observed in the efficiency of CAP extraction from muscle tissue of various animals (bovine, porcine, and poultry) by the MSPD technique.

Evaluation of sample preparation for control of chloramphenicol residues in porcine tissues by enzyme-linked immunosorbent assay and liquid chromatography

The effects of sample preparation on the determination of chloramphenicol (CAP) residues by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography (LC) were investigated. First, the biological matrices were treated with extraction solutions, which were mixtures of acetate buffer and acetonitrile. The isolation of CAP from previously spiked samples was performed by homogenization or ultrasonication. For clean-up, the suitability of solid phase extraction columns (in reverse phase (RP) or mixed mode) was compared. The level of CAP in tested samples was determined by a commercial ELISA test (R-Biopharm) and LC with UV detection at 270 nm, after separation on a RP column. It was found that the best isolation from interfering, endogenous compounds was obtained after homogenization of sample with a mixture of acetate buffer (pH 5.0) and acetonitrile (2:8, v/v). The clean-up of the sample extracts in a mixed mode rather than reverse phase was more efficient. The recovery of CAP from spiked pig muscle was 82%, and from kidney, 85%. The same analytical protocol is suitable for CAP screening in animal tissues by ELISA as well as by LC.

Screening and confirmation of chloramphenicol in shrimp tissue using ELISA in combination with GC–MS 2 and LC–MS 2

According to the European Commission Decision 2001/699/EC and 2001/705/EC certain fishery and aquaculture products, imported from China, Vietnam or Indonesia and intended for human consumption, must be subjected to a test in order to ensure the absence of chloramphenicol residues. For that reason an analytical method has been developed and validated based on ELISA for screening and gas chromatography–tandem mass spectrometry (GC–MS 2) or liquid chromatography–tandem mass spectrometry (LC–MS 2) for confirmation. The chloramphenicol ELISA was carried out directly on an aqueous extract of the shrimps or after an extraction with ethyl acetate. Confirmation of suspect samples was performed after extraction with ethyl acetate and defatting with n-hexane. The clean-up was based on solid phase extraction using C 18 cartridges or reversed phase HPLC. After derivatisation with N-methyl- N-(trimethylsilyl)trifluoroacetamide (MSTFA), the final extracts were analysed by GC–MS 2 in the negative ion chemical ionisation mode. Confirmation of chloramphenicol was also possible with LC–MS 2 after the same clean-up. Both selective techniques made it possible to detect chloramphenicol residues at the 0.1 μg kg −1 level starting from 20 g of matrix for enzyme-linked immunosorbent assay (ELISA) with organic solvent extraction, or from 5 g of matrix for ELISA with aqueous extraction.

Stability of Sulfonamides, Nitrofurans, and Chloramphenicol Residues in Preserved Raw Milk Samples Measured by Liquid Chromatography

A stability study was made of 10 antimicrobials: 6 sulfonamides, 3 nitrofurans, and chloramphenicol residues in raw milk samples preserved with 0.1 % potassium dichromate (K2Cr2O7) and 0.05% mercuric bichloride (HgCl2) during cold storage for 7 days. Preserved milk samples fortified with 50 ppb of each antimicrobial were analyzed by liquid chromatography (modified AOAC Method 993.32). Drugs were extracted with chloroform-acetone after solvent evaporation residues were dissolved with aqueous sodium acetate buffer solution (0.02M, pH 4.8), and fat was removed with hexane. Sulfonamides and chloramphenicol were detected at 275 nm (UV) by using a gradient system of sodium acetate buffer solution-acetonitrile starting at 95 + 5 (v/v) and finishing at 80 + 20 (v/v). Nitrofurans were detected at 375 nm (UV) isocratically with sodium acetate buffer solution-acetonitrile (80 + 20, v/v). Residues stability was measured through recovery data. Sulfamethoxazole, sulfachloropyridazine, nitrofurazone, furazolidone, and furaltadone residues remained stable in the presence of either preservative for 7 days. Sulfamethazine and chloramphenicol were not affected by K2Cr2O7, but had significant losses (p <0.05) when HgCl2 was used: 26.2 and 13.4%, respectively. Average recoveries of sulfamonomethoxine, sulfamerazine, and sulfathiazole significantly decreased by Day 7, with losses of 17.1, 17.2, and 23.2% for K2Cr2O7, and 23.3, 20.7, and 48.0% for HgCl2, respectively. During 5 days of cold storage all antimicrobials tested, except sulfathiazole, remained stable in milk samples preserved with 0.1 % K2Cr2O7 or 0.05% HgCl2.

Analysis and monitoring of chloramphenicol residues in food of animal origin in Slovenia from 1991 to 2000

To prevent the illegal use of chloramphenicol (CAP), regulatory control of its residues in food of animal origin is essential. In Slovenia, the monitoring of CAP residues for statutory purpose started in 1991. The results of a 10-year period are presented. CAP residues were determined by capillary gas chromatography (GC) with electron capture detection (ECD) using meta-CAP as an internal standard (ISTD). Before chromatographic determination, analytes were derivatized by silylation. Overall, CAP recovery, adjusted for ISTD, was for bovine muscle tissue and raw cow's milk (in the region of 2–10 µg kg-1) 89 and 102%, respectively, and for whole eggs, 87% (in the region of 1–10 µg kg-1). The use of meta-CAP improved significantly the precision of the method. The detection limit for CAP was 1 µg kg-1, which was sufficiently sensitive for routine use. A total of 1308 random samples of Slovenian origin were analysed from 1991 to 2000, covering all parts of the country. CAP was found only in one milk sample in 1997 at a concentration of 4.6 µg kg-1.

DETERMINATION OF CHLORAMPHENICOL RESIDUES IN GILTHEAD SEABREAM (SPARUS AURATA L.) TISSUES BY HPLC-PDA

A high performance liquid chromatographic method for the determination of chloramphenicol residues in muscle tissue of the cultured fish gilthead seabream (Sparus aurata L.) was developed. Chloramphenicol is extracted with ethyl acetate and after centrifugation and solvent evaporation the oily extract is partitioned between 3% sodium chloride solution and n-pentane, and chloramphenicol is extracted back into ethyl acetate. After evaporation to near dryness, the residue is dissolved in n-hexane and is cleaned up on a Silica gel SPE mini column. Chloramphenicol was analyzed on a ZORBAX SB - C18 column at a temperature of 50°C, with the mobile phase being methanol:water 30 + 70 v/v delivered isocratically. Detection was performed using a Photo Diode Array detector monitored at λmax ∼ 278 nm. The mean recovery (R%) achieved was 88.62 ± 9.65% for a range of 10, 25, 50, 100, 200 μg/kg blank fortified samples (n = 4). The limit of detection (LOD) was 1.87 ng corresponding to 5 μg/kg chloramphenicol in muscle and the limit of quantification (LOQ) was 10 μg/kg.