Confirmation Of Chloramphenicol Research Articles

QuEChERS and HPLC-MS/MS Combination for the Determination of Chloramphenicol in Twenty Two Different Matrices.

A simple method for the determination of chloramphenicol in 22 matrices was prepared based on the QuEChERS and HPLC-MS/MS combination. Following a hydrolysis step, the homogenized samples were extracted and partitioned after adding sodium chloride with acetonitrile. Chloramphenicol was analysed by HPLC-MS/MS in negative electrospray mode by monitoring the daughter ions m/z: 321→194 and 321→152. The limit of decision (CCα) was calculated at the range of 0.10 μg kg−1 to 0.15 μg kg−1 and detection capability (CCβ) from 0.12 μg kg−1 to 0.18 μg kg−1. Validation results showed that this method is suitable for the determination and confirmation of chloramphenicol in various matrices.

Quantitation and Confirmation of Chloramphenicol, Florfenicol, and Nitrofuran Metabolites in Honey Using LC-MS/MS.

This paper describes a rapid and robust method utilizing a single liquid-liquid extraction for the quantitation and confirmation of chloramphenicol, florfenicol, and nitrofuran metabolites in honey. This methodology combines two previous extraction methods into a single extraction procedure and utilizes matrix-matched calibration standards and stable isotopically labeled standards to improve quantitation. The combined extraction procedure reduces the average extraction time by >50% when compared with previously used procedures. The drug residues were determined using two separate LC-tandem MS conditions. Validation of all the analytes was performed, with average quantitation ranging from 92 to 105% for all analytes and the RSDs for all analytes being ≤12%.

Analytical Strategy for Determination of Chloramphenicol in Different Biological Matrices by Liquid Chromatography - Mass Spectrometry.

IntroductionThe main problem in determination of chloramphenicol in food of animal origin is a large number of matrices. The main target of this study was to create a method for determination and confirmation of chloramphenicol in products and food of animal origin.Material and MethodsEach 5 g matrix sample was mixed with 5 mL of water and 10 mL of acetonitrile/ethyl acetate, homogenised, and centrifuged. The organic layer was evaporated and redissolved in 6 mL of 4% NaCl. The extract was cleaned up by SPE technique. Chloramphenicol was analysed by LC-MS/MS in electrospray mode.ResultsThe procedure was validated according to the Commission Decision No. 2002/657/EC. The apparent recoveries were in the range of 92.1% to 107.1% with a repeatability less than 11.0% (4.4%–11.0%) and within-laboratory reproducibility below 13.6% (4.7%–13.6%).ConclusionThe method was successfully validated and proved to be efficient, precise, and useful for quantification of chloramphenicol in more than 20 different matrices.

Quantitation of Chloramphenicol and Nitrofuran Metabolites in Aquaculture Products Using Microwave-Assisted Derivatization, Automated SPE, and LC-MS/MS.

This paper describes a rapid and robust method utilizing microwave-assisted derivatization, automated SPE, and LC-MS/MS for the quantitation and confirmation of chloramphenicol (CAP) and nitrofuran metabolites in various aquaculture matrixes. The use of equipment presented in this work allowed extractions to be completed on average within 6 h, with quantitation accuracy ranging from 89 to 107% and RSD≤8.3%. The demonstrated detection limits for all the nitrofuran metabolites of interest in three different matrixes were ≤0.06 ng/g, with a quantitation limit of ≤0.2 ng/g. Additionally, the method exhibited a CAP detection limit for all matrixes≤0.01 ng/g and an LOQ of ≤0.03 ng/g.

Transfer of chloramphenicol from milk to commercial dairy products – Experimental proof

The purpose of the study was to investigate the chloramphenicol (CAP) residue in dairy products after experimental CAP transferring from milk to butter, sour cream, white cheese and whey. In order to determine the CAP residue in dairy products, the new approach for the extraction process was developed. The original, simple and fast analytical method is based on QuEChERS and LC-MS/MS. The homogenized sample was extracted and partitioned after the adding of sodium chloride with acetonitrile. The experiment was conducted to check if CAP is transferred from milk to dairy products, and also to check the extraction of CAP from the different dairy matrices. Average recovery ranged from 97.8 to 102.8% and within-laboratory reproducibility was lower than 8.7%. The suggested method is sensitive, the calculated limit of decision (CCα) was from 0.06 to 0.10 μg kg−1 and detection capability (CCβ) from 0.08 to 0.15 μg kg−1. The results of the experiment (butter 4.86 μg kg−1, sour cream 3.5 μg kg−1, white cheese 2.36 μg kg−1 and whey 0.14 μg kg−1) and validation demonstrated that this method is suitable for determination and confirmation of CAP in a variety of dairy product matrices.

Development of LC-MS/MS methodology for the detection/determination and confirmation of chloramphenicol, chloramphenicol 3-O-β-d-glucuronide, florfenicol, florfenicol amine and thiamphenicol residues in bovine, equine and porcine liver.

A method for the detection and confirmation of organic solvent extractable residues of the neutral, acidic, and basic analytes of the amphenicol class veterinary drugs and selected metabolites was developed and validated. Using a modified QuEChERS extraction with SPE cleanup and LC-MS/MS analysis, limits of detection and confirmation for the different analytes in bovine, equine, and porcine liver ranged from 0.1ng/g for chloramphenicol to 1ng/g for florfenicol amine. Tissue homogenization with an ammonium formate/EDTA solution and subsequent analyte partitioning against 7:3 acetonitrile:isopropanol solution and mixed-mode strong-cation exchange solid-phase extraction cartridge cleanup allowed for the extraction of all compounds from tissues with mean recoveries ranging from 50% (chloramphenicol 3-O-β-d-glucuronide) to 90% (thiamphenicol). Matrix effects ranged from greater than 85% suppression for florfenicol amine to 70% matrix enhancement for chloramphenicol 3-O-β-d-glucuronide. Quantitation and confirmation were accomplished using commercially available penta-deuterated chloramphenicol as internal standard and multiple reaction monitoring (MRM) of two or three transitions per target analyte. Method accuracy was greater than 15% for all compounds except the glucuronide metabolite. Intra-lab method repeatability estimates ranged from 73% RSD for chloramphenicol 3-O-β-d-glucuronide to 14% RSD for chloramphenicol. Only chloramphenicol 3-O-β-d-glucuronide and florfenicol amine at the low end of their calibration ranges (0.25 and 1ng/g, respectively) did not meet AOAC recommended HorRatr guidelines for intra-lab repeatabilities. Preliminary tests show that the method's extraction protocol can be used to recover analytes of the β-agonists, corticosteroids, fluoroquinolones, sulfonamides, and tetracycline drug classes from the same matrices. Requirements for use in national chemical monitoring programs as a detection/confirmatory (florfenicol amine and chloramphenicol 3-O-β-d-glucuronide) and determinative/confirmatory (chloramphenicol, florfenicol, thiamphenicol) analytical methodology are met.

Determination and confirmation of chloramphenicol in honey, fish and prawns by liquid chromatography–tandem mass spectrometry with minimum sample preparation: validation according to 2002/657/EC Directive

A reliable, simple and sensitive liquid chromatography–electrospray ionisation-tandem mass spectrometry (LC–ESI-MS/MS) confirmation method has been developed for chloramphenicol (CAP) determination in honey, fish and prawns. For honey, samples were extracted with ethyl acetate, an aliquot was evaporated to dryness and re-dissolved in mobile phase. For fish and prawns, tissues were extracted with acetonitrile and chloroform. The organic layer was evaporated to dryness and the residue was re-constituted with water: acetonitrile (90:10). LC separation was achieved on a C18 column with gradient elution using a mobile phase of acetonitrile and water. Analysis was carried out on a triple–quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via electrospray interface operated in negative ionisation mode, with deuterated chloramphenicol-d5 (d5-CAP) as internal standard. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Four identification points were obtained for CAP with one precursor ion and two product ions. The limit of detection (LOD) was 0.02 µg kg−1. Linear calibration curves were obtained over concentration ranges of 0.1–1.0 µg kg−1 in tissues. Mean recoveries ranged from 85.5% to 115.6%, with the corresponding intra- and inter-day variation ranging from 1.0% to 22.5%, depending on matrix type and level of concentration. The decision limit (CCα) and detection capability (CCβ) of the method were obtained for all matrices: 0.04 and 0.06 µg kg−1, respectively, for prawns and fish and 0.05 and 0.09 µg kg−1 for honey.

Validation of a rapid and sensitive routine method for determination of chloramphenicol in honey by LC–MS/MS

Chloramphenicol (CAP) is a broad spectrum antibiotic used in the treatment of human and animal diseases. However, CAP can exhibit toxic effects in certain susceptible individuals, causing bone marrow depression, including fatal aplastic anemia. As this condition is dose-independent, CAP has been banned for use in food-producing animals, including honeybees. In this study, a quick, simple and low-cost routine analytical method was developed for the screening and confirmation of chloramphenicol in honey by LC–MS/MS. Sample clean-up takes only two steps without SPE procedure and with recoveries >97%. Honey samples were selected from several producers in Brazil and diluted in a small amount of water. After fortification and addition of d s-chloramphenicol as internal standard, the samples were extracted with ethyl acetate. Complete validation of the method was performed on the basis of EU decision 2002/657. Within-laboratory CV reproducibility at the lowest concentration was <10%. An evaluation of two different methods to calculate the decision limit and detection capability gave 0.08 µg kg−1 for CCα and 0.12 µg kg−1 for CCβ.

Simultaneous determination and confirmation of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscle by liquid chromatography–tandem mass spectrometry

A reliable and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) confirmation method has been developed for the simultaneous determination of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in chicken muscle. Samples were extracted with basic ethyl acetate, defatted with hexane, and cleaned up on Oasis MCX cartridges. LC separation was achieved on a XTerra C 18 column with gradient elution using a mobile phase composed of acetonitrile and water at a flow rate of 0.20 mL/min. The analysis was carried out on a triple–quadrupole tandem mass spectrometer in the multiple reaction monitoring (MRM) mode via electrospray interface operated in the positive and negative ionization modes, with deuterated chloramphenicol-d5 (d 5-CAP) as the internal standard. The method validation was performed according to the criteria of Commission Decision 2002/657/EC. Four identification points were obtained for each analyte with one precursor ion and two product ions. Limits of detection (LODs) were 0.1 μg/kg for CAP, 0.2 μg/kg for FF and 1 μg/kg for TAP and FFA in chicken muscle. Linear calibration curves were obtained over concentration ranges of 0.3–20 μg/kg for CAP, 0.5–20 μg/kg for FF and 3–100 μg/kg for TAP and FFA in tissues. Mean recoveries of the 4 analytes ranged from 95.1% to 107.3%, with the corresponding intra- and inter-day variation (relative standard deviation, R.S.D.) less than 10.9% and 10.6%, respectively. The decision limit (CCα) and detection capability (CCβ) of the method were also reported.

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%.

Food safety evaluation: Detection and confirmation of chloramphenicol in milk by high performance liquid chromatography-tandem mass spectrometry

A simple and rapid procedure for extraction of chloramphenicol (CAP) in milk and analysis by high-performance liquid chromatography coupled with quadrupole mass spectrometry in tandem was developed. The method consisted of one step of liquid–liquid extraction using ethyl acetate and acidified water (10 mmol L −1 formic acid) and HPLC-MS/MS detection. CAP-D5 was used as internal standard. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear, with typical r 2 values higher than 0.98. Absolute recovery of CAP from milk proved to be more than 95%, however CAP-D5 absolute recovery was 75%. The method was accurate and reproducible, being successfully applied to the monitoring of CAP in milk samples obtained from the Brazilian market. Decision limit (CCα) was 0.05 ng mL −1 and detection capability (CCβ) was 0.09 ng mL −1.

Liquid Chromatography/Tandem Mass Spectrometry Analysis of Chloramphenicol in Cooked Crab Meat

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method is described for the extraction, cleanup, determination, and confirmation of chloramphenicol (CAP) in cooked crab meat. The method involves pulverization of cooked crab meat with dry ice; extraction of the CAP into ethyl acetate (EtOAc); evaporation (by N2) of the EtOAc; addition of methanol, aqueous NaCl, and heptane; extraction of the lipids into the heptane, followed by extraction of the aqueous phase with EtOAc; evaporation (by N2) of the EtOAc; dissolution into methanol-water; filtration; and separation/detection/confirmation using LC/MS/MS. Crab meat was fortified at 0.25, 0.50, and 1.0 ng/g (ppb) chloramphenicol. Average absolute recoveries were 67, 84, and 86%, respectively, with relative standard deviation values all less than 1%. Four daughter ions (m/z 152, 176, 194, and 257) were monitored off the m/z 321 precursor ion. Determination was based on a standard curve using the peak areas of the m/z 152 daughter ion (the base peak) for standard solutions equivalent to 0.10, 0.20, 0.50, and 1.0 ppb in tissue (made with control crab extract). A set of 6 matrix controls (unfortified crab meat) was also analyzed, in which no chloramphenicol was detected. For identification purposes, the ion ratios (of each daughter ion versus the base daughter ion) of the fortified crab versus those of the chloramphenicol standards agreed within 10% (relative) at fortified chloramphenicol concentrations of 0.25-1.0 ppb.

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%.

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 in animal tissues and urine: Liquid chromatography–tandem mass spectrometry versus gas chromatography–mass spectrometry

A very sensitive method was developed for detection and confirmation of chloramphenicol (CAP) in equine, porcine and bovine muscle and urine. The method included ethyl acetate extraction of CAP followed by a two-step C18 solid-phase clean up; recovery was >80%. Extracted CAP was determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in negative atmospheric pressure chemical ionization mode. LC–MS/MS gave superior sensitivity and selectivity compared to that shown by gas chromatography–mass spectrometry (GC–MS) in electron impact mode. Even in a “dirty” matrix, such as urine, the estimated CAP detection limit for LC–MS/MS detection was 0.02 μg kg −1 while the corresponding value for GC–MS was only 2 μg kg −1.

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.

High Performance Liquid Chromatography with UV Detection and Scanning UV Confirmation of Chloramphenicol in Fatty Liver

Abstract A liquid chromatographic (HPLC) method has been developed to detect chloramphenicol (CAP) in fatty liver. After addition of water, samples were defatted with hexane partition and CAP was extracted by ethyl acetate. A solid-phase extraction protocol was used to recover CAP from the fatty liver extract. CAP was analyzed using a mobile phase of acetonitrile-0.005 M phosphate buffer, pH 7.9 (19:81), an ultraviolet detection wavelength of 278 nm and confirmation by scanning. The column used was a Nova-Pak C18 (150 x 3.9 mm). The limit of detection was < 1 ng/g in fatty liver and the mean recovery of CAP was found to be 71% over the concentration range 5–20 ng/g. The analyte identity can be confirmed by the use of scanning detection and by LC/MS method for concentrations less than 10 ng/g.

International Validation Study for the Determination of Chloramphenicol in Bovine Muscle

A gas chromatographic (GC) procedure for the quantitation and GC/negative ion chemical ionization mass spectrometric (NICIMS) confirmation of chloramphenicol in calf muscle tissue was the subject of a validation study. Five analysts representing 5 laboratories in 4 countries participated in the quantitative method and analyzed 7 randomly numbered blind triplicates at 4 fortified and 3 incurred tissue concentrations on 3 separate days. The chloramphenicol concentrations ranged from 0 to 2.5 ppb. All data were reported to 3 significant figures. The coefficients of variation were 9.5-28.7% for repeatability and 14.6-38% over the study range for reproducibility. NICIMS data representing 3 laboratories in 3 countries successfully confirmed chloramphenicol in samples at 0.6 ppb or greater with no false positives in blank tissues.