Determination of Chloramphenicol using the ACQUITY UPLC and the Quattro Premier XE in ES Negative Ion Mode MS/MS

This application note describes an extraction method from chicken and a LC-MS/MS method for the quantification and confirmation of CAP in chicken.

Chloramphenicol (CAP), shown in Figure 1, is an inexpensive, broad spectrum antibiotic, which has very effective antibacterial properties. It was isolated from the soil bacterium Streptomyces venezuelae in 1947 but, unlike many antibiotics derived from bacteria and fungi, it is readily synthesised and inexpensive to produce. Due to CAP’s low cost and high availability, the antibiotic has been used to treat food-producing animals, which is of some concern since it is reportedly a cause of the potentially fatal blood condition idiosyncratic aplastic anemia. Additionally, hypersensitivity to the drug affects around one in thirty thousand people, regardless of the dosage.¹ Due to the various adverse effects associated with the use of CAP in the treatment of infections, its use in humans is restricted to cases where safer antibiotics have proved ineffective and the benefits of the drug outweigh the risks associated with toxicity.

In the EU, no Maximum Residue Limit (MRL) has been set for CAP in animal derived food and it is listed in Annex IV of EU Council regulation 2377/90/EEC. It has been banned from use in food-producing animals since 1994. EU Decision 2003/181/EC, sets a Minimum Required Performance Limit (MRPL) of 0.3 ppb for CAP.

There is a requirement to achieve the lowest possible levels of quantification and confirmation. Since its ban in 1994, CAP has been reported to be found in food like shrimp, honey, and chicken.^2,3

This paper describes an extraction from chicken and a LC-MS/MS method for the quantification and confirmation of CAP in chicken.

Extraction of CAP from Chicken

• Weigh minced tissue (3 ± 0.1 g) into 30 mL screw capped glass boiling tube.
• Add 200 μL of ISTD, D₅-CAP to the tubes and let stand for 10 min.
• Add 4 mL PBS and homogenise for 1 min.
• Add 1 mL of sodium chloride solution and mix for 15 seconds.
• Add 4 mL acetonitrile, vortex and sonicate for 10 min. Centrifuge at 2,200 rpm for 20 min.
• Transfer supernatant to a clean 35 mL screw capped tube and add 10 mL water.
• Add 10 mL hexane and shake gently for 30 seconds.
• Centrifuge the tubes at 2,200 rpm for 20 min. and discard the upper hexane layer. Add 8 mL of ethyl acetate.
• Mix by inversion of tube for 1 min. and centrifuge tubes at 2,200 rpm for 20 min.
• Transfer organic solvent layer (upper layer) to 9 mL clean tubes and evaporate to dryness at 65 °C under nitrogen.
• Reconstitute residue in 5 mL water, then carry out SPE using a C₁₈ cartridge.
• Evaporate the eluant at 65 °C under a stream of nitrogen and reconstitute sample residue in 100 μL of 50% methanol and transfer to micro vials for analysis.
• Reconstitute standards in 200 μL of 50% methanol prior to LC analysis.

MS Conditions

System: Waters Micromass Quattro Premier XE in electrospray mode with negative polarity

MRM transitions along with the cone voltages and collision energies are listed in Table 1. For CAP, two transitions were chosen, one for quantification (bold type) and another as confirmation (regular type), in accordance with European guidelines.⁴ The transitions were optimized with argon as the collision gas. D₅-CAP was used as the internal standard for the method. Since the calibration standards were prepared in mobile phase solvents, a comparison of peak areas between calibration and recovery experiments allowed an estimation of matrix suppression effects to be made.

Software

Data were acquired with Waters MassLynx Software and processed with Waters TargetLynx Application Manager.

The absolute sensitivity for the ACQUITY UPLC/Quattro Premier XE is shown in Figure 2, with a 0.01 pg/μL standard giving a signal-to-noise ratio of approximately 45:1.

The three MRM transitions used for CAP and D₅-CAP are shown in Figure 3 at a concentration of 0.3 μg/kg. For the data acquired using the ACQUITY UPLC/Quattro Premier XE, calibration was performed using solvent standards and matrix-spiked chicken samples were analyzed.

Figures 4 and 5 illustrate the typical linearity and repeatability that is obtained by using the extraction method discussed with the ACQUITY UPLC/Quattro Premier XE.

A rapid method for the determination and quantification of chloramphenicol in chicken has been described. The Waters ACQUITY UPLC/Quattro Premier XE provides a sensitive, selective, and reproducible analysis method. The limits of determination achieved are below that required by legislation for any country in the European Union.

1. Roybal, J, in: Turnipseed, S. and Long, A (Eds.), Analytical Procedures for Drug Residues in Food of Animal Origin, Science Technology System, West Sacramento, CA (1998) 227–260.
2. The New York Times. World Briefing: Asia. P-6A, March 20, 2002.
3. Food Production Daily Web Site Article, Dutch Destruction of Contaminated Meat and Seafood Angers China: www.foodproductiondaily.com/news/ng.asp?id=27078
4. Commission Decision (EC) 96/23, Official Journal of the European Communities, No. L221 (2002) 8.