Analysis of Residual Antimicrobials in Meat Using HPLC

Gesa J. Schad¹ and Natsuki Iwata²,¹Shimadzu Europa GmbH, Duisburg, Germany, ²Shimadzu Corporation, Kyoto, Japan

Analysis of residual antimicrobials in animal products is crucially important to ensure food safety. This article presents a simple, fast, and highly sensitive high performance liquid chromatography (HPLC) assay of regulated antimicrobials, as well as sample preparation and purification methods. Twenty-four different analytes of interest were investigated in beef, pork, and chicken meat samples. 

The presence of antimicrobial residues in animal products resulting from treatment of livestock with pharmacological drugs can pose a severe risk to consumer health. It can lead to allergic reactions, toxicological effects, or, most importantly, to resistant pathogenic bacteria (1). To ensure food safety, contamination with antimicrobial residues must be monitored strictly, and so sensitive methods are essential to assay these compounds in complex matrices. As long as the maximum residue limits (MRL) are complied with, residues pose only a low risk to consumer health (2).

MRLs for drug residues in different animal products were set by European legislation (3). Food products of animal origin must be tested for contamination to guarantee that these MRLs are not exceeded. Antimicrobials do not share a common chemical structure, so detection of various groups of drugs using one method can be challenging. In this application, a simple high performance liquid chromatography (HPLC) screening system equipped with photodiode array (PDA) as well as fluorescence (RF) detection was used to determine the presence of 24 antimicrobials from different classes (quinolones, sulfonamides, and antifolates) in meat (beef, pork, and chicken muscle) with a limit of detection below the EU MRL’s.

Method 

Analytical Conditions: System: LC-2040C 3D, RF-20Axs; column: 150 × 4.6 mm, 3-μm Shim-pack FC-ODS; mobile phase: A) 20 mM (sodium) phosphate buffer containing 0.1 M sodium perchlorate, B) acetonitrile–methanol (90:10 v/v) (quinolones), C) acetonitrile–methanol (80:20 v/v) (sulfonamides, antifolates); time program: gradient elution; flow rate: 1.0 mL/min; column temperature: 40 ˚C (quinolones), 50 ˚C (sulfonamides, antifolates); injection volume: 5 μL (quinolones), 20 µL (sulfonamides, antifolates); PDA detection: 280 nm (quinolones), 240, 270, 280, 285, 350, and 380 nm (sulfonamides, antifolates); RF detection (quinolones): Ex at 290 nm, Em at 495 nm, Ex at 325 nm, Em at 365 nm

Sample Preparation: Sample pretreatment for sulfonamides and antifolates was performed using the QuEChERS method for extraction and fat removal followed by evaporation and reconstitution. Samples for determination of quinolone substances were prepared using a protocol based on the “Simultaneous Analysis Method I for Veterinary Drugs by HPLC” (4,5). After acetonitrile extraction and fat removal by acetonitrile–hexane partitioning, a sample solution was prepared by evaporation and reconstitution. Figure 1 shows the different sample pretreatment protocols.

Antimicrobial Screening: An antimicrobial screening system was used to determine if levels of antimicrobials, subject to regulation in various countries, were above a maximum residue level of 10 µg/kg. The system uses an i-Series integrated HPLC instrument (Shimadzu) and RF-20Axs high-sensitivity fluorescence detector (Shimadzu). 

Results

Analysis of Sulfonamides and Antifolates in Meat: Samples of chicken and beef were prepared as shown in Figure 1. Analytical conditions for the analysis of sulfonamides and antifolates are described above. Figure 2 illustrates chromatograms of the pretreated meat sample solution (blue line), the matrix solution spiked with antimicrobial standard to create matrix standard solutions at a concentration of 10 µg/kg (red line), and neat standard solution with a concentration of 25 µg/L (black line). All target compounds were detected using the PDA detector at six different wavelengths. The 12 target compounds (Table 1) were separated and eluted in approximately 25 min.

Analysis of Quinolones in Meat: Samples of chicken and pork were prepared as described in Figure 1. Analytical conditions for the analysis of quinolones are described above. Chromatograms of the pretreated meat sample solution (blue line), the matrix solution spiked with antimicrobial standard to create matrix standard solutions at a concentration of 10 µg/kg (red line), and neat standard solution with a concentration of 25 µg/L (black line) are shown in Figure 3. Analysis was performed with the fluorescence detector in dual wavelength mode. New quinolones (compounds 1 to 8 in Table 1) were detected at an excitation wavelength of 290 nm and fluorescence wavelength of 495 nm; old quinolones (compounds 9 to 11 in Table 1) were detected at an excitation wavelength of 325 nm and fluorescence wavelength of 365 nm. Piromidic acid (compound 12 in Table 1) differs from other quinolones in exhibiting no fluorescence characteristics and was detected using the PDA detector. The 12 target compounds were separated and eluted in approximately 22 min.

Similarity Calculation Using UV Spectral Library: All target compounds detected by PDA were analyzed qualitatively based on UV spectra as well as retention times. Sample spectra were checked for similarity against library spectra. Figure 4 shows a UV spectrum of sulfaquinoxaline in a beef matrix spiked with standard solution at threshold concentration. The degree of similarity with the library spectrum was 0.997.

Conclusion
 

A new multiclass method has been developed for the rapid screening of residues of 24 antimicrobials in meat samples by HPLC with a combination of PDA and RF detection. Sample preparation was performed using the QuEChERS and liquid–liquid extraction (LLE) methods respectively for different compound classes. The method was successfully applied to meat samples from different animal species. None of the meat samples tested showed contamination with the antimicrobials under investigation. Results are obtained faster than with conventional tests, like the paper disc method, and provide higher determination accuracy. 

References

1. Sanz et al., EJNFS 5(3), 156–165 (2015).
2. http://www.r-biopharm.com/news/news-en/antibiotics-in-meat-5-facts-about-residues-in-food
3. European Commission Council Regulation No 37/2010
4. Multiresidue Method I for Veterinary Drugs, etc. by HPLC (Animal and Fishery products) Director Notice about Analytical Methods for Residual Compositional Substances of Agricultural Chemicals, Feed Additives and Veterinary Drugs in Food (Syoku-An No. 0124001, 24 January 2005. Final amendments were made on 26 May 2006), Japan’s Ministry of Health, Labor and Welfare.
5. “Standard methods of analysis in food safety regulation (for veterinary drugs and animal feed additives)” p.26–43, Japan Food Hygiene Association (2003), edited under the supervision of Japan’s Ministry of Health, Labor and Welfare.

Gesa Johanna Schad graduated with a diploma in chemical engineering from the Technical University NTA in Isny, Germany in 2004 and an MSc in pharmaceutical analysis from the University of Strathclyde in Glasgow, UK in 2005. She worked until 2006 as a consultant in HPLC method development and validation in an analytical laboratory of the FAO/IAEA in Vienna, Austria. She gained her doctorate for research in pharmaceutical sciences at the University of Strathclyde in 2010 and was employed as an HPLC specialist in the R&D department at Hichrom Ltd. in Reading, UK, from 2009. Since 2013, she has worked as a HPLC product specialist and since 2015 as HPLC Product Manager in the analytical business unit of Shimadzu Europa in Duisburg, Germany.

Natsuki Iwata graduated with a diploma in chemistry and materials technology from the Kyoto Institute of Technology in Kyoto, Japan, in 2010 and a MEng in chemistry and materials technology from the graduate school of Science and Technology, Kyoto Institute of Technology in 2012. She has worked as a HPLC product specialist in global application development centre of Shimadzu Corporation in Kyoto, Japan since 2012.

E-mail:shimadzu@shimadzu.euWebsite:www.shimadzu.eu