Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
권호 표지
DOI QR코드

DOI QR Code

Analysis of Aminoglycoside Antibiotics in Meat and Cell Culture Medium Coupled with Direct Injection of an Ion-pairing Reagent

이온쌍 시약 직접 주입법을 활용한 육류 및 세포배양액 내 아미노글리코사이드계 항생제 분석

  • Kyung-Ho Park (Food Safety Center, Food Safety division, Pulmuone Co. Ltd.) ;
  • Song-Yi Gu (Food Safety Center, Food Safety division, Pulmuone Co. Ltd.) ;
  • Geon-Woo Park (Food Safety Center, Food Safety division, Pulmuone Co. Ltd.) ;
  • Jong-Jib Kim (Food Safety Center, Food Safety division, Pulmuone Co. Ltd.) ;
  • Jong-soo Lee (Food Safety Center, Food Safety division, Pulmuone Co. Ltd.) ;
  • Sang-Gu Kim (Food Safety Center, Food Safety division, Pulmuone Co. Ltd.) ;
  • Sang-Yun Lee (Food Safety Center, Food Safety division, Pulmuone Co. Ltd.) ;
  • Hyang Sook Chun (Department of Food Safety and Regulatory Science, Chung-Ang University)
  • 박경호 ((주)풀무원 풀무원기술원 식품안전실) ;
  • 구송이 ((주)풀무원 풀무원기술원 식품안전실) ;
  • 박건우 ((주)풀무원 풀무원기술원 식품안전실) ;
  • 김종집 ((주)풀무원 풀무원기술원 식품안전실) ;
  • 이종수 ((주)풀무원 풀무원기술원 식품안전실) ;
  • 김상구 ((주)풀무원 풀무원기술원 식품안전실) ;
  • 이상윤 ((주)풀무원 풀무원기술원 식품안전실) ;
  • 전향숙 (중앙대학교 식품안전규제과학과)
  • Received : 2023.08.29
  • Accepted : 2023.10.04
  • Published : 2023.10.30
Download PDF
⟨ Previous Next ⟩

Abstract

Aminoglycoside antibiotics, also known as aminoglycosides (AGs), are veterinary drugs effective against a wide range of gram-negative and gram-positive bacteria. Owing to their recent use in cultured meats, it has become essential to establish an analytical method for safety management. AGs are highly polar compounds, and ion-pair reagents (IPRs) are used to ensure component separation. Owing to the high possibility of potential mechanical problems resulting from IPR addition to the mobile phase, an analytical method in which IPRs are added directly to the vial was explored. In this study, methods for analyzing 10 AGs via liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the addition of two IPRs were validated for selectivity, detection limit, quantitation limit, recovery, and precision. The detection limit was 0.0001-0.0038 mg/kg, the quantification limit was 0.004-0.011 mg/kg, and the linearity (R2) within the concentration range of 0.01-0.5 mg/kg was over 0.99. Recovery and precision (expressed as relative standard deviation) evaluated in the two matrices (beef and cell culture media) ranged from 70.7% to 120.6% and 0.2% to 24.7%, respectively. The validated AG analytical method was then applied to 15 meats prepared from chicken, beef, and pork, and 6 culture media and additives used in cultured meat. No AGs were detected in any of the 15 meats distributed in Korea; however, streptomycin and dihydrostreptomycin were detected at levels ranging from 695.85 to 1152.71 mg/kg and 6.35 to 11.11 mg/kg, respectively, in the culture media additives. The LC-MS/MS method coupled with direct addition of IPRs to the vial can provide useful basic data for AG analysis and safety evaluation of meats as well as culture media and additives for cultured meats.

아미노글리코사이드계 항생제(Aminoglycosides, AGs)는 그람음성균과 양성균에 광범위하게 작용하는 동물용 의약품으로, 최근 배양육에 사용된다고 알려져 있어, 안전성 관리를 위한 분석법 마련이 반드시 필요하다. AGs는 고극성 화합물로 성분 간의 분리를 위해 이온쌍 시약(ion-pairing reagent, IPR)을 사용하고 있으나 IPR을 이동상에 첨가하는 기존 분석방법의 경우 용매가 흐르는 동안 질량분석기로 주입되는 IPR로 인해 기기적인 문제가 발생할 가능성이 높아, IPR를 바이알에 직접 첨가하는 분석방법을 검토하였다. 본 연구에서 10종 AGs 성분에 대한 분석방법을 확인하고 유효성을 검증하였다. 검출한계와 정량한계는 각각 0.0001-0.0038 mg/kg 와 0.004-0.011 mg/kg의 범위로 나타났으며, 0.01-0.5 mg/kg 범위 내의 직선성(R2)은 0.99 이상이었다. AGs의 시료 회수율을 확인하고자 소고기와 세포배양배지(cell culture medium) 매질에서 회수율과 상대표준편차로 나타낸 정밀도를 확인한 결과 각각 70.7-120.6% 및 0.2 to 24.7%로 나타났다. 기존의 이동상에 IP 첨가 방법과 비교하였을 때 유사한 수준으로 양호하였다. 검증된 AGs 분석법은 국내 유통되는 닭고기, 소고기, 돼지고기 15품목과 배양육 배지 첨가제 6품목에 적용해보았다. 그 결과 국내 유통되는 육류 15품목 모두 AGs 성분이 검출되지 않았으나, 세포배양배지에서 streptomycin은 695.85-1152.71 mg/kg, dehydrostreptomyci은 6.35-11.11 mg/kg로 검출되었다. 따라서 IRR을 바이알에 직접 첨가하는 LC-MS/MS 방식은 육류, 세포배양배지, 배지첨가제 중 AGs 분석 및 안전성 평가를 위한 기초자료로 활용될 것으로 기대된다.

Keywords

Acknowledgement

이 연구는 식품의약품안전처의 2021년도 연구비 지원(21153MFDS605) 및 (주)풀무원, 풀무원기술원 지원에 의해 수행되었으며, 이에 감사드립니다.

References

  1. Font-I-Furnols, M., Guerrero, L., Consumer preference, behavior and perception about meat and meat products: an overview. Meat Sci., 98, 361-371 (2014).  https://doi.org/10.1016/j.meatsci.2014.06.025
  2. Moe, S.M., Zidehsarai, M.P., Chambers, M.A., Jackman, L.A., Radcliffe, J.S., Trevino, L.L., Donahue, S.E., Asplin, J.R., Vegetarian compared with meat dietary protein source and phosphorus homeostasis in chronic kidney disease. Clin. J. Am. Soc. Nephrol., 6, 257-264 (2011).  https://doi.org/10.2215/CJN.05040610
  3. Gerhardt, C., Suhlmann, G., Ziemssen, F., Donnan, D., Warschun, M., Kuhnle, H.J., How will cultured meat and meat alternatives disrupt the agricultural and food Industry? Ind. Biotechnol., 16, 262-270 (2020).  https://doi.org/10.1089/ind.2020.29227.cge
  4. Bryant, C.J., Culture, meat, and cultured meat. J. Anim. Sci., 98, 1-7 (2020).  https://doi.org/10.1093/jas/skaa172
  5. Njoga, U.J., Njoga, E.O., Nwobi, O.C., Abonyi, F.O., Edeh, H.O., Ajibo, F.E., Azor, N., Bello, A., Upadhyay, A.K., Okpala, C.O.R., Korzeniowska, M., Guine, R.P.F., Slaughter conditions and slaughtering of pregnant cows in southeast Nigeria: Implications to meat quality, food safety and security. Foods, 10, 1298 (2021). 
  6. Britt, J.H., Cushman, R.A., Dechow, C.D., Dobson, H., Humblot, P., Hutjens, M.F., Jones, G.A., Ruegg, P.S., Sheldon, I.M., Stevenson, J.S., Invited review: Learning from the future-A vision for dairy farms and cows in 2067. J. Dairy Sci., 101, 3722-3741 (2018).  https://doi.org/10.3168/jds.2017-14025
  7. Kumar, P., Chatli, M.K., Mehta, N., Singh, P., Malav, O.P., Verma, A.K., Meat analogues: health promising sustainable meat substitutes. Crit, Rev, Food Sci, Nutr., 57, 923-932 (2017).  https://doi.org/10.1080/10408398.2014.939739
  8. Bryant, C.J., Barnett, J.C., What's in a name? consumer perceptions of in vitro meat under different names. Appetite, 137, 104-113 (2019).  https://doi.org/10.1016/j.appet.2019.02.021
  9. Djisalov, M., Knezic, T., Podunavac, I., Zivojevic, K., Radonic, V., Knezevic, N.Z., Bobrinetskiy, I., Gadjanski, I., Cultivating multidisciplinarity: manufacturing and sensing challenges in cultured meat production. Biology, 10, 204 (2021). 
  10. Lee, S.Y., Kang, H.J., Lee, D.Y., Kang, J.H., Ramani, S., Park, S., Hur, S.J., Principal protocols for the processing of cultured meat. J. Anim. Sci. Technol., 63, 673-680 (2021).  https://doi.org/10.5187/jast.2021.e40
  11. Bhat, Z.F., Kumar, S., Fayaz, H., In vitro meat production: challenges and benefits over conventional meat production. J. Integr. Agric., 14, 241-248 (2015).  https://doi.org/10.1016/S2095-3119(14)60887-X
  12. Choudhury, D., Tseng, T.W., Swartz, E., The business of cultured meat. Trends Biotechnol., 38, 573-577 (2020).  https://doi.org/10.1016/j.tibtech.2020.02.012
  13. Ong, K.J., Johnston, J., Datar, I., Sewalt, V., Holmes, D., Shatkin, J.A., Food safety considerations and research priorities for the cultured meat and seafood industry. Compr. Rev. Food Sci. Food Saf., 20, 5421-5448 (2021).  https://doi.org/10.1111/1541-4337.12853
  14. Choi, K.H., Yoon, J.W., Kim, M., Lee, H.J., Jeong, J., Ryu, M., Jo, C., Lee, C.K., Muscle stem cell isolation and in vitro culture for meat production: A methodological review. Compr. Rev. Food Sci. Food Saf., 20, 429-457 (2021).  https://doi.org/10.1111/1541-4337.12661
  15. Lalitha Aishwarya, K.V., Venkataramana Geetha, P., Eswaran, S., Mariappan, S., Sekar, U., Spectrum of aminoglycoside modifying enzymes in gram-negative bacteria causing human infections. J. Lab. Physicians, 12, 27-31 (2020).  https://doi.org/10.1055/s-0040-1713687
  16. Reemtsma, T., Berger, U., Arp, H.P.H., Gallard, H., Knepper, T.P., Neumann, M., Quintana, J.B., Voogt, P.D., Mind the gap: Persistent and mobile organic compounds- water contaminants that slip through. Environ. Sci. Technol., 50, 10308-10315 (2016).  https://doi.org/10.1021/acs.est.6b03338
  17. Hong, Y.M., Lee, S.K., Kim, H.A., Hwang, Y.K., Simultaneous analytical method for the neomycin, gentamicin residues in seafood. J. Appl. Biol. Chem. 53, 25-30 (2010).  https://doi.org/10.3839/jabc.2010.005
  18. Kang, Y.W., Joo, H.J., Kim, Y.S., Cho, Y.J., Kim, H.Y., Lee, G.H., Kim, M.H., Analysis and monitoring of residues of aminoglycoside antibiotics in livestock products. Korean J. Food Sci. Technol., 43, 1-5 (2011).  https://doi.org/10.9721/KJFST.2011.43.1.001
  19. Cho, Y.J., Choi, S.J., Kim, M.A., Kim, M.K., Yoon, S.J., Chang, M.I., Lee, S.M., Kim, H.J., Jeong, J.Y., Rhee, G.S., Lee, S.J., Simultaneous determination of aminoglycoside antibiotics in meat using liquid chromatography tandem mass spectrometry. J. Food Hyg. Saf., 29, 123-130 (2014).  https://doi.org/10.13103/JFHS.2014.29.2.123
  20. Jo, H.W., Sun, J.H., Heo, H.M., Lee, S.H., Kim, J.E., Moon, J.K., Development and validation of Dithiocarbamates fungicide analytical method using CS2 trap method in livestock product. Korean J. Environ. Agric., 40, 127-133 (2021).  https://doi.org/10.5338/KJEA.2021.40.2.15
  21. McCalley, D.V., Understanding and manipulating the separation in hydrophilic interaction liquid chromatography. J. Chromatogr. A, 1523, 49-71 (2017).  https://doi.org/10.1016/j.chroma.2017.06.026
  22. Asakawa, D., Uemura, M., Sakiyama, T., Yamano, T., Sensitivity enhancement of aminoglycosides in hydrophilic interaction liquid chromatography with tandem mass spectrometry by post-column addition of trace sodium acetate in methanol. Food Addit. Contam.: Part A, 35, 1116-1126 (2018).  https://doi.org/10.1080/19440049.2017.1388543
  23. Bijleveld, Y., de Haan, T.R., Toersche, J., Jorjani, S., van der Lee, J., Groenendaal, F., Dijk, P., van Heijst, A., Gavilanes, A.W.D., de Jonge, R., Dijkman, K.P., van Straaten, H., Rijken, M., Zonnenberg, I., Cools, F., Nuytemans, D., Mathot, R., A simple quantitative method analysing amikacin, gentamicin, and vancomycin levels in human newborn plasma using ion-pair liquid chromatography/tandem mass spectrometry and its applicability to a clinical study. J. Chromatogra. B, 951-952, 110-118 (2014).  https://doi.org/10.1016/j.jchromb.2014.01.035
  24. Joseph, A., Rustum, A., Development and validation of a RP-HPLC method for the determination of gentamicin sulfate and its related substances in a pharmaceutical cream using a short pentafluorophenyl column and a charged aerosol detector. J. Pharm. Biomed. Anal., 51, 521-531 (2010).  https://doi.org/10.1016/j.jpba.2009.09.002
  25. Guironnet, A., Sanchez-Cid, C., Vogel, T. M., Wiest, L., Vulliet, E., Aminoglycosides analysis optimization using ion pairing liquid chromatography coupled to tandem mass spectrometry and application on wastewater samples. J. Chromatogr. A, 1651, 462133 (2021). 
  26. Wang, X., Yang, S., Li, Y., Zhang, J., Jin, Y., Zhao, W., Zhang, Y., Huang, J., Wang, P., Wu, C., Zhou, J., Optimization and application of parallel solid-phase extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry for the determination of 11 aminoglycoside residues in honey and royal jelly. J. Chromatogr. A, 1542, 28-36 (2018).  https://doi.org/10.1016/j.chroma.2018.02.029
  27. Mokh, S., Jaber, F., Kouzayha, A., Budzinski, H., Al Iskandarani, M., Optimization and comparisons for separation, detection and quantification of 12 aminoglycosides using 2 chromatographic conditions by LC-MS/MS. Am. J. Anal. Chem., 5, 982-994 (2014).  https://doi.org/10.4236/ajac.2014.514105
  28. Gustavsson, S.A., Samskog, J., Markides, K.E., Langstrom, B., Studies of signal suppression in liquid chromatography-electrospray ionization mass spectrometry using volatile ion-pairing reagents. J. Chromatogr. A, 937, 41-47 (2001).  https://doi.org/10.1016/S0021-9673(01)01328-0
  29. Ministry of Food and Drug Safety (MFDS), (2023, May 30). Guidelines on standard procedures for preparing analysis method. Retrieved from http://www.nifds.go.kr/brd/m_15/view.do?seq=8 215 
  30. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), (2023, May 30). Validation of analytical procedures: text and methodology Q2(R1). Retrieved from https://database.ich.org/sites/default/files/Q2_R1_Guideline.pdf 
  31. Bae, J.Y., Yun, D.Y., Kang, N.S., Choe, W.J., Jeong, Y.H., Jang, G.H., Moon, G., Investigation on pesticide residues in agricultural products in domestic markets using LC-MS/MS and GC-MS/MS. J. Food Hyg. Saf., 38, 131-139 (2023). https://doi.org/10.13103/JFHS.2023.38.3.131