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13C NMR detection of non-protein nitrogen substance adulteration in animal feed

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Abstract

Illegal adulteration of melamine in animal feed and food has been widely studied. However, the risk of using substitute non-protein nitrogen substances still exists. In this study, we developed the 13C NMR method for the detection of non-protein nitrogen substance adulteration in animal feed. Three compounds, i.e., urea, melamine, and biuret, were used for method development. We found that the chemical shifts of the characteristic peaks in the carbon spectra of high-nitrogen adulterants were all between 150 and 170 ppm, whereas the chemical shifts of real protein peptide bonds (-CO–NH-) were between 170 and 180 ppm, demonstrating a good distinction between non-protein nitrogen and authentic protein. The method for analyzing melamine, urea, and biuret was validated. The R2 values were all above 0.99 within the calibration range of 0.05–2% (w/w). The limits of quantification of urea, melamine, and biuret were 0.0120%, 0.0660%, and 0.0806%, respectively. This method involves simple sample pretreatment and rapid detection while also providing high accuracy. All the sample information obtained by NMR detection does not require strict impurity removal. Compared with a previously reported 1H NMR method, the developed 13C NMR method does not require strict moisture removal to avoid active hydrogen exchange, and the interfering peak overlap is mitigated.

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References

  1. Andersen WC, Turnipseed SB, Karbiwnyk CM, Clark SB, Madson MR, Gieseker CM, et al. Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry. J Agric Food Chem. 2008;56:4340–7. https://doi.org/10.1021/jf800295z.

    Article  CAS  PubMed  Google Scholar 

  2. Xu XM, Ren YP, Zhu Y, Cai ZX, Han JL, Huang BF, et al. Direct determination of melamine in dairy products by gas chromatography/mass spectrometry with coupled column separation. Anal Chim Acta. 2009;650:39–43. https://doi.org/10.1016/j.aca.2009.04.026.

    Article  CAS  PubMed  Google Scholar 

  3. Koebel M, Elsener M. Determination of urea and its thermal decomposition products by high-performance liquid chromatography. J Chromatogr A. 1995;689:164–9. https://doi.org/10.1016/0021-9673(94)00922-V.

    Article  CAS  Google Scholar 

  4. Patterson BW, Carraro F, Wolfe RR. Measurement of 15N enrichment in multiple amino acids and urea in a single analysis by gas chromatography/mass spectrometry. Biol Mass Spectrom. 1993;22:518–23.

    Article  CAS  Google Scholar 

  5. Dai XH, Fang X, Su FH, Yang MR, Li HM, Zhou J, et al. Accurate analysis of urea in milk and milk powder by isotope dilution gas chromatography-mass spectrometry. J Chromatogr B. 2010;878:1634–8. https://doi.org/10.1016/j.jchromb.2010.04.005.

    Article  CAS  Google Scholar 

  6. Filazi A, Sireli UT, Ekici H, Can HY, Karagoz A. Determination of melamine in milk and dairy products by high performance liquid chromatography. J Dairy Sci. 2012;95:602–8. https://doi.org/10.3168/jds.2011-4926.

    Article  CAS  PubMed  Google Scholar 

  7. Finete VdLM, Gouvêa MM, Marques FFdC, Netto ADP. Validation of a method of high performance liquid chromatography with fluorescence detection for melamine determination in UHT whole bovine milk. Food Control. 2015;51:402–7. https://doi.org/10.1016/j.foodcont.2014.12.001.

    Article  CAS  Google Scholar 

  8. Pibarot P, Pilard S. Analysis of urea in petfood matrices: comparison of spectro-colorimetric, enzymatic and liquid chromatography electrospray ionization high resolution mass spectrometry methods. Am J Anal Chem. 2012;3:613–21. https://doi.org/10.4236/ajac.2012.39080.

    Article  CAS  Google Scholar 

  9. Abbas O, Lecler B, Dardenne P, Baeten V. Detection of melamine and cyanuric acid in animal feed ingredients by near infrared spectroscopy and chemometrics. J Near Infrared Spectrosc. 2013;21:183–94. https://doi.org/10.1255/jnirs.1047.

    Article  CAS  Google Scholar 

  10. Balabin RM, Smirnov SV. Melamine detection by mid- and near-infrared (MIR/NIR) spectroscopy: a quick and sensitive method for dairy products analysis including liquid milk, infant formula, and milk powder. Talanta. 2011;85:562–8. https://doi.org/10.1016/j.talanta.2011.04.026.

    Article  CAS  PubMed  Google Scholar 

  11. Okazaki S, Hiramatsu M, Gonmori K, Suzuki O, Tu AT. Rapid nondestructive screening for melamine in dried milk by Raman spectroscopy. Forensic Toxicol. 2009;27:94–7. https://doi.org/10.1007/s11419-009-0072-3.

    Article  CAS  Google Scholar 

  12. Shen GH, Fan X, Yang ZL, Han LJ. A feasibility study of non-targeted adulterant screening based on NIRM spectral library of soybean meal to guarantee quality: the example of non-protein nitrogen. Food Chem. 2016;210:35–42. https://doi.org/10.1016/j.foodchem.2016.04.101.

    Article  CAS  PubMed  Google Scholar 

  13. Li QB, Kang X, Shi DD, Liu QS. Determination of melamine in soybean meal by near-infrared imaging and chemometrics. Anal Lett. 2016;49:1564–77. https://doi.org/10.1080/00032719.2015.1118482.

    Article  CAS  Google Scholar 

  14. Li J, Qi HY, Shi YP. Determination of melamine residues in milk products by zirconia hollow fiber sorptive microextraction and gas chromatography–mass spectrometry. J Chromatogr A. 2009;1216:5467–71. https://doi.org/10.1016/j.chroma.2009.05.047.

    Article  CAS  PubMed  Google Scholar 

  15. Li ZY, Welbeck E, Wang RF, Liu Q, Yang YB, Chou GX, et al. A universal quantitative 1H nuclear magnetic resonance (qNMR) method for assessing the purity of Dammarane-type Ginsenosides. Phytochem Anal. 2015;26:8–14. https://doi.org/10.1002/pca.2527.

    Article  CAS  PubMed  Google Scholar 

  16. Simmler C, Napolitano JG, McAlpine JB, Chen S-N, Pauli GF. Universal quantitative NMR analysis of complex natural samples. Curr Opin Biotechnol. 2014;25:51–9.

    Article  CAS  Google Scholar 

  17. Walker GS, Bauman JN, Ryder TF, Smith EB, Spracklin DK, Obach RS. Biosynthesis of drug metabolites and quantitation using NMR spectroscopy for use in pharmacologic and drug metabolism studies. Drug Metab Dispos. 2014;42:1627–39. https://doi.org/10.1124/dmd.114.059204.

  18. Rizzo V, Pinciroli V. Quantitative NMR in synthetic and combinatorial chemistry. J Pharm Biomed Anal. 2005;38:851–7. https://doi.org/10.1016/j.jpba.2005.01.045.

    Article  CAS  PubMed  Google Scholar 

  19. Lachenmeier DW, Humpfer E, Fang F, Schutz B, Dvortsak P, Sproll C, et al. NMR-spectroscopy for nontargeted screening and simultaneous quantification of health-relevant compounds in foods: the example of melamine. J Agric Food Chem. 2009;57:7194–9. https://doi.org/10.1021/jf902038j.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Manzano MR, Colnago LA, Aparecida FL, Bouchard D. Fast and simple nuclear magnetic resonance method to measure conjugated linoleic acid in beef. J Agric Food Chem. 2010;58:6562–4. https://doi.org/10.1021/jf100345e.

    Article  CAS  Google Scholar 

  21. Turbitt JR, Colson KL, Killday KB, Milstead A, Neto CC. Application of 1H-NMR-based metabolomics to the analysis of cranberry (Vaccinium macrocarpon) supplements. Phytochem Anal. 2020;31:68–80. https://doi.org/10.1002/pca.2867.

    Article  CAS  PubMed  Google Scholar 

  22. Duquesnoy E, Castola V, Casanova J. Identification and quantitative determination of carbohydrates in ethanolic extracts of two conifers using 13 C NMR spectroscopy. Carbohydr Res. 2008;343:893–902. https://doi.org/10.1016/j.carres.2008.01.001.

    Article  CAS  PubMed  Google Scholar 

  23. Stubbe B, Graulus GJ, Reekmans G, Courtin T, Martins JC, Vlierberghe SV, et al. A straightforward method for quantification of vinyl functionalized water soluble alginates via 13 C-NMR spectroscopy. Int J Biol Macromol. 2019;134:722–9. https://doi.org/10.1016/j.ijbiomac.2019.05.015.

    Article  CAS  PubMed  Google Scholar 

  24. Barding GA, Salditos R, Larive CK. Quantitative NMR for bioanalysis and metabolomics. Anal Bioanal Chem. 2012;404:1165–79. https://doi.org/10.1007/s00216-012-6188-z.

    Article  CAS  PubMed  Google Scholar 

  25. Graham SF, Kennedy T, Chevallier O, Gordon A, Farmer L, Elliott C, et al. The application of NMR to study changes in polar metabolite concentrations in beef longissimus dorsi stored for different periods post mortem. Metabolomics. 2010;6:395–404. https://doi.org/10.1007/s11306-010-0206-y.

    Article  CAS  Google Scholar 

  26. Li QQ, Meng XH, Zhu D, Pang XM, Wang KQ, Frew R, et al. Determination of nonprotein nitrogen components of milk by nuclear magnetic resonance. Anal Lett. 2016;49(18):2953–63. https://doi.org/10.1080/00032719.2016.1164180.

    Article  CAS  Google Scholar 

  27. Asakura T, Matsuda H, Naito A. Acetylation of Bombyx mori silk fibroin and their characterization in the dry and hydrated states using 13C solid-state NMR. Int J Biol Macromol. 2020;155:1410–9. https://doi.org/10.1016/j.ijbiomac.2019.11.116.

    Article  CAS  PubMed  Google Scholar 

  28. McBride BW. 13C CP-MAS NMR spectroscopy of cellulose and forages. N Z J Agric Res. 1991;34:257–62.

    Article  CAS  Google Scholar 

  29. Donato P, Cacciola F, Cichello F, Russo M, Dugo P, Mondello L. Determination of phospholipids in milk samples by means of hydrophilic interaction liquid chromatography coupled to evaporative light scattering and mass spectrometry detection. J Chromatogr A. 2011;1218:6476–82. https://doi.org/10.1016/j.chroma.2011.07.036.

    Article  CAS  PubMed  Google Scholar 

  30. Naz S, Tufail S, Sherazi H, Talpur FN, Mahesar SA, Huseyin K. Rapid determination of free fatty acid content in waste deodorizer distillates using single bounce-attenuated total reflectance-FTIR spectroscopy. J AOAC Int. 2012;95:1570–3. https://doi.org/10.5740/jaoacint.11-034.

  31. Chen L, Zhang HF, Liu Q, Pang XY, Zhao X, Yang HS. Sanitising efficacy of lactic acid combined with low-concentration sodium hypochlorite on Listeria innocua in organic broccoli sprouts. Int J Food Microbiol. 2019;295:41–8. https://doi.org/10.1016/j.ijfoodmicro.2019.02.014.

    Article  CAS  PubMed  Google Scholar 

  32. James H, Mamula BM, Adams KM, Xie ZH, Moore JC. Variance of commercial powdered milks analyzed by proton nuclear magnetic resonance and impact on detection of adulterants. J Agric Food Chem. 2018;66:8478–88. https://doi.org/10.1021/acs.jafc.8b00432.

    Article  CAS  Google Scholar 

  33. Jablonski JE, Moore JC, Harnly JM. Nontargeted detection of adulteration of skim milk powder with foreign proteins using UHPLC-UV. J Agric Food Chem. 2014;62:5198–206. https://doi.org/10.1021/jf404924x.

    Article  CAS  PubMed  Google Scholar 

  34. Limm W, Karunathilaka SR, Yakes BJ, Mossoba MM. A portable mid-infrared spectrometer and a non-targeted chemometric approach for the rapid screening of economically motivated adulteration of milk powder. Int Dairy J. 2018;85:177–83. https://doi.org/10.1016/j.idairyj.2018.06.005.

    Article  CAS  Google Scholar 

  35. Scholl PF, Bergana MM, Yakes BJ, Xie ZH, Zbylut S, Downey G, et al. Effects of the adulteration technique on the near-infrared detection of melamine in milk powder. J Agric Food Chem. 2017;65:5799–809. https://doi.org/10.1021/acs.jafc.7b02083.

    Article  CAS  PubMed  Google Scholar 

  36. Karunathilaka SR, Yakes BJ, He K, Brückner L, Mossoba MM. First use of handheld Raman spectroscopic devices and on-board chemometric analysis for the detection of milk powder adulteration. Food Control. 2018;92:137–46. https://doi.org/10.1016/j.foodcont.2018.04.046.

    Article  CAS  Google Scholar 

  37. Karunathilaka SR, Yakes BJ, He K, Chung JK, Mossoba MM. Non-targeted NIR spectroscopy and SIMCA classification for commercial milk powder authentication: a study using eleven potential adulterants. Heliyon. 2018;4(9):e00806. https://doi.org/10.1016/j.heliyon.2018.e00806.

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Funding

This work was supported by the National Key Research and Development Program of China (no. 2018YFC1602304) and the Natural Science Foundation of China (31871891).

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Authors and Affiliations

  1. College of Chemistry and Chemical Engineering, Tianjin University of Technology, Tianjin, 300384, China

    Chengxiang Zhao & Yongyue Sun

  2. Institute of Agricultural Quality Standards and Testing Technology, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Chengxiang Zhao, Tongtong Wang, Furong Chen & Gang Chen

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Zhao, C., Wang, T., Chen, F. et al. 13C NMR detection of non-protein nitrogen substance adulteration in animal feed. Anal Bioanal Chem 414, 2453–2460 (2022). https://doi.org/10.1007/s00216-022-03886-y

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