Forgó, Péter

Kiss, Attila

EKE Repository of Publications

PÉTER FORGÓ*, ATTILA KISSDEVELOPMENT AND ADAPTATION OF NOVEL HPLCPROCEDURE TO CHARACTERIZE BIOGENIC AMINES INFOOD SAMPLESAbstract: A high performance chromatographic (HPLC) method for the si-multaneous determination of seven biogenic amines (histamine (HI), tryptamine(TR), tyramine (TY), putrescine (PUT), cadaverine (CAD), spermidine (SPD)and spermine (SPM)) in food products has been developed. The technique wasapplied to determine the biogenic amine content of wine and bear samples,which were treated with dansyl-chloride to form dansyl derivatives of biogenicamines. A gradient elution program was applied in the separation process in-volving water (A) and acetonitrile (B) solvents. Quantitative detection of thecompounds was carried out with light absorbance at 336 nm, the componentswere identified with a mass-spectrometer detector by their molecular mass. Thesensitivity of the applied method was 3-5pM for the studied derivatives. Themethod was tested on commercially available beer and wine samples, wherehistamine was proven to have the highest level (0.25-0.32 mM in beers, whilewines showed higher histamine level 0.33-0.41mM). Beers contained detectableamount of tryptamine (around 0.01 mM), while wines had notable tyramine andtryptamine content.Keywords: food products, biogenic amine, HPLC-MS, derivatization, diode-array detection.IntroductionBiogenic amines (BAs) represent a unique class of natural compounds andcan be characterized as small or medium sized organic amine based derivativeswith aliphatic, aromatic and heterocyclic moieties.1 Biogenic amines are pro-duced in biochemical metabolism during fermentation or brewing food produc-tion processes2 by microbial degradation2, 3 and decarboxylation process fromamino acids associated with food spoilage. Moreover, biogenic amines can beformed “in vivo” from the corresponding aldehydes4. The members of this mo-lecular class can be found in a wide range of food products including fish, meat,vegetables, fruits, nuts, dairy products, brewed drinks and fermented food prod-ucts. However, the total biogenic amine content is strongly correlated to theEszterházy Károly College, EGERFOOD Regional Knowledge Centre, Leányka str. 6, H-3300, Eger, Hungary. Corresponding author: Dr. Peter Forgo, forgop@ektf.hu45microorganisms present in the sample. Several studies have pointed out theirhigh biological activity that can increase hypertension as tryptamine5, can affectcardiovascular, muscle, neural, gastric functions6, 7 and can cause food poisoningas histamine. Furthermore, the members of the family have a potential to formcarcinogen nitrosamines with nitrite.8 Nevertheless, the putrescine based polya-mines play an important role in cellular growth and metabolism.9As a result of the high biological relevance, several analytical methods havebeen developed for the qualitative and quantitative detection of biogenic amines.Fluorescence based protocols have been developed for the detection of histaminein fish10, ", cheese and other fermented food products12. Thin layer chromato-graphic methods have been applied to determine natural13 and derivatized com-pounds14 involving densitometric detection13. Gas-chromatographic and HPLC15methods coupled with diode array and mass spectroscopic detection16 have beenutilized to study the biogenic amine content in brewed17 and other food prod-ucts18, 19, 20. Capillary electrophoretic21, 15 separation and pulsed amperometric22detection methods have also been used to determine biogenic amine contents inseveral food products. Electrochemical biosensors23 have also been widely usedin the detection of biogenic amines.24, 25Our purpose was to develop an efficient HPLC-MS method in order to detectbiogenic amines in food products and brewed drinks. The method was optimizedfor the detection and quantitative analysis of seven biogenic amines: histamine(HI)(1), tryptamine (TR)(2), tyramine (TY)(3), putrescine (PUT)(4), cadaverine(CAD)(5), spermidine (SPD)(6) and spermine (SPM)(7), the experiments wererun with the presence of diamino-heptane (DAH)(8) as internal standard.f f 'H\ ih 2C t f '~H/?—\  /^NH2h o —f y— /Histamine (HIS) (1) Tryptamine (TR) (2) Tyramine (TY) (3)k ^ N H 2k ^ N H 2C m' fPutrescine (PUT) (4) Cadaverine (CAD) (5) Spennidine (SPD) (6)r|1H2 r - ANH 20 ^ 0Spermine (SPM) (7) Diamino-heptane (DAH) (8) Dansyl-chloride (9)Figure 1. The structure o f the investigated compounds.46Experimental procedureApproximately 9 mg of the standard seven biogenic amines and diamino-heptane (DAH) were dissolved in a 25 ml volumetric flask. A standard dansyl-chloride(9) solution was also prepared in acetone solution (23.6 mg dansyl-chloride in 1.18 ml acetone). Standard solutions (250, 200, 150, 100 and 50 plfrom the stock solution were used to dilute the solution to 0.85 ml) were pre-pared by the dilution of the stock solution and adding sodium-hydroxide anddansyl-chloride. The standard solutions were kept in 60°C for one hour, then theexcess dasyl-chloride was decomposed by the addition of ammonia (50 pl). Af-ter filtration and addition of acetonitrile (0.5 ml) 10pl of the standard solutionwas injected for HPLC analysis. Real samples were treated the same way asstandard solutions, 250 pl of wine and beer samples were used for the HPLCanalysis with the addition of sodium-hydroxide and dansyl-chloride stock solu-tions. The derivative producing reaction took place during one hour at 60°C andthe excess dasyl-chloride was decomposed by the addition of ammonia (50 pl).After filtration and addition of acetonitrile (0.5 ml) 10pl of the sample solutionwas injected for HPLC analysis. A Shimadzu LCMS-2010 HPLC instrumentwas used for the separation, which was coupled to a mass detector with atmos-pheric pressure chemical ionization (APCI) in the m/z range of 100-1200 amuwith positive ion detection mode and 1.5 kV detector voltage. The diode-arraydetector was used for quantitative analysis and it covered a 190-800 nm wave-length range, the biogenic amine content was analyzed at 336 nm. The chroma-tographic column was a YMC-Pack ODS-AQ 250x4.6 mm, 5pm. A gradientelution was carried out with acetonitrile (B) and water(A) solvents at a flow rateof 0.4 ml/min and with the following gradient profile: 0 min (65% B), 2 min(65% B), 20 min (80 % B), 24 min (90 % B), 32 min (100 % B), 40 min (100 %B), 42 min (90 % B), 44 min (80 % B), 47 min (65 % B).Results and discussionSpecific chromatographic conditions have been elaborated and adjustedthroughout our experiments. Variable parameters have been tested such as elu-ents and flow rates. Acetonitrile-water solvent system with gradient elutionproved to be the most efficient, while 0.4 ml/min was found to be most appro-priate flow rate.The seven biogenic amine derivatives and the reference diamino-heptane ap-peared separately in the chromatogram with the following retention times: HIS:11.19 min, TR: 18.83 min, PUT: 22.71 min, CAD: 24.30 min, DAH: 28.61 min,TY: 32.55 min, SPD: 33.58 min, SPM: 38.96 min. (Figure 2). The signals in thechromatograms were assigned by their mass spectrum extracted from the totalion chromatogram (Figure 3). The calibration curve of the signal intensities as a47function of the injected amount of eight derivatives showed linear characteristicsin the 5X10"10-5X10"9 mole concentration region (figure 4). The calculated sensi-tivity using the calibration curves fall to the 3-5pM concentration region. Theseexperimental conditions were used to determine the biogenic amine content ofcommercially available ten beer and five wine samples The main biogenic aminecomponent was histamine (HIS), the detected level varied between 0.25 mM and0.31 mM. The tryptamine content was around 0.01 mM in the ten beer samples,tyramine, putrescine, cadaverine, spermine and spermidine could not be deter-mined quantitatively in beer samples. The histamine content of the five winesamples were a little higher than in beers, the values varied between 0.33 mMand 0,41 mM. Tryptamine content was proven to be between 0.05mM and0.06mM, while the thyramine concentration was even more lower (0.006-0.008mM). Putrescine contrentrations in wine samples were between 0.03 mM and0.04 mM. Cadaverine, spermine and spermidine could not be determined quanti-tatively in wine samples.Figure 2. Chromatogram o f the biogenic-amine reference solution with the highest con-centration detected with a diode-array detector at 336 nm.48A12i&tjL Jiiifj.ffu,],,7fco' ' ' ' M  ' ' ’M  ' ' idoó ' ' ’nbdC.uiVf. m  I i.TO.ii1Í0 H a  300 4M SCO1-lTt? I j a    |J1I . . iW ta I W , I Wft JW , Ilo Tbo BÓO 900 1M0 1lt» liFigure 3. Mass-spectra offour selected biogenic amine derivatives,A- SPM, B- SPD, C- CAD, D- DAH.Figure 4. The calibration curves for the biogenic amine derivatives, signal intensities at336 nm as a function o f the injected number o f moles.49Table 1. The obtained values o f biogenic amine contentfor commercially availablelight beers and wines.Beer #1 0,28 0,009 nd ndBeer #2 0,31 0,010 nd ndBeer #3 0,25 0,008 nd ndBeer #4 0,27 0,009 nd ndBeer #5 0,32 0,010 nd ndBeer #6 0,27 0,009 nd ndBeer #7 0,26 0,008 nd ndBeer #8 0,27 0,009 nd ndBeer #9 0,29 0,009 nd ndBeer#10 0,31 0,010 nd ndWine #1 0,38 0,061 0,007 0,042Wine #2 0,41 0,066 0,008 0,046Wine #3 0,33 0,053 0,006 0,037Wine #4 0,37 0,060 0,007 0,041Wine #5 0,39 0,063 0,007 0,043ConclusionsA reliable chromatographic method was developed to determine the biogenicamine content in food products, the seven biogenic amine derivative can be reli-ably determined in food matrices. The method uses a dansyl derivative makingstep which allows the uv/vis detection of the amine derivatives. The studied tenbeer products had histamine levels between 0.25 mM and 0.32 mM, the seconddetectable biogenic amine was tryptamine, but with much lower levels. In caseof wine samples the histamine level was found to be higher (0.33-0.41 mM) andtryptamine, tyramine and putrescine were also in detectable amount. The methodcan be generally used to detect biogenic amines in food product with the use ofappropriate sample preparation protocol.AcknowledgementsThis work was financially supported by the Pázmány Péter Program (RET09/2005) financed by Hungarian National Office on Research and technology.50References1. M. H. Silla Santos, Biogenic Amines: their importance in foods, International Journalo f FoodMicrobiology 29 (1996) 213-231.2. Brink, B.; Damink, C; Joosten H. M. L. J.; Huis in’t Veld, J. H. J.; (1990) Occurrenceand formation of biologically active amines in food. Int. J. Food Microbiol, 11, 73-84.3. Halász, A.; Baráth, A.; Simon-Sarkadi, L.; Holzapfel, W. (1994), Biogenic amines andtheir production by microorganisms in food. Trends FoodSci. Technol. 5. 42-48.4. Maijala, R. L.; Eerola, S. H.; (1993) Contaminant lactic bacteria of dry sausages pro-duce histamine and tyramine. Meat Sci. 35, 387-395.5. Shalaby, A. R. (1996). Significance of biogenic amines to food safety and humanhealth. Food Research International, 29, 675-690.6. Soll, A. H., Wollin, A. Gastroenter, 1977, 72, 1166.7. Taylor, S. L., Hui, J. Y., Lyons, D. E., in Seafood toxins, Vol. 262 (Ed.: Regalis E. P.),Washington DC 1984, 417-430.8. Shahidi F., Pegg R. B., Sen, N. P. (1994) Absence of volatile N-nitrosamine in cookednitrite free cured muscle foods. Meat Sci. 37, 327-336.9. Bardócz, S.; Poliamines in tissue regeneration. In: U. Barhrach and Y. M. Heimer(Eds.), Physiology ofpolyamines. Vol. 1, C.R.C. Press, Boca Ratón, FL. USA, pp 96-106.10. Williams, S. (ed) (1984), Official methods of analysis of the Association of OfficialAnalytical Chemists, 14th edn. Association of Official Analytical Chemists, Ar- lington, VA, p. 341.11. Taylor, S. L. (1985), Histamine poisoning associated with fish, cheese, and otherfoods. World Health Organization. WPH/FOS/85(1), 1-47.12. Stratton, J.e., Hutkins, R. W., Taylor, S. L., (1991), Biogenic amines in cheese andother fermented foods. A review. J. FoodProt. 54, 460-470.13. Shalaby, A. R. (1999). Simple, rapid and valid thin layer chromatographic methodfor determining biogenic amines in foods. Food Chemistry, 65, 117-121.14. Lapa-Guimaraes, J., Pickova, J. (2004), New solvent systems for thin layer chroma-tographic determination of nine biogenic amines in fish and squid. Journal o fChromatography A, 1045, 223-232.15. Lange T., Wittmann C., (2002). Comparison of a capillary electrophoresis methodwith high-performance liqiud chromatography for the detection of biogenic amines in various food samples. Journal o f Chromatography B: Analytical Technology Biomedical Life Science, 779, 229-239.16. Gaboriau F., Havouis R., Moulinoux J-P., Delcros J-G, Atmospheric pressurechemical ionization-mass spectrometry method to improve the determination of dansylated polyamines, Analytical Biochemistry, 318 (2003) 212-220.17. Fernandes, J. O., Ferreira, M. A. (2000), Combined ion pair extraction and gaschromatography mass spectrometry for the simultaneous determination of di- amines, ployamines and aromatic amines in Port wine and grape juice. Journal ofChromatography A, 886, 183-195.18. Hwang, B. S., Wang, J.-T., Choong, Y.-M. (2003), A rapid gas chromatographicmethod for the determination of histamine in fish and fish products. Food chemi-stry, 82, 329-334.5119. E. H. Soufleros, E. Bouloumpasi, A. Zotou and Z. Loukou (2007) Determination ofbiogenic amines in Greek wines by HPLC and ultraviolet detection after dansylation and examination of factors affecting their presence and concentration,Food Chemistry 101, 704-716.20. F. Gosetti, E. Mazzucco, V. Gianotti, S. Polati and M.C. Gennaro (2007) High per-formance liquid chromatography/tandem mass spectrometry determination of biogenic amines in typical Piedmont cheeses, Journal o f Chromatography A,1149,151-15721. Kvasnicka, F., Voldrich, M., (2006), Determination of biogenic amines by capillaryzone electrophoresis with conductometric detection. Journal o f ChrokmatographyA. 1103, 145-149.22. Sun X., Yang X., Wang, E., (2003), Determination of biogenic amines by capillaryelectrophoresis with with pulsed amperometric detection. Journal o f Chromato-graphy A, 1005, 189-195.23. Mello L. D., Kubota L. T., Review of the use of biosensors as analytical tools int hefood and drink industries, Food chemistry, 77 (2002) 237-256.24. Draisci R., Volpe G., Lucentini L., Cecilia A., Federico R., Palleschi G., (1998)Determination of biogenic amines with an electrochemical biosensor, and its application to salted anchovies. Food Chemistry, 62, 225-232.25. Lande J., Wittmann C. (2002), Enzyme sensor array for the determination ofbiogenic amines in food samples, Analytical and bioanalytical chemistry, 372, 276-283.52

Development and adaptation of novel HPLC procedure to characterize biogenic amines in food samples

http://publikacio.uni-eszterhazy.hu/1355/1/45-52_Forgo.pdf

Development and adaptation of novel HPLC procedure to characterize biogenic amines in food samples

Abstract

Similar works

Full text

Available Versions

EKE Repository of Publications