Development and Validation of a High-Performance Liquid Chromatography Method for the Determination of Histamine in Fish Samples Using Fluorescence Detection with Pre-column Derivatization


Histamine level determination in fish and fish products is important for human safety, fish quality and food industry. For this reason, a rapid, robust, and precise method is needed. To achieve this objective, an HPLC method with fluorescence detection was developed and validated. Histamine in fish samples was efficiently extracted with perchloric acid, and purified with ion-exchange solid-phase extraction cartridge. A pre-column derivatization was adopted with ortho-phthalaldehyde (OPA) in the presence of the reducing agent 2-mercaptoethanol, and the stability of the histamine-OPA derivatives was achieved with the acidification of the reaction medium. In terms of validation, besides the excellent linear correlations, satisfactory recoveries at all spiking levels ranging between 0 and 200 mg kg−1 were attained, with limit of detection calculated at 1.8 mg kg−1, whereas limit of quantification determined at 5 mg kg−1. The proposed method was successfully used in the analysis of reference materials and proficiency tests, and was found to be suitable, accurate, and rapid for detection and quantification of histamine in various fish samples.

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  1. 1.

    Khan S, Carneiro L, Vianna MS, Romani EC, Aucelio RQ (2017) Determination of histamine in tuna fish by photoluminescence sensing using thioglycolic acid modified CdTe quantum dots and cationic solid phase extraction. J Lumin 182:71–78

    CAS  Article  Google Scholar 

  2. 2.

    Silva TM, Sabaini PS, Evangelista WP, Gloria MB (2011) Occurrence of histamine in Brazilian fresh and canned tuna. Food Control 22:323–327

    CAS  Article  Google Scholar 

  3. 3.

    Muscarella M, Magro SL, Campaniello M, Armentano A, Stacchini P (2013) Survey of histamine levels in fresh fish and fish products collected in Puglia (Italy) by ELISA and HPLC with fluorimetric detection. Food Control 31:211–217

    CAS  Article  Google Scholar 

  4. 4.

    Altieri I, Semeraro A, Scalise F, Calderari I, Stacchini P (2016) European official control of food: determination of histamine in fish products by a HPLC–UV-DAD method. Food Chem 211:694–699

    CAS  Article  Google Scholar 

  5. 5.

    Maldonado M, Maeyama K (2013) Simultaneous electrochemical measurement method of histamine and Nτ-methylhistamine by high-performance liquid chromatography–amperometry with o-phthalaldehyde–sodium sulfite derivatization. Anal Biochem 432:1–7

    CAS  Article  Google Scholar 

  6. 6.

    Chen HC et al (2010) Determination of histamine and biogenic amines in fish cubes (Tetrapturus angustirostris) implicated in a food-borne poisoning. Food Control 21:13–18

    CAS  Article  Google Scholar 

  7. 7.

    Elik A, Altunay N, Gürkan R (2019) Ultrasound-assisted low-density solvent-based dispersive liquid–liquid microextraction coupled to spectrophotometry for the determination of low levels of histamine in fish and meat products. Food Anal Methods 12:489–502

    Article  Google Scholar 

  8. 8.

    Evangelista WP et al (2016) Quality assurance of histamine analysis in fresh and canned fish. Food Chem 211:100–106

    CAS  Article  Google Scholar 

  9. 9.

    Xie Z et al (2017) Tuneable surface enhanced Raman spectroscopy hyphenated to chemically derivatized thin-layer chromatography plates for screening histamine in fish. Food Chem 230:547–552

    CAS  Article  Google Scholar 

  10. 10.

    Alberto MR, Arena ME, Nadra MC (2002) A comparative survey of two analytical methods for identification and quantification of biogenic amines. Food Control 13:125–129

    CAS  Article  Google Scholar 

  11. 11.

    Hwang BS, Wang JT, Choong YM (2003) A rapid gas chromatographic method for the determination of histamine in fish and fish products. Food Chem 82:329–334

    CAS  Article  Google Scholar 

  12. 12.

    Peng J et al (2008) Development of an automated on-line pre-column derivatization procedure for sensitive determination of histamine in food with high-performance liquid chromatography–fluorescence detection. J Chromatogr A 1209:70–75

    CAS  Article  Google Scholar 

  13. 13.

    Tahmouzi S, Khaksar R, Ghasemlou M (2001) Development and validation of an HPLC-FLD method for rapid determination of histamine in skipjack tuna fish (Katsuwonus pelamis). Food Chem 126:756–761

    Article  Google Scholar 

  14. 14.

    Kovacova-Hanuskova E, Buday T, Gavliakova S, Plevkova J (2015) Histamine, histamine intoxication and intolerance. Allergol Immunopathol (Madr) 43:498–506

    CAS  Article  Google Scholar 

  15. 15.

    EC (2005) Commission Regulation (EC) No 2073/2005 of 15 Novembre 2005 on the microbiological criteria for foodstuffs. Off J Eur Comm 338:1–26

    Google Scholar 

  16. 16.

    EU, European Union (2014) Report EUR 26605 EN. Equivalence testing of histamine methods—Final report. Administrative Arrangement NL SANCO/2011/G4/JRC32515/

  17. 17.

    Hungerford JM (2010) Scombroid poisoning: a review. Toxicon 56:231–243

    CAS  Article  Google Scholar 

  18. 18.

    FAO (2012) Codex alimentarius Commission. Joint FAO/WHO Food Standar programme. Codex Committee on Fish and Fishery Products, 32 session Discussion paper histamine, pp 1–14

  19. 19.

    AOAC (2005) Association of official analytical chemists. AOAC Off Method 977:13

    Google Scholar 

  20. 20.

    Latorre-Moratalla ML et al (2009) Validation of an ultra-high-pressure liquid chromatographic method for the determination of biologically active amines in food. J Chromatogr A 1216:7715–7720

    CAS  Article  Google Scholar 

  21. 21.

    Önal A, Tekkeli SE, Önal CA (2013) Review of the liquid chromatographic methods for the determination of biogenic amines in foods. Food Chem 138:509–515

    Article  Google Scholar 

  22. 22.

    Santos MHS (1996) Biogenic amines: their importance in foods. Int J Food Microbiol 29:213–231

    CAS  Article  Google Scholar 

  23. 23.

    ISO (2017) International Organization for Standardization, Microbiology of the food chain-detection and quantification of histamine in fish and fishery products, ISO 19343

  24. 24.

    Duflos G et al (2019) Validation of standard method EN ISO 19343 for the detection and quantification of histamine in fish and fishery products using high-performance liquid chromatography. Int J Food Microbiol 288:97–101

    CAS  Article  Google Scholar 

  25. 25.

    Köse S et al (2011) Commercial test kits and the determination of histamine in traditional (ethnic) fish products-evaluation against an EU accepted HPLC method. Food Chem 125:1490–1497

    Article  Google Scholar 

  26. 26.

    Dang A, Pesek JJ, Matyska MT (2013) The use of aqueous normal phase chromatography as an analytical tool for food analysis: determination of histamine as a model system. Food Chem 141:4226–4230

    CAS  Article  Google Scholar 

  27. 27.

    Park JS et al (2010) Monitoring the contents of biogenic amines in fish and fish products consumed in Korea. Food Control 21:1219–1226

    CAS  Article  Google Scholar 

  28. 28.

    Bomke S, Seiwert B, Dudek L, Effkemann S, Karst U (2009) Determination of biogenic amines in food samples using derivatization followed by liquid chromatography/mass spectrometry. Anal Bioanal Chem 393:247

    CAS  Article  Google Scholar 

  29. 29.

    Önal A (2007) A review: current analytical methods for the determination of biogenic amines in foods. Food Chem 103:1475–1486

    Article  Google Scholar 

  30. 30.

    Directive EC (2002) 657/EC of 12 August, 1990 on implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. J Eur Communit L221:8–36

    Google Scholar 

  31. 31.

    Duflos G, Dervin C, Malle P, Bouquelet S (1999) Relevance of matrix effect in determination of biogenic amines in plaice (Pleuronectes platessa) and whiting (Merlangus merlangus). J AOAC Int 82:1097–1101

    CAS  Article  Google Scholar 

  32. 32.

    Malle P, Valle M, Bouquelet S (1996) Assay of biogenic amines involved in fish decomposition. J AOAC Int 79:43–49

    CAS  Article  Google Scholar 

  33. 33.

    Frattini V, Lionetti C (1998) Histamine and histidine determination in tuna fish samples using high-performance liquid chromatography: derivatization with o-phthalaldehyde and fluorescence detection or UV detection of “free” species. J Chromatogr A 809:241–245

    CAS  Article  Google Scholar 

  34. 34.

    Alvarez-Coque MG, Hernández MM, Camanas RV, Fernandez CM (1989) Formation and instability of o-phthalaldehyde derivatives of amino acids. Anal Biochem 178:1–7

    Article  Google Scholar 

  35. 35.

    Roth M (1971) Fluorescence reaction for amino acids. Anal Chem 43:880–882

    CAS  Article  Google Scholar 

  36. 36.

    Dorresteijn RC et al (1996) Determination of amino acids using o-phthalaldehyde-2-mercaptoethanol derivatization effect of reaction conditions. J Chromatogr A 724:159–167

    CAS  Article  Google Scholar 

  37. 37.

    Wang Z, Wu J, Wu S, Bao A (2013) High-performance liquid chromatographic determination of histamine in biological samples: the cerebrospinal fluid challenge—a review. Anal Chim Acta 774:1–10

    CAS  Article  Google Scholar 

  38. 38.

    Lerke PA, Bell LD (1976) A rapid fluorometric method for the determination of histamine in canned tuna. J Food Sci 41:1282–1284

    CAS  Article  Google Scholar 

  39. 39.

    Cinquina AL et al (2004) Validation and comparison of analytical methods for the determination of histamine in tuna fish samples. J Chromatogr A 1032:79–85

    CAS  Article  Google Scholar 

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Correspondence to Ayoub Kounnoun.

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Kounnoun, A., EL Maadoudi, M., Cacciola, F. et al. Development and Validation of a High-Performance Liquid Chromatography Method for the Determination of Histamine in Fish Samples Using Fluorescence Detection with Pre-column Derivatization. Chromatographia 83, 893–901 (2020).

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  • Histamine
  • Fish
  • Pre-column derivatization
  • Optimization
  • Validation