🐟 Histamine in Fish and Fishery Products — HPLC with Dansyl Chloride Derivatization

Food Chemistry PN-EN ISO 19343

Overview

STATUS (as of 02.11.2026). PN-EN ISO 19343:2017-08 — English version with a Polish title (in translation: “Microbiology of the food chain — Detection and quantification of histamine in fish and fishery products — HPLC method”), implements EN ISO 19343:2017 and ISO 19343:2017 [IDT]; no withdrawal note in the PKN catalogue (PKN search, 02.11.2026). ISO 19343:2017 is the first edition of the standard (published on 28.06.2017), current according to Standard Norge; it was prepared by CEN/TC 275 in collaboration with ISO/TC 34/SC 9. The description is based on the preview of ISO 19343:2017 (iTeh: introduction, clauses 1–9 without Table 2) and on the validation publication of the method (G. Duflos et al., Int. J. Food Microbiol. 2019, 288: 97–101).

INTRODUCTION OF THE STANDARD. Histamine causes scombroid poisoning (histamine fish poisoning). It occurs mainly in fish of the families Scombridae (tuna, mackerel) and Clupeidae (herring, sardine), which contain a lot of free histidine; it is formed by decarboxylation of histidine by microbial histidine decarboxylase. Consumption of food with a significant histamine concentration can cause symptoms similar to those of food allergy. The standard was developed to harmonize the method for the detection and quantification of histamine in fish and fishery products, in particular for the purposes of Regulation (EC) No 2073/2005.

SCOPE AND PRINCIPLE (clauses 1 and 4). An HPLC method for the determination of histamine in fish and fishery products (e.g. fish sauces, fish that has undergone enzyme maturation treatment in brine) intended for human consumption. The method separates histamine from other biogenic amines: the sample is extracted with perchloric acid, pre-column derivatization is carried out with dansyl chloride, the amines are separated by HPLC on a suitable column with UV detection, and the histamine concentration is calculated from the ratio of the histamine peak area to the internal standard peak area, using a calibration curve.

REAGENTS (clause 5). Water of grade 1 according to ISO 3696 (unless otherwise specified), HPLC-grade solvents: acetone, acetonitrile, toluene; nitrogen. Perchloric acid 0.2 mol/l (recommended): 19.5 ml of 65 % acid or 17.2 ml of 70 % acid to 1000 ml of water (stable for 6 months at 15 °C to 25 °C). Saturated sodium carbonate solution: 110 g in about 150 ml of water (stable for 3 months at 5 °C ± 3 °C). Dansyl chloride 7.5 mg/ml: 0.375 g in 50 ml of acetone (stable for 3 weeks in the dark below −18 °C). L-proline 100 mg/ml: 1 g in 10 ml of water (3 weeks at 5 °C ± 3 °C). Histamine stock solution 12.5 mg/ml: 1.034 g of histamine dihydrochloride in 50 ml of water (one year below −18 °C). Internal standard (recommended) — 1,7-diaminoheptane 6.4 mg/ml: 0.320 g in 50 ml of water (3 weeks at 5 °C ± 3 °C).

APPARATUS (clause 6). Grinder (mixer, blender), balances accurate to 0.1 g and 0.001 g, homogenizer with metal rods, refrigerated centrifuge up to 8 000 g, plastic screw-cap centrifuge tubes, pipettes from 20 µl to 200 µl and from 100 µl to 1 000 µl, screw-cap tubes of heat-resistant glass, vortex mixer, water bath at 60 °C ± 1 °C with a dark cover, refrigerator at 5 °C ± 3 °C, freezer below −18 °C, nitrogen evaporator, 20G needles (0.9 mm), 0.2 µm filters (PTFE or PP), 2 ml syringes, 2 ml glass vials with a 200 µl insert. Chromatograph: pump, refrigerated sample injector, column oven at 25 °C ± 2 °C, UV detector at 254 nm; C18 column, 5 µm, 100 Å, 25 cm × 4.6 mm or equivalent. HPLC and UHPLC instruments can be used; indicative separation conditions are given in informative Annex A.

PROCEDURE (clause 7). The fish flesh is homogenized in a mixer; a test portion of 5 g ± 0.1 g is transferred to a centrifuge tube. 10 ml of perchloric acid and 100 µl of the 1,7-diaminoheptane solution are added, the mixture is homogenized and then centrifuged for 5 min at 8 000 g at 4 °C. 100 µl of the supernatant, 300 µl of the sodium carbonate solution and 400 µl of the dansyl chloride solution are pipetted into a tube, mixed and incubated for 5 min in the dark at 60 °C; after cooling under cold water, 100 µl of the L-proline solution are added, mixed and left for 15 min in the dark. 500 µl of toluene are added and mixed, as much as possible of the upper organic phase (containing the histamine derivative) is transferred to a new tube and evaporated to dryness under a stream of nitrogen in a fume hood; separation of the organic phase is made easier by freezing the aqueous phase (below −18 °C, at least 30 min). The dry residue is dissolved in 200 µl of a mixture of acetonitrile and water 60/40 (V/V), mixed, filtered through a 0.2 µm filter into a vial and injected. The supernatant, or the sample after addition of toluene, can be stored below −18 °C for at most one week. Calibration samples are prepared by adding histamine stock solution to a homogenate of a histamine-free matrix — for example 0, 25, 50, 100, 250 and 500 mg/kg (0, 10, 20, 40, 100 and 200 µl) — and taking them through the whole procedure; calibration on the same matrix as the sample prevents matrix effects and bias in the result.

RESULT (clauses 8–9). The calibration function is determined by linear regression of the ratio of the peak areas of histamine and the internal standard. Histamine concentration in the sample: C = (A_H / A_IS) · 5 / (a · m) in mg/kg, where a is the slope of the calibration line and m the mass of the test portion (usually 5 g); if the result falls outside the range of the calibration samples, the analysis is repeated with a smaller test portion. For complex matrices or matrices that are difficult to obtain free of histamine (e.g. fishmeal, fish sauce), the standard addition method is used. The repeatability and reproducibility limits were determined in an interlaboratory study — according to the validation publication carried out in 2013 in 9 laboratories from 7 European countries for fish that had undergone enzyme maturation, fish without maturation and fish sauce; the method was standardized under mandate M381 of the European Commission.

REGULATIONS. Regulation (EC) No 2073/2005 (consolidated text of 01.07.2026, Annex I, Chapter 1) specifies EN ISO 19343 as the reference method for the criteria: 1.26 — fishery products from fish species associated with a high amount of histidine: n = 9, c = 2, m = 100 mg/kg, M = 200 mg/kg; 1.27 — such products that have undergone enzyme maturation treatment in brine (except 1.27a): n = 9, c = 2, m = 200 mg/kg, M = 400 mg/kg; 1.27a — fish sauce produced by fermentation of fishery products: n = 1, c = 0, limit 400 mg/kg; the criteria apply to products placed on the market during their shelf life. This concerns in particular species of the families Scombridae, Clupeidae, Engraulidae, Coryfenidae, Pomatomidae and Scombresosidae. The result for criteria 1.26–1.27 is satisfactory if the mean value does not exceed m, at most c of the n values lie between m and M and no value exceeds M; single samples may be taken at retail level.

WHAT WE DO NOT GIVE HERE. The indicative HPLC separation conditions (Annex A), the repeatability and reproducibility values (Table 2) or the performance characteristics of the method, e.g. the limit of quantification (Annex B) — these parts of the standard are not in the preview.

Method principle

A test portion of homogenized fish flesh (5 g) is extracted with 10 ml of perchloric acid 0.2 mol/l with 1,7-diaminoheptane added as the internal standard and centrifuged. The biogenic amines in the supernatant are derivatized with dansyl chloride in saturated sodium carbonate (5 min at 60 °C, in the dark), L-proline is then added, and the derivatives are extracted with toluene, evaporated and dissolved in a mixture of acetonitrile and water.

The derivatives are separated by HPLC on a C18 column and detected with a UV detector at 254 nm. The histamine concentration is calculated from the ratio of the peak areas of histamine and the internal standard, using a calibration prepared on the same histamine-free matrix (or by the standard addition method).

Applications

• Fish and fishery products intended for human consumption, including fish sauces and fish that has undergone enzyme maturation treatment in brine — histamine (clause 1)
• Food safety criteria 1.26, 1.27 and 1.27a of Regulation (EC) No 2073/2005 — EN ISO 19343 is the reference method
• Species associated with a high amount of histidine: families Scombridae, Clupeidae, Engraulidae, Coryfenidae, Pomatomidae and Scombresosidae (Regulation (EC) No 2073/2005, footnote 17)
• Complex matrices or matrices difficult to obtain free of histamine (e.g. fishmeal, fish sauce) — calibration by the standard addition method (note to 8.2)

Key parameters

Standard

Standard number

PN-EN ISO 19343:2017-08

Title (PL)

Mikrobiologia łańcucha żywnościowego — Wykrywanie ilościowe i oznaczanie histaminy w rybach i produktach rybnych — Metoda HPLC

Title (EN)

Microbiology of the food chain — Detection and quantification of histamine in fish and fishery products — HPLC method

Step-by-step procedure

1. Preparation of the sample

   Homogenization of the fish flesh in a mixer; test portion of 5 g ± 0.1 g into a centrifuge tube (7.1).

2. Extraction

   10 ml of perchloric acid 0.2 mol/l and 100 µl of 1,7-diaminoheptane; homogenization; centrifugation for 5 min at 8 000 g at 4 °C (7.2).

3. Derivatization

   100 µl of supernatant, 300 µl of saturated sodium carbonate, 400 µl of dansyl chloride; 5 min in the dark at 60 °C; cooling, 100 µl of L-proline, 15 min in the dark (7.3).

4. Clean-up

   500 µl of toluene, mixing; upper organic phase into a new tube, evaporation with nitrogen in a fume hood; residue in 200 µl of acetonitrile with water 60/40; 0.2 µm filter into the vial (7.4).

5. HPLC analysis

   Separation on a C18 column at 25 °C ± 2 °C, UV detection at 254 nm; indicative conditions — Annex A (6.16–6.17, 7.5).

6. Calibration

   Calibration samples from a histamine-free matrix (e.g. 0–500 mg/kg) through the whole procedure; linear regression of the ratio of the peak areas of histamine and the internal standard (7.6, 8.1).

7. Result

   C = (A_H / A_IS) · 5 / (a · m) in mg/kg; outside the range — repetition with a smaller test portion; assessment against the criteria of Regulation (EC) No 2073/2005 (8.2).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The 65–70 % perchloric acid from which the 0.2 mol/l solution is prepared is a strong oxidizer and corrosive — follow the safety data sheet
• Toluene is flammable and harmful — the organic phase is evaporated under a stream of nitrogen in a fume hood (7.4); follow the safety data sheets

Frequently asked questions