🐟 Histamine in fish (HPLC)
In short
Determination of histamine in fish and fishery products by HPLC with dansyl chloride derivatization and UV detection. The test is performed according to PN-EN ISO 19343:2017; Calibration range: 10–500 mg/kg. The procedure comprises 12 steps (about 3 h 27 min in total); it is used for: Mandatory histamine control in Scombridae fish (EC Reg. 2073/2005), Testing of tuna, mackerel, bonito, anchovy, herring, sardine, Fish canned goods and products control (pastes, fish sauces).
At a glance
- Standard: PN-EN ISO 19343:2017
- Category: Food Chemistry
- Procedure steps: 12
- Total time of stages: 3 h 27 min
- Analyte: Histamine (2-[4-imidazolyl]ethylamine)
- LOQ: 5–10 mg/kg
- Calibration range: 10–500 mg/kg
Overview
Histamine is a biogenic amine formed from histidine decarboxylation by bacterial enzymes (histidine decarboxylase) in fish and fishery products during improper storage (too high temperature). Histamine poisoning (scombroid fish poisoning) manifests with headaches, urticaria, nausea, and diarrhea, and in severe cases — breathing difficulties and blood pressure drop.
EU regulations (EC Reg. 2073/2005) require histamine testing in fish from families with high histidine content (Scombridae — tuna, mackerel, bonito; Clupeidae — herring, sardine; Engraulidae — anchovy). Limits: 9 sample units, with m = 100 mg/kg (max average) and M = 200 mg/kg (no sample may exceed). For fish ripened in brine (fish sauces, anchovies): m = 200, M = 400 mg/kg.
ISO 19343 method is based on histamine extraction with perchloric acid, derivatization with dansyl chloride (5-(dimethylamino)naphthalene-1-sulfonyl chloride), separation on HPLC C18 column in reversed phase and UV detection at 254 nm. Dansyl derivatization is necessary because histamine has no strong chromophore for direct UV detection. Alternatively, OPA (o-phthalaldehyde) derivatization with fluorescence detection or direct LC-MS/MS is used.
Histamine is thermostable — it is not decomposed during cooking, frying, or preservation. Therefore, the only way of prevention is maintaining the cold chain from catch to consumer. Histamine testing is a key element of HACCP system in fish processing.
Method principle
Fish sample (10 g) is extracted with 0.4 mol/L perchloric acid by homogenization and centrifugation. Supernatant is cleaned and subjected to pre-column derivatization with dansyl chloride (dansyl-Cl) in alkaline environment (pH 9–10, carbonate buffer). Dansyl-Cl reacts with histamine amino groups, forming fluorescent/UV-absorbing dansyl-histamine derivative. Excess dansyl-Cl is removed by reaction with proline. Biogenic amine derivatives are separated on C18 HPLC column in acetonitrile/water gradient and detected by UV (254 nm). Histamine concentration is calculated from calibration curve using internal standard (1,7-diaminoheptane).
Applications
- Mandatory histamine control in Scombridae fish (EC Reg. 2073/2005)
- Testing of tuna, mackerel, bonito, anchovy, herring, sardine
- Fish canned goods and products control (pastes, fish sauces)
- Anchovy and brine-ripened fish monitoring
- HACCP system in fish processing
- Fish cold chain control (freshness indicator)
- Food testing under RASFF (rapid alert) program
Key parameters
| Parameter | Value |
|---|---|
| Analyte | Histamine (2-[4-imidazolyl]ethylamine) |
| LOQ | 5–10 mg/kg |
| Calibration range | 10–500 mg/kg |
| EU limit (m) | 100 mg/kg (fresh fish), 200 mg/kg (fish sauces) |
| EU limit (M) | 200 mg/kg (fresh fish), 400 mg/kg (fish sauces) |
| Derivatization | Dansyl chloride (pre-column) or OPA (post-column) |
Standard
- Standard number
- PN-EN ISO 19343:2017
- Title (PL)
- Mikrobiologia łańcucha żywnościowego — Wykrywanie i oznaczanie ilościowe histaminy w rybach i przetworach 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
-
Sample preparation
Homogenize fish sample. Weigh 10 g into 50 mL centrifuge tube.
-
Extraction
Add 20 mL 0.4 mol/L perchloric acid. Homogenize 2 min (Ultra-Turrax). Centrifuge 10 min at 3000 rcf.
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Derivatization preparation
Take 1 mL supernatant into screw-cap tube. Add 200 µL internal standard solution (1,7-diaminoheptane).
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Alkalization
Add 300 µL saturated NaHCO₃ and 200 µL 2 mol/L NaOH. Mix.
-
Dansyl-Cl derivatization
Add 2 mL dansyl chloride (10 mg/mL in acetone). Mix on vortex. Incubate 45 min at 40°C.
-
Excess dansyl-Cl removal
Add 100 µL L-proline solution (100 mg/mL). Incubate 15 min at room temperature.
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Derivative extraction
Add 2 mL toluene or ethyl acetate. Shake 1 min. Collect organic phase. Evaporate to dryness under N₂.
-
Sample reconstitution
Dissolve dry residue in 200 µL acetonitrile/water (60:40). Mix on vortex. Filter into HPLC vial.
-
HPLC analysis
Inject 20 µL onto C18 column. Gradient: 50→95% MeCN in 25 min. UV detection 254 nm. Flow 1 mL/min.
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Quantification
Calculate histamine/IS peak area ratio. Read concentration from calibration curve. Include dilution.
-
Result interpretation
Compare with EU limits: n=9, c=2, m=100 mg/kg, M=200 mg/kg (fresh fish). No sample may exceed M.
-
Quality control
Every series: blank, spike (50 and 200 mg/kg), CRM (FAPAS). Recovery 80–110%, RSD < 15%. PT participation.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| HPLC system with UV/DAD detector | Shimadzu Nexera + SPD-M40, Agilent 1260 + DAD, Waters Alliance + PDA | 120 000–300 000 PLN |
| HPLC C18 column | Waters Symmetry C18 (250 × 4.6 mm, 5 µm), Phenomenex Luna C18 | 1 500–3 000 PLN |
| Homogenizer | IKA Ultra-Turrax T25, Kinematica Polytron PT 2500E | 5 000–15 000 PLN |
| Laboratory centrifuge | Hettich Rotina 380 R, Eppendorf 5810 R (≥3000 rcf) | 15 000–40 000 PLN |
| Water bath 40°C | Memmert WNB 7, Julabo TW 12 | 3 000–8 000 PLN |
| Fume hood / fume cupboard | For work with dansyl chloride and perchloric acid | 15 000–40 000 PLN |
| Analytical balance 0.1 mg | Mettler Toledo ME204, Radwag AS 220.R2 | 8 000–20 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Histamine dihydrochloride (standard) | 56-92-8 | Calibration standard, certified CRM, 1 g |
| Dansyl chloride (dansyl-Cl) | 605-65-2 | Derivatizing reagent, 5-(dimethylamino)naphthalene-1-sulfonyl chloride, 1 g |
| Perchloric acid 0.4 mol/L | 7601-90-3 | Histamine extraction from sample, 500 mL |
| 1,7-diaminoheptane (internal standard) | 646-19-5 | Internal standard IS, 5 g |
| Carbonate buffer pH 9–10 | — | Derivatization reaction environment (NaHCO₃/Na₂CO₃) |
| L-proline | 147-85-3 | Removing excess dansyl chloride after derivatization, 25 g |
| Acetonitrile HPLC grade | 75-05-8 | Mobile phase and solvent, 2.5 L |
| HPLC grade water (ultrapure) | — | Mobile phase, Milli-Q system (18.2 MΩ·cm) |
Health and safety (OHS)
- Perchloric acid — strong oxidizer (H271, H314). Explosion risk in contact with organic materials. Use gloves and goggles.
- Dansyl chloride — irritating (H315, H319). Work under fume hood. Avoid inhaling vapors.
- Acetonitrile — flammable and toxic. Work under fume hood.
- Toluene — flammable and toxic (H225, H304, H315, H336, H361d, H373). Work under fume hood.
- Fish samples — may contain pathogens. Use gloves. Dispose as biological waste.
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 19343:2017 — “Microbiology of the food chain — Detection and quantification of histamine in fish and fishery products — HPLC method”.
How does this method work?
Fish sample (10 g) is extracted with 0.4 mol/L perchloric acid by homogenization and centrifugation.
What is the measuring range and accuracy?
Analyte: Histamine (2-[4-imidazolyl]ethylamine); LOQ: 5–10 mg/kg; Calibration range: 10–500 mg/kg; EU limit (m): 100 mg/kg (fresh fish), 200 mg/kg (fish sauces).
How long does the test take?
The times given for the individual stages add up to approximately 3 h 27 min. The procedure comprises 12 steps.
What equipment is required?
HPLC system with UV/DAD detector, HPLC C18 column, Homogenizer, Laboratory centrifuge, Water bath 40°C, Fume hood / fume cupboard, Analytical balance 0.1 mg. Indicative cost of the core instrument (HPLC system with UV/DAD detector): 120 000–300 000 PLN.
Where is this test used?
Mandatory histamine control in Scombridae fish (EC Reg. 2073/2005); Testing of tuna, mackerel, bonito, anchovy, herring, sardine; Fish canned goods and products control (pastes, fish sauces); Anchovy and brine-ripened fish monitoring; HACCP system in fish processing; Fish cold chain control (freshness indicator); Food testing under RASFF (rapid alert) program.
What safety precautions apply?
Perchloric acid — strong oxidizer (H271, H314). Explosion risk in contact with organic materials. Use gloves and goggles.; Dansyl chloride — irritating (H315, H319). Work under fume hood. Avoid inhaling vapors.; Acetonitrile — flammable and toxic. Work under fume hood.; Toluene — flammable and toxic (H225, H304, H315, H336, H361d, H373). Work under fume hood.; Fish samples — may contain pathogens. Use gloves. Dispose as biological waste.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 19343.