Liquid Chromatography Determination of

Histamine in Traditionally Salted, Smoked and

Frozen Fish with Relation to Microbial Load.

Presented

By

Mohamed M. Deabes S.A.Ahmed, Hayam A. Mansour, Laila A. Mohamed, and Doha A. Salah El din

*Department of Food Toxicology and Contaminants , National Research Centre, Egypt

.

Introduction Histamine Formation

Histamine or β-aminoethylimidazole

Histamine (or scombroid) fish poisoning (HFP) is a

foodborne chemical intoxication caused by eating

spoiled, or bacterially contaminated, fish.

The fish are harmless when fresh, and after they have become toxic they

may still have a normal appearance and odour.

Foods Incriminated in Histamine

Poisoning

Scombroid poisoning (scombrotoxicosis) is a worldwide

problem of food borne intoxication caused by the

consumption of seafood containing large quantities of

histamine.

Scombroid fish including tuna, mackerel, saury.

Non-scombroid fish including bluefish, mahi-mahi,

sardine, anchovy, herring, marlin and swordfish.

Most of these fish species are rich in free histidine with

salmon and swordfish being exceptions.

The histamine poisoning is a chemical intoxication that

occurs after the ingestion of foods usually containing high

levels of histamine.

The symptoms can be cutaneous (rach, urticaria,

edema, Localized inflammation), gastrointestinal

(nausea, vomiting, diarrhea), hemodynamic

(hypertension) and neurological (headache, palpitations

tingling, flushing or burning, itching).

Symptomatology of Histamine Poisoning

Histamine toxicity and its

potentiators

The detoxification system is composed of two

distinct enzymes:

Diamineoxidase (DAO)

Histamine -N- methyltransferase (HMT)

Regulatory Limits of Histamine

Foods.

Toxicity

Histamine concentrations mg/ 100 g

Safe for consumption < 5

Possibly toxic 5 - 20

Probably toxic 20 -100

Toxic and unsafe for

consumption

> 100

In Germany, maximum permissible limits of biogenic amines for

fish and fish products 200mg /Kg of food where as it is only 100mg

/Kg in Canada, Finland and Switzerland (Lange and

Wittmann,2002).

A maximum histamine content of 200 mg/kg has been established in

the (EC) for acceptance of tuna and other fish .

EU suggested that in the future, a maximum of 300 mg/kg for total

biogenic amines in fish and fish products.

While according to the Egyptian Standard Specifications (2005) the

histamine content not exceed 200 mg/kg for smoked, salted and

frozen fish products.

The study by Deabes(2000) found that smoked herring fish

were collected from great Cairo safe for human health

regarding to histamine poisoning.

Tao et al., (2011) investigated histamine content in some fresh

scombroid fish including tuna ,mackerel , and non-scombroid

fish including mahi–mahi sardine, herring and fish products

(sauce, dried fish) which came from Fiji, Germany, the

Netherlands, Norway, Thailand, Cambodia, the Philippines,

Japan and China. They found histamine detected in 35 of the

159 fish samples tested for a detection rate of about 21%. Fish

samples exceeding 50 ppm histamine occurred in about 9% of

the samples with 5 samples exceeding 500 ppm of which 2 were

above 1000 ppm.

Histamine Exposure

Detection and determination of Histamine in food

Thin layer chromatographic (TLC)

Bioassay methods .

Calorimetric methods.

Gas Liquid Chromatography .

High Performance Liquid

Chromatography (HPLC).

TLC separation of the tested dansylamines using Chloroform:

Benzene: Triethylamine (6:4.5:1)Deabes,(2000)

1-tryptamine, 2-putrescine, 3-cadaverine, 4-spermidine, 5-histamine, 6- spermine, 7-tyramine and 8- β-phenylethylamine.

• Can assay numerous samples with a

minimum of time

• effort and cost and also it is simpler has

satisfactory accuracy.

Therefore it can be available in all food

testing laboratories for quantitative and

qualitative determination of all tested

biogenic amines.

The aim of the work

This work was planned to give some

additional scientific data on Histamine and

its relation to microbial load in

traditionally fish products in Egypt.

To monitor the effectiveness of the controls

in place to limit consumer exposure to the

hazard level of histamine poisoning.

Materials and Methods

Materials:

(a) Collection of fish product samples

A total of 50 random samples of smoked (Herring),

salted (Fesikh and Moloha) and frozen fish products

(Fish fillet and Fish fingers) ten from each product were

collected randomly from different supermarkets in

different localities in Cairo and Giza governorates

Egypt.

Samples were transferred to the laboratory in ice box

subjected to bacteriological examination, measurement

of pH-value and determination of histamine by HPLC.

Methods

(b) Bacteriology examinations:

1-Aerobic plate counts (APC),Enterobacteriaceae count ,

Psychrophilic count and Presumptive Coliforms test

according to (ICMSF, 1978).

2-Confirmatory test for Coliforms (APHA, 1992) and 5-

Enumeration of Fecal Coliform Bacteria 'MPN' (FAO,

1992).

Equipment and chromatographic

conditions

High-performance liquid chromatograph (Agilent 1100 series

)equipped with a photodiode array DAD (G 1315 B)

Reverse-phase analytical column packed with C18 material

(Agilent ZORBA,X DB- 5 μm, 150 mm × 4.6 mm ) .

Separation was performed at ambient temperature at a flow rate of

1.0 ml min-1

Injection volume was 50 µl for both standard solutions and sample

extracts by auto sampler(G1329A).

The Dad detector was operated at an 254 nm .

Table:(1)Solvent A and B were used in gradient elution

program as follow

The gradient program used for

histamine separation is illustrated in the following table (1).

Mobile phase solvents consists of:

Solvent A : Acetonitrile : 0.02 N acetic acid ( 1:9) and

Solvent B : 0.02 N acetic acid : acetonitrile : methanol (1 : 9 : 9).

25 g food sample + 125 ml of TCA 5% in warring blender (3min)

Filtration

10 ml Extract + 4 g NaCl + 1 ml NaOH 50% in culture tube

Shaking by vortex

Centrifugation

Extraction by 3 x5 ml CHCL3 + butanol (1:1)

+ 15 ml n-heptane

Extraction by 1 x 3 ml HCl 0.2 N

Collection 3 ml extract

Evaporation at 95°C

0.5 ml NaHCO3 +1 ml dansyl chloride

Incubation 45 min. at 55°C

Extraction by 10 ml distillated water + 3 x 5 ml diethylether

Evaporation of diethylether extract at 35°C

Dissolving in methanol

Injection in HPLC Spotting on TLC

Schematic Diagram of biogenic amines determination

(Deabes 2000).

Results and Discussion

Table(2):Statistical analysis of histamine content (μg/100g) in

examined fish products

Fig (1):Histamine (μg/ 100g) in examined fish

products

It is evident from the result recorded in table (2) that the

mean values of histamine among fesikh was the highest

followed by moloha, smoked herring, frozen fish fillet and

finally frozen fish fingers which was the lowest.

The mean values for fesikh, moloha, smoked herring,

frozen fish fillet and frozen fish fingers were 117.6, 110.8,

3.3, 2 and 1.74μg / 100g respectively.

Table (3):Correlation between Histamine with the

other studied parameters

The highest histamine content was among fesikh

there was a significant correlation between

histamine content and APC, Enterobacteriaceae

count, Pseudomonas count, Coliform count.

However, there was inverse correlation between

histamine and pH.

Fig(2): Enterobacteriaceae counts in examined fish

products

From these results its worthy to mention that the mean values

of Enterobacteriaceae count among frozen fish fingers was the

highest followed by frozen fish fillet, smoked herring, fesikh

and finally moloha which was the lowest in this products as

many types of bacteria cannot

tolerate the high salt concentration and reduced moisture

content also smoking reduce growth and presence of many

types of bacteria.

Figure (3):Dansylated of Histamine standard and fish samples

overviewed by HPLC

Conclusions

Our results improved that, the use of good quality fish raw material is required to produce a safe and a good quality salted, smoked and frozen fish products.

It is clearly that histamine level in all the investigated fish products samples was much lower than the hazard level ( for both EU and Egyptian Standards allows 200 mg/kg) and this could be attributed to the types of examined fish which were non-scombroids.

Thank you for your

attention!! Mohamed M. Deabes, Ph.D

National Research Centre Food Toxicology and Contaminants Dept.,

Dokki, Cairo, EGYPT , 12311

Mobile : +2-010057-43514 or +201200922779

Fax : +202-3337-0931 Institute: +202 - 3337-0499

Email: mydeabes@yahoo.com or

mydeabes@hotmail.com