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Histamine TestHistamine Test for Food Safetyfor Food Safety

Easy, Rapid and Specific!

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Histamine Histamine causes Allergycauses Allergy--like food poisoninglike food poisoning

tuna，sardine, Mahi-Mahi, herring, etc.

Bacteria

Allergy-like food poisoning

Poor handlingTemperature abused

HistamineHistidine (amino acid)

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Regulation ofRegulation of HistamineHistamine in Fish and Fisheries productsin Fish and Fisheries products

US (FDA/HACCP) 500ppm based on toxicity 50ppm defect action level

EU (EC 91/493/EEC) 9samples for each batch ≦200ppm, AV≦100ppm,

JAPAN (MHLW) 100ppm for control

*MHLW=the Ministry of Health and Welfare

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Performance comparison with other methodPerformance comparison with other method

Histamine Test Commercial test kit HPLC method AOAC method

Analytical Methodology Enzymatic method EIA method HPLC method Fluorometric method

Detection range 20 - 300 ppm 1 - 100 ppm 0.1 ppm 50 ppm

Total Test Time 1 h 1 - 1.5 h >6 h >6 h

Reproducibility Good Variation may occur. Excellent Variation is large.

Sensitivity Quite Accurate Accurate Very Accurate Accurate

Standard 1 point is required. ５ points are required. Required ４ points are required.

Complex procedure

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Performance comparison with other methodPerformance comparison with other method

Analytical Methodology Enzymatic method EIA method HPLC method Fluorometric method

Detection range 20 - 300 ppm 1 - 100 ppm 0.1 ppm 50 ppm

Total Test Time 1 h 1 - 1.5 h >6 h >6 h

Sensitivity Quite Accurate Accurate Very Accurate Accurate

Standard 1 point is required. ５ points are required. Required ４ points are required.

Simple Somewhat complicated Complicated Very complicated

Many stepsPreparation of analytical

apparatusMany steps, Reagent :

ready to use

Procedure

Histamine Test

Reproducibility Good Variation may occur. Excellent Variation is large.

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Comparison with other methodsComparison with other methods

10% trichloroacetic acidextraction

75% methanol extractionBoiling or water extraction

Histamine test HPLC method AOAC methodSample Sample Sample

Sample preparationSample preparation(Extraction) Neutralization(Extraction)

Add 0.4N acetic acid

Ion-exchange treatment Ion-exchange treatment

Enzymatic reaction

Fluorescence reaction

Fluorescence reactionAssayAssay

Assay using HPLC Assay using fluorometerAssay using spectrophotometer

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Histamine TestHistamine Test

Specific for Histamine

Rapid results

Easy procedure

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Principle of Principle of Histamine TestHistamine Test（colorimetric enzyme assay）

(reduced form）

Histamine dehydrogenase (HDH)*

Histamine

4-Imidazolylaldehyde 1-methoxy PMS

1-methoxy PMS

WST- 8

WST- 8 formazan（at 460nm）

* patent pending

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Composition of Composition of Histamine TestHistamine Test

60 assays / kit

Histamine standard solution 30ml×1

Colorimetric reagent(desiccated)　×6

Bufffer24ml×2

Enzyme reagent(desiccated)×6

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Assay procedureAssay procedure of of Histamine Test (1)Histamine Test (1)

2. Sample 1 g, and0.1M EDTA/Na (pH8) buffer 24 ml.

4. Filtrate.3. Boil for 20 min and then, cool the tube on ice （until it becomes <20ºC）. *

1. Homogenize a sample.

* You may skip this step for canned fish and fish meal.

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Assay procedureAssay procedure of of Histamine Test (2)Histamine Test (2)

8. Measure the absorbance at 460 nm.

7. Incubate at 25-37ºC for 15 min.

6. Add reagents and sample to test tube and mix well.

5. Add distilled water to vial of reagents.

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Linearity of Linearity of Histamine TestHistamine Test

y = 0.0378x + 0.0127r = 1

0.00

1.00

1.20

0 5 10 15 20 25 30 35

(2.5)

(1.9)

Histamine concentration (Histamine concentration (mMmM/assay)/assay)

0.20

0.40

0.60

0.80

(2.3)

(1.7)

(CV:%)Abs

A

bs 46

046

0

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Recovery of histamine added to canned tunaRecovery of histamine added to canned tuna

Canned tuna in oil 　Canned tuna in soup

Added 　　　　　　　　Recovered ( ppm )histamine(ppm )

10 11.3 10.8

20 21.6 21.2

50 51.0 49.5

75 72.0 73.5

　　　　　　　　Recovered ( )histamine(ppm )

10 11.3 10.8

20 21.6 21.2

50 51.0 49.5

75 72.0 73.5

5 5.0 5.05 5.0 5.0

Recovery Recovery 98 – 108 %RecoveryRecovery 96 – 103 %

Good recovery!

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Comparison of Comparison of Histamine TestHistamine Testand other methods resultsand other methods results

5662,545H PLC m ethod

5302,646AO AC m ethod※1

5742,728V e ※2

4632,764H istam arine※2

5372,886H istam ine Test

C anned tuna (ppm )

R aw tuna (ppm )

5662,545H PLC m ethod

5302,646AO AC m ethod※1

5742,728V eratox ※2

4632,764H istam arine※2

5372,886H istam ine Test

C anned tuna (ppm )

R aw tuna (ppm )

Storaged at 20℃　for 2 daysS toraged at 20℃　for 2 days

※※11　　fluorometric fluorometric analysis established as AOAC official method analysis established as AOAC official method ※※22　　commercial EIA test kitcommercial EIA test kit

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Performance comparison with other methodPerformance comparison with other method

Histamine Test Commercial test kit HPLC method AOAC method

Analytical Methodology Enzymatic method EIA method HPLC method Fluorometric method

Detection range 20 - 300 ppm 1 - 100 ppm 0.1 ppm 50 ppm

Reproducibility Good Variation may occur. Excellent Variation is large.

Sensitivity Quite Accurate Accurate Very Accurate Accurate

Standard 1 point is required. ５ points are required. Required ４ points are required.

Simple Somewhat complicated Complicated Very complicated

（Many steps）(Preparation of analytical

apparatus)（Many steps, Reagent :

ready to use）

Procedure

Histamine Test

Total Test Time 1 h 1～ 1.5 h >6 h >6 h

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Histamine TestHistamine Test for Food Safetyfor Food Safety

Easy, Rapid and Specific!