Histamine Test for Food Safety

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1 Histamine Test for Food Safety Easy, Rapid and Specific! 1

2 Histamine causes Allergy-like like food poisoning tuna,sardine, Mahi-Mahi, herring, etc. Histidine (amino acid) Bacteria Histamine Allergy-like food poisoning Poor handling Temperature abused 2

3 Regulation of Histamine in 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 3

4 Performance 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 ppm ppm 0.1 ppm 50 ppm Total Test Time 1 h 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 4

5 Performance comparison with other method Histamine Test Analytical Methodology Enzymatic method EIA method HPLC method Fluorometric method Detection range ppm ppm 0.1 ppm 50 ppm Total Test Time 1 h 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. Procedure Simple Somewhat complicated Complicated Very complicated Many steps Preparation of analytical apparatus Many steps, Reagent : ready to use 5

6 Comparison with other methods Histamine test HPLC method AOAC method Sample Sample Sample Sample preparation (Extraction) Boiling or water extraction 10% trichloroacetic acid extraction Neutralization Add 0.4N acetic acid 75% methanol extraction Ion-exchange treatment Ion-exchange treatment Assay Enzymatic reaction Fluorescence reaction Fluorescence reaction Assay using spectrophotometer Assay using HPLC Assay using fluorometer 6

7 Histamine Test Specific for Histamine Rapid results Easy procedure 7

8 Principle of Histamine Test(colorimetric enzyme assay) Histamine dehydrogenase (HDH)* Histamine 1-methoxy PMS WST- 8 formazan (at 460nm) 4-Imidazolylaldehyde 1-methoxy PMS (reduced form) WST- 8 * patent pending 8

9 Composition of Histamine Test 60 assays / kit Histamine standard solution 30ml 1 Bufffer 24ml 2 Enzyme reagent (desiccated) 6 Colorimetric reagent (desiccated) 6 9

10 Assay procedure of Histamine Test (1) 1. Homogenize a sample. 2. Sample 1 g, and 0.1M EDTA/Na (ph8) buffer 24 ml. 3. Boil for 20 min and then, cool the tube on ice (until it becomes <20ºC). * 4. Filtrate. * You may skip this step for canned fish and fish meal. 10

11 Assay procedure of Histamine Test (2) 5. Add distilled water to vial of reagents. 6. Add reagents and sample to test tube and mix well. 7. Incubate at 25-37ºC for 15 min. 8. Measure the absorbance at 460 nm. 11

12 Linearity of Histamine Test y = x r = 1 (1.9) Abs (1.7) (CV:%) 0.40 (2.3) 0.20 (2.5) Histamine concentration (mm( mm/assay) 12

13 Recovery of histamine added to canned tuna Added histamine(ppm ) Recovered ( ppm ) Canned tuna in oil Canned tuna in soup Recovery % % Good recovery! 13

14 Comparison of Histamine Test and other methods results Histam ine Test H PLC m ethod AOAC method 1 R aw tuna (ppm ) S toraged at 20 fo r 2 d ays 2,886 2,545 2, Histam arine 2 2, Veratox 2 2, Canned tuna (ppm ) 1 fluorometric analysis established as AOAC official method 2 commercial EIA test kit 14

15 Performance comparison with other method Histamine Test Commercial test kit HPLC method AOAC method Histamine Test Analytical Methodology Enzymatic method EIA method HPLC method Fluorometric method Detection range ppm 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. Procedure Simple Somewhat complicated Complicated Very complicated ( Many steps) (Preparation of analytical apparatus) ( Many steps, Reagent : ready to use) 15

16 Histamine Test for Food Safety Easy, Rapid and Specific! 16

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