Animal Health Research Institute

Alex food inspection lab

Microbiology of the food chain — Detection and

quantification of histamine in fish and fishery products
— HPLC method

Code No. Test Procedure ( )

Version No. 1
Issue Date 16/01/2022
No. of Pages 10

Preparation Revision Approval

Name Dr\ahmed aiad Dr\mohamed Dr\ahmed aiad
elsendiony
Position Technical manager Quality manger General manager
Sign
Date

) ‫( جدول التعديالت‬

‫اعتمادات الوثيقة‬ ‫التاريخ‬ ‫رقم الصفحة‬ ‫بيان التعديل‬ ‫مراجعة‬ ‫إصدار‬

\ ‫إصدار‬
‫مراجعة و اعتماد‬ ‫إعداد‬ ‫مراجعة‬

CONTENTS

Version : 1 Issue date: 09/01//2019 Page 1/9

Animal Health Research Institute
Alex food inspection lab

1. Purpose:
2. Scope:
3. Applicability.
4. Environment.
5. Equipment And Media.
6. Precautions.
7. Procedure.
8. Retention And Disposal.
9. Method Validation.
10. Test Report
11 Assuring the test result
12. Appendices.
13 Distribution List.

1. Purpose:
Detection and quantification of histamine in fish and fishery products
— HPLC method according to iso 19343:2017

Version : 1 Issue date: 09/01//2019 Page 2/9

Animal Health Research Institute
Alex food inspection lab

2. Scope:

This document specifies a high performance liquid chromatography (HPLC)

method to analyse histamine in fish and fishery products (fish sauces, fish
maturated by enzyme in brine, etc.) intended for human consumption.
3. Applicability.
This procedure shall be applied by all personnel who authorized to
Carry out this test.
4. Environment.
 No air currents
 No dusty atmosphere
 Atmospheric confederation .22±2 and 45-50% humidity
5. Equipments and Media.
5.1. Equipments
Grinder, e.g. mixer, blender.

Balances, (precisions 0,1 g and 0,001 g).

Crusher, (homogenizer) with metallic rods.

Centrifuge, refrigerated, capable of a centrifugal force of 8 000g.

Centrifuge tubes, (plastic) with closing caps.

Pipettes, ranges 20 µl to 200 µl and 100 µl to 1 000 μl.

Tubes, (temperature resistant glass) with closing caps.

Vortex.
Nitrogen evaporator
Needles
Syringes
Filters
LC system
LC column (c18 5µm , 100 A (25 cm x4.6 mm) or equivalent

Version : 1 Issue date: 09/01//2019 Page 3/9

Animal Health Research Institute
Alex food inspection lab

Reagents and materials

Acetone, CH3COCH3.

Acetonitrile, CH3CN.

Toluene, C7H8.

Water, HPLC grade.

Water, distilled or equivalent.

Nitrogen, gas.

Perchloric acid, c(HClO4) = 0,2 mol/l (recommended).

Dilute 19,5 ml of HClO4 65 % or 17,2 ml of HClO4 70 % to 1 000 ml of water . The solution is stable for six
months if stored at room temperature (15°C to 25°C).

Saturated sodium carbonate solution, Na2CO3.

Dissolve 110 g of sodium carbonate in about 150 ml of water . The solution is in excess and stable for three
months if stored at 5 °C ± 3°C.

Dansyl chloride solution, ρ(C12H12ClNO2S) = 7,5 mg/ml.

Dissolve 0,375 g of dansyl chloride in 50 ml of acetone . The solution is stable for three weeks if
stored in the dark at a temperature lower than < −18 °C.

L-proline solution, ρ(C5H9NO2) = 100 mg/ml.

Dissolve 1 g of L-proline in 10 ml of water . The solution is stable for three weeks if stored at 5 °C ±
3°C.

Histamine stock solution, ρ(C5H9N3) = 12,5 mg/ml.

Dissolve 1,034 g of histamine dihydrochloride in 50 ml of water . The solution is stable for one year if stored
at < −18 °C.

Internal standard (IS) 1,7-diaminoheptane stock solution, ρ(C7H18N2) = 6,4 mg/ml.

(recommended).

Version : 1 Issue date: 09/01//2019 Page 4/9

Animal Health Research Institute
Alex food inspection lab

Dissolve 0,320 g of 1,7-diaminoheptane in 50 ml of water .The solution is stable for three weeks if stored at
5 °C ± 3°C.

6. Precautions.
 Avoid unnecessary talking
 Wear a laboratory coat to protect your cloths
 Do not put any thing in or near your mouth
 Wash your hands with soap and water.
 Test portion temperature should not exceed 22± 2 oC
 Operation should not be carried out in direct sunlight.
 Mouth pipetting is prohibited.

7. Procedure.
7.1 Sample preparation
Homogenize the sample (fish flesh) by grinding in mixture. transfer test portion of 5 g ±0.1
g of fish homogenate to centrifuge tube

7.2 extraction
- add 10 ml of perchloric acidand 100 µl 1,7diaminoheptan to 5 g fish in centrifuge tube
and mix
- after complete homogenization centrifuge 8000 g for 5 min at 4 º c

Version : 1 Issue date: 09/01//2019 Page 5/9

Animal Health Research Institute
Alex food inspection lab

7.3 derivatization
transfer 100 µl of supernatuant into a tube , add 300 µl of sodium
-
carbonate solution and 400 µl of dansyl chloride solution

- vortex and incubate at 60 º c in the dark for 5 min

- Cool under cold tape water , add 100 µl of L -proline solution

- vortex and place it in the dark for 15 min
note: supernatant can be stored at -18 ºc

7.4 purification

- add 500 µl of toluene and vortex .

note : manipulation can be stopped at this stage with storage at at -18 ºc for
one week maximum .
- transfer as much as possible of upper phase into new tube and dry it in a fume
of hood with nitrogen stream.
note: derivatized histamin found in organic phase of toluene and not the non
organic (aqueous phase ) , freezing at -18 º c for 30 min increase quality of
organic phase .
- resuspend dry tube with 200 µl of acetonitrile / water (60/40 volume
fraction )and vortex .
- filter the solution in a glass autosampler vial and fill vial
7.5 LC condition
Adjust hplc and column
7.5 range of standard sample
Standard samples should be prepared by supplementation of volumes of histamine stock solutions
to homogenate samples prepared according to 7.1 from histamin free matrix

- add volumes as specified in table 1 to histamine free samples and procced to extraction ,
derivatization , purification and LC

Version : 1 Issue date: 09/01//2019 Page 6/9

Animal Health Research Institute
Alex food inspection lab

Level Volume of histamin stock solution

Mg/kg µl
0 0
25 10
50 20
100 40
250 100
500 200
8 .Calculation
Calibration curve
Perform calibration by linear regression analysis using histamine standard samples and internal
standard with this formula :

Version : 1 Issue date: 09/01//2019 Page 7/9

Animal Health Research Institute
Alex food inspection lab

The mass m usually corresponding to 5g but if result fallout the standard sample repeat
test with smaller test portion .

9. Method Validation .

The test procedure validation is not needed because the procedure is

derived from ISO 19343:2017. .
10. Test Report .
The final laboratory report has a ( form 20-1)
11. Assuring the quality of test results
The laboratory applied the quality control program to insure the quality
11.1 Internal quality control :
 Spiked samples
 Repeatability
 Duplicate test
12. Appendixes :
ANNEX A : LC CONDITION
ANNEX B : CHROMATOGRAM

13. Distribution List :

 Technical Manger specialty
 Staff of HPLC UNIT

Version : 1 Issue date: 09/01//2019 Page 8/9

Animal Health Research Institute
Alex food inspection lab

Version : 1 Issue date: 09/01//2019 Page 9/9

Animal Health Research Institute
Alex food inspection lab

Version : 1 Issue date: 09/01//2019 Page 10/9