Determination of Histamine Originating from Aquatic Products

Method: SPE
Matrix: Aquatic Products
Application No.: 101721
Columns: ProElut PXC 150 mg / 6 mL 30/pk
Cat No.: 68204
Sample Pretreatment: 1. Sample preparation / extraction
1.1. Weighing
Smoked fish and other dried samples: Weigh 2.5 g of sample (accurate to 0.01 g) in 50 mL centrifuge tube.
Tuna and other wet samples: Weigh 5.0 g of sample (accurate to 0.01 g) in 50 mL centrifuge tube.
1.2. Extraction of histamine
Add 20 mL histamine extract* to centrifuge tube, vortex 1 min, shock in thermostatic water bath for 30 min at 60 ºC, centrifuge at 4,000 rpm for 10 min. Take 6 mL of of supernatant and adjust the pH to be between 2 - 3 with 50% H3PO4 as the sample solution to be purified.
*Histamine extract: MeOH:50 mM KH2PO4 = 1:1
2. Sample purification
ProElut™ PXC 150 mg / 6 mL (Cat#68204)
Condition: 6 mL MeOH / 6 mL H2O
Load: 6 mL supernatant
Wash 1: 6 mL 0.1 M HCl
Wash 2: 6 mL solution of NH4OH:MeOH:H2O = 5:5:90
Elute: 6 mL solution of NH4OH:MeOH:H2O = 5:60:35
Reconstitute: Reconstitute to 6 mL with elution solvent
Conditions: HPLC method
Column: Inspire™ 5 µm C18, 150 x 4.6 mm (Cat#81001)
Mobile Phase: A: MeOH, B: Phosphoric acid - triethylamine buffer*, A:B = 40:60
Flow Rate: 1.0 mL/min
Temperature: 30 ºC
Detection: FLD Ex: 345 nm, Em: 445 nm
Injection Volume: 10 µL
Injection Procedure: 7.5 µL o-Phthalaldehyde (OPA) + 10 µL sample + 7.5 µL OPA
* Phosphoric acid - triethylamine buffer: Add 12.5 mL triethylamine and 25.7 mL phosphoric acid to 1 L deionized water
Author: Dikma Technologies Inc.
Keyword: Histamine, Aquatic Products, SPE