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