Biochemical Techniques and Instrumentation

Procedure

Preparation of standard

Take 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the histamine working standard along with blank (2.0 ml of 0.1N HCL) in separate test tubes and make up the volume to 5ml with 0.1N HCl.

Preparation of sample

Homogenize 5 g of fish with 25 ml of 0.4 N HClO₄ in a homogeniser for 1 min. Centrifuge the homogenate for 10 min at 3000 rpm. Filter the supernatant through Whatman No. 1 filter paper under slight suction and make up to 50 ml with 0.4 N HClO₄.

Extraction of sample

Transfer 5 ml of the extract into 250 ml separating funnel, add 10 ml of distilled water and make alkaline with 5 ml of 1N NaOH. Then, add 1.5 – 2.0 g of NaCl (to saturation) and extract two times with 10 ml of n-butanol. Collect all the butanolic phases. Then, wash the butanolic phases with 10 ml of 1N NaOH and 2.0 g NaCl. Remove the aqueous phase and add 25 ml of petroleum ether and shake well. Discard the aqueous phase. Then, extract again the butanolic phases two times with 10 ml of 0.1N HCl. Collect the aqueous-acid phases (0.1N HCl) in a 50 ml volumetric flask and make up to 50 ml with 0.1N HCl.

Condensation reaction

Transfer 2.0 ml solution of each standard, sample and blank in separate test tubes, add 4.0 ml of 0.1N NaOH and shake well. Then, add 0.2 ml of OPT solution immediately. Shake to mix thoroughly and let stand for 5 min. Then, add 2.0 ml of 0.2 M citric acid to the tubes and shake well. Finally, measure the fluorescence at the excitation wavelength of 340 nm and emission wavelength of 425 nm in a spectrofluorimeter.