ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ...

B.A.l.5.4.5 Liquid Chromatography Mass-Spectroscopy
The extracts of Amadumbe extracted with hexane and methanol-chloroform mixture were
dissolved in methanol and the dichloromethane. 200 J-Ll ofthis solution was then made up
to 2 ml using the mixture of methanol and water. The analysis of the fatty acids was
conducted under the following conditions by LCMS whereby the mass spectrometer
conditions were kept in the capillary voltage of3500 kV, cone voltage, ISO kV, desolvation
temperature 250 DC and desolvation gas flow rate 300 DC. The solvents used was methanol
and water at the flow rate of 0.035 ml min· l . The dissolved extracts were then injected in
LCMS at an injection volume of5 J-Ll.
B.A.2 Determination ofanti-nutrients
B.A.2.1 Trypsin inhibitor [Smith et aL (1980»)
Ig Amadumbe powder was extracted by homogenizing it in 20 g/litre of NaCl at the ratio
1:10 (w/v). The homogenate was stirred for 24 hours at room temperature, passed through a
layer of cheese cloth and centrifuged at 9000 rpm for 15 minutes. The supernatant was
passed through glass wool to remove any floatiog lipid materials.
B.A.2.1.1 Enzyme inhibitor assay
The following additions were pipetted into a series of 10 ml test tubes containing:
• reagent blank: 2 ml ofdistilled water (test tube a);
• standard trypsin: 2 ml ofstandard trypsin solution, 2 ml ofwater distilled water
(test tube b);
• sample blanks: 1 ml ofdiluted sample extract, 1 ml ofdistilled water (test tube c);
• test samples: 1 ml ofdiluted sample extracts, 1 ml ofdistilled water, 2 ml of standard
trypsin solution (test tube d).
Afler mixing the solutions and heating them to 37 0 C for 10 minutes,S ml of BAPNA
solution (previously warmed to 37 0 C) was pipetted into each test tube and the resulting
solution was mixed. Afler an incubation of exactly 10 minutes at 37 0 C, the reaction was
stopped by adding 1 ml of 39 per cent (v/v) acetic acid. The absorbance was measured at
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