Determination Of Polyamines Content In Soybean And Bean Products By SPE-HPLC Method

The polyamines include putrescine, spermine, spermidine and so on. It is veryimportant for physiological activities in biological systems. At present, the polyaminesdetection methods include enzyme immunoassay, thin-layer chroroatosraphy, gaschromatography, capillary electrophoresis, ion chromatography and high performanceliquid chromatography and so on. The HPLC is widely used because of its metrits suchas the high efficiency，veracity and shortcut and so on.A method for determining polyamines was established in this thesis. Thepolyamines is pre-column derivatived by dansyl chloride, extracted by solid-phaseextraction, separated by reversed-phase HPLC and determined by fluorescencedetector. Firstly, The thesis studied the derivatives by dansyl chloride and theconditions of derivative. The reaction solution’s pH is adjusted to10, and reacted30minutes at the temperature of70℃. The optimalchromatographic conditions are thatthe derivatives are eluted with methanol and distilled water using a one-step linergradient (methanol: water=80:20to keep3min, from3min to14min, gradient ofmethanol ratio from80%to100%) and determined by fluorescence detector(excitationwavelength:340nm, emission wavelength:515nm). The chapter uses liquid-liquidextraction and draw the standard curve of SPD and SPM, and obtain the regressionequation of SPD is Y=-0.1229+0.2723X (R=0.9942), the SPM’s regression equationis Y=0.0633+0.1237X (R=0.9950).Secondly, the subject uses the solid-phase extraction to replace the traditionalliquid-liquid extraction and optimizes these conditions. The leacheate is acetone-waterand the acetone’s density is20%, the elution agents use3mL methanol. Draw thestandard curve of SPD and SPM in the optimal condition and obtain regressionequations, The SPD’s regression equation is Y=0.1548+0.2604X (R=0.9987) andThe SPM’s is Y=0.1565+0.1293X (R=0.9998). The relative standard deviation ofspermidine derivative’s peak area is0.93%and the detection limit is0.32pmol; Therelative standard deviation of spermine derivative’s peak area is0.87%and thedetection limit is0.29pmol.Finally, the soybean, soy sauce, sofu and chungkukiang was detected in the optimal condition. The results are that the content of spermidine is2.24mg/Kg andspermine’s is3.87mg/Kg in soybean, the content of spermidine is10.84mg/L andspermine’s is21.72mg/L in soy sauce, the content of spermidine is29.08mg/Kg andspermine’s is59.48mg/Kg in sofu, the spermidine’s content is14.99mg/Kg andspermine’s is30.03mg/Kg in chungkukiang. In addition, the determination of theaverage rate of recovery of spermidine respectively are85.2%,85.8%,90.6%,87.8%and the spermine’s average rate of recovery respectively are87.1%，88.1%，93.2%，89.6%in the soybean, soy sauce, sofu and chungkukiang.The results indicate that the method has many merits like the simple operation,improved selectivity, the shorter determination time, easy to widely apply and so on. Itcould be used to detect polyamines in soybean, soy sauce, sofu and chungkukiang andit provides the data for these parts of clinical medicine and food analysis or another.