| Polyamines exist in a wide variety of organisms, at physiological pH, they showed high density of positive charge. They combined with DNA, RNA and proteins and other biological macromolecules which carrying a large negative charge. Polyamines participate in many physiological activities, including the regulation of gene expression and translation, cell proliferation, cell signal transduction, cell membrane stability and so on. Polyamines are closely related to human health, which mainly from food, so the detection of polyamines in foods for people’s health is of great significance, and content of spermidine and spermine is the object often detected.The most common method of polyamines detection is pre-column perivatization-fluorescence detection; this paper has studied the reaction conditions of spermidine with several common fluorescent reagents, and determined spermidine and spermine content in the kelp and jellyfish by a better experimental program. The thesis mainly includes the following four parts:(1) Dansyl chloride as a derivatization reagent to react with spermidine and investigated Effect of the amount of derivative agent, buffer solution pH, reaction temperature on derivation reaction. The optimum reaction and HPLC conditions were obtained: reaction in the dark 30 min with 70℃water bath, sodium carbonate sodium bicarbonate(pH = 10) as a buffer solution. Later reaction mixture was extracted with toluene and dried by nitrogen, dissolved in methanol. The derivatives were eluted with methanol and distilled water using a one-step liner gradient (methanol: water = 80: 20 to keep 3 min, from 3 min to 14 min, gradient of methanol ratio from 80% to 100%). In this method, the standard calibration curve of spermidine derivatives is Y= 0.08136 + 0.3807X, the linear correlation coefficient R = 0.9991, detection limit of 0.0924 pmol. In the derivatives, the relative standard deviation of retention time was 0.47% and relative standard deviation of peak area was 1.37%.(2) In this part, experimental conditions of FMOC reacted with spermidine were studied and optimized best: reaction in 40℃water bath for 10 min, sodium borate(pH = 8) as a buffer solution. The reaction solution without treatment, was directly injected for HPLC analysis (methanol: water = 95:5 for mobile phase). In this method, the standard calibration curve of spermidine derivatives is Y=1.014 + 21.649 X, the linear correlation coefficient R = 0.9999, detection limit of 8.80×10-4 pmol. In the derivatives, the relative standard deviation of retention time was 0.15% and relative standard deviation of peak area is 1.42%.(3) O-phthalaldehyde-mercaptoethanol as derivatization reagent was used to derive spermidine. Optimum derivatization was obtained at room temperature shaking 60 seconds for in the presence of sodium borate(pH = 8). The reaction solution was directly injected for HPLC analysis using methanol: water (water, phosphoric acid, triethylamine volume ratio 360:9:6) = 90:10 as mobile phase. The standard calibration curve of spermidine derivatives is Y = 0.5260 + 1.1314X, R = 0.9981, detection limit of 0.0143 pmol.The derivative product with instability has bad repeatability by HPLC.(4) A method for determinating spermidine and spermine in kelp and jellyfish by pre-column privatization was developed. The polyamine were extracted by 5% perchloric acid from samples and then derived with FMOC and DNS-Cl respectively. The results of both methods are basically the same, while the recovery of the experimental results showed that the former than the latter one. |
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