Determination Of Polyamines Content In Marine Shellfish By High Performance Liquid Chromatography

Polyamine is a kind of small molecular aliphatic nonprotein nitrogen containingbases chemical compounds. It exists in cells widely, mainly including putrescine,spermine, spermidine etc. Polyamine is involved in many physiological andmetabolic activities of living organisms, and has very close ties with the people’shealth. A part of polyamine in the human body is synthesized, and the other part isfrom food. Therefore, determining the content of polyamine in food has impoetantsignificance. There are many mithods for determining the content of polyamine. Themost common application is the high performance liquid chromatography, which canefficiently, quickly and accuratly determine the content of polyamine.Polyamine has no fluorescence or UV absorption. So determining the content ofpolyamine need the derivatization reagent by the fluorescence determination. Thispaper uses the dansyl chloride, liquid-liquid extraction and fluorescence detector todetermining the content of polyamine by HPLC.First of all, The standard substance of spermine and spermidine react withdansyl chloride, so as to determine dansyl chloride dosage. On this basis, this paperselects the appropriate buffer solution system and the appropriate pH. Then thispaper determines the optimum reaction temperature and reaction time. Six kinds ofextraction agent extract the polyamine, so as to compare the extraction effect andselect suitable extractant. Then this paper determines polyamine under the optimalchromatographic. This paper has established the best derivation condition andchromatographic condition. The volume of dansyl chloride is500μL; Boric acidbuffer solution’s pH is8.0;60℃water bath heating react30min in dark. The mobilephase of HPLC is methanol and distilled water(methanol: water=80:20); Theproportion of mobile phase keep3min, after methanol gradient ratio began toincrease to100%from4min to14min at a flow rate of0.8mL·min-1. Under theoptimum conditions, we draw the standard curves of SPD and SPM and obtain theregression equation. The regression equation of SPD is Y=0.1729+0.2616X(R=0.9988); The regression equation of SPM is Y=0.0740+0.1704X (R=0.9992).Next, this paper uses nine kinds of abstracting agent(including differentconcentration of HCl solution and H2SO4solution, HClO4solution, methanol andwater) to abstract polyamine in clams, oysters, scallops and select the optimumabstraction agent, abstraction time and abstraction temperature. The optimum abstraction agent is6mol·L-1HCl solution and the optimum temperature is60C.The optimum time of clams and oysters is3h, and the optimum time of scallops is4h.Then, under the opitimum experimental conditions, the content of polyamine inabstraction solution of clams, oysters, scallops has been determined. The results arethat the content of SPD and SPM in clams respectively is39.59mg/Kg,47.73mg/Kg;the content of SPD and SPM in oysters respectively is24.26mg/Kg,55.90mg/Kg;the content of SPD and SPM in scallops respectively is23.66mg/Kg,32.74mg/Kg.Finally, Using6mol·L-1HCl solution at60℃water bath abstract polyaminefrom spanish mackerel, hairtail, small yellow croaker and sardines in3h. Also underthe opitimum experimental conditions, the content of polyamine in abstraction solu-tion of spanish mackerel, hairtail, small yellow croaker and sardines has beendetermined. The results are that the content of SPD and SPM in spanish mackerelrespectively is9.15mg/Kg,9.94mg/Kg; the content of SPD and SPM in hairtailrespectively is6.86mg/Kg,7.29mg/Kg; the content of SPD and SPM in smallyellow croaker respectively is6.50mg/Kg,6.78mg/Kg; the content of SPD andSPM in sardines respectively is6.03mg/Kg,7.14mg/Kg. The results of fishescompare with the results of shellfish, kelp, jellyfish, soybean and its products. Theresults show that the concent of spermine in jellyfish and fermented bean curd is thehighest; the concent of spermine and sperdimine in clams is relatively high; theconcent of spermine and sperdimine in kelp and soybean is very low; the concent ofspermine and sperdimine in fishes is relatively low, and their content is very similar,the difference of spermine and sperdimine is less in the levels.