植物中多胺含量超高效液相色谱法的建立

[目的] 植物体内的多胺对植物生长具有明显的促进作用,对体内抗逆能力的提高也具有重要意义。本试验基于高效液相色谱方法(HPLC),探讨了超高效液相色谱法(UPLC)测定植物中内源多胺,如腐胺(Put)、亚精胺(Spd)、精胺(Spm)的含量。[方法] 以小麦叶片、草坪草叶片作为试验材料,用液氮将其磨成粉末,加入冰预冷的5%(体积分数)高氯酸,涡旋混匀,置于冰浴中浸提1 h,然后4℃、15000g离心30 min,吸出上清液放入新的离心管中,经过衍生以后,选用色谱柱ACQUITY UPLC HSS T3(100 mm×2.1 mm×1.8 μm),进行多胺含量的测定,流动相为乙腈和水(体积比为44:56),柱温为30℃,流速为0.45 mL？min-1,等度洗脱,检测波长为230 nm,进样体积为2 μL。[结果] 样品中的Put、Spd和Spm保留时间分别为1.24、1.99、3.19 min,样品中的多胺测定一次可在5 min内完全分离;Put、Spd和Spm在1~100 μg？mL-1范围内,线性关系良好,相关系数分别为0.9993、0.9991、0.9990,重复性和稳定性试验的相对标准偏差(RSD)均小于2%,说明该测定方法快速、稳定、分析精密度高。[结论] 该试验方法简洁、快速、灵敏度高、重现性好,可有效测定植物中多胺的含量,为多胺在植物逆境代谢途径中的研究提供了基础。
[Objectives] Polyamines(PAs)such as putrescine(Put), spermidine(Spd), and spermine(Spm)play a positive role in plants growing and significantly enhance the tolerance to biotic and abiotic stresses. This study attempts to explore an appropriate methods in measuring the PAs in plants by the use of the ultra performance liquid chromatography(UPLC).[Methods] The fresh leaves of wheat and turfgrass were used as the plant materials in this study. Samples were grounded into powder with liquid nitrogen, then loaded into centrifuge tubes, 5% perchloric acid(which was pre-cooling in the ice)was added into the tubes, vortex for mixing. The tubes with mixture were ice bathed for one hour and then centrifuged for 30 minutes on 4℃, 15000 g, supernatant was transferred to new centrifuge tubes and fully derived for further use. UPLC separation was carried out in a ACQUITY UPLC HSS T3(100 mm×2.1 mm×1.8 μm)with acetonitrile and water(volume ratio is 44:56)as solvents proportions, isocratic elution, the column temperature was 30℃, the flow rate was 0.45 mL？min-1, the detection wavelength was 230 nm, and the injection volume was 2 μL.[Results] The results showed that the retaining time of Put, Spd and Spm in plant samples was at about 1.24, 1.99 and 3.19 minutes, respectively, and the analysis could be finished in five minutes. The concentration of Put, Spd and Spm was ranged from 1-100 μg？mL-1, all these three polyamines exhibited an excellent linearity and the correlation coefficient was above 0.999. The relative standard deviation of precision and stability experiments was lower than 2%, indicating that the method was a rapid and simple method with more accuracy and stability in measuring PAs.[Conclusions] This UPLC method is simple, and fast, with high sensibility and good repeatability. So, it was an effectively method in PAs contents measurement and analysis in plants, and provided a certain experimental way to the exploration on the metabolic pathway of PAs in plants