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目的建立测定褪黑素的反相高效液相色谱紫外分析方法,分析并测定通过组培获得的贯叶连翘不同转基因株系及其亲本植株的褪黑素。方法以SymmetryC18(150mm×4.6mm,5.0μm)为色谱柱;100mmol乙酸铵-甲醇(80∶20)为流动相,体积流量为0.8mL/min;检测波长为265.8nm,灵敏度为2.00AUFS。结果褪黑素浓度在20～500ng/mL线性关系良好(R2=0.9992),平均加样回收率为98.09%(n=6),RSD为2.21%,最小检出量为1.0ng/mL。褪黑素峰保留时间约为8.8min。结论该方法可以简便准确分析不同株系贯叶连翘中的褪黑激素的量;贯叶连翘YXu55(含庆大霉素抗性标记和AANAT-HIOMT基因)转基因株系的褪黑素的量均高于pZP122(仅含庆大霉素抗性标记,不含AANAT-HIOMT基因的空白质粒)转基因株系和未转基因的对照植株;而pZP122转基因株系褪黑素的量跟未转基因的对照植株的量几乎相等。
Objective To establish a method for the determination of melatonin by reversed-phase high-performance liquid chromatography (UV-HPLC) and analyze and determine melatonin in different transgenic lines and their parents plants obtained from tissue culture. Methods The column was composed of Symmetry C18 (150mm×4.6mm, 5.0μm). The mobile phase consisted of 100mmol ammonium acetate-methanol (80∶20). The volumetric flow rate was 0.8mL/min. The detection wavelength was 265.8nm and the sensitivity was 2.00AUFS. Results The melatonin concentration in the range of 20-500 ng/mL showed a good linear relationship (R2=0.9992). The average recovery was 98.09% (n=6), the RSD was 2.21%, and the minimum detectable amount was 1.0 ng/mL. The melatonin peak retention time is about 8.8 min. Conclusion The method can easily and accurately analyze the amount of melatonin in different strains of Hypericum perforatum; the quantity of melatonin in the transgenic lines of Hypericum perforatum YXu55 (including gentamicin resistance marker and AANAT-HIOMT gene) is higher pZP122 (Gentamicin-only marker, blank plasmid without AANAT-HIOMT gene) transgenic lines and non-transgenic control plants; and the amount of melatonin in the pZP122 transgenic line was the same as the amount of non-transgenic control plants. almost equal.