GPC-HPLC-LC/MS测定植物组织中的赤霉素

以胡杨叶片、烟草组培苗及萌发绿豆为材料,研发了利用凝胶渗透色谱(GPC)纯化、液相质谱(LC-MS)定性、 高效液相色谱(HPLC)定量测定植物组织中赤霉素(GAS )的方法。植物样品用80% 甲醇研磨后4℃浸提过夜,抽 滤离心(5 000 r/ min, 20 min)、滤液加2 滴浓氨水浓缩至水相,冻融离心,上清液,调pH 2.5 ~3.0 乙酸乙酯萃取,过 Sep-pak C18 小柱,用含5% 甲醇的CH2 Cl2 溶解样品,经0.22 m 滤膜过滤后,通过GPC 纯化、LC-MS 定性,利用 HPLC 外标曲线法定量。经检测:胡杨叶片中GA3 含量为1 162.789 8 ng/ g 鲜质量; 烟草中GA3 含量为920.906 7 ng/ g 鲜质量; 萌发绿豆中GA3 含量为700.923 6 ng/ g 鲜质量。 

Quantitative analysis of gibberellins in plant tissues by GPC-HPLC-LC/MS

We developed a protocol to determine the contents of gibberellins (GAs) in various plant tissues, such as Populus euphratica leaves, Nicotiana tabacum plantlets, and germinated green beans (Vigna radiata). GAs samples were purified with gel permeation chromatography (GPC), confirmed by liquid chromatography-mass spectrometry (LC-MS), and quantified by high performance liquid chromatography (HPLC). Plant samples were ground with 80% methanol and extracted at 4℃overnight. Crude extract was filtrated and then centrifuged at 5 000 r/ min for 20 min. Two drops of ammonia were added to the liquid sample which was concentrated to aqueous phase, then frozen, thawed and centrifuged. Upper liquids were re-extracted with ethyl acetate at pH 2.5 - 3.0 (x3), and purified by Sep-pak C18 columns. The samples were resolved by 5% methanol-CH2Cl2 and filtrated through 0.22 m filter. Then samples were purified through GPC and GAs fluid was collected for subsequent quantification. GAs in the GPC-purified samples was quantified with HPLC by means of external standard curves. The gibberellins detected in plant tissues were confirmed with LC-MS. The result showed that the GA3 content was 1 162.789 8 ng/g FW in poplar leaves, 920.906 7 ng/g FW in tobacco leaves and 700.923 6 ng/g FW in germinated green beans.