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.