Quantification of near-attomole gibberellins in floral organs dissected from a single Arabidopsis thaliana flower.

There remains a methodological bottleneck in the quantification of ultra-trace plant hormones in very tiny plant organs at fresh weights below a milligram. The challenge becomes even more serious in the determination of endogenous gibberellins (GAs), which are a class of compounds that are difficult to separate and detect. Herein, a quantification method using ultra-high-performance liquid chromatography-tandem mass spectrometry was developed, combined with a derivatization technique in which GAs react with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide in ethanol. The method was validated as capable of determining GAs in floral organs (about 80-250 μg) - pistil, stamens, petals, sepals and receptacle - which were dissected from only one flower of Arabidopsis thaliana. Substantially different abundance patterns of GAs were measured from the floral organs at floral stages 13, 14 and 15 along the non-13-hydroxylation pathway and the early 13-hydroxylation pathway in plants. This allows sub-flower-level insights into how GAs affect floral development. The method exhibited excellent limit of detection and limit of quantification down to 5.41 and 18.0 attomole, respectively, and offered a fairly wide linear range from 0.01 to 25 femtomole with linear coefficients above 0.9961. The precision of the method was evaluated with relative standard deviations below 10.6% for intra-day and 11.4% for inter-day assays, and recoveries ranged from 64.0% to 107%.