Simultaneous determination of 16 important biologically active phytohormones in Dendrobium huoshanese by pressurized capillary electrochromatography.

ABSTRACT

A rapid pressurized capillary electrochromatography (pCEC) method has been successfully developed for the simultaneous determination of 16 phytohormones in Dendrobium huoshanense. Effects of wavelength, mobile phase, the flow rate, pH value, concentration of buffer and applied voltage were investigated, respectively. The results showed that the 16 phytohormones could be baseline-separated rapidly in less than 21 min on a reversed-phase EP-100-20/45-3-C18 capillary column (total length of 45 cm, effective length of 20 cm, diameter of 100 µm, ODS packing inside for 3 µm) with ACN/5.0 mM ammonium acetate (containing 0.05% formic acid, pH = 3) as the mobile phase using gradient elution mode as follows 0.1-10.0 min 40%ACN,10-15.0 min 70%ACN, 15.0-20 min 80% ACN, 20-21.0 min 80% ACN at a flow rate of 0.12 mL/min, applied voltage of -5 kV and a UV detection wavelength of 210 nm. The method validation howed that the established method is precise and stability, and the RSDs of intra- and inter-day precision based retention time and peak area were all below 5%. Employed the established method, in our experimental conditions, total 6 endogenous hormones including IAA, IBA, NAA, GA, ABA, t-Z were detected in D. huoshense. However, a relative larger amount of exogenous hormone 2,4-D (25.3 ~ 4.2 µg/kg) and 6-BA (79.5 ~ 35.4 µg/kg) were detected in 1 ~ 4 year old cultivated D. huoshense, suggesting there were still a certain amount of exogenous hormone residue in tissue-cultured D. huoshanese though they had been transplanted to field cultivation from the test-tube plantlets for several years.