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

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.