METHOD OF THE RAPID ANALYSIS OF 20-HYDROXYECDYSONE CONTENT IN PLANTS AND FERNS USING SOLID-PHASE EXTRACTION ON POLYAMIDE AND MICROCOLUMN HPLC-UV
Abstract
A method for the rapid quantitative analysis of 20-hydroxyecdysone in plants was developed using the microcolumn reversed-phase HPLC with UV detection (246 nm) on the ProntoSIL-120-5-C18 column (2×75 mm) and the gradient eluent system LiClO4/HClO4-acetonitrile. The chromatographic stage of the analysis was 5 minutes long, which made it possible to characterize the technique as the fastest. For preliminary purification of the plant extracts, solid phase extraction on polyamide was used which led to an increase in the sensitivity of the analysis. Validation studies showed that the technique characterized by satisfactory metrological data. The values of the limit of detection (LOD) and the limit of quantification (LOQ) of 20-hydroxyecdysone were 3.3 and 10 μg/ml, respectively. The accuracy indices for various levels of 20-hydroxyecdysone content (80–120%) did not exceed 98.57–101.38%. The method applied to the analysis of 20-hydroxyecdysone in 359 species of flowering plants and 12 ferns species growing on the territory of the Buryatia Republic. The presence of 20-hydroxyecdysone was established in 22 species including 18 flowering plants and 4 ferns. The concentration levels of 20-hydroxyecdysone in plants varied from trace (Athyrium filix-femina, Diplazium sibiricum, Pteridium aquilinum) to very high (25.40 mg/g in Rhaponticum uniflorum and 25.87 mg/g in Silene jeniseensis). The presence of 20-hydroxyecdysone was firstly detected in three species including Gastrolychnis gracilis, G. saxatilis and Silene violascens. The developed technique is fast, simple, sensitive and stable and can be recommended for searching purpose to evaluate the new plant sources of 20-hydroxyecdysone.
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