OBJECTIVETo develop a high performance liquid chromatography method for simultaneous determination of tour active constituents, dactylorhin B, loroglossin, dactylorhin A, militarine present in a Tibetan herb Gymnadenia conopsea.

METHODThe analysiswas achieved on a Pravil C18 column (4.6 mm x 150 mm, 5 microm) eluted with the mobile phases of methanol (A)-water (B) in gradient elution. The initial condition was 15% A, chsnhrf to 20% A in 10 min, to 45% A in 10 min, to 70% A in 15 min. The detection wavelength was set at 222 nm. The flow rate was 0.7 mL x min(-1). Orthogonal test was adopted to optimize extraction process of four active compounds from Gymnadenia conopsea.

RESULTThe assay displayed good linearity over the concentration ranges of 0.20-4.05 microg (r = 0.9999), 0.10-5.11 micro g (r = 0.9999), 0.10-5.11 microg (r = 0.9999) and 0.09-1.79 microg (r = 0.9999), respectively. The average recoveries (n=9) were 99.19%, 99.16%, 99.52% and 99.00% for dactylorhin B, loroglossin, dactylorhin A and militarine respectively.

CONCLUSIONThe method is simple, sensitive, reliable and reproducible which can be used for the quality study of Gymnadenia conopsea.