ABSTRACT
OBJECTIVE:To establish a method for the determination of dendrobine and its metabolites M-250 and M-280 in mice plasma for the first time. METHODS:Mice were given dendrobine 60 mg/kg by intragastric administration,1 h later plasma were collected and treated. Using pseudoephedrine hydrochloride as internal standard and dendrobine reference substance as control, the plasma concentrations of dendrobine and its metabolites M-250 and M-280 were determined by UPLC-MS combined with quantitative analysis of multi-components by single marker. The separation was performed on Hypersil Gold C18 column with 0.1％formic acid-acetonitrile(gradient elution)at the flow rate of 0.3 mL/min. The column temperature was set at 40℃,and sample size was 5 μL. Heatable electrospray ionization (HESI) source, scan/ESI + were applied and operated in positive ion mode with atomization temperature of 300℃,ion transmission tube temperature of 350℃,the sheath gas velocity of 35 arb,the auxiliary air velocity of 15 arb,the spray voltage of 3.5 kV,the collision voltage of 30,40,50 eV. The mass-to-charge ratio of detection range were 100-1500. RESULTS:The endogenous substances of mice plasma had no interference with the content determination of dendrobine and its metabolites M-250 and M-280. The linear range of dendrobine were 9.13-912.94 ng/mL(r=0.9996). The limit of quantitation was 3.04 ng/mL. RSDs of intra-day and inter-day were all less than 7.5％(n=5 or n=3). The accuracy were 96.8％-107.5％(n=5). Matrix effects were 97.1％-106.0％(RSD=1.8％-4.7％,n=5). RSDs of the content of sample at 15℃ for 24 h,at -70 ℃ after three times freeze-thaw,at -70 ℃ for 15 d were lower than 12.8％ (n=3). The content of dendrobine in plasma sample of mice was (41.3 ± 5.7) ng/mL (n=12). The contents of its metabolites M-250 and M-280 were (493.0 ± 73.1) and (41.4 ± 3.0) ng/mL (n=12) with Relative correction factor of 1.0. CONCLUSIONS: The method is sensitive and accurate,and can be used for content determination of dendrobine and its metabolites M-250 and M-280 in mice plasma.