Preparative Chromatographic Isolation of Caulophine from Radix caulophylli and its Metabolism In Vivo and In Vitro

Abstract

Caulophine, as a novel alkaloid, was found in Radix caulophylli and has anti-myocardial ischemia activities. To conduct product development research on Radix caulophylli and caulophine, a preparative high-performance liquid chromatography method for the preparation of caulophine was investigated. Preparative HPLC was optimized to allow fraction I to be separated first and the caulophine was isolated following the second round of preparative HPLC. The purity of caulophine was >98%, which was assessed using analytical HPLC. Then, a highly sensitive and specific liquid chromatography-mass spectrometric method for the excretion and metabolism of caulophine in vivo was investigated. The metabolism to caulophine glucuronide conjugate was studied in rat liver microsomes or dog liver microsomes in vitro systems. Biosamples were pretreated by solid phase extraction (SPE) and analyzed by LC-MS with electrospray ionization (ESI) interface. Method validation results showed within-day and between-day precision was 1.14–6.21% and 5.45–9.78%, respectively, and average recoveries for all matrices were greater than 80%. The limits of detection for this method were determined to be up to 1 ng · mL−1 of caulophine. Excretion of caulophine in rat results indicated that the total cumulative excretion of caulophine was high, with greater than 50% of the treatment dose being excreted. Two metabolites including glucuronide conjugate and N-oxide of caulophine were found in rat urine and feces by LC-MS. Moreover, the same caulophine glucuronide conjugate was observed in rat liver microsomes system.