Purification, Quantification, and Qualitative Analysis of Cyclopamine from Extracts

HPLC was used to purify cyclopamine from the alkaloid mixture using a Dionex UltiMate ^® 3000 uHPLC system coupled to a diode array detector (DAD) and an automated fraction collector. A semi-preparative Agilent Zorbax SB-C₁₈ column (9.4 × 250 mm, 5 μm) was used to achieve separation. The mobile phase was 0.1% TFA in water (Buffer A) and acetonitrile (Buffer B) with a flow rate of 3.0 ml/min. The linear gradient method was used to separate alkaloids from the mixture starting at 5% acetonitrile and ending at 90% acetonitrile over 25 minutes. Cyclopamine isolated by this procedure was stored as a dried solid at −20 °C for use in bioactivity studies and mass spectrometry analysis.

Aliquots of crude alkaloid extracts were used to quantitate cyclopamine content using a charge aerosol detector (CAD), and MSQ Plus mass spectrophotometer equipped with a Thermo Acclaim 120 C₁₈ column (2.1 × 150 mm, 3 μm). Buffer and gradient conditions were the same as stated above, but the flow rate was decreased to 0.3 ml/min. Cyclopamine standard was used to create a calibration curve at concentrations of 0.5, 1.0, 2.5, 5.0 and 10.0 mM with detection recorded by a Corona Veo RS CAD with the power function set to 1.70. The quantity of cyclopamine was determined from the alkaloid mixtures obtained from each extraction method performed in triplicate, and the extraction efficiency and the standard deviation was calculated.

Cyclopamine isolated from each alkaloid mixture was analyzed by mass spectrometry using an ultra-high resolution Quadrupole Time of Flight (QTOF) instrument (Bruker maXis). The electrospray ionization (ESI) source was operated under the following conditions: positive ion mode; nebulizer pressure: 0.8 Bar; flow rate of drying gas (N₂): 4 L/min; drying gas temperature: 200 °C; voltage between HV capillary and HV end-plate offset: 3000 V to −500 V; mass range was set from 80 to 1000 m/z; and the quadrupole ion energy was 4.0 eV. Samples were analyzed by direct infusion with a syringe pump at a flow rate of 240 μL/hr. Sodium formate was used to calibrate the system in the mass range. Spectra were collected for intact parent ions followed by isolation and fragmentation using collision induced decay MS/MS over a range of collision energies (0-40 eV). Fragmentation patterns were compared to cyclopamine and veratramine standards. Data were analyzed using the Compass Data Analysis software package (Bruker Corporation, Billerica, Massachusetts).