Quantification of Taspine and Magnoflorine by liquid chromatography-mass spectrometry (LC-MS)

Quantification and identity confirmation of magnoflorine and taspine were performed in a tandem LC-MS platform. The same samples used for the LC-HRMS assays were analysed but an additional methanolic extract was generated from C. draco latex which was harvested from small incisions at intervals of 5–10 cm on branches and on the bark of the main trunk. The latex was lyophilized, and a 1 mg was dissolved in 1 mL of methanol with 0.1% of formic acid (MS grade, Sigma-Aldrich) to analyse by LC-MS-MS. Chromatographic separation and detection of taspine and magnoflorine was performed using an Agilent Technologies 1290 UPLC coupled to a 6460 triple quadrupole (QqQ) mass spectrometer with a Jetstream electrospray (ESI) source. The column used was an Agilent Zorbax SB-C18, 2.1 × 50 mm, 1.8 μm and the column temperature was 40 °C. The mobile phases were water (A) and acetonitrile (B), both with 0.1% formic acid (MS grade, Sigma Aldrich) at a flow rate of 0.3 mL/min. The solvent gradient was 0–10 min, 5–30% of B; 10–11 min, 30–95% of B; 11–12 min, 95% of B and finally 12–14 min, 95–5% of B and remained unchanged for 1 min. The injection volume was 1 μL and each extract pool was injected in triplicate. The mass spectrometer settings were gas temperature 300 °C with flow of 5 L/min. The nebulizer pressure was set at 0.31 MPa, and the sheath gas temperature and flow were 250 °C and 11 L/min, respectively. The capillary and nozzle voltages used were 3500 and 500 V, respectively. The polarity used for the analysis was positive with a fragmentor voltage of 135 V, and a collision energy of 10 V. The acquisition method used was dynamic multiple reaction monitoring (dMRM) with the transitions 342.1 > 296.7 and 370.1 > 325.1 for magnoflorine and taspine, respectively. The magnoflorine commercial standard was purchased from Sigma-Aldrich and taspine was purified in-house by using the protocol described previously [17]. A calibration curve was prepared for each compound with 10 calibration points (0.25, 0.5, 1, 3, 5, 7, 9, 11, 13, 15 and 17 μM). Each calibration point was injected twice, and the areas were plotted against concentration. Quadratic regression was performed to obtain an r² value of > 0.99 for each compound and quantities were established by using MassHunter Workstation Software vB.06.00 (Agilent Technologies). The results are expressed as μg/g of sample (dry weight).