Analyst software v 1.6.2

HPLC analysis was performed on a Shimadzu LC-30AD system (Shimadzu, Kyoto, Japan) which consist of two interconnected pump units: one with an integrated degasser and the other with a mixer, and is comprised of a UHPLC gradient system, a refrigerated autosampler, and a column oven compartment. Mass spectrometric detection was performed on an AB SCIEX QTRAP^® 5500 (AB SCIEX instruments, Foster, CA, USA) in MRM mode and EPI mode. A Turbo V™ Ion Source (ESI) interface in positive ionization mode was used. Both the UHPLC and mass spectrometer were controlled remotely using Analyst^® software v. 1.6.2 (AB SCIEX instruments, Foster City, CA, USA). A Waters BEH C18 column (1.7 µm 2.1 mm × 100 mm, Waters, Milford, MA, USA) was applied for analysis. The mass spectrometer was equipped with an electrospray ionization source and spectra were acquired in the positive ion multiple reaction monitoring (MRM) mode and enhanced product ion (EPI) scan modes. MS was optimized using a capillary voltage of 5.50 kV and desolvation temperature of 500 °C. The cone gas pressure and desolvation gas pressure were 50 psi. Nitrogen was used as the cone and collision gasses, respectively. The raw data were analyzed using an Analyst 1.6.2 workstation (AB SCIEX, Foster, CA, USA).

The analysis was conducted on a SHIMADZU Prominence LC system (Kyoto, Japan) coupled with a 5500 QTRAP mass spectrometer (AB SCIEX, Foster City, CA, USA), which includes a LC-20AD_XR solvent delivery system, a DGU-20A_3R automatic degasser, a SIL-20A_XR autosampler and a CTO-20AC column oven. Chromatographic separation was performed at 40 °C on a Waters UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) The mobile phase consisted of a binary solvent system with ACN (A) and 0.1% formic acid in water (B), and run under the following parameters: 35% A at 0.01–0.5 min, 35–90% A at 0.5–9 min, 90% A at 9–11 min, 90–35% A at 11–12 min and 35% of A for 4 min to re-equilibrate the column. The injection volume was 2 μL and the flow rate was set to 0.3 mL/min.
The mass spectrometric detection was operated in the multiple reaction monitoring (MRM) mode with the negative electrospray ionization (ESI). The optimized ESI parameters included ion spray voltage of 4500 V, temperature of 550 °C, curtain gas of 0.24 Mpa, nebulizer gas of 0.38 Mpa and heater gas of 0.38 Mpa. The parameters of MRM transitions, declustering potential (DP) and collision energy (CE) of nine analytes were optimized using a syringe infusion pump and are listed in Table 2. Data acquisition and analysis were processed by the Analyst software (V.1.6.2) from AB SCIEX (Concord, ON, Canada).