The experiments were performed in an Agilent HPLC system coupled to an AB Sciex API 5500 QTRAP triple quadrupole mass spectrometer (AB Sciex, Foster City, CA, USA). A Poroshell 120 EC-C18 (3.0 x 50 mm, 2.7 μm) column (Agilent, Milford, MA, USA) with a C18 pre-column (Phenomenex, CA, USA) was used for the chromatographic separation. The column temperature was set at 40°C. Mobile phase A consisted of water with 0.5% acetic acid and 1 mM ammonium acetate, and mobile phase B consisted of acetonitrile with 0.5% acetic acid and 1 mM ammonium acetate. The flow rate was set at 0.5 mL min-1. Initial conditions were set at 98% A with a linear gradient from 98% A to 5% A for 1 min. Then, 5% A was held for 8 min with an immediate return to 98% A from 9 min to 10 min. The total run time for each injection was 12 min, and the injection volume was 4 μL.
The operational conditions of the mass spectrometer were established by direct infusion of the standards. Two MRM transitions were established and monitored for each analyte (Table 1 ). Nitrogen was used as collision gas at 55 psi. Ion source gas 1 (GS1) and ion source gas 2 (GS2) were used at 55 psi and 20 psi, respectively. The ion source was operated in the positive and negative mode. Capillary voltage was set at 5.5 kV in the positive mode and -4.5 kV in the negative mode. The temperature of the source was set at 500°C.
The operational conditions of the mass spectrometer were established by direct infusion of the standards. Two MRM transitions were established and monitored for each analyte (