2.3. LC–MS instrumentation and methods
LC-MS analysis was performed using an API4000 mass spetrometer (Milwaukee, WI, USA) equipped with an electrosprayionization (ESI) source interface coupled to an Agilent 1100-LC system (Palo Alto, CA, USA). The mobile phase and the LC gradientprogram were the same as the HPLC chromatographic conditionsdescribed in section 2.2. The flow rate was maintained at 1 mL/minand was split at the column outlet to allow 0.2 mL of eluent to flowinto the mass spectrometer. The MS was in positive-ion electrospray mode, and the operating parameters were as follows: ionspray voltage, 5000 V; declustering potential, 70 V; entrance potential, 10 V; turbo ion spray temperature, 500◦C; collision energy,40 V; collision cell exit potential, 15 V; interface heater, on; and mass range, 100–800 Da. Nitrogen was used as both curtain and auxiliary gas. The system was operated using an Analyst Softwareworkstation, version 1.5.2