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The LC-MS/MS system consisted of a NANOSPA … The LC-MS/MS system consisted of a NANOSPACE SI-II HPLC (Shiseido) and a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a heated-electrospray ionization-II (HESI-II) source. The positive and negative HESI-II spray voltages were 3,500 and 2,500 V, respectively, the heated capillary temperature was 350°C, the sheath gas pressure was 60 psi, the auxiliary gas setting was 40 psi, and the heated vaporizer temperature was 350°C. Both the sheath gas and auxiliary gas were nitrogen. The collision gas was argon at a pressure of 1.5 mTorr. The LC-MS/MS system was controlled by Xcalibur software (Thermo Fisher Scientific) and data were collected with the same software.
LC separation was performed using a reverse-phase column [Capcell Pak ACR (250 mm × 1.5 mm inner diameter, 3 μm particle size; Shiseido)] with a gradient elution of solvent A (5 mM ammonium formate in water, pH 4.0) and solvent B (5 mM ammonium formate in 95% (v/v) acetonitrile, pH 4.0) at 150 μl/min. Solvent A was prepared by mixing 95 ml of Milli-Q (Millipore) water and 5 ml of 100 mM ammonium formate, and adjusting to pH 4.0 with formic acid (approximately 9.6 μl). Similarly, solvent B was prepared by mixing 95 ml of acetonitrile and 5 ml of 100 mM ammonium formate, and adjusting to apparent pH 4.0 with formic acid (approximately 1,160 μl). The initial condition was set at 55% B. The following solvent gradient was applied: maintained 55% B for 10 min and then followed by a linear gradient to 85% B from 10 to 30 min, and hold at 85% B for 7 min. Subsequently, the mobile phase was immediately returned to the initial conditions and maintained for 3 min until the end of the run. Column effluent was introduced into the mass spectrometer between 3 and 37 min after injection. eter between 3 and 37 min after injection.
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