Table 5 LC/MS settings

 1. HPLC elution solvents

 The anionic ion-pair reagent HFBA was added to the mobile phase to improve analyte interactions with the stationary phase. An aqueous solution of HFBA (approximately 0.5 M) was diluted in water (mobile phase A) and methanol (mobile phase B) to a final concentration of 0.5 mM.

 2. Gradient system (pair of two alternating columns)

 The gradient system used two identical columns (Agilent Zorbax SB-C18; 2.1 mm × 50 mm, 1.8 μm; Waldbronn, Germany) connected to a 10-port two-position switching valve. Using the column switching valve and a second binary pump, the two columns were applied alternately. When one column was used for the analytical gradient, the other column was cleaned and re-equilibrated. From the prepared samples, 5 μL was injected into the HPLC system. The analytical gradient (pump 1; flow rate: 0.5 mL/min) increased linearly from 10% B to 50% B within 11 min. After isocratic elution for 0.5 min, the gradient returned to starting conditions until 11.6 min, and isocratic flow was performed for 10% B from 11.6 to 12.5 min at 0.5 mL/min. This short return of pump 1 to starting conditions was necessary for flushing the tubing from the pump to switching valve before the freshly equilibrated second column was switched in for the next injection. Parallel to the analytical gradient, the other column was cleaned and equilibrated by pump 2; within 1.0 min, the gradient was increased from 10% B to 95% B. The isocratic flow of 95% B was held for 4.5 min. From 5.5 to 6.5 min, initial conditions of the analytical gradient (10% B) were achieved and retained at isocratic flow until 12.5 min for flushing and equilibrating the column. Total time from injection to injection was 13.3 min (including the autosampler operation time of 0.8 min).