4.23. LC–MS analysis for products of enzyme reactions

Products from enzyme reactions in methanol (10 μl) were injected on an Agilent 1,290 Infinity II with a Xterra C18 reverse phase column (2.1 x 250 mm; 5 μm) set to 45°C. The 35 min run used the solvents .1% (v/v) formic acid in water (A) and .1% (v/v) formic acid in acetonitrile (B) with the following gradient: 1 min, 95% A; 2 min, 87% A; 8 min, 87% A; 14 min, 70% A; 19 min, 60% A; 24 min, 50% A; 30 min, 30% A; 31 min, 5% A; 32 min, 5% A; 33 min, 95% A; 35 min, 95% A with a flow rate of .45 ml/min. The MS was acquired in negative mode from ESI + Agilent Jet Stream in MS2 scan.

To separate kaempferol glucosides, 10 μl samples were injected on an Agilent 1,290 Infinity II with a reverse phase C18 Symmetry column (4.6 x 75 mm; 3.5 μm) set to 30°C. The 15 min run used the solvents .1% (v/v) acetic acid in acetonitrile (A) and .1% (v/v) acetic acid in water (B) with the following gradient: 1 min, 98% B; 7 min, 42% B; 9 min, 20% B; 11 min, 10% B; 13 min, 10% B; 13.1 min, 98% B; 15 min 98% B with a flow rate of .8 ml/min. The MS was acquired in negative mode from ESI + Agilent Jet Stream in MS2 scan.