2.6. Analysis of the flavonol glycosides and their microbial metabolites
using HPLC-DAD
The Agilent HPLC-System consisted of a quaternary pump (1050 series),
autosampler (1050 series) and a DAD (1100 series). A gradient
elution based on Rohn et al. (2007) using acidified water (A; water/
acetic acid; 99.5/0.5, v/v) and acetonitrile (B) was carried out on a
250 ~ 4.6 mmi.d., 5 ƒÊm, Fluofix 120E column (Wako Pure Chemical Industries,
Osaka, Japan) connected to a 10 ~ 4.6mmi.d. guard column of
the same material. Gradient elution was performed as follows: 0% B (5
min); 0.4% B in 4 min; 4.2% B in 1 min; 2.4% B in 5 min; 4.8% B in
15 min; 8.22% B in 15 min; 22.28% B in 5 min; 28% B (5 min); 28.
45% B in 10 min; 45.0% B in 1 min; at a flow rate of 1.0 mL/min and a
column temperature of 30 ‹C. The detection was performed simultaneously
at 325 nm, 350 nm and 280 nm. Spectra were recorded
from 200 to 500 nm. All compounds were identified by their retention
times and UVspectra in comparison to reference substances. Calibration
curves were used for quantification.