2.4. Anthocyanin analysis by HPLC
All extracts were analyzed by HPLC (Knauer K-1001) on a
250 mm 4.6 mm i.d. reversed-phase C18 column (Merck);
detection was performed at 520 nm using a diode array detector
(Knauer K-2800). The solvents were (A) H2O/HCOOH (9:1) and (B)
H2O/CH3CN/HCOOH (6:3:1). The gradient was 20–85% B for 70 min
at a flow rate of 1.0 mL/min. The column was washed with 100% B
for 20 min and then stabilized in the initial conditions for another
20 min. The quantification was performed using calibration curves
of cyanidin-3-glucoside, and the results were expressed as
cyanidin-3-glucoside equivalents. The range of the linear calibration
curves (r2 > 0.98) was 0.05 (limit of detection) to 0.5 mg/L for
the lower concentration compounds and 0.5–1000 mg/L for the
higher concentration compounds. The reproducibility of this
method from extraction to HPLC analysis for four samples of the
same sample gave a coefficient of variation < 5%.