The HPLC analytic was performed using an Agilent 1200 Series Gradient HPLC with a diode array detector (Agilent Technologies Inc., Santa Clara, USA) with an Agilent Poroshell 120 column EC-C18-C18 4.6, 150 mm, 4 μm with a AJO-4286 SecurityGuard C18 4x2.0 mm (Phenomenex Ltd., Aschaffenburg, Germany). As an eluent, methanol/acetonitrile/water (40/15/45 wt. %) containing 1 wt. % acetic acid was used with a flow rate of 0.8 ml min−1. The oven temperature was 35°C and the diode array detector was set to 368 nm. As a standard, rutin trihydrate was used, Figure 1 (analytical grade, CAS-No. 153-18-4, Sigma Aldrich Chemie GmbH, Germany).
The concentrations used for calibration were 1, 5, 10, 20, 50, and 100 mg l−1. The retention time for rutin was 2.7 min. Injection volumes for rutin and the extraction solutions were 10 μl and the extraction solutions were diluted 1 : 10 with water before analysis (deionized water, Milli-Q®, Type I).
The concentrations used for calibration were 1, 5, 10, 20, 50, and 100 mg l−1. The retention time for rutin was 2.7 min. Injection volumes for rutin and the extraction solutions were 10 μl and the extraction solutions were diluted 1 : 10 with water before analysis (deionized water, Milli-Q®, Type I).