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Abstract
An HPLC method with DAD was developed to quantify some flavonoids (rutin, quercetin, and kaempferol) in hop extracts. Quercetin and kaempferol were determined in the free forms and they can also be found in the form of glycosides (rutin and others). The glycosides were hydrolyzed during the extraction step with 0.1 mol/L HCl or 1.5 mol/L HCl. If 1.5 mol/L HCl was used for extraction, the lower results of flavonoid determination were achieved due to decomposition of hydrolysis products. Free forms of quercetin and kaempferol were quantified in the water extract because the hydrolysis of glycosides in water was negligible. Pre-column in off-line mode was used for purification of extracts. The limits of detection were 0.25 µg/mL for rutin, 0.3 µg/mL for quercetin, 0.25 µg/mL for kaempferol, 0.2 µg/mL for hesperidin, 0.34 µg/mL for hesperetin, 0.23 µg/mL for naringenin, 0.42 µg/mL rhamnetin, 0.16 µg/mL for patuletin, 0.25 µg/mL for patulitrin, and 0.32 µg/mL for isorhamnetin-3-O-glc. The limits of quantification were 0.5 µg/mL for rutin, 0.6 µg/mL for quercetin, 0.38 µg/mL for hesperidin, 0.7 µg/mL for hesperetin, 0.4 µg/mL for naringenin, 0.8 µg/mL rhamnetin, 0.38 µg/mL for patuletin, 0.5 µg/mL for patulitrin, and 0.4 µg/mL for isorhamnetin-3-O-glc 0.5 µg/mL for kaempferol. The concentration of quercetin was 165.3 ± 5.0 µg/g of dry sample; the concentration of kaempferol was 125.9 ± 4.3 µg/g of dry sample after 2 hr of extraction using 0.1 mol/L HCl in methanol solutions. The concentrations of the other flavonoids were under the limit of quantification. If pure water and methanol were used for extraction, the lower concentrations were determined, which indicated that higher amounts of flavonoids under study existed in glycoside forms.
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ACKNOWLEDGMENT
This work was supported by VEGA grants no. 1/0436/08, 1/1005/09, and 1/0164/11.
Notes
*Under the limit of quantification.
Column: Grace Smart RP 18 (150 × 4.6 mm, 5 µm);
Mobile phase: 55% water with 0.5% H3PO4 and 45% methanol;
Wavelength of detection 360 nm;
Volume of injection: 20 µL; flow rate: 0.5 mL/min.
*Under the limit of quantification.
Column: Grace Smart RP 18 (150 × 4.6 mm, 5 µm);
Mobile phase: 55% water with 0.5% H3PO4 and 45% methanol;
Wavelength of detection 360 nm;
Volume of injection: 20 µL; flow rate: 0.5 mL/min.
Column: Grace Smart RP 18 (150 × 4.6 mm, 5 µm);
Mobile phase: 55% water with 0.5% H3PO4 and 45% methanol;
Wavelength of detection 360 nm;
Volume of injection: 20 µL; flow rate: 0.5 mL/min.
*Under the limit of quantification.
Column: Grace Smart RP 18 (150 × 4.6 mm, 5 µM);
Mobile phase: 55% water with 0.5% H3PO4 and 45% methanol;
Wavelength of detection 360 nm;
Volume of injection: 20 µL; flow rate: 0.5 mL/min.
*Under the limit of quantification.
Column: Grace Smart RP 18 (150 × 4.6 mm, 5 µm);
Mobile phase: 55% water with 0.5% H3PO4 and 45% methanol;
Wavelength of detection 360 nm;
Volume of injection: 20 µL; flow rate: 0.5 mL/min.
*Under the limit of quantification.
Column: Grace Smart RP 18 (150 × 4.6 mm, 5 µm);
Mobile phase: 55% water with 0.5% H3PO4 and 45% methanol;
Wavelength of detection 360 nm;
Volume of injection: 20 µL; flow rate: 0.5 mL/min.
Column: Grace Smart RP 18 (150 × 4.6 mm, 5 µm);
Mobile phase: 55% water with 0.5% H3PO4 and 45% methanol;
Wavelength of detection 360 nm;
Volume of injection: 20 µL; flow rate: 0.5 mL/min.