2.11. Determination of Quercetin and Rutin by HPLC

Guava juice samples were prepared and analyzed to determine the concentrations of rutin and quercetin. Extraction of flavonoids from guava juice samples for HPLC analysis was carried out by taking samples of guava juice (10 mL) in 50 mL Eppendorf tubes; 25 mL of 100% methanol was added to each tube [33]. The samples were sonicated for 20 min, then centrifuged at 10,000 rpm for 10 min at 25 °C. After centrifugation, the supernatants were collected, and methanol (5 mL) was added to the residues, which were centrifuged again at 10,000 rpm; the supernatants were again collected. The combined supernatants were filtered using Whatman No. 42 filter paper, and 5 g of anhydrous sodium sulfate was added to each supernatant to remove excess water.

Qualitative and quantitative determination of quercetin and rutin was conducted using HPLC (LC-10A, Shimadzu, Kyoto, Japan). Two solvents (A: 3% trifluoroacetic acid (TFA) and B: acetonitrile and methanol (80:20 v/v) were prepared. An equal mixture of these solvents (50:50 v/v) was used for the separation of flavonoids in isocratic mode with the following parameters:

HPLC system: LC-10 A, with a UV-Vis detector (SPD 10-A (λ 360 nm); flow rate: 0.8 mL/min; pressure: 67 kgf/cm²; injection volume: 20 µL; column temperature: 30 °C (CTO 10-A); analytical column: C18 (250 × 4.6 mm, 5 µm particle size, Supelco, Bellefonte, PA, USA); delivery pump: LC-10AS; system controller: SCL-10A; acquisition software: CLASS LC-10. The determination of individual flavonoids was made by comparing the retention times and peak areas to those of certified reference standards (quercetin, rutin, myricetin, and kaempferol).

Flavonoids (rutin and quercetin) were purchased from Sigma-Aldrich, USA. Different concentrations of standard were prepared in 100% methanol. A series of solutions (0, 10, 20, 50, 100, 200 and 300 µg/mL) was prepared to determine the limit of detection (LOD) and linearity of detector (response).