3.7. Determination of Coumarin Concentration by High Performance Liquid Chromatography

Determination of coumarin in the obtained extracts was performed using reverse phase (RP)-HPLC method with UV detection. The analysis was performed on a Varian ProStar system (Varian Analytical Instruments, Palo Alto, CA, USA) containing Varian ProStar 230 Solvent Delivery Module, ProStar 500 Column Valve Module (Varian Analytical Instruments), and ProStar 330 Photodiode Array detector (Varian Analytical Instruments) and coupled to a computer with the ProStar 5.5 Star Chromatography Workstation and PolyView 2000 V 6.0. (Varian Analytical Instruments). COSMOSIL 5C18-MA-II (NacalaiTesque, Inc., Kyoto, Japan) column, 150 mm long with internal diameter of 4.6 mm was used for chromatographic separation. Gradient elution with distilled water as phase A and methanol as phase B was used for separation, with the following gradient: 0–15 min, 60% A and 40% B phase; 15–20 min, increasing the share of phase B to 80% and decreasing phase A to 20%; 20–40 min, holding 20% A and 80% B phase; 40–41 min decreasing of B phase to 40% and increasing A phase to 60%, 41–50 min, holding 60% A and 40% B phase. The analyses were performed at room temperature, with flow rate 1.0 mL/min, injection volume 20 µL, and UV detection wavelength 279 nm. The stock solutions of coumarin standard were prepared in a solvent, and calibration was obtained at six concentrations (concentration range 1.0, 2.0, 5.0, 10.0, 20.0, 30.0 mg/L). Linearity of the coumarin calibration curve was confirmed by R² = 0.9997. Coumarin limit of detection (LOD) was 0.035 mg/L, limit of quantification (LOQ) 0.345 mg/L, and compound retention time was 22.1 min. The extracts were weighted, diluted in HPLC grade methanol, filtered through 0.45 μm polytetrafluoroethylene (PTFE) filters, and subjected to HPLC analyses. Coumarin concentration in the plant extracts (μg/mL) determined by HPLC analysis (in triplicate) was recalculated to mg of coumarin/100 g of the plant sample.