Liquid chromatography system

To determine the plasma level of luteolin and its metabolites, a 10 μL sample of the extract was subjected to an HPLC–MS/MS system consisting of a liquid chromatography system (Agilent, Tokyo, Japan) and a 4000 QTRAP HPLC–MS/MS (SCIEX, Tokyo, Japan), with analytical parameters that were similar to our previous study^{11 (link),12 (link)}. Chromatographic separation of luteolin and its metabolites were done on a C18 column (CAPCELLPAK C18 MGII S3, 4.6 × 150 mm; Shiseido, Tokyo, Japan) at a flow rate of 0.8 mL/min and temperature maintained at 40 °C. Gradient elution was performed using water containing 0.1% trifluoroacetic acid (mobile phase A) and acetonitrile (mobile phase B). The gradient profile was as follows: 0–20 min, 10–30% B linear; 20–25 min, 30–50% B linear. The mobile phase was split so that the eluate entered the HPLC–MS/MS system at a flow rate of 0.2 mL/min. Luteolin, luteolin-3'G, luteolin-4'G, and luteolin-7G were analyzed in negative ion mode and detected by multiple reaction monitoring (MRM) for the transition of precursor ions to productions: luteolin (m/z 284 > 133), luteolin-3'G (m/z 461 > 285), luteolin-4'G (m/z 461 > 285), and luteolin-7G (m/z 461 > 285).

HPLC was performed with an Agilent liquid chromatography system, consisting of UV-VIS Spectra-Focus detector (220 nm) and injector-auto sampler. Sample (10 mg/ml) were filtered through a 0.45 μm PVDF-filter and injected in to the HPLC column. The injection volume was 10 μl and the column was 20RBAX ECLIPSE, XDB-C18, (5 μm; 4.6 × 150 mm, Agilent USA). The method involved the use of a binary gradient with mobile phases containing: solvent A (0.05% trifluoroacetic acid) and solvent B (0.038% trifluoroacetic acid in 83% acetonitrile (v/v) with the following gradient: 0–5 min, 15% B in A, 5–10 min, 50% B in A, 10–15 min, 70% B in A. The flow rate was kept constant at 1 ml/min. Identifications were based on retention times in comparison with authentic standards. The crude extract was partitioned three times with 25% hydrochloric acid and methanol. The percolate was concentrated in a rotary evaporator and dissolved in HPLC grade methanol. Calibration curves for standard analytes at 10, 20, 50, 100 and 200 μg/ml concentrations were found to be linear (Supplementary File 1). Quantification of each constituent was completed by means of integration of peaks using the external standard scheme.