Agilent 1200 series diode array detector

Purity of the synthesised compounds was determined by HPLC using an Agilent 1200 HPLC system equipped with a quaternary pump and an Agilent 1200 series diode array detector. HPLC grade acetonitrile (Merck) and Milli-Q water (Millipore) were used for the chromatography. A Venusil XBP C18 column (4.60 × 150 mm, 5 µm) was used with acetonitrile (30%) and Milli-Q water (70%) as the initial mobile phase at a flow rate of 1 mL/min. At the start of each injection, a solvent gradient programme was initiated by linearly increasing the composition of the acetonitrile in the mobile phase to 85% acetonitrile over a period of 5 min. Each run lasted 15 min and a time period of 5 min was allowed for equilibration between runs. A volume of 20 µL of solutions of the test compounds in acetonitrile (1 mM) was injected into the HPLC system, and the eluent was monitored at wavelengths of 210, 254 and 300 nm.

An aliquot of 10 mg of the 70% ethanol extract of safflower seed was dissolved in 10 mL of 50% methanol by repeated vortexing, and passed through a Dismic-25 JP membrane filter (Advantec Toyo, Tokyo, Japan; pore diameter: 5 µm). Next, 5 µL of the sample was injected into a reverse-phase HPLC system with an INNO C18 column (4.6 × 250 mm, 5 µm pore size, YoungJin Biochrom, Korea) at 35°C. The mobile phase comprised (A) a 0.1% aqueous solution of formic acid and (B) acetonitrile, and the following gradient conditions were used: 0 min, 0% B; 5 min, 6% B; 10 min, 22% B; 50 min, 28% B; 66 min, 50% B; and 70 min, 100% B. The flow rate was 0.8 mL/min. The UV absorbance at 254 nm was monitored by using an Agilent 1200 series diode array detector (Agilent Technologies, Waldbronn, Germany). All peaks were assigned through co-injection tests with authentic samples and comparison of the UV spectral data; through this process, compounds including serotonin and its derivatives were detected from safflower seed extract. All measurements were repeated in triplicate. Representative HPLC results are illustrated in Figure 1. The quantification of serotonin, N-(p-coumaroyl)serotonin, N-feruloyl serotonin, luteolin, and luteolin-7-O-glucoside was achieved by peak area measurement. The quantity of each identified compound is shown in Table 1.