Selection of optimal HPLC conditions is important for the determination and separation of suforaphane. In this study, different wavelengths (205 nm, 235 nm and 254 nm) were investigated, and the results showed sulforaphane had largest absorbance under 205 nm. Therefore, 205 nm was chosen for further analyses.

Different kinds of mobile phases such as methanol, acetonitrile, and different concentrations of methanol/H₂O and acetonitrile/H₂O were investigated. Under 205 nm, methanol has strong background absorbance, which is difficult to balance the C₁₈ column. The 20% acetonitrile/H₂O (v/v) was proved to provide the best separation, because there is no interference of other impurities and the retention time of sulforaphane is short. The chromatogram of sulforaphane is shown in Figure 2.

To optimize the hydrolysis of glucoraphanin to sulforaphane in fresh broccoli, preliminary trials were conducted with different pHs of acidic water (3, 4, 5 and 6), and hydrolysis times (2, 4 and 6 h). The incubation temperature was constant at 35 °C. Table 1 shows that the resulting sulforaphane amount was highest with pH 3.0 and hydrolysis time 4 h.

Sulforaphane was obtained from Sigma-Aldrich (USA), and used without further purification. Acetonitrile, methanol, ethyl acetate and dichloromethane were obtained from Duksan Pure Chemical Co., LTD (Ansan, Korea). All the other reagents used in the experiment were HPLC or analytical grade. Double distilled water was filtered with a vacuum pump (Division of Millipore, Waters, USA) and filter (HA-0.45, Division of Millipore, Waters, USA) before use. All the samples were filtered by using a filter (MFS-25, 0.2 μm TF, WHATMAN, USA) before injection into the HPLC system. Stock standard solutions of sulforaphane were prepared by dissolving 10 mg of standards in 10 mL of acetonitrile. Commercial C₁₈, amino and silica cartridge (200 mg/3 mL) were purchased from Alltech (USA).

Chromatography was performed with a Waters 600 s multisolvent delivery system, a Waters 616 liquid chromatography, and a Waters 2487 variable wavelength, dual-channel, UV detector (Waters Associates, Milford, MA, USA). A six-port Rheodyne injector (20 μL sample loop) was also used. Data processing was performed with Millennium 3.2 software resident in an HP Vectra 500PC. Compounds were separated on a 250 mm × 4.6 mm, 5-μm particle, OptimaPak C₁₈ column (RS Tech, Daejeon, Korea). HPLC separation of sulforaphane was conducted by using acetonitrile/H₂O (20/80, v/v) as mobile phase at a flow rate of 0.5 mL/min and the detection was carried out at a wavelength of 205 nm. Distilled water was filtered with a vacuum pump and filter (HA-0.45 μm; Millipore, Waters, USA) before use.

Fresh broccoli was pulverized and 5 g of the resultant powder was weighed and extracted with 20 mL of different pH of hydrochloric acid (HCl) for 2 h. The resulting mixture was extracted 3 times with 20 mL of dichloromethane, which was combined and salted with anhydrous sodium sulfate. The dichloromethane fraction was dried at 30 °C under vacuum on a rotary evaporator. The residue was dissolved in acetonitrile and was then filtered through a 0.22 mm membrane filter for the following study. The sulforaphane was purified with different SPE cartridge. Prior to use, the silica cartridge was conditioned with 4 mL of dichoromethane, the C₁₈ cartridge was conditioned with 4 mL methanol and amino cartridge was conditioned with 4 mL ethyl acetate. First, 200 μL of organic extract was loaded through the three different kinds cartridges, then the silica cartridge was washed with 4.0 mL of ethyl acetate (which was then discarded) and the sulforaphane eluted with 4 mL of dichloromethane; the C₁₈ cartridge was washed with 4 mL of water (which was then discarded) and the sulforaphane eluted with 4 mL of 0.1 mol/L of acetic acid; and the amino cartridge was washed with 4 mL of ethyl acetate (which was then discarded) and the sulforaphane eluted with 4 mL dichloromethane. The obtained extractants were evaporated to dryness in a vacuum oven at 45 °C for 2 h, and redissolved with 2 mL of acetonitrile. The resulting solutions were vortexed for 30 s and filtered with a membrane of 0.45 μm. 10 μL samples of these solutions were then injected into the column of the HPLC system. All samples were analyzed in duplicate.