L 7300 column oven

Analysis of α-tocopherol was carried out as described in a previous report [14] (link) with some modification. The concentration of α-tocopherol was determined using an HPLC system equipped with an L-6200 pump, an L-7300 column oven and an F-1050 fluorescence spectrophotometer (HITACHI, Tokyo, Japan). One hundred μl of a sample with 100 μl of 0.1 M Na 2 HPO 4 buffer and 200 μl of methanol with 1 μg/ml δ-tocopherol as an internal standard were added. Then 1,100 μl of n-hexane/dichloromethane (4/1, v/v) was added and the mixture was shaken vigorously for 1.5 min. After centrifugation at 800 × g for 10 min, 900 μl of the organic layer was taken and evaporated to dryness under a nitrogen gas stream. The residue was dissolved in 200 μl of mobile phase for HPLC injection. The column for HPLC was an Inertsil ® ODS-4 (3 mm in inside diameter × 150 mm) (GL Sciences Inc., Tokyo, Japan). A mobile phase containing methanol/distilled water (98/2, v/v) was used. Column temperature and flow rate were 30°C and 0.4 ml/min, respectively. The light excitation and emission wavelengths for detection were 298 and 325 nm, respectively. Fifteen μl of a sample was injected into the HPLC system.

The HPLC system consisted of an L-7100 pump, L-7300 column oven, and an L-7420 UV detector (Hitachi, Ltd.). The conditions were as follows: 5C18MS-II (Cosmosil, Nacalai Tesque Inc. Kyoto, Japan) column, 4.6 mm I.D. × 250 mm; solvent, MeOH-water: 20% (0 min) → 30% (60 min) → 45% (80 min) → 64% (140 min) MeOH; flow rate, 1ml/min, injection volume, 10 μl; detection at 254 nm. Agarotetrol in the samples was identified by comparing peak retention times with the retention time of agarotetrol purified from agarwood.