Lab solution lc software

High Performance liquid Chromatography (HPLC) was performed with a Jasco modular LC system equipped with a Rheodyne Injector, binary pump, column oven, auto sampler and thermo controller (set at 4°C) and detection was done using photodiode array detector. All these were controlled by Lab solution LC software (Shimadzu, Japan).
The needle was washed with acetonitrile after each injection to prevent residual samples from previous run. Injection volume was 10μl, flow rate was 0.3ml/min and column temperature was set at 26°C. Sample was applied on reverse phase column, Zorbax Eclipse plus C18 column (4.6X 250mm, 5 μm). Mobile phase A contained water and 0.1% Formic Acid. Mobile phase B was Acetonitrile containing 0.1% Formic Acid. Gradient programme of 15% at 0 min, 15% at 4min, 27% at 7 min, 30% at 10min, 35% at 13 min, 35% at 15 min, 45% at 18min, 100% at 20min, 15% at 25 min was done with flow rate of 0.3 ml/min. Chromatograms were analysed at 254nm.
Working solutions were prepared by mixing stock solution with water to the desired concentration. Fresh stock solution of β-ODAP was prepared before every run. To 1000μg of β-ODAP, 60μl of 0.5M NaHCO₃ was added and made up to 1ml with water. They were passed through 0.22μm PVDF filter prior to HPLC analysis.

Vitamin E congeners were quantified in triplicate as previously described (Grebenstein & Frank, 2012 (link)). In brief, about 200 mg flour was weighed into a glass tube and 2 ml of ethanol (containing 1% ascorbic acid (w/v)), 900 µl of H₂O, and 600 µl of KOH were added and samples saponified for 30 min at 70°C. Samples were then extracted three times with 2 ml of hexane and in total 5 of 6 ml added hexane was collected and evaporated. Samples were dissolved in 100 µl of ethanol and analyzed on a Shimadzu (Kyoto, Japan) Prominence HPLC equipped with an LC‐20 AT pump, a DGU‐14A degasser, a CTO‐10AS column oven (set to 40°C), cooled autosampler SIL‐20 AC HT (4℃), and an RF‐20A fluorescence detector. Tocopherols and tocotrienols were separated on a Phenomenex KinetexTM PFP column (2.6 µm, 150 × 4.6 mm; Phenomenex) using methanol/H₂O (85:15, v/v) as eluent at a flow rate of 1.2 ml/min. The fluorescence detector was operated at an excitation wavelength of 296 nm and an emission wavelength of 325 nm. Peaks were recorded and integrated using Lab solution LC software (Shimadzu, Kyoto, Japan) and quantified against external calibration curves.