Prominence lc 20ad

Cecal contents were obtained from 8-week-old mice that had been treated orally (or not) with an antibiotic cocktail (ampicillin, neomycin, and metronidazole each at 1 g/l, and vancomycin at 0.5 g/l) for 4 weeks. The samples were diluted in sterile water, mixed with 2-ethylbutyrate as an internal control, and then labeled with 2-nitrophenylhydrazine with the use of a YMC-Pack FA kit (YMC, Kyoto, Japan). SCFAs in the samples were analyzed by liquid chromatography (Prominence LC-20AD; Shimadzu, Kyoto, Japan).

After being thawed, the stored supernatants were centrifuged again (at 1,000 g and 5°C for 7 min), and the resultant supernatants were filtered through a polypropylene membrane (pore size: 0.20 μm; Toyo Roshi Ltd., Tokyo, Japan). The sample was analyzed by HPLC (high-performance liquid chromatography) (Shimadzu Prominence LC-20AD; Shimadzu Corporation, Kyoto, Japan) to determine the levels of acetic acid, lactic acid, formic acid, malic acid, fumaric acid, succinic acid, citric acid, α-ketoglutaric acid, oxalic acetic acid, and pyruvic acid.

Optical rotations were measured on a Rudolph IV Autopol automatic polarimeter. IR spectra were determined on a Thermo Nicolet Nexus 470 FT-IR spectrometer. 1D and 2D NMR spectra were recorded on a Bruker Avance 400 NMR spectrometer (400 MHz for ¹H and 100 MHz for ¹³C, respectively). Chemical shifts were referenced to the solvent peaks at δ_H 2.50 and δ_C 39.8 for dimethyl sulfoxide (DMSO-d₆), respectively, and coupling constants were in Hz. ESIMS and HRESIMS spectra were obtained from a Thermo Scientific LTQ Orbitrap XL instrument. Column chromatography was carried out with silica gel (160–200 mesh, 200–300 mesh), and HF₂₅₄ silica gel for TLC was obtained from Qingdao Marine Chemistry Co. Ltd. (Qingdao, China), ODS gel (50 μm), and Sephadex LH-20 (18–110 μm) were obtained from YMC (Kyoto, Japan) and Amersham Pharmacia Biotech AB, Uppsala, Sweden. HPLC chromatography was performed on an Alltech instrument (426-HPLC pump), equipped with an Alltech UVIS-200 detector and semipreparative reversed-phase columns (Grace Prevail C₁₈, 5 μm, 250 mm × 10 mm). Analytical HPLC chromatography was performed on Prominence LC-20AD (Shimadzu, Kyoto, Japan) equipped with pump LC-20AD, detector Prominence SPD-M20A PDA and Shimadzu VP-ODS column (150 mm × 4.6 mm), Thermo BDS Hypersil C₁₈ column (150 mm × 4.6 mm, 5 μm).

SEC analysis was performed using a Shimadzu Prominence LC-20AD (Shimadzu Corporation, Kyoto, Japan) system equipped with a UV (280 nm) and refractive index (RI) detector and a three-column sequence of Shodex KD802, KD803, and KD805. HPLC-grade N, N-dimethyl formamide (DMF) with 10 mM LiBr was used as the eluent, with a flow rate of 1.0 mL min⁻¹ at 40 °C. Fluka polyethylene glycol/poly(ethylene oxide) standard ReadyCal sets (Sigma-Aldrich Japan G. K., Tokyo, Japan) were used for molecular mass calibration. The standard and samples were completely dissolved in the eluent and filtered through a 0.45 μm PTFE syringe filter (ADVENTEC) prior to injection to the system. Some of the GL400 samples treated at ≥120 °C did not dissolve completely, thus, the soluble portion was filtered and subjected to SEC analysis.

A Shimadzu HPLC system equipped with a fluorescence detector (Prominence LC-20AD, Shimadzu, Kyoto, Japan) was used for the detection of tocotrienols. The separation was carried out with an isocratic flow of methanol/acetonitrile/dichloromethane (25:23:2, v/v/v) at a flow rate of 1 mL/min, and eluted through a reverse phase C18 column (250 × 4.6 mm, 5 µm, Phenomenex, Torrance, CA, USA). The temperature of the column was maintained at 30 °C and the injection volume was 25 µL. The excitation and emission wavelengths for the fluorescence detector were set at 290 and 330 nm, respectively.
Quantification of the three isomers of tocotrienols (α-, β-/γ-, and δ-) was done by referring to each of the external calibration curves (R² ≥ 0.99) established for the individual isomer using the reference standard mixture. The standard solutions of 2 to 10 µg/mL were prepared through serial dilution of the standard mixture with acetonitrile. Tocotrienols content was calculated from the triplicate injections by using the linear calibration curves. The final content was expressed in µg/g sample. The entrapment efficiency of tocotrienols was calculated based on Equation (2) as follow:
where T_sample refers to the tocotrienols content in the sample, whilst T_oil is the total tocotrienols amount in the oil (palm olein and TRF) added into the emulsion.