Rf 20axs

HPLC analyses were performed with a Shimadzu HPLC (Shimadzu, Kyoto, Japan) consisting of a binary pump capable of producing a flow rate at 1 mL/min, a syringe loading injection valve with a 100 µL loop. Chromatographic separation was performed by a reverse-phase column (Zorbax C18, 150 mm × 4.6 mm, 5 µm particles, Phenomenex, Torrance, CA, USA) preceded by a 0.5 µm filter and thermostated at 30 °C. The mobile phase was ACN: H2O: Acetic acid (99:99:2, v/v/v) eluted at rate of 1 mL/min. A fluorescence detector (Shimadzu RF-20A xs) was used and set at 333 nm and 460 nm for excitation and emission wavelengths, respectively. The injection volume was 30 µL for both purified urine extracts and purified food extracts. The run time was 10 min. The analytical method used in this study is the European Standard Method EN 14132:2003 also adopted by AOAC as Official Method 2000.03. This method was successfully validated with an interlaboratory study conducted on barley but resulted equally efficient for other cereals and foods by several laboratories worldwide and recommended by companies producing immunoaffinity columns specific for OTA. Successively the European Committee for Standardization (CEN) decided to update this method by editing EN 14132:2009 who incorporated a similar method specific for roasted coffee.

After 0.01 g of dried fractionated SFs in 6 M HCl_aq were treated for 18 h at 105 °C, the hydrolyzed solution was neutralized by 0.2 M sodium citric acid and filtered through a 0.45 µm filter (Hawach Scientific Co. Ltd., Shaanxi, China). The amino acid compositions were ascertained using a prominence amino acid analysis system (RF20AXS; Shimadzu Corp., Kyoto, Japan) and a Na-type amino acids mobile-phase kit (Shimadzu Corp., Kyoto, Japan). The amino acid compositions were glycine, alanine, serine, and tyrosine, which are the major amino acids in the SF molecule. They were calculated with normalization against the total concentrations of serine and tyrosine.

The dried extract was resuspended in 1 mL of mixture of ultra-pure H₂O: CH₃OH: CH₃CN (60:30:10, v/v/v). An aliquot of 10 µL was injected into HPLC equipped with an oven (CTO-10A), pump (LC20AD), degasser (DGU-20A3), auto sampler (SIL-20AHT), fluorescence detector (RF-20AXS), and reversed-phase column (XR-ODS/C8/Phenyl, 3 mm × 100 mm, 2.2 µm, Shimadzu, Kyoto, Japan). The excitation and emission wavelengths were 365 nm and 435 nm, respectively. The mobile phase was H₂O: CH₃OH: CH₃CN (60:30:10, v/v/v) at a flow rate of 0.5 mL/min at 50 °C, with a total run time of 10 min [54 ]. The AFM₁ concentration was expressed in ng of AFM₁/g.

Tocopherols were determined by normal phase HPLC under isocratic condition using a Phenomenex Luna silica column (150 mm × 4.6 mm, 3 µm, 100 A) fitted with a silica 4 × 3.0 mm Security Guard cartridge (Phenomenex Australia Pty, Sydney, Australia) on an HPLC system. Canola oil (260–750 mg, depending on the oil sample, weights of aged oil were increased to improve quantification) was dissolved in 10 mL hexane and filtered through 0.45 µm syringe filters into amber HPLC vials, with 10 µL being injected onto the column. The mobile phase was hexane: isopropanol (99.5:0.5 v/v) and the flow rate was 1.0 mL/min (total run time was 20 min). A Varian Star INERT 9012 binary pump was used with a Varian ProStar Model 410 autosampler. Tocopherols were determined using a Prominence Fluorescence detector (RF—20A XS, Shimadzu, Melbourne, Australia) with excitation wavelength, 290 nm and emission wavelength, 330 nm. Five-point calibration curves were constructed in the ranges of 0.50–6.00 mg/L and 1.00–15.00 mg/L for α- and γ-tocopherols, respectively, and used to quantify tocopherols.

Plasma was derivatized60 (link) with AccQ-Fluor Reagent Kit (Waters, Milford, MA, USA) according to the manufacturer’s instructions. HPLC was performed on Nexera sytem equipped with degasser DGU-20/A5, auto-sampler SIL-30AC, column oven CTO-20AC and fluorescence detector RF-20AXS (all obtained from Shimadzu, Kyoto, Japan). Chromatographic separation was performed on Reprospher 100 C18-DE 1.8 μm, 50 × 2 mm (Dr Maisch GmbH, Ammerbuch-Entringen, Germany) at 45 °C. Elution was performed using acetonitrile and sodium acetate buffer, pH 5.25, at flow rate of 0.8 ml min⁻¹ using a gradient. Detection was carried out using excitation wavelength of 250 nm and emission wavelength of 395 nm.

UHPLC analysis was realized using a Shimadzu Nexera (Shimadzu, Kyoto, Japan) coupled with the same components used for polyphenols analysis and a fluorescence detector RF-20Axs with double acquisition channels and a 12 µL cell. The detector was set at 296 nm and 325 nm for exciting and emission wavelengths, respectively. Oil samples were diluted in 2-propanol for reaching a 100 mg/mL concentration and 1μL injected on the column as a compromise between sensibility and column capacity.
The chromatographic separation was performed using an Agilent Eclipse PAH column (1.8 µm particle size, 4.6 × 50 mm) under isocratic conditions with solvent A (methanol) and B (acetonitrile) in the ratio 60/40 (v/v) and a total flow of 600 μL min⁻¹. The oven temperature was set to 30 °C. The injected volume for each sample was 1 μL.
Tocopherols were quantified using a calibration curve for α, β+γ and δ respectively in the range 0.05–100 ng on the column with R² values higher than 0.999.

FSEC was performed as described previously with modifications (Kawate & Gouaux, 2006 ▶ ). The C-terminally GFP-His₈-tagged YidC proteins were overproduced in Escherichia coli C41(DE3) or BL21(DE3) cells harbouring pRARE (Novagen) and the pCGFP-BC-based plasmid under a variety of growth conditions by changing key parameters such as culture temperature, duration and induction timing. The cells were grown in 5 ml LB medium supplemented with appropriate antibiotics. The cells were harvested, resuspended in buffer A (20 mM Tris–HCl pH 8.0, 300 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride) and disrupted by sonication with a Bioruptor (Cosmo Bio, UCW-310). After centrifugation at 13 000g for 30 min, the supernatant was solubilized with 2% n-dodecyl-β-d-maltoside (DDM) in buffer A. The insoluble material was removed by ultracentrifugation (Beckman Coulter, TLA55 rotor, 71 680g, 30 min) and the supernatant was loaded onto a Superdex 200 10/300 column (GE Healthcare) equilibrated in buffer B (20 mM Tris–HCl pH 8.0, 300 mM NaCl, 0.1% DDM). The fluorescence in the eluate was detected by a fluorometer (Shimadzu, RF-20Axs) with excitation at 480 nm and emission detection at 512 nm.