Sp 2330

GC of the alditol acetates were carried out on a Agilent 7890A gas-liquid chromatograph (Avondale, PA, United States) equipped with a flame ionization detector and fitted with a fused silica column (0.25mm i.d. × 30 m) WCOT-coated with a 0.20 mm film of SP-2330 (Supelco, Bellefonte, PA, United States). Chromatography was performed: from 200 to 240°C at 2°C min^-1, followed by a 10-min hold for alditol acetates. For the partially methylated alditol acetates, the initial temperature was 160°C, which was increased at 1°C min^-1 to 210°C and then at 2°C min^-1 to 230°C. N₂ was used as the carrier gas at a flow rate of 1 mL min^-1 and the split ratio was 80:1. The injector and detector temperature was 250°C.

Individual sugars were assessed according to Mrabet et al. [17 (link)]. Prior to determining the neutral sugars, the sugar fraction was hydrolyzed with trifluoro acetic acid (TFA) for 1 h at 121 °C. The released sugars were reduced and then acetylated and, finally, identified and quantified as alditol acetates using gas chromatography (HP 6890 Plus, Hewlett-Packard, Palo Alto, CA, USA) equipped with a capillary column (30 m × 250 μm × 0.20 mm, SP-2330, Supelco, Bellefonte, PA, USA). The flow of the vector gas (Helium) was 2.2 mL/min at a pressure of 148.24 kPa. The run time was 40.7 min, during which the injector and flame ionization detector (FID) temperatures were 250 and 300 °C, respectively. After 2 min of the injection (splitless mode), the oven temperature was 50 °C; it was then held progressively at 180 °C and finally increased to 220 °C for 22 min. The internal standard was Myo-inositol. The results were expressed as milligrams per kg of dry weight (mg/kg DW).

Neutral monosaccharide composition of the digested wheat brans were determined as their alditol acetate derivatives using separation by gas chromatography on a capillary column (SP2330; SUPELCO, Bellefonte, PA) coupled with mass spectrometry (GC/MS; models 7890A and 5975C inert MSD with a Triple-Axis detector, Agilent Technologies, Inc., Santa Clara, CA), as previously described^{68 (link)}. Helium was used as a carrier gas. The GC/MS run conditions are as follows: injection volume of 2 µl with a split ratio of 1:2; injector temperature at 240 °C; detector temperature at 300 °C; the gradient temperature program set was 160 °C for 6 min, then 4 °C /min to 220 °C for 4 min, then 3 °C /min to 240 °C for 5 min, and then 11 °C /min to 255 °C for 5 min.
The total starch contents of the samples were determined using a total starch assay kit (Product Code: K-TSTA) according to the manufacturer instructions (Megazyme International, Wicklow, Ireland).

A representative aliquot from the pulverized freeze-dried tissue was used for fat analysis. Fat content and fatty composition was determined in duplicate by quantitative determination of the individual fatty acids by gas chromatography [45] (link). Fatty acid methyl esters were directly obtained by transesterification using a solution of 20% boron trifluoride in methanol and then determined by gas chromatography using a capillary column SP2330 (30 m × 0.25 mm, Supelco, Bellefonte, PA). Quantification was carried out through area normalization after adding into each sample 1,2,3-tripentadecanoylglycerol as internal standard. Fatty acids were identified by comparing their relative retention times with those of the external standard and confirmed by comparing their mass spectra to the computer library of the GC/MS database Wiley 275.L and NBS 75 K.L. The proportion of individual fatty acids, as well as that of SFA (14∶0; 16∶0; 18∶0; and 20∶0), MUFA (16∶1; 18∶1; and 20∶1), and PUFA (18∶2; 18∶3; 20∶2; and 20∶4), were calculated as percentages relative to total fatty acid content. Blood triglycerides, cholesterol, leptin and insulin-like growth factor-1 were determined using available kits [46] (link).

The derivatized sugars were separated using a Thermo Trace GC Ultra gas chromatography system (GC) with a capillary fused silica SP-2330 (30 m × 0.25 mm layer thickness of 20 microns internal diameter, Supelco^®- Sigma–Aldrich) GC column and detected by mass spectrometry (MS). After an initial temperature of 80°C for 3 min, the temperature was gradually increased to 160°C at a rate of 30°C/min, then increased gradually to 210°C at a rate of 2°C/min, and finally increased to 240°C at a rate of 5°C/min and held for 10 min. The separated permethylated alditol acetates (PMAA) were detected by a Finnigan Polaris Q mass spectrometer (Thermo) and analyzed using the XcaliburTM Data System. Identification of the derivatives and deduction of the glycosidic linkages were based on the mass spectrum and retention time of previously established standards, and by its ion patterns according to the spectral database PMAA of the Complex Carbohydrate Research Center at the University of Georgia, USA (CCRC) and as described by (Carpita and Shea, 1989 ) for interpreting data derived from the analysis of linkages. The described PMAA analyses were performed in triplicates and the results were averaged. The results are presented as the percentage of composition of each sample, calculated by normalizing to the total ion chromatogram peak areas identified.