2.3.3. Sugars and Glycosidic-Linkage Composition

The fillet sample was dialysed with a membrane cut-off of 12–14 kDa to recover the high molecular weight (HMW) compounds. Neutral sugars of the initial and dialysed samples were analysed by gas chromatography-flame ionization detection (GC-FID) after conversion to their alditol acetates [21,22]. The quantification was carried out using 2-deoxyglucose as internal standard. Monosaccharides were released from polysaccharides with pre-hydrolysis of the samples using 0.2 mL of 72% (w/w) H2SO4 for 1 h at room temperature followed by 2.5 h hydrolysis in 1 M H2SO4 at 100 ◦C. After hydrolysis, the reduction (NaBH4) and acetylation (acetic anhydride using methyl imidazole as catalyst) of the monosaccharides were performed. The alditol acetates were analysed using a DB-225 column (30 m, 0.25 mm i.d., 0.25 μm film thickness) and a GC-FID PerkinElmer-Clarus 400 [23,24]. Free sugars were also quantified on the initial sample (not dialysed) using the same method, by omitting the hydrolysis in the abovementioned steps. The oven temperature program was as follows: 220 ◦C, hold for 7 min, to 240 ◦C at a rate of 5 ◦C/min. The temperature of injector was 220 ◦C, and the detector was 240 ◦C. Hydrogen was used as the carrier gas.

Glycosidic-linkage composition of the dialysed sample (HMW) was determined by GC-qMS of the partially methylated alditol acetates as previously described [22]. The polysaccharides were methylated using CH3I, hydrolysed (TFA 2M) and the resultant monosaccharides were reduced (NaBD4) and acetylated. The partially methylated alditol acetates (PMAAs) obtained were analysed by gas chromatography mass spectrometry (GC-qMS) on a Shimadzu GCMS-QP2010 Ultra [25]. The GC was equipped with an SGE HT5 (Supelco, Bellefonte, PA, USA) fused silica capillary column (30 m length, 0.25 mm i.d., and 0.10 μm of film thickness).
