*2.6. Analysis of Sugars and Inhibitors in Liquid Samples*

Analysis of monosaccharides (glucose, xylose, arabinose, galactose, and mannose) was performed using HPAEC (as described in Section 2.3). Acid posthydrolysis of pretreatment liquids with 4% (*w*/*w*) sulfuric acid was performed in order to hydrolyze oligosaccharides to allow their quantification.

Determination of furfural and HMF was carried out using an HPLC system (Dionex UltiMate 300; ThermoFisher, Waltham, MA, USA) with a diode-array detector and a 3 × 50 mm, 1.8-µm Zorbax RRHT SB-C 18 column. The temperature was set to 40 ◦C.

Determination of formaldehyde, acetaldehyde, *p*-benzoquinone, vanillin, coniferyl aldehyde, syringaldehyde, *p*-hydroxybenzaldehyde, and acetovanillone was performed using UHPLC-ESI-QqQ-MS after derivatization with 2,4-dinitrophenylhydrazine (DNPH). A 1290 Infinity system (Agilent Technologies, Santa Clara, CA, USA) coupled to a 6490 triplequadrupole mass spectrometer (QqQ-MS) was used. The MS parameters were set as follows: gas temperature 290 ◦C, gas flow 20 L/min, nebulizer 30 psi, sheath gas temperature 400 ◦C, sheath gas flow 12 L/min, capillary voltage—3000 V, and nozzle voltage—2000 V. A 2.1 × 150 mm XTerra MS C18 column was used. Eluent A consisted of aqueous 0.1% (*v*/*v*) formic acid, and Eluent B was a 75:25 (*v*/*v*) mixture of acetonitrile and 2-propanol with 0.1% formic acid. Elution was performed using a gradient profile containing the following fractions of Eluent B: 0.0–4.5 min 30–40%, 4.5–9.0 min 40–50%, 9.0–11.0 min 50–70%, 11.0–11.01 min 70–95%, 11.01–15.0 min 95%, 15.0–15.01 min 95–30%, 15.01–18.0 min 30%, and, at the end, two min post-time with 30% for further re-equilibration. Data evaluation was done with MassHunter Quant software. The calibration and derivatization processes were based on previous studies [19,21].

The determination of aliphatic carboxylic acids (formic acid, acetic acid, and levulinic acid) was performed by MoRe Research Örnsköldsvik AB, Sweden. Acid determination was done using HPAEC.

Total aromatic content (TAC) in pretreatment liquids was determined by measuring the absorbance at 280 nm using a UV1800 spectrophotometer (Shimadzu, Kyoto, Japan). Total carboxylic acid content (TCAC) was determined by titration from pH 2.8 to pH 7.0 using an aqueous solution of sodium hydroxide (200 mM).

The determination of total phenolic compounds was carried out by using the Folin– Ciocalteu method [29], with vanillin as the calibration standard. A SpectraMax i3x (Molecular Devices, LLC, San Jose, CA, USA) multimode microplate reader was used for reading the absorbance at 760 nm of the color generated after the incubation of reaction mixtures containing the sample and Folin–Ciocalteu reagent at 23 ◦C for two hours. Reactions were performed in triplicates.
