Syringaldehyde

Halloysite nanoclay (diameter × length- 30–70 nm × 1–3 μm, nanotube), sulfamethoxazole (SMX, analytical standard), laccase from Trametes versicolor (form- powder), sodium sulfite (Na₂SO₃, ACS reagent grade), sodium hydroxide (NaOH, ACS reagent grade), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, assay- ≥98% high-performance liquid chromatography (HPLC)), syringaldehyde (SA, assay- ≥98%), guaiacol (GUA, assay- ≥98%), methanol (HPLC grade), glutaraldehyde (GTA, grade II, 25% in H₂O), and chitosan (75–85% deacetylated, low molecular weight, molecular weight- 50,000–190,000 Da) were obtained from Sigma-Aldrich (St. Louis, MO, USA). FeCl₃-6H₂O was obtained from JUNSEI (Kyoto) Japan. laccase from Trametes versicolor have a molecular mass of 70 kDa and an isoelectric point (pI) of 3.5 [23 (link)].

The solvents used for polyphenol extraction and liquid chromatographic mass spectrometry (LC-MS/MS) analysis, methanol, acetonitrile and formic acid, were purchased from Merck (Darmstadt, Germany). 2,2-diphenyl-1picrylhydrazyl (DPPH), used to determine the antioxidant capacity, was purchased from Thermo Fisher (Kandel, Germany). The polyphenols used as standards, aminobenzoic acid, acetylsalicylic acid, caffeic acid, chlorogenic acid, ellagic acid, gallic acid, p-coumaric acid, protocatechuic acid, salicylic acid, trans-ferulic acid, vanillic acid, apigenin, epicatechin, aesculetin, catechin hydrate, isorhamnetin, kaempferol, luteolin, polydatin, quercetin, resveratrol, rutin, syringaldehyde and viniferin, were purchased from Sigma-Aldrich (Madrid, Spain). The Mili-Q water used in all the solutions was purified with the Merck Millipore Milli-Q™ Reference Ultrapure Water Purification System model Z00QSVC01 (Darmstadt, Germany).

Samples (40 mg) were added to 10 mL of a 4.0 M NaOH solution and incubated at 170 °C for 2 h for the determination of the total phenolic monomers content while the quantification of the ester-linked monomers was performed using 10 mL of a 2.0 M NaOH solution for 2 h at 35 °C [37 (link), 60 (link)]. After cooling to room temperature, pH was adjusted to ~1 with hydrochloric acid and phenolic compounds were extracted three times with diethyl oxide. Ether fractions were pooled and evaporated under reduced pressure (800 mbar). Residues were finally added to 1.5 mL of a H₂O/methanol solution (1:1 v/v) and quantified using HPLC high-performance liquid chromatography using a Sperisorb S5ODS2 (Waters, RP-18, 250 × 406 mm) column equipped with Waters photodiode array UV detector at 302 nm. A gradient method was used as follows: A (acetonitrile–orthophosphoric acid 15 mM, 10:90 v/v) 100–92% for 6 min; then 92–0% A with solvent B (methanol–orthophosphoric acid 15 mM, 80:20 v/v) 0–50% and C (acetonitrile–orthophosphoric acid 15 mM, 80:20 v/v) 0–50% for 29 min. Internal reference was 3,4,5-trimethoxycinnamic and pure standards of vanillin, vanillic acid, 4-hydroxybenzoic acid, syringic acid, syringaldehyde, p-coumaric acid, ferulic acid and sinapic acid (Sigma-Aldrich, St. Louis, MO, USA) were used for the validation of peak compound identification and for the quantification.

Methanol (LC-MS grade) and sodium hydroxide (>99%) were purchased from Merck (Darmstadt, Germany). Ammonium acetate (≥99% for HPLC) and formic acid (LC-MS Ultra) were purchased from Fluka (Buchs, Switzerland). Isopropanol was obtained from Fisher Scientific (Geel, Belgium). Ultrapure water was provided by a Milli-Q purification apparatus (Millipore Direct-Q UV, Bedford, MA, USA). Regarding the standards that were used, syringic acid 95% was purchased from Extrasynthèse (Genay, France). Gallic acid 98%, ferulic acid 98%, epicatechin 97%, p-coumaric (4-hydroxycinnamic acid) 98%, homovanillic acid 97%, quercetin 98%, oleuropein 98%, pinoresinol 95%, caffeic acid 99%, taxifolin 98%, vanillic acid 97% and syringaldehyde 98% (internal standard) were obtained from Sigma-Aldrich (Steinheim, Germany). Hydroxytyrosol 98% and luteolin 98% were acquired from Santa Cruz Biotechnologies. Vanillin 99%, apigenin (4,5,7-trihydroxyflavone) 97% and tyrosol (2-(4-hydroxyphenyl) ethanol) 98% were purchased from Alfa Aesar (Karlsruhe, Germany). Stock standard solutions of individual compounds (1000 mg/L) were solubilized in methanol and stored at −20 °C in dark brown glass bottles. Intermediate standard working solutions containing the analytes were prepared by appropriate dilution of the stock solutions with methanol:water (80:20, v/v) over the concentration range 0.1–8 mg/L.

The solvents used were: HPLC methyl alcohol (J. T. Baker), formic acid (Dynamics), and ultrapure water obtained from a Milli-Q system. Gallic acid, catechol, catechin, chlorogenic acid, caffeic acid, vanillin, syringaldehyde, p-coumaric acid, coumarin, rutin, myricetin, and quercetin were acquired by the Sigma-Aldrich Brasil LTDA. All other reagents were of analytical grade.