Syringic acid

A previously LC-MS/MS method [38 (link),39 (link),40 ,41 (link)] was slightly modified (replacing of sodium phosphate with acetic acid in the mobile phase) and applied for the identification of 18 polyphenols in the sample WS extracts: caftaric acid, gentisic acid, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, sinapic acid, hyperoside, isoquercitrin, rutozid, myricetol, fisetin, quercitrin, quercetin, patuletin, luteolin, kaempferol, and apigenin. In brief, chromatographic separation was performed on a reverse-phase analytical column (Zorbax SB-C18, 100 mm × 3.0 mm i.d., 3.5 µm) with a mixture of methanol: 0.1% acetic acid (v/v) as mobile phase and a binary gradient. The elution started with a linear gradient, beginning with 5% methanol and ending at 42% methanol at 35 min; isocratic elution followed for the next 3 min with 42% methanol; rebalancing in the next 7 min with 5% methanol. The flow rate was 1 mL/min, the column temperature 48 °C and the injection volume was 5 μL.
The detection of the compounds was performed on both UV and MS mode. The UV detector was set at 330 nm until 17 min (for the detection of polyphenolic acids, then at 370 nm until 38 min to detect flavonoids and their aglycones. The MS system operated using an electrospray ion source in negative mode (capillary +3000 V, nebulizer 60 psi (nitrogen), dry gas nitrogen at 12 L/min, dry gas temperature 360 °C). The chromatographic data were processed using ChemStation and DataAnalysis software from Agilent, USA.
Another LC-MS method was used to identify other six polyphenols in WS extracts: epicatechin, catechin, syringic acid, gallic acid, protocatechuic acid, and vanillic acid. The chromatographic separation was performed on the same analytical column as mentioned before (Zorbax SB-C18, 100 mm × 3.0 mm i.d., 3.5 µm) with a mixture of methanol: 0.1% acetic acid (v/v) as mobile phase and a binary gradient (start: 3% methanol; at 3 min: 8% methanol; at 8.5 min: 20% methanol; keep 20% methanol until 10 min then rebalance column with 3% methanol). The flow rate was 1 mL/min and the injection volume was 5 μL. The detection of the compounds was performed on MS mode (Table 2). The MS system operated using an electrospray ion source in negative mode (capillary +3000 V, nebulizer 60 psi (nitrogen), dry gas nitrogen at 12 L/min, dry gas temperature 360 °C). All identified polyphenols were quantified both in the WS extracts and hydrolyzed WS extracts (equal quantities of extract and 4 M HCl kept 30 min on 100 °C water bath) on the basis of their peak areas and comparison with a calibration curve of their corresponding standards (epicatechin, catechin, syringic acid, gallic acid, protocatechuic acid, vanillic acid, hyperoside, isoquercitrin, quercitrin). The results were expressed as milligrams of phenolic per gram of dry weight of septum extract.

In this study, most of the chemicals, reagents, and standards were analytical grade and purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Gallic acid, L-ascorbic acid, vanillin, hexahydrate aluminium chloride, Folin-Ciocalteu’s phenol reagent, sodium phosphate, iron(III) chloride hexahydrate (Fe[III]Cl₃.6H₂O), hydrated sodium acetate, hydrochloric acid, sodium carbonate anhydrous, ammonium molybdate, quercetin, catechin, 2,2′-diphenyl-1-picrylhy-drazyl (DPPH), 2,4,6tripyridyl-s-triazine (TPTZ), and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from the Sigma-Aldrich (Castle Hill, NSW, Australia) for the estimation of polyphenols and antioxidant potential. Sulfuric acid (H₂SO₄) with 98% purity was purchased from RCI Labscan (Rongmuang, Thailand). HPLC standards including gallic acid, p-hydroxybenzoic acid, caftaric acid, caffeic acid, protocatechuic acid, sinapinic acid, chlorogenic acid, syringic acid, ferulic acid, coumaric acid, catechin, quercetin, quercetin-3-galactoside, diosmin, quercetin-3-glucuronide, epicatechin gallate, quercetin-3-glucoside, kaempferol and kaempferol-3-glucoside were produced by Sigma-Aldrich (Castle Hill, NSW, Australia) for quantification proposes. HPLC and LC-MS grade reagents including methanol, ethanol, acetonitrile, formic acid, and glacial acetic acid were purchased from Thermo Fisher Scientific Inc. (Scoresby, VIC, Australia). To perform various in vitro bioactivities and antioxidant assays, 96 well-plates were bought from the Thermo Fisher Scientific (VIC, Australia). Additionally, HPLC vials (1 mL) were procured from the Agilent technologies (VIC, Australia).

All prepared sample extracts were analysed by LC-HRMS as described earlier [13 (link)]. In brief, a UHPLC system (Accela, Thermo Fisher Scientific, San Jose, CA, USA) equipped with a reversed-phase XBridge C₁₈, 150 × 2.1 mm i.d., 3.5 µm particle size (Waters, Milford, MA, USA) analytical column as well as water containing 0. % FA (v/v) (eluent A) and MeOH containing 0.1% FA (v/v) (eluent B) were used for linear gradient elution starting with 90% A and continuous increase of B up to 100% in 30 min after an initial hold time of 2 min. The UHPLC system was coupled to an LTQ Orbitrap XL (Thermo Fisher Scientific) equipped with an ESI source. All measurements were performed in the positive ionisation mode with a scan range of m/z 100–1000 and a resolving power setting of 60,000 full width at half maximum (FWHM) at m/z 400.
For quality control (QC), a standard solution consisting of 15 authentic reference standards (N-methylanthranilate, ferulic acid, 2.5-dihydroxybenoic acid, syringic acid, methyl-indole-3-carboxylate, indole-3-acetonitrile, kaempferol, 4-triacetate lactone, l-tryptophan, alpha linolenic acid, galangin, 3′,4′,5′-O-trimethyltricetin, orientin, schaftoside and reserpine) each in a concentration of 1 mg/L, dissolved in MeOH + H₂O 1 + 1 (v/v) + 0.1% formic acid was prepared and measured every eighth injection throughout each LC-HRMS sequence. Retention time stability, peak area precision as well as mass accuracy were determined for all QC samples to verify proper measurement performance throughout the whole sequence with the help of QCScreen [35 (link)] (data not shown).

Compound 2: HRESI-MS, m/z 235.0580 (calc. for C₁₀H₁₂O₅Na m/z 235.0582). ¹H NMR, δ: 7.32 (2H, s, H-2 and H-6), 3.94 (6H, 2xOCH₃), 3.90 (3H, CH₃); ¹³C NMR, δ: 166.7 (CO₂Me), 146.0 (C-3, C-5), 136.1 (C-4), 121.0 (C-1), 106.3 (C-2, C-6), 56.4 (2xOCH₃), 51.9 (CH₃).

Minor phenolic compounds with polar characteristics were extracted and subsequently quantified using high-pressure liquid chromatography (HPLC) according to the standard method of the IOC [58 ]. Some modifications were made based on Türkay et al. [16 (link)] to improve accuracy. HPLC (Agilent 1260 model infinity II, Santa Clara, CA, USA) was equipped with a Spherisorb ODS-2 C18 reverse-phase column (4.6 mm × 25 cm), a 100 A° spectrophotometric UV detector, and an integrator at 280 nm. Two grams of the sample were carefully weighed and transferred into glass tubes, after which 1 mL of a solution containing syringic acid was introduced as an internal standard. The resulting mixture underwent vortexing for 30 s, and then 5 mL of a methanol and water solution in an 80/20 (v/v) ratio was added. Subsequently, the samples underwent a 15 min ultrasonic treatment in a bath, followed by centrifugation at 5000 × rpm for 25 min. The injection volume was 40 μL. The mobile phases were water (0.2% H3PO4 v/v) (A), methanol (B), and acetonitrile (C), and the flow rate was 0.8 mL/min with a gradient flow composition. The gradient elution was as follows, starting from 96% A, 2% B, and 2% C; 50% A, 25% B, and 25% C at 40 min; 40%, 30%, and 30% at 5 min; 50% B and 50% C at 15 min with a 10 min standby; and 96% A, 2% B, and 2% C at 2 min with 10 min standby. The peaks were identified according to the relative retention time of the internal standard peak (syringic acid) described in the standard method. To quantify and express the identified phenolics as tyrosol, the respective response factor (RRF value) was found to be 4.7 (the ratio of the response factor of syringic acid to tyrosol), as described in the standard method. The peaks were identified using the RRT of phenolic compounds against syringic acid, which served as the internal standard of the method.