P hydroxybenzaldehyde

Caffeine was purchased from Merck (Darmstadt, Germany). Fluoranthene, ibuprofen, p‐hydroxybenzaldehyde, p‐coumaric acid, 3‐methoxycinnamic acid, ferulic acid, sulfanilamide, and diclofenac were purchased from Sigma Chemical (St. Louis, Missouri, USA). Carbon dioxide of 5.3 purity grade was purchased from Linde (Guildford, UK). Methanol and acetonitrile (HPLC grade) used were purchased from VWR (Radnor, Pennsylvania, United States). Milli‐Q water was used in all experiments. The compound concentration in all standard solutions was 50 µg/mL dissolved in both acetonitrile and water (Fluoranthene and ibuprofen in water were not studied because of poor solubility). Approximately 3 mL honey purchased from a local market was extracted with 3 mL acetonitrile and spiked with sulfanilamide to 500 ng/mL. Ground water sample was collected from a small lake near the lab, filtrated and then spiked with diclofenac to 1 µg/mL.

HPLC grade methanol, ethanol, and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Standards of phenolic compounds, including, quinic acid, caffeic acid, vanillic acid, syringic acid, ferulic acid, arbutin, adenosine, tachioside, vanillic alcohol, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillin, p-coumaric acid, coniferol, syringaldehyde, acetovanillone, sinapic acid, coniferylaldehyde, sinapaldehyde, syringaresinol, and balanophonin; standards of furfural (FF) and 5-hydroxymethylfurfural (HMF) were obtained from Sigma-Aldrich (MO, USA) with purity ≥ 98%. Folin–Ciocalteu (FC) reagent, 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were provided by Yuanye (Shanghai, China). FRAP test kit was purchased from Solibao (Beijing, China). Other chemicals (analytical grade) were supplied by Sinopharm (Beijing, China). Purified water was obtained using a PALL Lab Water Purification System (Port Washington, NY, USA).

The standards
used for the identification of some of the compounds present in the
analyzed fraction were gallic acid (certified reference material);
5-hydroxymethylfurfural (≥99%); furfural (≥98.5%, GC);
vanillic acid (≥97.0%, HPLC); p-hydroxybenzaldehyde
(95.0%, HPLC); 5-hydroxymethylfurfural (≥99%); syringic acid
(≥95.0%, HPLC); vanillin (≥97%); syringaldehyde (≥98%);
coniferaldehyde (≥98%); and sinapaldehyde (≥98%), all
of which were supplied by Sigma-Aldrich (Saint Louis, MO).
The
hydroalcoholic mixtures used for the identifications were made using
99.8% ethanol supplied by Sigma-Aldrich (Saint Louis, MO) and ultrapure
water (EMD-Milipore, Bedford, MA).

The phenolic standards comprising p-hydroxybenzaldehyde, protocatechuic aldehyde, 2,4-dihydroxybenzoic acid, p-coumaric acid, ferulic acid, catechin, quercetin and naringenin, as well as the chemicals 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and fluorescein disodium salt, were acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Liquid chromatography-mass spectrometry (LCMS)-grade methanol, acetic acid, formic acid, LCMS-grade acetonitrile, Folin-Ciocalteu phenol reagent, hydrochloric acid, sodium hydroxide, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium carbonate, sodium iodide, ethyl acetate, and acetone were procured from Thermo Fisher Scientific (West Palm Beach, FL, USA).

Following methods by (Lopes et al., 2011 (link)), 200 mg of extracted cells were transferred to 5 ml glass tubes with 2 ml 2N NaOH and 200 µl of nitrobenzene, sealed with PTFE caps and heated to 170°C for 3 h. After cooling, excess nitrobenzene was extracted three times with 1-ml ether and the residue acidified with concentrated HCl to pH 2.0. Phenolic compounds were extracted with diethyl ether, dried under nitrogen stream, and re-dissolved in 50% methanol. Separation of phenolics was achieved via a Perkin Elmer Flexar HPLC with a photo diode array detector (PDA). The system was equipped with a Prevail C₁₈ column (250 mm × 4.6 mm, 5µm particle size, Alltech Assoc., Deerfield, IL) and 10-µl injection volume and eluent of 95% water (pH 2.0 adjusted with acetic acid) and acetonitrile 5% v/v at 1 ml/min. A gradient run was created where acetonitrile was increased from 5% to 15% v/v over 10 min, 20% over 30 min, and to 60% over 40 min. Standards used for identification and quantification included p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid, coumaric acid, p-hydroxybenzaldehyde, vanillin, and syringaldehyde, all purchased from Sigma (Sigma-Aldrich Corp., St. Louis, MO).