Hydroxylamine hydrochloride

All chemicals were used without further purification. Sigma‐Aldrich: (3‐aminopropyl) trimethoxysilane (APTMS, 99 %), sodium silicate solution (27 %), Rhodamine 6G, RhCl₃ (98 %), hydroxylamine hydrochloride (>98 %); Alfa Aesar: HAuCl₄ ⋅ 3H₂O (99.99 % metal basis); Merck: hydrochloric acid (37 %); and Acros Organics: trisodium citrate dihydrate (99 %). Water was purified with Milli‐Q system (18.2 MΩ) before use. Argon (99.999 %), hydrogen (99.999 %), oxygen (99.999 %), and nitric oxide (10 % NO in He), quality>4.5 (>99.995 %) were purchased from Linde Gas.

All reagents were used as received from commercial vendors and used as such. Ethanol and hydroxylamine hydrochloride were purchased from Alfa Aesar (Ward Hill, MA). Ethyl glyoxylate and heparin sodium salt were purchased from Sigma-Aldrich (St. Louis, MO). Collagenase Type IV was obtained from Gibco, (Waltham, MA). ¹⁴C-labeled Ethyl glyoxylate was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Deuteriochloroform (CDCl₃), methanol-_d4 and DMSO-_d6 were purchased from Cambridge Isotope Laboratories (Tewksbury, MA). 1-(Azepan-1-yl)propan-2-amine, 1-(piperidin-1-yl)propan-2-amine, (2-aminopropyl)dimethylamine, (2-aminopropyl)diethylamine, α-methyl-1-pyrrolidineethanamine, α,4-dimethyl-1-piperidineethanamine, were purchased from Enamine Building Blocks (Cincinnati, OH). 2-(S)-2-(Boc-amino)propanal (Boc-ala-aldehyde), 4-Piperidinopiperidine, were purchased from Combiblocks (San Diego, CA.). Aminocyclopropane, aminocyclobutane, 1-aminobutane, benzylamine, homopiperazine and (S)-( +)-α-Methoxy-α-(trifluoromethyl)phenylacetyl chloride (Mosher’s acyl chloride) were purchased from Sigma-Aldrich (St Louis, MO). Ethyl glyoxylate oxime and all RS-based compounds were synthesized according to Sit et al.^{14 (link)} and obtained as a pale-yellow liquid that was stored at 4 °C and only taken out when used.

Double distilled water was used for obtaining solutions in this work. Nitric acid (65%) from Merck, (Darmstadt, Germany), hydrogen peroxide from Chempur (Piekary Śląskie, Poland), hydrochloric acid and acetonitrile from Honeywell Fulka (Seelze, Germany), ammonia (25%) from VWR Chemicals, (Fontenay-sous-Bois, France), ethylenediaminetetraacetic acid (EDTA) in the form of a disodium salt dihydrate from Raral (Budapest, Hungary), and 4-nitro-1,2-phenylenediamine from Sigma Aldrich (St. Louis, MO, USA) were used. Hydroxylamine hydrochloride, sodium selenate (anhydrous, >99.8%, metal basis), and sodium selenite (anhydrous, >99.75%, metal basis) were obtained from Alfa Aesar (Kandel, Germany), and dimethyl sulfoxide was purchased from VWR International (Solon, OH, USA).

In this study, HgII was provided as HgCl₂ while MeHg was provided as CH₃HgCl. Since all experiments were performed individually for either HgII or MeHg, we only performed total-Hg analysis to examine the changes in HgII or MeHg levels in samples. Cell pellets containing HgII or MeHg were fully digested by concentrated TMG nitric acid and reagent-grade hydrogen peroxide (4:1; v-v) in a Teflon digestion vessel (Savillex, Eden Prairie, MN, USA) at 80 °C overnight^{33 (link)}. Digestion of media samples was strengthened by adding an acidic mixture of permanganate and persulfate, and heated at 80 °C overnight^{34 (link)}. All digested samples were cooled, and neutralized with aliquots of 30% hydroxylamine hydrochloride (Alfa Aesar, Haverhill, MA, USA). Sample Hg was quantified by double amalgamation technique and cold vapor atomic fluorescence spectrophotometer (Brooks Rand Model III, Bothell, WA, USA) as described elsewhere³⁵ . Throughout the digestion of samples, we included reagent blanks, washing solutions, buffer solutions and a standard reference material (SRM; i.e., National Research Council Canada DORM-4 fish protein). The reagent blanks, HBSS, PBS, PBS + 1 mM DMPS and PBS + 2 mM GSH showed an average of 2.9, 29.7, 30.0, 30.5 and 30.4 pg/ml of Hg, respectively. Digestion of DORM-4 yielded an average recovery of 96.1% of certified total-Hg value.

Using the same amount of extract as the methoxyimino penta-trimethyl silyl Derivatization for each sample group, the samples were dried in 0.5 mL V-vials and reconstituted in 50 μL of 20 mg/mL hydroxylamine hydrochloride (CAS# 5470-11-1; Acros Organics, Fair Lawn, NJ, USA) in pyridine (CAS#25104, Thermo Fisher Scientific, Waltham, MA, USA) and agitated with a microstirbar at 90 °C for 1.5 h. 100 μL of propionic anhydride (CAS# 240311-50G; Sigma Aldrich, St. Louis, MO, USA) was then added and samples were agitated with a microstirbar at 70 °C for 30 min. The samples were dried down by a gentle stream of air and then reconstituted in 100 μL of ethyl acetate for GC-MS analysis.