Identification of Hydrogen Disulfanes and Hydrogen Trisulfanes in

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Identification of Hydrogen Disulfanes and Hydrogen Trisulfanes in H2S Bottle, in Flint and in Dry Mineral White Wine Christian Starkenmann, Charles Jean-François Chappuis, Yvan Niclass, and Pascale Deneulin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03938 • Publication Date (Web): 06 Nov 2016 Downloaded from http://pubs.acs.org on November 12, 2016

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Journal of Agricultural and Food Chemistry

Identification of Hydrogen Disulfanes and Hydrogen Trisulfanes in H2S Bottle, in Flint and in Dry Mineral White Wine Christian Starkenmann,*,† Charles Jean-Francois Chappuis, Yvan Niclass, Pascale Deneulin‡ †

Firmenich SA, Corporate R&D Division, P.O. Box 239, CH-1211 Geneva 8, Switzerland

‡

Changins – Viticulture and Oenology,University of Applied Sciences and Arts Western

Switzerland, Switzerland City, Country *To whom correspondence should be addressed. Tel: +41 22 780 34 77, Fax: +41 22 780 33 34 e-mail [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Through the accidental contamination of a gas cylinder of H2S, the importance of polysulfanes

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for flint, gun powder, and match odors was discovered. The hydrogen disulfane was prepared

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from disulfanediylbis[methyl(diphenyl)silane] and its odor descriptor was evaluated in gas phase

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from a gas chromatography coupled to an olfaction port. The occurrence of this compound in

6

flint and pebbles was confirmed by analyses after derivatization with pentafluoro bromobenzene.

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The occurrence of this sulfane was also confirmed in two dry white Swiss Chasselas wines,

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sorted by a large scale sensory analysis from 80 bottles and evaluated by 62 wine professionals.

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The occurrence of disulfane was confirmed for the two wines described as the most mineral. The

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polysulfane is a class of compounds contributing to the flint odor and that may contribute to the

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wine mineral odor descriptor. Due to the high volatility and instability HSSH pure was not

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isolated but kept in solution and its odor profile was described by gas chromatography coupled to

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an olfaction port as flint, matches, fireworks with an higher odor intensity compared to H2S.

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KEYWORDS

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Disulfane and trisulfane; wine minerality, flint odor, bis(methyl diphenyl silane) disulfane

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INTRODUCTION Volatile organic sulfur compounds are important odorant contributors found in the

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environment, in food, and in the chemical signals of animals.1-4 Odorant molecules used to flavor

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food matrices are now well understood, but some pieces are still missing, mainly in terms of

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understanding unstable compounds.

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In the course of our research on toilet malodors, a gaseous reconstitutions of malodors was

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delivered in olfactometers.5,6 Diluted H2S in N2 was blown into olfactometers with toilet malodor

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constituents.5 Over time, we noticed that the smell of the hydrogen sulfide (H2S) changed. When

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H2S was the only gaseous compound in the olfactometer, the odor was no longer eggy or sewage,

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but changed to a flint-like odor, similar to the cold smell of fireworks or the smell associated

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with a dentist drilling teeth. Our hypothesis was that the odor originated from oxidized H2S

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because the source of organic compounds should be limited in a certified H2S/N2 gas cylinder.

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Di- and trisulfanehave been reported in the literature since the early 1900s but not their odors.

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Publications associated with these compounds are linked to the rubber, latex science, sensor

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development, and atmospheric and geological chemistry industries, but to the best of our

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knowledge not to the food products industry. The well-known odors of flint have thus not been

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explained in detail.7-12

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The misleading definition of minerality has been stressed by several authors13-14 and a sensory

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consensus seems difficult to find.15-17 This descriptor is used mainly for white dry wines from

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cool climates such as Loire Valley and Burgundy, as well as for Riesling wine from Alsace and

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Germany. A mineral wine may associated with odors such as stony, wet rock, chalky, or like

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rocks being scraped together.18-22 Flint odor is not well understood despite being widely used by

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enologists.17 The root of the problem is that the only odor reference for gun flint aroma is 3



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benzene methanthiol.23 T. Tominaga discussed the minerality linked to a smoky, empyreumatic

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aroma and the contribution of benzene methanethiol and 4-methyl-4-sulfanyl-pentan-2-one,

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which constitute the boxwood smell in their definition of gun flint aroma.23 No other reference

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exists outside of scraping rocks together for the flinty odor. South African and New Zealand

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enologists agree with this definition of the flinty/mineral odor, which is mainly used for Corban

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and Sauvignon blanc wines.17-20 Green et al. are studying the influence of mercaptans on

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minerality,21 whereas Esti et al. associate mineral odor with flint, sulfur, and tuff, which is a type

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of volcanic rock.22 Regarding the taste aspect, the perceived acidity driven by the ratio between

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malic and lactic acid is one type of minerality.17

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Flint nodules are formed in an anaerobic environment. The sediments are rich in H2S produced

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by the bacterial reduction of sulfates. The presence of iron from clay minerals then immobilizes

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the H2S as iron sulfides.24 We can speculate that other sulfur forms, such as H2Sn and a range of

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polysulfides, are encapsulated and released when these rocks are scraped together.

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This research aimed to analyze the odor contaminants in a pressurized H2S bottle. We also

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searched for sulfanes in flint and conglomerate pebbles, as well as in Swiss dry white Chasselas

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wine.

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MATERIALS AND METHODS Chemicals. Commercially available reagents and solvents of adequate quality were used

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without further purification. The H2S for reactions was in a 227 g cylinder. C2H5SH (97%), 1,8-

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diazabicyclo[5.4.0]undec-7-ene (DBU) 99%, trifluoroacetic acid (TFA) 98%, NaH 55% in oil,

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elemental sulfur (S8), the internal standard (I.S.) methyl octanoate, ethane thiol, propanethiol,

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iron powder (Fe°) (cat 44900), salts for buffers, triethyl amine, 1H-pyrrole-2,5-dione, and 14


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ethyl-(N-ethyl maleimide) (NEM) were from Sigma-Aldrich (Buchs, Switzerland). CH3SH in

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ethanol (1%) was made from in-house ingredients (Firmenich S.A., Geneva, Switzerland).

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Pentafluorobenzyl bromide (PFBBr) and methyldiphenylsilane were from Alfa Aesar (Heysham,

68

England). The contaminated H2S pressurized cylinder was a dilution containing 52.5 µL/m3 H2S

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and 15.4 × 103 µL/m3 N2 (Carbagas, Carouge, Switzerland). Solid phase extraction OASIS HLB

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cartridges (1 g) were purchased from Waters (Montreux-Chailly, Switzerland).

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Wines. A generic Chasselas wine was used for method development: Chasselas de Romandie

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12%, (Coop Basel, Switzerland). Four other dry white Chasselas wines were selected on the basis

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of their minerality exemplarity: Brez La Colombe 2012, Raymond Paccot (VDm54); Sélection

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Comby Héritage, 2012, Yann Comby (VSm72); Lu, 2012 Guy et Mathurin Ramu (GEa14); Le

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Cellier du Mas, Grand Cru, Mont-sur-Rolle 2012, David et Françoise Blanchard (VDa60

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Gas Chromatography (GC)/Electron Impact Mass Spectrometry (EI-MS). The analytical

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GC-MS was an Agilent-GC-6890 system connected to an Agilent-MSD-5973 quadrupole mass

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spectrometer (Palo Alto, California) operated at ca. 70 eV. Helium was the carrier gas set at a

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constant flow rate of 0.7 mL/min. Separations were performed on fused-silica capillary columns,

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coated with SPB-1 (Supelco, Buchs, Switzerland, 30 m × 0.25 mm i.d., 0.25 µm). The standard

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oven program was as follows: 40 °C for 8 min, increased to 250 °C at 15 °C/min; for derivatives

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100 °C for 5 min, increased to 250 °C at 10 °C/min, and then held at 250 °C.

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Gas Chromatography Mass Spectrometry Olfaction (EI-MS-O). The GC-MS-olfaction

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analyses were carried out using an Agilent GC-7890A system equipped with an Agilent 5975C

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mass spectrometer and a Gerstel Olfactometry Port (ODP3)., The column was a DB-1 capillary

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column (60 m × 0.25 mm), with film thickness 0.25 µm (Agilent). Temperature program: 45 °C,

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to 120 ºC by 5 °C/min, then 10 °C/min to 250 °C, 5 °C isotherm, then 15 °C/min to 300 °C, 5



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isotherm 20 mn. Carrier gas flow (He): 1.1 mL/min; split ratio 1:10, injection volume 1 µL of

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neat sample. The crosspiece was installed in the GC oven to provide a 1/1 ratio between the

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sniffing port and the MS. Mass spectra were generated at 70 eV at a scan range from m/z 40 to

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450. The compound identifications were made by using Firmenich MS and linear retention index

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(LRI) libraries, and by comparison with reference samples. LRIs were determined after injection

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of a series of n-alkanes (C5-C31) under identical conditions on the first dimension (non-polar).

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The LRIs were calculated by linear interpolation from the retention times of the analytes and the

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two closest alkanes. Mass spectra are available in the Supporting Information.

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Gas Chromatography Quadrupole Time of Flight (QTOF), The high mass resolutions were

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measured with an Agilent 7200 GC–quadrupole time-of-flight (QTOF) mass spectrometer

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operated in positive chemical ionization mode and controlled by MassHunter Acquisition B.07.

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The GC system was equipped with a DB-1 MS Ultra Inert capillary column (60 m, 250 µm i.d.,

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0.25 µm thick). The GC oven temperature was programmed to begin at 50 °C (held for 5 min) to

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increase to 120 °C by 5 °C /, and then to increase to 300 °C by 10 °C /min (held for 15 min). The

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MMI inlet was set at 250 min °C in split mode with a 50:1 split ratio and an injection volume of

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1 µL. Helium was used as a carrier gas at a flow rate of 1.5 mL/min. The QTOF MS was

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operated at 2 spectra/s in an m/z range of 30–500, with a resolution of approximately 15,000 at

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m/z 150–500. The column was a DB-1 UI (60 m, 250 µm i.d., 0.25 µm thick), operated at 50 °C

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(5 min), increased by 5 °C/min to 120 °C, increased by 10 °C/min to 300 °C (15 min); split 50:1,

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injection 1 µL; MS emission tune fixed in µA; Cl, 5.1/100 (source temperature 300 °C).

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Gas Chromatography Flame Photometric Detector Plus (FPD Plus). The GC was an

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Agilent 6890 was equipped with Flame Photometric Detector Plus (FPD Plus). The column was

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a HP-5 capillary column (30 m × 0.25 mm), with film thickness 0.32 µm (Agilent). Temperature 6


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program: 45 °C, 5 min., then 10 °C/min to 250 °C, Carrier gas flow (He): 1.1 mL/min; split ratio

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1:10, injection volume 1 µL of neat sample.

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High Pressure Liquid Chromatography HPLC-UV: The acids were quantified using an

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HPLC Agilent 110 equipped with detector UV (DAD G1315B). The column was a Phenomenex

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Aqua® 5 µm C18 125 Å, LC Column 250 x 2.0 mm. The elution type was isocratic with 50 mM

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KH2PO4, pH 2.9 with a flow rate of 0.25 mL/min. The flow and column diameter were the only

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changed accordingly to the application note: Phenomenex App ID: 14171. The dosage was

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performed exactly as described in the application note.

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Gas Analysis via N-Ethyl Maleimide (NEM) Derivatization. The gas was bubbled (350 L, 200

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L/h) in the same conditions as described previously5 in a 500 mL solution of N-ethyl maleimide

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(NEM) (5 mg/L) with a KH2PO4/K2HPO2 buffer at pH 8 (0.01 M). The organic compounds were

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trapped on a solid phase extraction cartridge (OASIS) and desorbed with 10 mL Et2O containing

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1 µg methyl octanoate as the I.S.

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Preparation of HSSH via disulfanediylbis[methyl(diphenyl)silane].25The methyl diphenyl

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silane (25 g, 126 mmol) was heated at 185 °C for 24 hours in the presence of elemental sulfur (4

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g, 126 mmol). The crude mixture was cooled and distilled (125-135°C, 0.1 mm) to give 10.4 g

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(yield 35.8%). This compound was diluted in toluene (40 mL) and added dropwise (30 min) to

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NaH (2.2 g, 55% in oil, 50 mmol) in toluene (90 mL) in an ice bath (5-10 °C). The reaction

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mixture was filtered. Iodine (5.1 g, 20 mmol) in toluene (200 mL) was added dropwise (2 h),

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keeping the temperature at 20 °C with a cool water bath. As soon as the yellow color

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disappeared, the reaction was stopped. The solution was filtered and the solvent removed at 40

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°C under vacuum on a rotary evaporator. The oil (4 g) (pH 6-7) was kept at -18°C.

7



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To release HSSH, the crude oil (1 g, 2.2 mmol) was diluted in toluene (5 mL) and TFA (0.22 g,

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2.0 mmol) was added and stirred for 12 h. A Vigreux column (1 cm) was then placed above the

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reactor and about 1.0 mL of toluene enriched in HSSH was distilled under a 10 mmHg vacuum.

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The distillate was collected in a flask cooled to -78 °C. GC-MS-high resolution (HR):HSSH

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M+H = 65.9653 ∆ = 1.97 ppm.

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Preparation of H2S mother solution. Pure H2S from a cylinder was bubbled to saturation in

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EtOH (100 mL). The outlet flask was connected to a bleach scrubber. When H2S absorption

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stopped, the flask was closed and weighed. The weight gain at 22 °C was precise and

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reproducible at 1.4 g/ 100 mL. This solution used for quantitation. The solution was typically

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prepared Monday, used on Tuesday and Wednesday, and then discarded in diluted bleach

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solution. The saturated solution of H2S in toluene was prepared the same way. The solubility was

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0.85 g/ 100 mL.

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Preparation of H-Sn-H and R-Sn-H in EtOH.

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The H2S/EtOH saturated solution (2 mL) was placed in a 20 mL vial and about 200 mg Fe° was

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added. The solution can be used after 15 h at 22 °C as such or extracted with pentane (20 mL)

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and HCl (0.1 M) 2x10 mL. The ratio between H2S, HSSH, and HSSSH was not stable.

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The H2S/EtOH saturated solution (10 mL) was also mixed with CH3SH (1% EtOH, 4.8 mL, 1

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mmol), C2H5SH (62 mg, 1 mmol), Fe° powder (100 mg, 18 mmol), and H2O2 (2 mmol, 30% in

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H2O, 230 µL). After 15 h at 22 °C, the mixture was extracted with pentane and washed with HCl

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(0.1 M) dried on anhydrous Na2SO4, filtered and concentrated gently under argon. GC-MS-HR:

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CH3SSH M+H = 80.9831 ∆ = 4.7 ppm, CH3CH2SSH 94.9988 ∆ = 4.6 ppm.

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Sulfite Solution Treated with Iron. Na2SO3 (0.2 g, 1.6 mmol) and iron powder (10 g) were

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added to a white wine model system containing L-malic acid (1.5 g), DL-tartaric acid (1.5 g), 8


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water (900 mL), and EtOH (100 mL) adjusted to pH 3.3 with NaOH (1 M). Every hour, 100 mL

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of the solution was extracted as described below for analysis.

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Wine Analysis of H2S and HSSH after Derivatization.

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Wine (100 mL) was extracted with a mixture of pentane (50 mL), diethyl ether (20 mL), and

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methyl octanoate (0.1 mL) as the i.s. (solution: 1 g/100 mL EtOAc). The free thiols were

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derivatized by adding PFBBr (10 µL) and DBU (10 µL). The solvent was removed by distillation

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to 5 mL and washed with H2O (2 × 2 mL), the organic phase was dried by using anhydrous

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Na2SO4 and filtered, and the solvent volume was reduced to 0.3-0.5 mL.

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Analysis of flint and pebble. Two were stroke and immeditlay rinsed in a pentane solution

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containing PFBBr and DBU. The pentane was washed with water to remove DBU, dried and the

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solvent was injected on GC-MS.

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General method for quantitation estimations. The Chasselas de Romandie wine (100 mL) was

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spiked with 0.1 mL i.s. (solution: 1 g/100 mL EtOAc) and H2S, from the saturated solution in

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EtOH 1400 µg, 140 µg, 14 µg and no addition. The wine was extracted as described above. The

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extract was injected and the peak area in SIM of 394 was recorded as well as the peak area of 74

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(i.s.). The calibration curve was based on the ratio of 394/74 to give: y=0.0003x + 0.0004, RSQ

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0.997. The same wine was spiked with HSSH in toluene 10 µL; 1 µL and no addition. The wine

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was extracted as described above. The extract was injected and the peak area in SIM of 426 was

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recorded as well as the peak area of 74 (i.s.). The calibration curve was based on the ratio of

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426/74 to give: y=3.37 10-5x + 1.06 10-5, RSQ 0.992.

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HSSH sensory evaluation. Subjects for GC-MS-O and odor profiles of H2S compared to HSSH

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were. Firmenich S.A. (Geneva, Switzerland) employees from perfumery, flavor, and R&D. They 9



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were asked to participate in the GC-MS-O (12 subjects) study and to give a profile for the struck

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stones.

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Selection of two groups of Chasselas contrasted on their minerality level by sensory

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analysis. . The wines came from a specific sensory analysis about minerality in wine performed

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by researchers at Changins – University of Applied Sciences and Arts Western Switzerland. 80

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Chasselas wines (vintage 2012) were selected in equal numbers from the four French-speaking

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parts of Switzerland (Vaud, Valais, Genève, and Neuchâtel). and spread over two sessions (40

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wines per session). To perform this selection, 62 wine professionals participated at the two

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tasting sessions. For each wine presented, they had to answer the following question: “Do you

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think that this wine is a good example or a poor example of what a mineral wine is?” 26-28 The

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minerality level evaluation was scored ranked on a linear scale from 0 = “poor example” toor 10

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= “good example”.. Evaluation was global without any distinction between orthonasal and

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mouthfeel perception. A total of 30 mL of each wine was poured at 15°C ±2°C into an official

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Institut National d'Appellation d'Origine (INAO) glass and coded with three random digits. The

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presentation was monadic and balanced, based on a Williams Latin square design. A 2 minutes

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break was imposed after every 10 wines and a 10 minutes break in the middle of the session

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(after 20 wines). Two significant distinct groups were identified and selected, seven with a poor

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level of minerality exemplarity and seven with a high level of minerality.20 Within each

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subgroup, two wines were selected for this study (Table 1, Supporting Information).

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RESULTS AND DISCUSSION

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Analysis of the Contaminated H2S/N2 Cylinder. The GC-MS analysis of the contaminated gas

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cylinder displayed two major signals with a mass of 284, corresponding to the molecular formula 10


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C12H16N2O4S with m/z 127 (C6H9NO2), typical for the NEM moiety. These two signals were

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assigned to the NEM-S-NEM diastereoisomers.5,29,30 Additional compounds with a fragment

203

corresponding to m/z 127 were detected with masses corresponding to 173, 187, 205, 219, and

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316 (MS in Supporting Information). The possible chemical structures should be limited to

205

compounds that are closely related to H2S. From the masses and the fragmentation patterns of

206

NEM derivatives, M+ 173 was attributed to methylmercaptan, M+ 187 to ethylmercaptan, and M+

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205 and 219 to their disulfides; M+ 316 could well be attributed to NEM-S-S-NEM. A GC-MS

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signal with a mass of M+ 159 and a fragmentation of m/z 126 and 60 was also obtained. This MS

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spectrum was never observed during our previous work when we focused on quantifying H2S and

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methylmercaptan in latrine headspace in developing countries. The mass of M+ 159,

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corresponding to C6H9NO2S, was tentatively assigned to NEM-SH (Figure 1 and S2 - 7). The

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flint smell could not come from methylmercaptan and ethylmercaptan because their odor profile

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does not correspond to the smell liberated by the cylinder in toilet model, therefore to only

214

possible origin of the smell was from the disulfane. In order to verify this hypothesis we

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prepared the disulfane.

216 217

Preparation of Hydrogen Sulfanes (H2Sn). The disulfane was prepared in 19309 dissolving

218

elemental sulfur in boiling water with Na2S and then this solution was added to concentrate HCl.

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A mixture of sulfane was obtained by distillation. Reproducing this procedure failed in our labs,

220

maybe because we worked on a too small scale. A cleaner method was described by Becker and

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Wojnowski,31 a and Hahn and Altenbach (Figure 2).25 The disulfanediylbis

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[methyl(diphenyl)silane] was not stable on SiO2 and decomposed during flash chromatography to

223

give silane oxide derivatives. For this reason, the filtered solution was kept at -18 °C without 11



224

further purification. Sulfanes HSSH (H2S2) (LRI 561) was generated when needed by using TFA

225

and co-distilled with toluene as described.25 It was not possible to avoid the formation of

226

HSSSH (H2S3) (LRI 817). The proportion of trisulfane was around 10% of the disulfane in terms

227

of peak area. The di and tri-sulfanes are stable in crude distilled toluene solution. They can be

228

injected on GC apolar column but decomposed on polar column (Figure 3).

229 230

To avoid having the disulfane in toluene and TFA, we investigated a Fenton-type oxidation

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method. The H2S/EtOH saturated solution was added to a suspension of iron (Fe°) or a solution

232

of FeSO4 in water at pH 3.5 containing 1 equivalent molar of H2O2. The addition of this solution

233

to H2S instantaneously produces a white solid, likely corresponding to the formation of elemental

234

sulfur. Using a catalytic amount of H2O2 (1% mol) promoted the formation of sulfanes, but over

235

time they disappeared. The other problem was that ethanol was slightly oxidized in acetaldehyde

236

with the Fenton solution. Acetaldehyde reacts with H2S and gives a range of sulfur compounds

237

having a garlic character. Therefore, the preparation of H2Sn in ethanol as an authentic sample

238

was abandoned. The GC-MS obtained after the addition of iron powder (Fe°) to ethanol

239

saturated with H2S, followed by extraction and derivatization, is disclosed in the(Figure S8-10).

240

The addition of methylmercaptan and ethylmercaptan to H2S and iron gave a very complex

241

mixture having a garlic odor profile. From this complex mixture, we obtained references for

242

CH3SnH and C2H5SnH (n = 1, 2, 3). These compounds were characterized by MS, HRMS, GC-

243

olfaction (O) and LRI measurements. CH3SSH (LRI 634) was described as matches, burnt eggs,

244

and sewage. CH3CH2SSH (LRI 726) eluted just after dimethyldisulfide (LRI 715) and was

245

described as garlic, rubber, matches, and green (Figures S11-18).

12


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Derivatization with Pentafluorobenzyl Bromide (PFBBr). Derivatization with NEM is not

247

appropriate for H2S or for H2Sn.5,29 For this reason, we changed the derivatization agent and used

248

PFBBr.32 The HSSH freshly distilled in toluene was derivatized with PFBBr and DBU (Figure

249

4). . This method is preferred over that of NEM, mainly for chromatographic reasons (Figure

250

4A), as the peaks are better defined. A typical mass spectrum fragment of PFBBr in MS is m/z

251

181, which should allow easy tracking in single ion monitoring mode (Figure 4B), but the

252

problem is that PFBBr is a hard electrophile and in can react with electrophiles. In the case of

253

wine, it reacts with organic acids, and the ester between octanoic acid and PFBBr has a close LRI

254

(1580 ±5) on an apolar column compared to PFB-S-PFB (LRI 1617 ±5). For this reason the

255

molecular ion was recorded for the quantifications and not m/z 181 (Figure 4B). Different

256

options have been considered for preparing H2S and H2Sn derivatives in wine. The first option

257

was to raise wine pH to 8 with NaOH and then add PFBBr/DBU, but changing the pH, could

258

harm H2S and HSSH by oxidation or lost due to other reactions. Therefore the wine was

259

extracted with a mixture of pentane and diethylether and the derivatization was performed in the

260

organic extract. On polar GC column the ratio of PFB-S-PFB (LRI 2082 ±5), PFB-S-S-PFB (LRI

261

2345 ±5) were different and PFB-S-S-S-PFB decomposed.

262

An authentic sample was also prepared mixing H2S, CH3SH and C2H5SH in ethanol in presence

263

of iron (Fe°) and H2O2 to confirm the occurrence of these compounds in flint and pebble (Figure

264

5).

265

Odor impact estimation of HSSH over H2S by GC-O. HSSH couldn’t be obtained pure, it is

266

unstable and it always contains traces of H2S and H2S3 When the contaminated gas was blown in

267

the toilet model system, the flint smell was obvious even if H2S was the major compound in the

268

headspace. The only way to evaluate the odor profile of HSSH pure was by GC-O. Its retention 13



269

time on the GC apolar column is the same as EtOH and it decomposes on polar column. When

270

pentane is used as an extraction solvent, the hydrogen disulfane (LRI of 561) is in the peak

271

tailing of the solvent. The pentane solution obtained by the extraction of the H2S oxidation in

272

EtOH is not stable, probably because of the co-extraction of radical intermediate species from the

273

reaction in ethanol. But the crude HSSH solution obtained from the silanyl precursor in toluene is

274

stable for several weeks at 4 °C. Therefore we reasoned that the best estimate of odor intensity

275

could be obtained by comparing the odor impact of HSSH with that of H2S by GC-O. We also

276

supposed that smelling highly diluted H2S over milliseconds by GC-O did not affect our sense of

277

smell, as we assumed that the time is too short for habituation. A saturated solution of H2S in

278

toluene was prepared (8 ± 0.3 mg/mL) from the H2S cylinder and determined the concentration

279

by weight. We added up to 500 µL of this H2S solution, portion wise, to 400 µL of HSSH

280

toluene solution and 100 µL of toluene to get the same peak area for H2S and HSSH by GC-MS

281

in TIC. The same solution injected on a GC equipped with a pulsFPD gave a peak ratio of H2S

282

65% and HSSH 35%. This was our best guess for obtaining approximatively quantitative data,

283

and we assumed that the concentrations of H2S and HSSH was 2/1 (4 mg/mL H2S and 2 mg/mL

284

in toluene). The GC-O was performed with eight subjects. The sniffing time were predetermined

285

for H2S (6.08 min) and HSSH (7.09 min) and the sniffing was stopped just before the elution of

286

toluene. Only one sniffing per day was performed by each subject. The sniffing was repeated for

287

four dilution steps corresponding to 1.3, 0.44, 0.15, and 0.05 mg/mL. The focus was to describe

288

the odor intensity on a linear scale from 0 to 5 and to describe the odor profile of hydrogen

289

disulfane. The descriptors were flint, matches, firework, and cold ashes for HSSH. The intensity

290

of HSSH, in considering all the difficulties, was roughly estimated as about 5-10 times higher

14


Page 14 of 36

Page 15 of 36


291

than H2S by GC-O (Figure 6). This value indicates that HSSH has a stronger impact than H2S but

292

in our conditions it was not possible to determine an odor threshold.

293

Disulfane from Struck Conglomerate Pebbles and Flint. Flint is a hard sedimentary

294

cryptocrystalline form of the mineral quartz. We obtained two pieces from Dordogne, France,

295

close to the “Grotte de Lascaux.” When two rocks are struck against themselves, sparks can be

296

produced along with a typical smell. Twenty subjects were asked to describe the odor

297

immediately after striking. The descriptors are listed in order of frequency of use by the panelists:

298

flint, struck matches, cold fireworks, and dentist drill odor.

299

Dark pebbles from a conglomerate, a common and widely distributed type of rock formed by

300

metamorphic processes, were obtained from Mont Pelerin (Canton de Vaud, Switzerland). The

301

odor was close to that obtained from flint. No blind sensory panels were organized to assess

302

whether the differences observed by direct comparison are significant. The odor descriptors are

303

listed in order of frequency of use by the panelists as follows: gun powder, skunks, burnt hairs,

304

mushroom shiitake, struck matches, cold fireworks. The struck pebble odor was more culinary

305

and more complex compared with flint, for which fewer descriptors were used. The smell

306

disappeared in less than 1 min, indicating that odor active compounds are highly volatile or

307

unstable.

308

The stones were struck several times and immediately rinsed with pentane in an Erlenmeyer flask

309

containing PFBBr and DBU. From the pentane alone, no peak could be seen. After

310

derivatization, we observed the occurrence of H2S, CH3SH, C2H5SH, CH3SSH, C2H5SSH,

311

HSSH, and HSSSH (Figures S1 - 18). The strength of the struck and the contact surface were

312

not normalized therefore a relative apparent concentration of sulfur compounds is displayed and

313

the same pattern was repetitively observed (Figure 7). 15



314

Occurrence of Disulfane in Wine. If the minerality is associated with the flint odor, therefore

315

HSSH should be more abundant in mineral wine. The white wine Chasselas de Romandie was

316

used as a model system in our development method. To start, H2S in EtOH was spiked at

317

different concentrations in the Chasselas de Romandie wine with a constant amount of i.s.

318

Extraction and derivatization were performed in the organic phase, then the ratio of m/z 394 / 74

319

in SIM mode was recorded to establish the calibration curve. The wine is a difficult matrices,

320

thiol can be oxidized, complexed, or reacts with electrophiles.33,34 Ferreira noticed that in some

321

wine 97% of H2S is complexed.33 The calibration curve was directly determined in white wine

322

and the semi-quantitative values obtained are not considering if it is free or bound H2S. The

323

same calibration curve was established for HSSH and the ratio m/z 426 / 74 was plotted and we

324

obtained a perfect linearity. We experienced a lot of difficulties, for instance we used the

325

propane thiol as standard29 but unfortunately HSSH reacted immediately to give H2S and

326

C3H7S2H.

327

The goal of this paper is not to develop a precise quantitative method but to demonstrate that the

328

olfactives differences observed between mineral and non-mineral wines can be seen at the level

329

of H2S and HSSH.

330

Due to the importance reported of the malic acid for the mineral character, the concentrations of

331

malic acid-lactic acid were measured. The most mineral wine had indeed a higher proportion of

332

malic acid compared to the non-mineral wines. The non-mineral wines like VDa60 was 0.06 g/L

333

malic acid -3.3 g/L lactic acid(pH 3.9) and GEa14 was 0.25-2 g/L (pH 3.6) for the least mineral

334

wines. The and 2 g/L malic acid -0.2 lactic acid g/L (pH 3.7) for VDm54, the most mineral wine

335

and 1.38-0.75 g/L (pH 3.3) for VSm72.

16


Page 16 of 36

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336

This analysis confirmed that HSSH is more abundant in wines defined as mineral and that H2S

337

was present in all wines (Figure 8).

338

Is it possible to promote the formation of HSSH by adding iron (Fe°)? When the white wine

339

Chasselas de Romandie was treated with iron, both H2S and HSSH concentrations increased by a

340

factor of 10. This was difficult to explain if it was due to oxido-reductions reaction or any other

341

interaction with sulfur complexes. To gain some understanding, Na2SO3 was added to a wine

342

model system containing L-malic acid, DL-tartaric acid, water, and EtOH, adjusted to pH 3.3.

343

When iron was added, H2S was first generated, but after 6-8 hours, HSSH was the major species

344

and after 24 hours, only traces of sulfanes could be detected.

345

The occurrence of HSSH in wine is a result of complex equilibriums. The oxido-reduction

346

reaction mechanisms of copper Cu (II), Cu (I) or iron Fe (III), Fe (II) with H2S, cysteine, 3-

347

sulfanylhexan-1-ol, and other sulfur compounds in wine has recently been discussed. Sulfuric off

348

odors, described as having a reductive character originating from H2S, CH3SH, and C2H5SH, can

349

be controlled with copper; the formation of inorganic CuI&II or Fe II&III and organic polysulfane

350

was demonstrated but not the formation of HSSH or HSSSH, nor their odor contribution.35,36

351 352

In conclusion, the oxidation of H2S in a gas cylinder helped us to discover the odor impact of

353

sulfanes and describe their odor profile. The same sulfanes were present in both flint and in

354

pebbles however the extract was unstable. Thus it was not possible to describe in details the

355

impact of each compound, including other mercaptans. This may explain why this common is

356

smell still not very well understood. This could also help to better understand the nature of “flint

357

odor” in wine. When wine tasters associate flint odor with wine minerality, the di- and trisulfanes

358

could be the compounds responsible for this odor. Precisely defining “wine minerality” was not 17



359

the objective of this paper; rather, we aimed to investigate the potential link between the

360

occurrence of sulfanes and the minerality of wine. This discovery opens the door to new

361

investigations into this unstable class of compounds.

362 363

ABBREVIATIONS USED

364

DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; EI-MS, electron impact-mass spectrometry; GC, gas

365

chromatography; HR, high resolution; INAO, Institut National d'Appellation d'Origine; i.s.,

366

internal standard; LRIs, linear retention indices; NEM, 1-ethyl-(N-ethyl maleimide); O, olfaction;

367

PCA, principal component analysis; PFBBr, pentafluorobenzyl bromide; QTOF, quadrupole

368

time-of-flight; TFA, trifluoroacetic acid; TIC, total ion current, SIM single ion monitoring.

369

ACKNOWLEDGMENT

370

We would like to express our appreciation to the following people: Mrs Myriam Broggi-Praz,

371

former president of the “Association suisse des sommeliers professionnels (ASSP)”; M. Anthony

372

Morris, retired perfumer and wine dealer; M. Eric Praz, flavorist, for pretasting and discussion;

373

Prof. Pascal Kindler, Département de Géologie et Paléontologie from the University of Geneva;

374

Dr. Laurent Wunsche for his helpful discussions and support; Dr. Emilie Belhassen for high-

375

resolution MS measurements; Philippe Merles and Mme Lucie Barroux for running the GC-MS-

376

O; Mrs Laetitia Schaller for GC-FPD and Dr. Isabelle Cayeux for setting up the contact with

377

Changins.

378

ASSOCIATED CONTENT

18


Page 18 of 36

Page 19 of 36


379

Supporting Information: MS spectral data of NEM derivatives, GC-MS trace as a fingerprint of

380

EtOH/H2S solution in the presence of iron. Experimental procedures and MS spectral data of

381

acetaldehyde gemdithiol derivatized with PFBBr and PFBBr esterified with octanoic acid. MS

382

spectral data of CH3SS-R, C2H5SS-R, CH3SSS-R, CH3SSH, C2H5SSH, CH3SSSH, and

383

C2H5SSSH and two wine selections and attributes. Chasselas dry white wines selected for this

384

analysis based on their minerality or non-minerality

385 386 387 388

19



389

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31. Becker, B.; Wojnowski, V. Contribution to the chemistry of silicon sulfur compounds.

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32. Mateo-Vivaracho, L.; Cacho, J.; Ferreira, V. Quantitative determination of wine

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mass spectrometric analysis of their pentafluorobenzyl derivatives. J. Chromatogr. A 2007,

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33. Franco-Luesma, E., Ferreira V., Quantitative analysis of free and bonded forms of volatile

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34. T. E. Siebert, M. R. Solomon, A. P. Pollnitz, D. W. Jeffery. Selective Determination of

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Chemiluminescence Detection. J. Agric. Food Chem. 2010, 58, 9454-9462

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35. Kreitman, G. Y.; Danilewicz, J. C.; Jeffery, D. W.; Elias, R. J. Reaction mechanisms of

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36. Kreitman, G. Y.; Danilewicz, J. C.; Jeffery, D. W.; Elias, R. J. Reaction mechanisms of

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485

oxidation. J. Agric. Food Chem. 2016, 64, 4105–4113.

486

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487

Figure captions

488

Figure 1.

489

Structural formulas of NEM derivatives present in a contaminated H2S gas pressurized bottle.

490

Figure 2.

Synthetic route used to prepare HSSH from its silanyl precursor.

491

Figure 3.

Mass spectra (EI-MS) of HSSH and HSSSH obtained via the silanyl precursors.

492

Figure 4.

(A) GC-MS trace of pentafluoro benzene HSSH and HSSSH derivatives obtained

493

via the silanyl precursor deprotected toluene solution (R = pentafluoro benzene).

494

(B) Mass spectra (EI-MS) of RSR, RSSR, and RSSSR.

495

Figure 5.

496 497

Structural formulas of PFBBr sulfur derivatives of main other compounds found in stones and wine.

Figure 6.

498

GC-O sensory evaluation of HSSH pure and comparative odor intensity rated on a linear scale from 0 to 5 between H2S and HSSH.

499

Figure 7.

GC-MS trace of sulfur compounds from flint and pebbles.

500

Figure 8.

Estimated approximate concentration of H2S and HSSH in wine and model

501

systems based on a GC-TIC ratio of IS/PFB-S(n)-PFB. The bars representing the

502

Chasselas de Romandie was repeated 4 times on 100 mL with 4 different bottles

503

and the standard deviation was 26%.

504

25



505 506

.

507

26


Page 26 of 36

Page 27 of 36


Figure 1

O

O

O

SH

S

N

N

O MW 159

CH3

O

MW 173

MW 187

S N O

O

O

O

MW 219

O

N

O O

S

O

S

O

MW 205

N

N

N

O

O S

S

S

N

O

N

O S

N

O

O O

O S

N S

S N

O

N S O

O

O MW 316

MW 284


27


Page 28 of 36

Figure 2


28

Page 29 of 36


Figure 3

0 m/z-->

64.0

33.0 36.0

63.9

Relative Abundance

Relative Abundance

66.0

97.9

32.0

68.0

30 34 38 42 46 50 54 58 62 66 70

0 m/z-->

20

30


81.0 40

50

60

70

80

90

100

29


Page 30 of 36

Relative Abundance TIC

Figure 4

A 16.7 min. LRI 1804

14.5 min. LRI 1616

6.0

7.0

8.0

9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 Time (min.)

B

R-S-S-R

181

R-S-S-S-R

181

Abundance Scan 820 (16.7 min)


R-S-R

181


5.0

18.6 min. LRI 1988

394 45

93 131

20 60 100 140 180 220 260 300 340 380

393 458

426

211 93 131 0

213

297 343

93 131

210

60 100 140 180 220 260 300 340 380 420 40 80 120 160 200 240 280 320 360 400 440


30

Page 31 of 36


Figure 5


31


Page 32 of 36

1

2

3

H2S HSSH

0

Intensity [0-5]

4

5

Figure 6

-2.0

-1.5

-1.0

-0.5

0.0

0.5

Log of mass injected [µg]


32

Page 33 of 36


R-S3-R

R-S2-R

R-S2-C2H5

R-S2-CH3

R-S-C2H5

R-S-CH3

Relative abundance: Ion 181.00 (180.70 to 181.70)

R-S-R

Figure 7

Flint pebble blank

Time--> min.

5

6

7

8

9

10

11

12

13

14

15


16

17

18

19

33



Page 34 of 36

34

Page 35 of 36


Figure 8


35


Page 36 of 36

HSSH H2S

Chasselas

C2H5SSH

CH3SH

HSSSH

Flint

CH3SSH

C2H5SH


CH3SSSH

Pebble

Identification of Hydrogen Disulfanes and Hydrogen Trisulfanes in

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