# "High-throughput LC-MS/MS discrimination of musts and wines contaminated by fresh mushroom off-flavor on the basis of reliable and easily quantifiable analytical markers: 1-hydroxyoctan-3-one and its derivate octane-1,3-diol"

Sarah Ployon, Lucas Suc, Christine Le Guernevé, Eléonore Augès, Héloïse Mahé, Marion Hervé, Audrey Bloem, Aurélie Roland

► To cite this version:

Sarah Ployon, Lucas Suc, Christine Le Guernevé, Eléonore Augès, Héloïse Mahé, et al. "High-throughput LC-MS/MS discrimination of musts and wines contaminated by fresh mushroom off-flavor on the basis of reliable and easily quantifiable analytical markers: 1-hydroxyoctan-3-one and its derivate octane-1,3-diol". Food Chemistry, 2025, 482, pp.143454. 10.1016/j.foodchem.2025.143454 hal-05497762

HAL Id: hal-05497762

https://institut-agro-montpellier.hal.science/hal-05497762v1

Submitted on 6 Feb 2026

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<figure><img src="image_1.png" /></figure>

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The olfactory perception thresholds for FMOff compounds vary widely, ranging from a few ng/L for trace compounds like 1-octen-3-one and 1-nonen-3-one (Darriet et al., 2002; La Guerche et al., 2006) to hundreds of µg/L for 2-heptanol (La Guerche et al., 2006) and 1-hydroxyoctan-3-one (Delcros, Costis, et al., 2023). These thresholds can also vary slightly depending on the matrix studied, such as water (Delcros, Costis, et al., 2023), musts (Darriet et al., 2002), red wines (La Guerche et al., 2006) or white wines (Boutou & Chatonnet, 2007; Pons et al., 2011). Other compounds, such as 1-octen-3-ol, 3-octanone and 3-octanol, have intermediate olfactory perception thresholds in water, with values of 7 µg/L, 23 µg/L (Darriet et al., 2002) and 0.25 µg/L (Xu et al., 2019), respectively.

The complexity of the FMOff defect in wine lies in its variability during the vinification process (Meistermann & Villaumé, 2022), making detection difficult. To date, there is no clear, easily analyzable and reliable analytical marker. Indeed, 1-octen-3-ol has been identified in Meunier, Pinot gris and Sauvignon blanc wines elaborated with grapes affected by bunch rot, and selected for their strong fresh mushroom flavor but at concentration below its sensory perception threshold and without correlation with defect intensity (Pons et al., 2011), while it was not detected in musts and wines contaminated by *Crustomyces subabruptus* (Delcros, Collas, et al., 2023) and in FMOff wines from the Champagne area (Delcros, Costis, et al., 2023). 3-Octanol was detected in these FMOff base wines but was not a reliable marker to differentiate healthy from contaminated matrix (Delcros, Costis, et al., 2023). 1-Nonen-3-one was identified as a trace component in mushroom odoriferous zone in red and white wines in concentrations close to its recognition threshold (30 ng/L) in similar amount among altered wines having different mushroom off-flavor intensity (Pons et al., 2011), which suggests synergistic role of this compound to FMOff of these wines or involvement of other compounds.

1-Octen-3-one, considered to be a major compound impacting FMOff due to its low sensory threshold (40 ng/L in neutral white wines, (Pons et al., 2011)), has been detected in Cabernet Sauvignon and Sauvignon blanc grapes exhibiting a mushroom flavor and affected by powdery mildew (Darriet et al., 2002), in Cabernet Sauvignon, Merlot, Gamay, Pinot Noir, Sauvignon, and Semillon grapes visibly affected by bunch rot (La Guerche et al., 2006) and in Pinot must contaminated by *C. subabruptus* (Delcros, Collas, et al., 2023). While Pons et al. found a correlation (R² = 0.76) between 1-octen-3-one concentration and FMOff perception in wines from the 2006 harvest in Champagne (Pons et al., 2011), Delcros, Collas, et al. (2023) did not observed a similar correlation in base wines from Champagne area. Interestingly, in the same study, 1-hydroxyoctan-3-one appeared to correlate more closely with FMOff perception in selected base wines.

The aim of the current study was first to identify relevant FMOff markers using untargeted analysis of volatile compounds in musts made from grapes naturally affected by grey rot, then to quantify these markers in various oenological matrices and finally, to develop a diagnosis tool of FMOff alteration in both musts and wines based upon a high-throughput, reliable and simple LC-MS/MS method.

# 2. Materials & methods

## 2.1. Chemicals

Dichloromethane (≥99.9 %) and pentane (≥99 %) were purchased from Honeywell (Saint-Germain-en-Laye, France). Glucose (≥99.5 %), sodium chloride (≥99 %) sodium sulfate anhydrous (≥99 %) Tert-butyldimethylsilyl chloride (1 M in THF), 3-hydroxypropionitrile (≥97 %), imidazole (99 %), pentylmagnesium bromide (2 M in diethyl ether), hydrochloric acid (37 %) and ethyl acetate (>99.5 %) were purchased from Sigma Aldrich (St Quentin Fallavier, France). Ethanol absolute (≥99.9 %) was purchased from Carlo Erba (Val-de-Reuil, France). Tartaric acid (≥99.5 %), n-hexane (≥98.5 %) came from VWR (Rosny-sous-Bois, France). Methanol (>99.98 %), water (LC-MS grade)

and formic acid (99 %) were brought from Biosolve (Dieuze, France).

1-Octen-3-ol (98 %), 3-octanol (99 %), 2-octanol (≥ 99.5 %), 2 heptanol (98 %), 3-octanone (≥98 %) were purchased from Sigma Aldrich (St Quentin en Fallavier, France). 1-octen-3-one (97 %) came from Fisher Scientific (Illkirch-Graffenstaden, France). Deuterated internal standards [1,2-²H]-1-octen-3-ol (48 %) and [1,2-²H]-1-octen-3-one (46 %) were synthetized according to Delcros, Costis, et al. (2023) protocol. 1-hydroxyoctan-3-one was synthesized following the protocol of Husain et al. (1999). Development was required for other standards syntheses described in paragraph 4.2.1.

# 3. Samples

## 3.1. Production of experimental musts and wines

Meunier and Pinot noir grapes were collected in Champagne vineyard during the 2023 harvest. Considering visible symptoms, nine batches of 10 kg of grapes were composed with grapes contaminated by grey rot, with a global visually attack degree ranging from 0 % (healthy grapes, i.e. control grapes) to 100 % (i.e., 100 % of affected grape) (see Table 1).

Batches of grapes were pressed using manual air presses with central membrane, following the pressing defined by Champagne appellation (25 hL of must/4000 kg of grape). The musts obtained were sulfited at 7 g/hL and treated overnight at 15 °C with pectolytic enzymes (E Catal® Clarif, Station Oenotechnique de Champagne, 1 g/hL).

The nine clarified musts (4.5 L) were inoculated with 10 g/hL of *Saccharomyces cerevisiae* (Levuline® CHP, Oenofrance). Alcoholic fermentation (AF) was conducted using 5 L flasks and was monitored by aerometry. Malolactic fermentation (MLF) was conducted with a "pied de cuve" inoculum (3 % v/v). After MLF, flasks were kept a week at 10 °C then raked and 10 mg/L of SO₂ was added before bottling in 37.5 cL glass bottles. Experimental wines were stored in bottle at 18 °C until analysis and were tasted 3 months after bottling.

## 3.2. Collection of FMOff musts and base wines

To study the relevance of FMOff markers, 33 FMOff and 29 control base wines were collected in Champagne in 2023. Original musts, used to make these FMOff wines and the control wines, were also collected.

## 3.3. Sensory analysis of wines

A panel of eleven expert judges from Comité Champagne (CIVC) was trained to detect and identify FMOff. The tests took place with black INAO glasses (according to the NF ISO 3591 standard) containing 30 mL of wine and served in a tasting room complying with the NF ISO 8589 standard (temperature regulation between 18 and 22 °C). Wines from the collection were tasted during 9 sessions of 7 samples and experimental wines were tasted during another session, where panelists were asked to score samples from 0 (not detected) to 4 (strong intensity) in terms of fresh mushroom intensity. Scores were collected using FIZZ computerized data acquisition system (FIZZ software, Biosystemes, Couternon, France). Base wines with no FMOff (FMOff score = 0) were categorized as the "control group" and base wines with FMOff (FMOff scores >0) were categorized as the "FMOff group".

# 4. Untargeted analysis (GC-MS) of aroma profile of experimental musts

## 4.1. Liquid-liquid extraction for untargeted analysis

Samples were prepared in duplicate as follows. Musts were centrifuged (5 min at 4500 rpm). Two internal standards were added to 15 mL of must: [1,2-²H]-1-octen-3-ol at 50 µg/L, [1,2-²H]-1-octen-3-one at 500 ng/L. The working solutions of internal standards were prepared in
<table><caption>**Table 1**

C8 quantification in experimental musts (M) and wines (W) affected by different visual percentages of grey rot and their evolution during alcoholic fermentation (AF) and their FMOff wine sensory score, depending on the degree of rot.</caption><thead><tr><th rowspan="2">Sample must (M)/ wine (W)</th><th rowspan="2">Variety</th><th rowspan="2">Plot</th><th rowspan="2">Visual % Grey rot</th><th rowspan="2">FMOff wine sensory Score (/4)</th><th colspan="2">1-hydroxyoctan-3-one (µg/L)</th><th colspan="2">Octane-1,3-diol (µg/L)</th><th colspan="2">3-octanone (µg/L)</th><th colspan="2">1-octen-3-one (ng/L)</th><th colspan="2">3-octanol (µg/L)</th><th colspan="2">1-octen-3-ol (µg/L)</th></tr><tr><th>M</th><th>W</th><th>M</th><th>W</th><th>M</th><th>W</th><th>M</th><th>W</th><th>M</th><th>W</th><th>M</th><th>W</th></tr></thead><tbody><tr><td>M-1 / W-1</td><td>PN</td><td>A</td><td>0</td><td>0.00 <sup>a</sup></td><td>&lt; LOD</td><td>&lt; LOD</td><td>0.70</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td></tr><tr><td>M-2 / W-2</td><td>PN</td><td>F</td><td>NE</td><td>0.45</td><td>4.74</td><td>&lt; LOD</td><td>2.13</td><td>2.42</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td></tr><tr><td>M-3 / W-3</td><td>PN</td><td>A</td><td>10</td><td>0.73 <sup>a</sup></td><td>2.71</td><td>3.22</td><td>4.61</td><td>5.93</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOD</td></tr><tr><td>M-4 / W-4</td><td>MN</td><td>B</td><td>15</td><td>1.45 <sup>b</sup></td><td>6.68</td><td>2.67</td><td>2.48</td><td>2.67</td><td>&lt; LOD</td><td>&lt; LOD</td><td>0.44</td><td>&lt; LOQ</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOQ</td></tr><tr><td>M-5 / W-5</td><td>PN</td><td>C</td><td>50</td><td>1.91</td><td>17.30</td><td>11.85</td><td>11.74</td><td>18.87</td><td>&lt; LOD</td><td>&lt; LOQ</td><td>&lt; LOQ</td><td>&lt; LOD</td><td>&lt; LOD</td><td>&lt; LOQ</td><td>5.85</td></tr><tr><td>M-6 / W-6</td><td>MN</td><td>D</td><td>100</td><td>2.45 <sup>b</sup></td><td>6.81</td><td>6.59</td><td>4.85</td><td>8.89</td><td>&lt; LOD</td><td>1.23</td><td>&lt; LOQ</td><td>&lt; LOQ</td><td>&lt; LOQ</td><td>&lt; LOQ</td><td>5.21</td></tr><tr><td>M-7 / W-7</td><td>PN</td><td>A</td><td>60</td><td>3.91 <sup>c</sup></td><td>251.4</td><td>187.74</td><td>84.82</td><td>190.70</td><td>NE</td><td>6.97</td><td>750.00</td><td>238.00</td><td>&lt; LOQ</td><td>0.85</td><td>12.15</td><td>15.53</td></tr><tr><td>M-8 / W-8</td><td>MN</td><td>B</td><td>100</td><td>4.00 <sup>c</sup></td><td>472.35</td><td>289.63</td><td>53.87</td><td>149.66</td><td>0.66</td><td>4.98</td><td>620.00</td><td>978.00</td><td>NE</td><td>0.83</td><td>12.12</td><td>10.35</td></tr><tr><td>M-9 / W-9</td><td>No data</td><td>No data</td><td>10</td><td>4.00 <sup>c</sup></td><td>4,616.79</td><td>4,105.78</td><td>431.06</td><td>924.47</td><td>NE</td><td>96.00</td><td>14,010.00</td><td>2,260.00</td><td>NE</td><td>1.73</td><td>26.31</td><td>27.97</td></tr></tbody></table>

PN = Pinot Noir; MN = Meunier; wine perceived as a no FMOff, b presence of a FMOff or c presence of an intense FMOff (Fisher exact test, α = 0.05); LOD = Limit of detection and LOQ = Limit of Quantification; NE = not evaluated.

Concentrations were measured from a single injection; analytical repeatability for each analyte is given in Table 2.

<table><caption>**Table 2**

Detection parameters and analytical performances of FMOff targeted analytical methods by GC-MS/MS and LC-MS/MS. LOD = limit of Detection; LOQ = Limit of Quantification; CID = Collision Induced Dissociation values.</caption><thead><tr><th>Analytical method</th><th>Compound</th><th>Internal standard (IS)</th><th>RT (min)</th><th>Quantitative ion</th><th>CID (V)</th><th>Qualitative ion</th><th>CID (V)</th><th>Linear range (µg/L)</th><th>R<sup>2</sup></th><th>LOD (µg/L)</th><th>LOQ (µg/L)</th><th>Repeatability</th><th>Reproducibility</th><th>Accuracy</th></tr></thead><tbody><tr><td rowspan="5">HS-SPME-GC-MS/MS</td><td>3-octanone</td><td>[1,2,<sup>2</sup>H]-1-octen-3-ol</td><td>10.1</td><td>99.1 - &gt; 81.1</td><td>10</td><td>57.0 - &gt; 42.0</td><td>35</td><td>0-35</td><td>0.96</td><td>0.06</td><td>0.21</td><td>22 %</td><td>21 %</td><td>115 %</td></tr><tr><td>1-octen-3-one</td><td>[1,2,<sup>2</sup>H]-1-octen-3-one</td><td>11.1</td><td>97.0 - &gt; 43.0</td><td>20</td><td>97.0 - &gt; 79.0</td><td>10</td><td>0-1</td><td>0.94</td><td>0.02</td><td>0.08</td><td>7 %</td><td>8 %</td><td>89 %</td></tr><tr><td>2-heptanol</td><td>[1,2,<sup>2</sup>H]-1-octen-3-ol</td><td>11.6</td><td>83.1 - &gt; 55.1</td><td>10</td><td>70.1 - &gt; 55.1</td><td>10</td><td>0-35</td><td>0.97</td><td>1.2</td><td>1.2</td><td>15 %</td><td>9 %</td><td>67 %</td></tr><tr><td>3-octanol</td><td>[1,2,<sup>2</sup>H]-1-octen-3-ol</td><td>11.7</td><td>59.0 - &gt; 41.1</td><td>10</td><td>83.0 - &gt; 55.1</td><td>10</td><td>0-35</td><td>0.99</td><td>0.11</td><td>0.38</td><td>6 %</td><td>3 %</td><td>105 %</td></tr><tr><td>1-octen-3-ol</td><td>[1,2,<sup>2</sup>H]-1-octen-3-ol</td><td>14.2</td><td>72.1 - &gt; 43.0</td><td>10</td><td>85.1 - &gt; 43.0</td><td>10</td><td>0-35</td><td>0.98</td><td>1.43</td><td>4.05</td><td>15 %</td><td>15 %</td><td>106 %</td></tr><tr><td rowspan="5">LC-MS/MS</td><td>1-hydroxyoctan-3-one</td><td>[8,8,<sup>2</sup>H]-1-hydroxyoctan-3-one</td><td>6.0</td><td>145.0 - &gt; 127.1</td><td>4</td><td>145.0 - &gt; 109.1</td><td>8</td><td>0-500</td><td>0.99</td><td>0.82</td><td>2.45</td><td>13 %</td><td>14 %</td><td>103 %</td></tr><tr><td>octane-1,3-diol</td><td>[8,8,<sup>2</sup>H]-1-hydroxyoctan-3-one</td><td>6.2</td><td>147.0 - &gt; 129.1</td><td>0</td><td>150.0 - &gt; 114.0</td><td>4</td><td>0-1000</td><td>1.00</td><td>0.32</td><td>0.86</td><td>12 %</td><td>12 %</td><td>104 %</td></tr><tr><td>(IS)</td><td>-</td><td>6.0</td><td>148.0 - &gt; 130.1</td><td>8</td><td>148.0 - &gt; 112.1</td><td>8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>[8,8,<sup>2</sup>H]-octan-1,3 diol</td><td>-</td><td>6.2</td><td>150.0 - &gt; 132.0</td><td>0</td><td>150.0 - &gt; 114.0</td><td>4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>(IS)</td><td>-</td><td>-</td><td>6.2</td><td>150.0 - &gt; 132.0</td><td>0</td><td>150.0 - &gt; 114.0</td><td>4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>
absolute ethanol. Samples were extracted three successive times with 5 mL of pentane-dichloromethane (2/1, v/v). Organic extracts were dried over Na₂SO₄ were then rectified up to 150 µL using a Dufton column at 40 °C. Few drops of hexane were added to samples before GC-MS analysis. The samples were kept at 10 °C in an autosampler until injection.

## 4.2. GC-MS conditions

Untargeted (MS in full scan mode) volatile compounds analyses were performed using a Trace GC ULTRA gas chromatograph coupled with a triple quadrupole mass spectrometer TSQ 8000 (Thermo Scientific).

The GC was equipped with a DB-HeavyWAX column (fused silica capillary column, 30 m × 0.250 mm, 0.25 µm film thickness). The injector temperature was set at 250 °C. Analyses were performed in splitless mode. The oven temperature gradient was set at 40 °C for 3 min then increase to 131 °C at 7 °C/min and to 280 °C at 20 °C/min with a 5 min holding at 280 °C. Helium was used as the carrier gas with a flow of 1.2 mL/min. The electron impact ionization source (EI) temperature and the transfer line were set up at 250 °C. MS ionization energy was 70 eV.

## 4.3. Deconvolution of GC-MS chromatograms

GC-MS scan were loaded and deconvoluted with MassHunter Un-
knowns Analysis software 10.1 (Agilent Technologies France, les Ulis).
Deconvoluted mass spectrum were compared to NIST database, WILEY
database and to INRAMASS and AROMLIB databases (INRAe internal
databases).

# 5. Targeted analysis of FMOFF compounds in musts and wines

## 5.1. GC-MS/MS analysis

### 5.1.1. Sample preparation: HS-SPME extraction of volatiles FMOFF aroma in must and wine samples

5 mL of wine were diluted with 5 mL of milliQ water and two internal standards ([1,2-²H]-1-octen-3-ol at 5 µg/L [1,2-²H]-1-octen-3-one at 500 ng/L) were spiked in the samples. The salting out of volatile compounds was promoted by the addition of 1 g of NaCl. Samples were placed in an autosampler with temperature set at 10 °C. The sampling was carried out by a PAL-system (CTC Analytics, Zwingen, Switzerland). HS-SPME autosampling was performed using a DVB/CAR/PDMS fiber (Supelco, 50/30 µm, Stableflex (2 cm, 23Ga). Samples were pre-incubated for 15 min at 40 °C with 250 rpm shaking. Extraction was then performed for 30 min (Needle penetration of 16 mm; fiber penetration of 20 mm). Desorption was performed for 3 min with a penetration of 32 mm. Fiber was conditioned 15 min at 270 °C.

### 5.1.2. GC-MS/MS conditions

Several C7 and C8 volatile compounds, namely 2-heptanol, 3-octa-
none, 1-octen-3-one, 3-octanol, 1-octen-3-ol were analyzed by GC-MS/
MS. Targeted GC-MS/MS analysis (MS/MS in Multiple Reaction Moni-
toring mode) of volatile compound were performed using the same
system as GC-MS untargeted analyses, with the same settings and using
the same chromatographic conditions. Analyses were performed in split
mode with a split flow set at 20 mL/min, a split ratio of 8 and a splitless
time of 3.1 min. Nitrogen was used as collision gas for MS/MS frag-
mentation. MS/MS parameters for detection of FMOFF are given in
Table 2.

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Synthesis of natural and labelled analytical standards of 1-hydroxyoctan-3-one and octane-1,3-diol.</figcaption></figure>
## 5.2. SIDA-LC-MS/MS analysis of 1-hydroxyoctan-3-one and octane-1,3-diol in musts and wines

### 5.2.1. Synthesis of analytical standards

Syntheses are resumed in Fig. 1.

#### 5.2.1.1. Synthesis of natural analytical standard of octane-1,3-diol.
Octane-1,3-diol (5) was obtained by chemical reduction of 1-hydroxyoctan-3-one (1) previously obtained according to Husain et al. and Delcros et al. (Delcros, Costis, et al., 2023; Husain et al., 1999). A NaBH₄ in MeOH solution (65.7 mg, 1.74 mmol, 0.5 eq) was slowly added to 1-hydroxyoctan-3-one (1) (500 mg; 3.47 mmol; 1 eq). The mixture was stirred for 90 min at room temperature until the end of the reaction. Then, 10 mL of miliQ water were added to the reaction mixture and methanol was evaporated under vacuum. The remaining aqueous mixture was extracted three time with 30 mL of ethyl acetate. The organic phase was washed with brine (50 mL), dried over MgSO₄ and concentrated to dryness. The residual product, containing octane-1,3-diol (5) was weighed, and purity was assessed by ¹H NMR (305 mg, 63 % yield, 44 % ¹H NMR purity).

¹H NMR (CD₃OD, 400 MHz) δ 3.75–3.64 ppm (3H, m), 1.75–1.53 ppm (2H, m), 1.50–1.26 ppm (8H, m), 0.91 ppm (3H, t, *J* = 6.9 Hz).

¹³C NMR (101 MHz, CD₃OD) δ 70.01, 60.37, 40.77, 38.74, 33.08, 26.40, 23.71, 14.39 ppm.

#### 5.2.1.2. Synthesis of deuterated/labelled analytical standards: [8,8,8²H]-1-hydroxyoctan-3-one and [8,8,8²H]-octane-1,3-diol.
3-(Tert-butyldimethylsilyloxy)propionitrile (2) was synthetised as described by Husain et al. (1999). Magnesium (331 mg, 13.6 mmol, 1.05 eq) in diethyl ether (2 mL) and 3-(tert-butyldimethylsilyloxy)propionitrile (2.27 g, 13 mmol, 1 eq) in diethyl ether (3 mL) were added to a solution of [5,5,5²H]-1-bromopentane (1.64 mL, 13 mmol, 1 eq) in diethyl ether (2 mL). The mixture was kept under stirring for 15 min at 35 °C in order to start the reaction, then cooled down to room temperature and stirred overnight. The mixture was diluted with diethyl ether (10 mL) and was carefully poured into a 100 mL flask with 20 mL of cold distilled water. HCl (12 M) was added to the mixture to decrease pH to 5 and the mixture was stirred for 2 h at room temperature. The mixture was then extracted three times with ethyl acetate (3 × 20 mL). The organic phases were washed with brine (50 mL), dried over MgSO₄ and then concentrated to dryness to give the intermediate product, 1-((tert-butyldimethylsilyl)oxy)octan-3-one (3) (1.61 g, 48 % yield). (3) was dissolved in 15 mL of a HCl methanolic solution (10 %). The solution was stirred for 90 min at room temperature. 50 mL of an aqueous solution of 5 % Na₂CO₃ were added, and methanol was evaporated under vacuum. The remaining aqueous mixture was extracted three time with 50 mL of ethyl acetate. The organic phases were washed with 100 mL of brine, dried over MgSO₄, and concentrated to dryness. The final product obtained was [8,8,8²H]-1-hydroxyoctan-3-one (4) (552 mg, 61 % yield, 36 % NMR purity).

¹³C NMR (101 MHz, CD₃OD) δ 57.86, 45.78, 43.68, 32.24, 24.02, 23.06 ppm.

¹H NMR (400 MHz, CD₃OD) δ 3.80, 2.64, 2.48, 1.57, 1.31, 1.30 ppm.

[8,8,8²H]-1-Hydroxyoctan-3-one (4) (272 mg, 1.85 mmol, 1 eq) was reduced with an aqueous solution of NaBH₄ (35 mg, 0.92 mmol, 0.5 eq) during 90 min at room temperature, under stirring. At the end of the reaction, 10 mL of miliQ water were added to the reaction mixture and methanol was evaporated under vacuum. The remaining aqueous mixture was extracted three time with 30 mL of ethyl acetate (30 mL). The organic phases were washed with 50 mL of brine then dried over MgSO₄. Solvent was removed using a rotatory evaporator. Final product was [8,8,8²H]-octane-1,3-diol- (6) (164 mg, 59 % yield, 75 % NMR purity).

¹H NMR (400 MHz, MeOD) δ 3.72, 3.70, 1.69, 1.63, 1.47, 1.47, 1.36, 1.35, 1.34, 0.93 ppm.

¹³C NMR (101 MHz, MeOD) δ 68.63, 58.97, 39.47, 39.39, 37.37, 31.65, 25.05, 24.96, 22.31, 13.04 ppm.

#### 5.2.1.3. NMR spectroscopy for structural characterization of analytical standards and absolute quantification.
NMR spectra were recorded at 25 °C using a 400 MHz Bruker Advance NEO spectrometer operating at 400.17 MHz for 1H and 100.63 MHz for ¹³C equipped with a 5 mm BBI probe. NMR data were processed and analyzed using both TopSpin 4.0 (Bruker) and MestReNova 15.0.1 software (Mestrelab Research, Spain). All spectra were referenced to the solvent CD₃OD signals at 3.31 and 49.0 ppm for ¹H and ¹³C respectively. Absolute concentration measurements were carried by quantitative ¹H 1D NMR (qNMR) using an external calibration method as previously described (Ferreira-Lima et al., 2016) allowing purity determination of each compound.

### 5.2.2. Sample preparation

1.5 mL of sample were spiked with [8,8,8²H]-1-hydroxyoctan-3-one and [8,8,8²H]-octane-1,3-diol at 100 µg/L both. Internal standards working solutions were prepared in absolute ethanol. Samples were centrifuged at 15000 rpm for 15 min before injection.

### 5.2.3. LC-MS/MS analysis

Analyses were performed on a 1290 Infinity II reversed phase UHPLC (Agilent Technologies, CA, USA) hyphenated to a 6470B Agilent Triple Quadrupole. Analytes were separated on a Hypersil gold column (1.9 µm, 100 mm × 2.1 mm) (ThermoFisher, Waltham, MA, USA) with a 40 °C oven temperature and a 17 min total run time. The mobile phases were composed of (A) water or (B) methanol each with 0.1 % formic acid. The gradient started at 1 % of B for 1 min, increased to 99 % of B over 9 min, held at 99 % of B for 3 min then decreased to 1 % B in 1 min and held for 2 min at a flow rate of 0.5 mL/min. The injection volume was 10 µL. Source parameters were set as follows: drying gas temperature at 260 °C, drying gas flow at 4 L/min, nebulizer at 35 psi, sheath gas temperature at 200 °C, sheath gas flow at 10 L/min, capillary voltage at 4000 V in positive mode, nozzle voltage at 1500 V and positive Delta EMV at 400 V. Ionization was carried out using positive electrospray (ESI+) and detection was made using Multiple Reaction Monitoring (MRM). MRM parameters are given in Table 2.

## 5.3. Quantification of odorant potentially contributing to FMOFF in musts and wines

1-Octen-3-one, 1-octen-3-ol, 3-octanone, 2-heptanol, 3-octanol were quantified by GC-MS/MS analysis (see paragraph 4.1.) while 1-hydroxyoctan-3-one and octane-1,3-diol were quantified using LC-MS/MS analysis (see paragraph 4.2.). 1-Octen-3-one, 1-octen-3-ol, 1-hydroxyoctan-3-one and octane-1,3-diol were quantified by stable isotope dilution assay (SIDA) using [1,2²H]-1-octen-3-one, [1,2²H]-1-octen-3-ol, [8,8,8²H]-1-hydroxyoctan-3-one and [8,8,8²H]-octane-1,3-diol as internal standards. Other compounds (3-octanone, 2-heptanol, 3-octanol) were quantified using [1,2²H]-1-octen-3-ol as internal standard.

Calibrations were performed in must and wine matrix using a model wine (5.5 g of tartaric acid and 120 mL of absolute ethanol, qs 1 L of milliQ® water, pH adjusted to 3.5 with NaOH solution) or a model must (220 g of glucose, 4 g of tartaric acid, qs 1 L of milliQ® water, pH adjusted to 3.5 with sodium hydroxide solution). Samples for calibration points were prepared as samples. All the concentration ranges are detailed in Table 2.

## 5.4. Analytical method validation

The LC-MS/MS and the HS-SPME-GC-MS/MS analytical method were validated prior to sample analysis: linearity, accuracy, repeatability, intermediate reproducibility (different days), limit of detection (LOD) and limit of quantitation (LOQ) were evaluated. The linearity
range was evaluated by spiking model wine or must samples at 9 con-
centrations for all analytes (including a zero), with constant internal
standards concentrations. The concentration ratios as function to the
respective area ratios were calculated for each analyte. Accuracy (n = 5)
and repeatability (n = 3) and intermediate reproducibility (n = 5) were
determined by spiking a must and a Champagne wine at medium con-
centration level (0.1 µg/L for 1-octen-3-one, 5.4 µg/L for 1-octen-3-ol,
5.2 µg/L for 3-octanol, 5.1 µg/L for 3-octanone, 5.4 µg/L for 2-heptanol,
50 µg/L for 1-hydroxyoctan-3-one and 50 µg/L for octane-1,3-diol) and
were expressed as the recovery and mean RSD (%) respectively. The
LOD and LOQ were calculated for each compound for a signal to noise
(S/N) ratio equal to 3 and 10 respectively.

5.5. Statistical analysis

Statistical analyses were performed using XLStat (Microsoft, US).
The difference of FMOff intensity (no FMOff, presence of FMOff or
intense FMOff) between wines was evaluated using a Fisher's exact test
(α = 0.05). Correlation between the FMOff compound concentrations
and/or sensory scores were assessed using non-parametric Spearman

test (significance level α = 0.01). Significance of differences in FMOff
concentrations between the Control group and the FMOff group was
assessed using non-parametric Kruskal-Wallis test (significance level α
= 0.01) followed by a post hoc Conover-Iman test for pairwise
comparison.

6. Results & discussion

6.1. Screening of potential sensory related compounds with FMOff in
experimental contaminated musts and wines

6.1.1. *Sensory analysis*

The experimental wines (W-1 to W-9), made with musts obtained
from visually grey rot affected grapes (M-1 to M-9), were tasted by a
panel of trained experts and scored according to their FMOff intensity,
where 0 means no FMOff and 4 an intense FMOff (Table 1). As expected,
the sample W-1 made from visually healthy grapes showed no FMOff (p
< 0.0001, α = 0.05). Two samples with high levels of grey rot (samples
60 % to 100 %) present an intense FMOff (score of 3.9 and 4.0,
respectively) (p = 0.015, α = 0.05). Interestingly, another sample made

<figure><img src="image_3.png" /><figcaption>Fig. 2. Comparison of GC-MS profile (black) and deconvoluted profile (green) of control sample (M-1) and strong FMOff sample (M-9); comparison of MS spectra with INRAMass database. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
with 100 % of visually attacked grapes (W-6) present a FMOff but with lower intensity (FMOff score below 3) (p = 0.034, α = 0.05), while the sample W-9, made with only 10 % of grey rot was scored as an intense FMOff (p < 0.0001, α = 0.05) with a score of 4. This suggests that not only the visual amount of rot could influence FMOff development, but also the fungal biomass contained in a grape (Steel et al., 2020).

### 6.1.2. Volatile compound analytical profile of musts using an untargeted GC-MS-FullScan analysis

To characterize the volatile compound analytical profile of musts contaminated by grey rot, volatile aroma compounds were extracted by liquid-liquid extraction and then analyzed by GC-MS. Deconvolution of the mass chromatograms revealed few intense and possibly coeluted peaks that could be present in the mass spectra. The extracted mass spectra were compared to various GC-MS databases (NIST, WILEY, INRAMASS and AROMLIB) to identify as many compounds as possible.

Fig. 2 shows the GC-MS full scan and deconvoluted chromatograms of samples M-1 (control) and M-9 (FMOff). The comparison of the control experimental sample M-1 (FMOff score of the associated wine of 0) with the samples M-7, M-8 and M-9 (FMOff scores of the corresponding wines of 3.9, 4.0 and 4.0, respectively) led to the identification of several C8 compounds. 1-Octen-3-one was identified only in sample M-9, while 1-octen-3-ol was detected in sample M-8 (data not shown). A high intensity peak was observed at a retention time of 21.2 min in two musts (M-8 and M-9) made with affected grapes and was attributed to 1-hydroxyoctan-3-one (Fig. 2). 1-Hydroxyoctan-3-one was recently identified as a FMOff compound in musts and wines obtained using a model of contamination by *Crystomyces subabruptus* and in selected FMOff base wines from Champagne area (Delcros et al., 2022). However, this is the first time the compound has been identified in musts made with grapes naturally affected by grey rot.

Several hypotheses have been proposed in the literature regarding

<figure><img src="image_4.png" /><figcaption>Fig. 3. 2D ¹H/¹³C edited-HSQC NMR spectra of A) 1-hydroxyoctan-3-one in CD₃OD, B) [8,8,8-²H]-1-hydroxyoctan-3-one in CD₃OD, C) octane-1,3 diol in CD₃OD and D) [8,8,8-²H]-octane-1,3 diol in CD₃OD.</figcaption></figure>
the origin of 1-hydroxyoctan-3-one in the FMOff Based on an aldol-type reaction demonstrated in orange juice (Mall et al., 2018), Wang et al. suggested that 1-octen-3-one could form 1-hydroxy-3-octanone during the drying of shiitake mushroom (Wang et al., 2021). Delcros et al. initially proposed that a similar reaction could occur during wine ageing. However, another hypothesis emerged from their studies, suggesting that 1-hydroxyoctan-3-one could also be a precursor of 1-octen-3-one (Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023).

A peak at 24.2 min (m/z 45, 57, 75, 83 and 99) was identified as octane-1,3-diol in two of the three must contaminated samples (M-7 and M-9) but not in M-1 and M-8 (Fig. 3). Interestingly, this compound, which has been identified in stored apple fruits, is formed by beta-oxidation of linoleic acid (Beuerle & Schwab, 1997), with a final step involving the reduction of 3-hydroxyoctanoic acid (Beuerle & Schwab, 1999). Hydroxy acids are intermediate metabolites in the lipid biodegradation pathway of fungi and are also produced by the enzymatic breakdown of linoleic acid (Tressl et al., 1982). The reduction of 3-hydroxyoctanoic acid to octane-1,3-diol has also been identified as a minor pathway in mushrooms (Tahara et al., 1977). Biologically, aliphatic diols, such as octane-1,3-diol, are known for their antimicrobial and antifungal properties (Akedo et al., 1977). Its structural similarity to FMOff compounds makes it a potentially interesting compound. 1-Hydroxyoctan-3-one and octane-1,3-diol have been linked in a de novo pathway of 3-hydroxyoctanoic acid synthesis (Schwab, 1989). However, to our knowledge, no detailed biological pathway between the two compounds has been demonstrated under oenological conditions to date. From a sensory perspective, there is limited data available in the literature. Chiral 1,3-octanediol is described differently, with (R)-octane-1,3-diol exhibiting sweet, astringent and body descriptors, while (S)-octane-1,3-diol was described as bitter and astringent (Nozaki et al., 2001). Octane-1,3-diol has been described as an odor-active compounds of 'Honeycrisp' apple fruit and related to green, fatty and mushroom notes (Yan et al., 2020).

## 6.2. Chemical synthesis of natural and deuterated standards

To quantify potential markers in the samples, it was necessary to synthesize standards that were not commercially available. Octane-1,3-diol was obtained by reducing the ketone group of 1-hydroxyoctan-3-one, which had previously been successfully synthesized by Delcros, Costis, et al. (2023). To ensure optimal selectivity and precision, it was decided to use a SIDA approach. Therefore, deuterated forms of 1-hydroxyoctan-3-one and octane-1,3-diol were also synthesized (Fig. 1).

NMR characterization was carried out for each synthesis and edited-HSQC spectra are presented in Fig. 3. The spectrum of compound 1 (Fig. 3A) showed signals with chemical shifts, multiplicity as well as relative integrations corresponding to those expected for 1-hydroxyoctan-3-one as previously described (Delcros, Costis, et al., 2023). Briefly, six multiplet signals corresponding to aliphatic protons were observed in the ¹H ¹D spectrum, three triplets and one pentet each integrated for 2 protons, and one triplet and one multiplet represented three and four protons respectively. Seven 1 J ¹H/¹³C correlations were observed in the edited-HSQC spectrum. One of them corresponded to a methyl (CH₃) whereas the six others originated from methylene (CH₂) groups. Besides, the ¹³C spectrum showed a bl Blinded signal at about 212 ppm corresponding to the carbonyl group.

The spectrum of compound 2 (Fig. 3B) was similar to that of compound 1 (Fig. 3A) except for the signals arising from the methyl group: neither the characteristic triplet signal in the ¹D ¹H spectrum nor the 1 J ¹H/¹³C correlation in the edited-HSQC spectrum could be observed. On the other hand, a signal was present in the 1D ¹³C spectrum at about 20 ppm which can be attributed to a CD₃. Altogether, these results confirm a deuteration of the methyl group and that the structure of the compound 2 is the [8,8,8²H]-1-hydroxyoctan-3-one. The purities, determined from the surface signal area of the methyl group at 3.83 ppm, were 39 % and 36 % for 1-hydroxyoctan-3-one (1) and [8,8,8²H]-1-

hydroxyoctan-3-one (4), respectively.

The structure of compounds 3 and 4 was also determined using both 1D and 2D NMR spectra (Fig. 3C and D). The two compound spectra display similar features including three multiplets centered at 1.38, 1.40 and 3.70 ppm integrating to three, two and eight protons respectively. In contrast, differences were observed in the HSQC spectra: compound 3 exhibited a correlation between a proton at δ 0.93 ppm and a carbon at δ 14.39 ppm which can be attributed to a methyl group (Fig. 3C). This correlation was not present in the HSQC spectrum of compound 4 (Fig. 3D). Moreover, a carbon signal was observed in the 1D ¹³C spectrum of compound 4 at 18.9 ppm which did not give correlation in the HSQC. This result is in accordance with a deuteration at a methyl position. The spectra showed that compound 3 contains six CH₂, one CH and one CH₃ which chemical shifts correspond to those expected for the octan-1,3 diol (5) and consequently, it can be deduced that compound 4 was the [8,8,8,8²H]-octan-1,3 diol (6). The purities of compounds 3 and 4, determined using the surface signal area of the multiplet between 3.57 and 3.64 ppm, were about 29 % and 49 % respectively.

## 6.3. Development and validation of quantitative targeted approach of compounds involved or potentially involved in FMOff in musts and wines

A quantitative analysis approach of the known FMOff C8 compounds and the newly identified compound, the octane-1,3-diol, was set up to study the involvement of each compound in FMOff.

The main challenge of this study lies in the ability to identify one or more reliable and easy to analyse FMOff compounds in oenological matrices. Indeed, the literature highlights the importance of 1-octen-3-one in FMOff, however, several limiting factors were identified in available studies, concerning the sampling volume, the sample preparation time and the sensitivity of analytical method. Delcros, Collas, et al. (2023) proposed a GC-MS method in SIM mode with LOD and LOQ of 30 ng/L and 220 ng/L, which is above detection and recognition threshold of the compound. More sensitive method, based on derivatization with pentafluorobenzylhydroxylamine coupled with GC-MS/MS analysis were developed (Culleré et al., 2006) and later adapted (GC-MS-SIM mode analysis) (Meistermann et al., 2021; Pons et al., 2011). While this approach offers advantages, such as an undeniable increase in sensitivity, with a LOQ of 0.75 ng/L achieved with GC-MS/MS analysis (Culleré et al., 2006) and 20 ng/L with GC-MS-SIM mode analysis (Pons et al., 2011), it also implies a more complex and time-consuming sample preparation process (derivatization step and use of SPE cartridge), in the same way as liquid-liquid extraction (Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023). Furthermore, the sample preparation required for the methods proposed in the literature also required an important sampling volume, ranging from 50 mL (Culleré et al., 2006) up to 500 mL (La Guerche et al., 2006) which are not compatible with high-throughput analysis. Therefore, a MS/MS approach has been proposed to try to achieve a better sensitivity and specificity with the smallest possible sampling volume.

Preliminary results showed that hydroxyoctan-3-one and octane-1,3-diol can be analyzed by LC-MS/MS with improved reliability. Indeed, phenomena of peak tailing, liner activation and non-specificity of the method were observed using a GC-MS/MS approach (data not shown). Consequently, 1-octen-3-one, 1-octen-3-ol, 3-octanone, 2-heptanol and 3-octanol were quantified by GC-MS/MS analysis while 1-hydroxyoctan-3-one and octane-1,3-diol were quantified using LC-MS/MS analysis. The analytical performance of both methods is detailed in Table 2.

Validation parameters of the GC-MS/MS and the LC-MS/MS methods were evaluated for each analyte. Depending on analytes, the correlation coefficient values of calibration curves ranged from 0.94 to 1.00. Repeatability ranged from 6 % to 22 % for all analytes. Reproducibility ranged from 3 % to 21 % for all analytes, which is considered quite acceptable for the analysis of trace compounds. Depending on the analyte, the accuracy of the methods was evaluated ranging from 67 % to 115 %.
The limits of detection (LOD) and limits of quantification (LOQ) for the analytes were calculated over a series of six samples by measuring a signal-to-noise ratio of 3 and 10, respectively. The objective was to obtain LOQs above the target sensory threshold. This was successfully achieved for all analytes except 1-octen-3-one and 3-octanol, where the sensitivity and accuracy allowed us to measure LOQs of 80 ng/l and 0.38 µg/L, respectively. These values are slightly above their perception thresholds of 40 ng/L in neutral white wine for 1-octen-3-one (but below its recognition threshold of 120 ng/L) (Pons et al., 2011) and 0.25 µg/L in water for 3-octanol (Xu et al., 2019).

## 6.4. Quantification of compounds involved or potentially involved in FMOff of experimental musts and wines

2-Heptanol was not detected in any of the samples (data not shown) and does not appear to be present as a free form in grape juice and wine samples. There are few occurrences in the literature about the involvement of this compound in the mushroom flavor in musts and wines. Indeed, it has been identified as contributing to mushroom aroma in red grapes and was quantified at levels below its sensory threshold in two musts made from rotten grapes (La Guerche et al., 2006). Its concentration increased significantly in Chardonnay grapes infected with powdery mildew (Rusjan et al., 2012).

Concerning other compounds, the concentration of 3-octanone, 1-octen-3-one, 3-octanol, 1-octen-3-ol, 1-hydroxyoctan-3-one and octane-1,3-diol generally seemed to increase in musts and wines with both the FMOff sensory level of the wines and the visual degree of grey rot on the grapes, with an exception with M/W-9 (Table 1).

3-Octanone was not detected in any of the musts whereas it was found in wines made with grapes with a visual attack of more than 10 %, with concentrations ranging from a few µg/L to up to 96 µg/L in the sample W-9.

3-Octanol was detected at very low concentrations (below 0.53 µg/L) in three musts made from highly contaminated grapes and in the experimental wines with FMOff scores above 1, with its concentration increasing in correlation with the FMOff sensory perception. This compound has a perception threshold of 0.25 µg/L (Xu et al., 2019), and concentrations above this threshold were measured in samples W-6 to W-9, which were also classified as medium to strong FMOff wines. Finally, these observations were consistent with previously reported values (Delcros, Collas, et al., 2023).

1-Octen-3-ol was present at low concentrations (below 5 µg/L) in must and wine samples with less than 50 % of contamination, but its concentration increased to levels close to the sensory threshold (25 µg/L) with high contamination levels.

1-Octen-3-one was present at concentrations below its LOQ (i.e. 80 ng/L) in M-2 to M-6, but was not detected (i.e. concentration below 24 ng/L) in the corresponding wines, although some of them being classified as FMOff wines. Among these, W-1 was not described as FMOff, but W-2, W-3, W-4 and W-5 had FMOff sensory values between 0.45 and 2.09. The wine 7 had a FMOff value of 2.45, though its concentration of 1-octen-3-one remained below the LOQ of our method (i.e., 80 ng/L). In contrast, 1-octen-3-one was found in high concentrations (up to 14 µg/L) in the samples M-7 to M-9 and W-7 to W-9, described with the highest FMOff sensory scores. These values agree with the analysis of naturally strong FMOff white wines (Delcros, Collas, et al., 2023; Pons et al., 2011). These results highlight the challenge of correlating 1-octen-3-one levels with FMOff, mainly due to the compound's very low sensory threshold and the difficulty of quantifying it at such low levels. The data available in the literature vary widely, with high concentrations of 1-octen-3-one measured both in control wines (Delcros, Collas, et al., 2023) and in FMOff wines (Delcros, Collas, et al., 2023; Pons et al., 2011). Recently, Destanque et al. (2024) reported the detection of FMOff by different sensory panels in laboratory-scale wines with 1-octen-3-one concentration below 20 ng/L, emphasizing the difficulty of using this compound as an analytical marker (Destanque et al., 2024). The

experimental musts represented a wide range of grape contamination levels and produced wines with different FMOff values, but none of these four C8 compounds stand out as useful markers for discriminating between samples.

The musts M-7, M-8 and M-9 also had the highest concentrations of 1-hydroxyoctan-3-one and octane-1,3-diol, with values ranging from 251 µg/L to over 4 mg/L for 1-hydroxyoctan-3-one, and from 53 µg/L to over 400 µg/L for octane-1,3-diol (Table 1). In contrast, the other five musts (M-2, M-3, M-4, M-5 and M-6), which produced wines with FMOff scores between 0.45 and 2.45 had lower concentrations of both compounds, ranging from less than 10 µg/L (M-2, M-3 and M-4) to a few tens of µg/L (M-5 and M-6). 1-hydroxyoctan-3-one was quantified for the first time by Delcros, Collas, et al. (2023) in experimental musts specifically contaminated by *Crustomyces subabruptus*. It was not detected in healthy Pinot noir musts, but its concentration reached several hundreds of µg/L in musts contaminated by *Crustomyces subabruptus*.

A similar trend was observed for 1-hydroxyoctan-3-one and octane-1,3-diol concentrations in wines as in the musts from which they were made. Contrary to the literature (Delcros, Collas, et al., 2023), 1-hydroxyoctan-3-one was not detected in the control sample W-1. Octane-1,3-diol was detected at an ultra-trace level of 0.7 µg/L in this sample. The wines with higher FMOff scores, W-7, W-8 and W-9, had higher concentrations of both compounds, similar to the musts from which they were made (from 187 µg/L up to 4.1 mg/L for 1-hydroxyoctan-3-one, and from 150 µg/L to 1 mg/L for octane-1,3-diol). In contrast, quite low concentrations (below 20 µg/L) were measured in the wines W-2 to W-6, which had a FMOff score between 0.45 and 2.45. In the literature, concentrations around 400 µg/L of 1-hydroxyoctan-3-one were reported for experimentally contaminated Pinot wines (Delcros, Collas, et al., 2023). A possible correlation between the concentration of 1-hydroxyoctan-3-one in wine and the intensity of its FMOff sensory descriptor was already evidenced by Delcros, Costis, et al. (2023). To the best of our knowledge, this is the first time that octane-1,3-diol is quantified in musts and wines samples.

## 6.5. Effect of alcoholic fermentation on FMOff compounds in experimental wine samples

Nine biological samples, consisting of different pairs of corresponding must and wine, were analyzed. It is therefore possible to suggest a trend in concentration evolution during alcoholic fermentation. A Spearman correlation test between the evolution of 1-hydroxyoctan-3-one and octane-1,3-diol concentration for the 9 observations (9 pairs M/W) was performed. A Spearman coefficient $ρ = -717$ ($α = 0.05$) was obtained, suggesting a possible correlation between the increase of octane-1,3-diol concentration and the decrease of 1-hydroxyoctan-3-one concentration.

Regarding the very good analytical performances of the LC-MS/MS method, we can assume that the concentration of 1-hydroxyoctan-3-one in the samples W-5, W-6, W-7, W-8, W-9 tended to decrease with the fermentation, suggesting a possible consumption of the molecule by the yeast (Delcros, Collas, et al., 2023). Additionally, there was no production of 1-hydroxyoctan-3-one during the fermentation of the control must M-1 and in the less contaminated musts, contrary to previous findings in the literature (Delcros, Collas, et al., 2023). In contrast, increasing trends in concentration were observed for the octane-1,3-diol during fermentation of the samples W-5, W-6, W-7, W-8 and W-9. The concentrations of both compounds in must and corresponding wine (M/W) were similar for the low FMOff score samples. Although drawing conclusions from a small number of observations is difficult, it should be noted that the variation in the concentration of the two compounds in the Must/Wine (M/W) pair sample is within the same range. This could indicate a conversion of the hydroxy ketone to the dihydroxy alcohol. During fermentation, the biosynthesis pathway of higher alcohols by *Saccharomyces cerevisiae*, partly due to the Ehrlich pathway, is the result of the multi-step catabolism of amino acids. After an intracellular
transamination reaction step, an alpha-keto-acid, which can also be
derived from the catabolism of sugars, is produced. It is then decar-
boxylated to form a fusel aldehyde, which is in turn reduced into a
higher alcohol (Hazelwood et al., 2008). Structurally, 1-hydroxyoctan-
3-one and octane-1,3-diol are closely related compounds. A possible
explanation for the opposite variation of these two compounds is the

reduction of the carbonyl group of 1-hydroxyoctan-3-one by a yeast
dehydrogenase. This reduction could occur as part of the Ehrlich
pathway, leading to the production of octane-1,3-diol as a higher
alcohol. An NAD-dependent oxidoreductase such as (R)-2,3-butanediol
dehydrogenase has been identified in *Saccharomyces cerevisiae* and is
able to catalyse the reversible reaction, reducing dihydroxy alcohol to

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Concentrations of FMOff markers measured in wines (A) and musts (B) from the 2023's harvest (*** p < 0.0001 and ** p < 0.01, Kruskal-Wallis test, significance level α = 0.01). Dot line indicates compound's LOQ.</figcaption></figure>
hydroxy ketone (Heidlas & Tressl, 1990). However, (R)-2,3-butanediol dehydrogenase can only catalyse the reduction of α-hydroxyketone, which suggest that another enzyme may be involved in the reduction of 1-hydroxyoctan-3-one. Another hypothesis is that the increase of octane-1,3-diol concentration in the most contaminated M/W pair samples –7, –8 and –9 is due to a release from precursor forms. Although the existence of glycosylated form of octane-1,3-diol has not yet been investigated in grapes, glycosidically bound forms have been reported in apple fruit (Schwab, 1989). Such bound forms have been observed in contaminated grapes for other C8 compounds, including as 1-octen-3-one, 1-octen-3-ol or 1-hydrooctan-3-one (Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023).

## 6.6. Screening of FMOff musts and base wines from the 2023 harvest

### 6.6.1. Sensory analysis

A sensory analysis session was carried out on a selection of 62 base wines collected in Champagne during the 2023 harvest. All the samples were tasted by a panel of trained experts from the Comité Champagne who rated the intensity of their FMOff, using the same scale applied to experimental wines, i.e., a score of 0 for wines with no FMOff detected and a score of 4 for wines having an intense FMOff.

Among these 62 samples, 29 samples received a score of zero and were combined to form the control group. The remaining 33 wines were classified as the FMOff group, with sensory scores ranging from 0.3 to 4 (supplementary data S1). Within the FMOff group, five samples were classified as “strong” FMOff wines, with scores between 3 and 4, while 11 samples were considered low FMOff (scores between 0.3 and 1) and 17 samples were scored intermediate, between 1.1 and 2.1 (supplementary data S1).

### 6.6.2. Quantification of FMOff potential markers in the 2023 harvest samples

The compounds identified as potential markers of FMOff in the experimental wines were then quantified in the 62 base wines described in the previous section. 2-heptanol, 2-octanol, 3-octanol and 3-octanone were not detected in any of the 62 samples. However, 1-hydroxyoctan-3-one, octane-1,3-diol, 1-octen-3-one and 1-octen-3-ol were detected and quantified in several samples from the 2023 harvest collection.

For each compound, the control group was compared with the FMOff group, and the results are shown in Fig. 4A. Values between the LOD and LOQ were also included in the statistical analysis (non-parametric Kruskal-Wallis test, $\alpha = 0.01$).

Four compounds significantly discriminated the control group from the FMOff group, namely 1-hydroxyoctan-3-one ($p < 0.0001$), octane-1,3-diol ($p < 0.0001$); 1-octen-3-one ($p = 0.002$), and 1-octen-3-ol ($p < 0.0001$). However, considering the samples individually, 1-octen-3-one was detected at concentrations above the LOQ (80 ng/L) in only 2 of the 33 FMOff samples, and in none of the control samples, representing just 6 % of the FMOff samples analyzed. Similarly, 1-octen-3-ol exceeded 4.05 µg/L in only one FMOff sample and in none of the control samples. In contrast, 1-hydroxyoctan-3-one was quantified above its LOQ (2.45 µg/L) in 22 of the 33 FMOff samples (66.7 %), with concentrations ranging from 2.47 µg/L to 33.16 µg/L, but was not detected in any of the control group samples (all concentrations were below the LOQ). Octane-1,3-diol was quantified in all the FMOff samples, with values ranging from 0.7 µg/L to 10.73 µg/L, and in only eight control samples, with an average concentration of 0.62 ($\pm$ 0.44) µg/L. Furthermore, correlations were observed between FMOff score and concentrations of 1-hydroxyoctan-3-one or octane-1,3-diol in the wine ($Q = 0.81$, $\alpha = 0.01$, for both compounds, supplementary data S2).

Relatively low concentrations of each target were measured, with concentrations of 1-octen-3-one below 120 ng/L, concentrations of 1-octen-3-ol below 5 µg/L and concentrations of hydroxyoctan-3-one and octane-1,3-diol below 35 µg/L. These results are consistent with a careful selection of healthy grapes required to produce quality wine.

Here again, the results showed difficulties in using common markers, such as octen-3-ol or octen-3-one, to analytically detect the FMOff. Indeed, 1-octen-3-one was detected at concentrations below 24 ng/L in twelve of the thirty-three FMOff samples (supplementary data S1). A possible explanation could be the establishment of interactions between carbonyl compounds with other molecules, leading to less volatile or not analytically investigated compounds such as bisulfite adducts (Allamy et al., 2017; Thibon et al., 2016), glutathione adducts (Pons et al., 2011; Thibon et al., 2016), or amino acid adducts, which were initially observed in tea (Cheng et al., 2008) and later in wine (Pons et al., 2011). Glutathione and glycine interactions resulted in a 70 % decrease in the amount of 1-octen-3-one after 65 days in base wines (Pons et al., 2011). In the same way, the addition of sulphur dioxide led to an 81 % decrease in the concentration of another unsaturated ketone, 1,5-octadien-3-one, in model wine (Allamy et al., 2017). Furthermore, it is likely that the sensory defect cannot be attributed to a single component. As already suggested (Delcros, Costis, et al., 2023), the intensity of the sensory defect could be due to a synergistic effect involving several compounds.

1-Hydroxyoctan-3-one and octane-1,3-diol were also quantified in 20 musts, 10 of which produced FMOff base wines (with a FMOff score between 0.5 and 1.8), and 10 of which produced wines without FMOff (i. e., control wines, with a 0 FMOff score) during the 2023's harvest. Both markers clearly distinguished between musts that produced control wines and those that produced FMOff wines ($p < 0.0001$, $\alpha = 0.01$) (Fig. 4B). 1-hydroxyoctane-3-one was quantified with concentrations above its LOQ (2.45 µg/L) in 18 of the 20 FMOff samples, with concentrations ranging from 1.39 µg/L to 10.91 µg/L, and in only one control must sample. Octane-1,3-diol was quantifiable in all the musts from the FMOff group, with values ranging from 0.89 µg/L to 3.69 µg/L, and in the control group it was found in eight samples, but at lower concentrations (mean concentration of $0.61 \pm 0.40$ µg/L).

As in wines, a strong correlation was also observed between 1-hydroxyoctan-3-one and octane-1,3-diol concentrations ($Q = 0.95$ in both musts and wines, supplementary data S2). This correlation could be due to a metabolic reaction or an interconversion mechanism between the two compounds.

## 7. Conclusion

The aim of the present study was to identify reliable analytical markers of the FMOff defect in oenological matrices. First, a qualitative study was carried out using untargeted GC-MS analysis on experimental samples obtained from naturally infected grapes, with increasing FMOff sensory defects. Two C8 compounds were detected in the contaminated musts: 1-hydroxyoctan-3-one, previously identified in base wines from Champagne area (Delcros, Costis, et al., 2023) and octane-1,3-diol which has been identified for the first time in musts and wines. Both 1-hydroxyoctan-3-one and octane-1,3-diol were quantified in increasing concentrations in all FMOff samples, suggesting their role of FMOff analytical markers.

This hypothesis was statistically validated in a broader analytical study involving 62 base wine samples collected during the 2023's harvest, which naturally developed, or not, an FMOff. Both compounds were also significantly discriminating at an earlier oenological stage: indeed, they allowed to distinguish musts that produced FMOff wines from musts that produced wines without FMOff. These results show that although FMOff is partially revealed during alcoholic fermentation, the defect is already present at an earlier stage, in the grapes.

In conclusion, this study shows that 1-hydroxyoctan-3-one and octane-1,3-diol are relevant analytical markers for FMOff-type alterations in oenological matrices, for both musts and base wines. Their early quantification in musts provides a reliable tool to identify a potential risk of FMOff development after fermentation and to prevent the use of risky raw materials in winemaking.
## Ethical statement

The sensory study did not require formal ethical approval from the French National Committee on Health Research Ethics. Professional panelists from the Comité Champagne participated in this study as part of their work assignments, with respect for their rights and privacy. All participants gave their consent to participate in the research project.

## CRediT authorship contribution statement

**Sarah Ployon:** Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. **Lucas Suc:** Writing – original draft, Formal analysis. **Christine Le Guernevé:** Writing – original draft, Formal analysis. **Eléonore Augès:** Resources, Project administration, Conceptualization. **Héloïse Mahé:** Resources, Project administration, Conceptualization. **Marion Hervé:** Resources, Project administration, Conceptualization. **Audrey Bloem:** Writing – review & editing, Supervision, Conceptualization. **Aurélie Roland:** Writing – review & editing, Supervision, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We thank Valerie Nolleau and Teddy Godet for their help with GC analyses at UMR SPO and Soline Caillé for advices with statistical data treatment.

## Financial support

We are grateful to the Comité Champagne and MHCS for the funding of this project.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at [https://doi.org/10.1016/j.foodchem.2025.143454](https://doi.org/10.1016/j.foodchem.2025.143454).

## Appendix B. Supplementary data

Supplementary data to this article can be found online at [https://doi.org/10.1016/j.foodchem.2025.143454](https://doi.org/10.1016/j.foodchem.2025.143454).

## Data availability

Data will be made available on request.

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