Jun 29, 2026

SPME-GC/MS Method for the quantification of 12 compounds associated with vegetal aromas in red wines (C6 alcohols, methoxypyrazines, salicylates)

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l2x58pv1y/v1
  • 1Univ. Bordeaux, Bordeaux INP, Bordeaux Sciences Agro, INRAE, UMR 1366 OENO, ISVV, F-33140 Villenave d’Ornon, France;
  • 2Tonnellerie Seguin Moreau, F-16103 Cognac, France
Pascaline Redon
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l2x58pv1y/v1
Protocol Citation: Pascaline Redon, Cécile Thibon, Alexandre Pons 2026. SPME-GC/MS Method for the quantification of 12 compounds associated with vegetal aromas in red wines (C6 alcohols, methoxypyrazines, salicylates). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2x58pv1y/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 05, 2026
Last Modified: June 29, 2026
Protocol  Integer ID: 318591
Keywords: vegetal aromas in red wine, vegetal aromas in wine, red wine, commercial red wine, wine, vegetal aroma, spiked neutral base wine, volatile compound, including c6 alcohol, c6 alcohol, ms method for the simultaneous quantification, ms method for the quantification, methoxypyrazine, loq values below sensory threshold
Abstract
This protocol presents a validated HS-SPME-GC/MS method for the simultaneous quantification of 12 volatile compounds associated with green/vegetal aromas in wines, including C6 alcohols, methoxypyrazines (IBMP, IPMP and sBMP), and salicylates. The procedure is detailed step-by-step for routine laboratory implementation, the validation section includes linearity (R² > 0.99), LOD/LOQ values below sensory thresholds, precision (RSD < 15%), and trueness (recoveries ranging from 87 to 116%) in spiked neutral base wine. The method is successfully applied to over 450 commercial red wines, and demonstrates robustness across diverse matrices.
Materials
Combi PAL autosampler (CTC Analytics)

DVB/CAR PDMS fiber (23 Ga, Supelco #57298-U)

GC system model 6890 (Agilent technologie)

Mass spectrometry model 5973 quadrupole system (Agilent technologie)

DB-Wax UI column (60 m × 0.25 mm × 0.25 µm, Agilent #122-7062UI)
SPME-GC/MS Method for the quantification of 12 compounds associated with vegetal aromas in red wines (C6 alcohols, methoxypyrazines, salicylates)
Introduction

Vegetal nuances in red wines represent a complex aromatic family, ranging from green bell pepper to herbaceous and minty notes, the control of which is a major quality issue for the wine industry. Traditionally, these perceptions are attributed to methoxypyrazines (IBMP, IPMP, sBMP) and C6 alcohols, whose sensory thresholds (ST) are approximately 15 ng/L for IBMP in red wines and near to 400 μg/L for cis-3-Hexenol, respectively. In 2016 and 2017, Poitou et al. demonstrated that these markers alone do not explain the diversity of observed green nuances. Their multidimensional GC approach revealed the key role of 1,8-Cineole (eucalyptol, ST : 1.1µg/L), detected up to 2.6 μg/L in Cabernet Sauvignon wines with an additive contribution to herbaceous perception, as well as that of Methyl salicylate, a cryptogamic stress marker present at concentrations reaching 70–130 μg/L [1-3].

Furthermore, several analytical methods have been developed and published for the quantification of a part of these compounds, including LLE, SPE, SPME, GC-MS, or GCxGC-TOF-MS [4-6]. However, the existing methods present significant trade-offs. Liquid-liquid extraction (LLE) is selective but consumes large solvent volumes and is time-consuming and difficult to automate. Solid-phase extraction (SPE) requires costly cartridges, multi-step elution protocols and cannot be used to extract different compounds simultaneously depending on the selectivity of the phase used. SPME, by contrast, is solvent-free, easily automated, and not time-consuming, with reduced preparation time.

The method presented here can quantify three chemical families (C6 alcohols, methoxypyrazines, and salicylates) in a single run.

This protocol is divided into two main sections: (1) a detailed step-by-step procedure for sample preparation, HS-SPME extraction, and instrumental analysis, and (2) method validation data (linearity, LOD/LOQ, precision, trueness) confirming analytical performance. Application results on over 450 red wines illustrate the applicability of method.
Download Fig. 1 Chemical structures of analytes.pdfFig. 1 Chemical structures of analytes.pdf70.5KB

1. Principle and Objectives

1.1 Principle

This method uses Headspace Solid-Phase Microextraction (HS-SPME) coupled with Gas Chromatography-Mass Spectrometry (GC/MS) to extract, separate, and quantify volatile compounds associated with green/vegetal aromas in wines at very low odour thresholds. The extraction is performed in a salted hydro‑alcoholic medium using a DVB/CAR/PDMS fiber, and analytes are detected in SIM mode for maximum sensitivity.

1.2 Objectives

The primary objective is to simultaneously quantify 12 key compounds related to vegetal and herbaceous characters (C6 alcohols, salicylates, and methoxypyrazines) in complex wine matrices. These target analytes are listed in Fig. 1. It is designed to be:

  • Simple and fast enough for high-throughput analysis, minimising sample preparation steps.
  • Robust and reproducible for routine analysis.
  • Sufficiently sensitive to detect concentrations below or close to sensory thresholds.
  • Applicable to a wide range of wines (red, white, various grape varieties, regions, and vintages).
2. Materials and Reagents

2.1. Chemicals

Ethanol (HPLC grade) and sodium chloride (>99.5%) are purchased from Fisher Scientific (Illkirch Graffenstaden, France). Water is purified through a Milli-Q purification system (Merck Millipore, Guyancourt, France).

2.2. Reference standards and stock solutions

2.2.1. Target analytes and internal standards

Analytical reference standards (1–15, Fig 1) are purchased from commercial suppliers: 1,4-Cineol (1, >99%, Sigma-Aldrich, Saint-Quentin-Fallavier, France), 1,8-Cineol (2, >99%, Sigma-Aldrich), Hexanol (3,>99%, Sigma-Aldrich), trans-3-Hexenol (4, >99%, Sigma-Aldrich), cis-3-Hexenol (5, >98%, Lancaster Synthesis Limited, Morecambe, Royaume-Uni), 3-octanol (Internal standard : IS) (6, >99%, Sigma-Aldrich), trans-2-Hexenol (7, >99%, Fluorochem Ltd, Hadfield, Royaume-Uni), cis-2-Hexenol (8, >90%, Sigma-Aldrich), 2-Isopropyl-3-methoxypyrazine (IPMP) (9, >99%, Sigma-Aldrich), 2-sec-Butyl-3-methoxypyrazine (sBMP) (10, ≥ 97 %, TCI, Zwijndrecht, Belgium), 2-Isobutyl-3-methoxy-d3-pyrazine (d3-IBMP) (IS) (11, >99%, Orga link Magny Les Hameaux, France), 2-Isobutyl-3-methoxypyrazine (IBMP) (12, >99%, Sigma-Aldrich), methyl-d3-salicylate (IS) (13, CIL,>98% Sainte-Foy-La-Grande, France), Methyl salicylate (14, >99%, Sigma-Aldrich), Ethyl salicylate (15, >99%, Thermo Scientific Chemicals).

2.2.2. Stock and working solutions

  • Prepare individual primary stock solutions of each reference standard and internal standard by accurately weighing the pure compound and dissolving it in ethanol to obtain a concentration of approximately 1 g/L.

  • Store primary stock solutions in amber glass vials at 4 °C for a maximum of 1 year.

  • Prepare mixed working standard solutions in ethanol by appropriate dilution of the primary stocks to cover the desired calibration ranges (see Table 1).

  • Store working standard solutions in amber glass vials at 4 °C and verify their stability periodically by GC‑MS.

2.2.3. Internal standard working solutions

Prepare the internal standard working solutions in ethanol at the following concentrations:
  • IS1: 3‑Octanol at 10 mg/L
  • IS2: d3-IBMP at 50 µg/L
  • IS3: Methyl-d3 Salicylate at 10 mg/L
Store in amber glass vials at 4 °C.

2.3. Wine samples

For method development and validation, use a neutral base wine (e.g., a red wine with minimal green character) as the matrix for calibration standards and spiking experiments as described in Sections 3.1 and 5.
For routine application of the method, collect red wine samples that are representative of the wines to be studied. Store bottles in the dark at cellar temperature (12-15 °C). To avoid excessive oxygen exposure, transfer wines after opening to appropriately sized glass bottles or vials filled as completely as possible (minimal headspace), and store them closed in the dark at low temperature (e.g., 4°C) until analysis.
For the application study presented in this protocol, the method was tested on more than 450 red wine samples from diverse geographical origins, grape varieties, and vintages, demonstrating its applicability to a wide range of wine styles.
3. Step-by-step protocol

3.1. Preparation of calibration standards in wine

Calibration standards are prepared in a neutral base wine to construct an 7-point calibration curve, including two blank (G0). (Table1)

Table1: Summary table of composition of the supplement mix and the composition of the calibration samples
3.2. Sample preparation for HS-SPME Analysis

For each 20 mL headspace vial (calibration standards and unknown wines):
  1. Weigh 4.0 g (± 0.1 g) of NaCl into the vial.
  2. Add 5.0 mL of Milli-Q water.
  3. Spike with 10 µL of each Internal Standard working solutions (IS1, IS2, IS3) using a precision syringe. This adds the IS to all samples (standards and unknowns) for internal standardization.
  4. Add 5.0 mL of the wine sample (calibration standard or an unknown wine).
  5. Immediately seal the vial tightly with a magnetic crimp cap with a PTFE/silicone septum.
  6. Homogenize sample
The vial is now ready for HS-SPME extraction

3.3. SPME-GC/MS analysis

Extractions are performed using a Combi PAL autosampler (CTC Analytics) equipped with a DVB/CAR PDMS fiber (23 Ga, Supelco #57298-U). For each vial, the following automated sequence is executed:
  1. Pre-incubation: 2 min at 40 °Cwith agitation (500 rpm, 5 s on / 2 s off)
  2. Extraction: 25 min at 40 °C with agitation (same conditions)
  3. Desorption: 6 min in the GC inlet at 240 °C(splitless mode)
  4. Fiber bake-out: 10 min at 270°C to minimize carry-over

GC separation is performed on an Agilent 6890 system equipped with a DB-Wax UI column (60 m × 0.25 mm × 0.25 µm, Agilent #122-7062UI). Helium carrier gas is set to a constant flow of 1.0 mL/min. The oven program is: 45 °C held for 5 min, ramped at 6 °C/min to 180 °C, then at 30 °C/min to 240 °C, with a final hold of 12 min (total runtime 41.5 min).

Mass spectrometry is performed on an Agilent 5973 quadrupole system operated in electron ionization mode (70 eV, 34.6 µA emission). The ion source and quadrupole are maintained at 230 °C and 150 °C,
respectively, with a transfer line temperature of 260 °C. Data acquisition is performed in SIM mode (gain factor 2).
Complete MS parameters, including retention times and selected ions for each analyte, are provided in Table 2.

Table 2: Retention time, internal standard and quantifier/qualifier ions of each compound

4. Quantification and Calculations

  • Integrate peak areas using quantifier ion SIM trace (MSD Chem, Agilent).
  • For each analyte, calculate the area ratio R=Ai/AIS with the corresponding internal standard.
  • Build calibration curves by plotting R versus nominal analyte concentration in the calibration standards (G0–G80).
  • Fit a linear regression and report slope, intercept, and coefficient of determination R2.
  • Calculate sample concentrations by interpolation from the calibration curve, correcting if needed for any dilution factor and expressing results in ng/L or µg/L depending on the compound.
5. Method validation

Perform a full validation according to ICH/OIV guidelines using the spiked base wine.

  • Linearity & Range: Establish calibration curves for each analyte using the prepared standards (G0-G80). Calculate mean slope, intercept, and correlation coefficients (R²).
  • Limit of Detection (LOD) & Quantification (LOQ): Determine via signal-to-noise ratio (S/N=3 for LOD, S/N=10 for LOQ) on spiked samples close to the lowest level (level G5, see table 1), or alternatively from the standard deviation of replicate blank measurements (G0) and the slope of the calibration curve (n=6).
  • Precision, recovery and robustness: Evaluate repeatability (intra-day) and intermediate precision (inter-day) by analysing spiked samples at low (level G5) and high (level G40) concentrations (n=6).
  • Assess intermediate precision (inter‑day) by repeating the same experiment on at least 2–3 different days. Expresses precision as relative standard deviation (RSD, %) for each level.
  • Trueness (recovery): Determine trueness by comparing the measured concentration with the nominal spiked concentration at low and high levels (G5, G40, n=6). Express recovery as percentage of nominal value.

Results are summarised in Table 3.
Table 3: Method validation result for the SPME-GCMS method developed of 12 compounds associated with green nuances in red wines.



6. Application to Wine Analysis

Quantitative assays were performed on a diverse set of over 450 red wines from various appellations, grape varieties, and vintages.   

The analysis successfully quantified all 12 target compounds, confirming the interest of the method. The measured concentrations ranged from several orders of magnitude, validating the applicability to wine analysis.

The data revealed distinct distribution patterns:

Concerning the distribution of C6 alcohols, hexanol is clearly predominant, with concentrations ranging from 800 to 1300 µg/L, while C6 unsaturated alcohols (cis-3-hexenol, trans-3-hexenol, trans-2-hexenol, and cis-2-hexenol) are detected at lower levels, with median concentrations of about 65, 20, 7, and 6 µg/L, respectively. All C6 alcohols are found at concentrations below or near their respective sensory thresholds in red wines.

Methoxypyrazines (IBMP, IPMP, sBMP) are present at very low levels (ng/L), with IBMP concentrations ranging from below the perception threshold (near 15 ng/L in red wine, [7]) to over 30 ng/L in pronounced "green" wines, demonstrating the method's critical sensitivity.

Among salicylates, methyl salicylate is detected across a wide range from 1 µg/L to over 70 µg/L, with substantial variability between wines. By contrast, the level of ethyl salicylate remains stable at close to 2 µg/L, regardless of the wine sampled.

Figure 2 : A box-and-whisker plot showing the concentrations obtained using this method for over 450 red wine samples from various geographical origins, grape varieties, and vintages

Conclusion

This HS-SPME-GC/MS protocol presented here is a rapid, robust and sensitive tool for the determination of green/vegetal aroma markers in red wines at trace levels.
Protocol references
1.         Poitou, X., C. Thibon, and P. Darriet, 1,8-Cineole in French Red Wines: Evidence for a Contribution Related to Its Various Origins. Journal of Agricultural and Food Chemistry, 2017. 65(2): p.383-393.

2.         Poitou, X., et al., Methyl salicylate, a grape and wine chemical marker and sensory contributor in wines elaborated from grapes affected or not by cryptogamic diseases. Food Chemistry, 2021. 360.

3.         Antalick, G., et al., Investigation and Sensory Characterization of 1,4-Cineole: A Potential Aromatic Marker of Australian Cabernet Sauvignon Wine. Journal of Agricultural and Food Chemistry, 2015. 63(41): p. 9103-9111.

4.         Sala, C., et al., Headspace solid-phase microextraction method for determining 3-alkyl-2-methoxypyrazines in musts by means ofpolydimethylsiloxane–divinylbenzene fibres. Journal of Chromatography A, 2000. 880(1): p. 93-99.

5.         Zhou, C., et al., Simple and Rapid Method for the Quantitation of Trace-Level 3-Alkyl-2-methoxypyrazines in Fragrant Vegetable Oils by Gas Chromatography–Tandem Mass Spectrometry. Journal of Agricultural and Food Chemistry, 2022. 70(20): p. 6247-6252.

6.         Ryona, I., B.S. Pan, and G.L. Sacks, Rapid Measurement of 3-Alkyl-2-methoxypyrazine Content of Winegrapes To Predict Levels in Resultant Wines. Journal of Agricultural and Food Chemistry, 2009. 57(18): p. 8250-8257.

7.     Roujou de Boubee, D., C. Van Leeuwen, and D. Dubourdieu, Organoleptic impact of 2-methoxy-3-isobutylpyrazine on red Bordeaux and Loire wines. Effect of environmental conditions on concentrations in grapes during ripening. Journal of Agricultural and Food Chemistry, 2000. 48(10): p. 4830-4834.