METHOD VALIDATION DATA
MATERIALS AND METHODS Chemicals:
Dichloromethane, absolute ethanol as well as sodium sulphate (anhydrous), ammonium sulphate and sodium hydroxide were purchased from Merck (Darmstadt, Germany). Tartaric acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). The water was purified through a Milli-Q purification system (Millipore, Bedford, MA, USA). The chemicals, all of which were of analytical grade, were purchased from Riedel de Haën (Seelze, Germany), Sigma-Aldrich (St. Louis, MO, USA), Fluka (Buchs, Switzerland), and Aldrich (Steinheim, Germany), are shown in Table 1.
Synthetic base wine simulant
A synthetic base wine simulant was prepared as described by Louw et al. (2009) and consisted of 2.5 gL-1 tartaric acid from Merck and 12 % (v/v) ethanol dissolved in purified water. The pH was adjusted to 3.5 using 0.1 M sodium hydroxide.
Gas
UPH hydrogen gas as carrier gas, UPH nitrogen as make up gas for the detector flow, and UPH air for the flame ionization detector (AFROX, South Africa).
Table 1 List of chemical standards used in this work, and their purity.
Isoamyl alcohol Aldrich >99%
1-Pentanol Fluka >99%
2-Phenylethanol Merck >99%
Hexanol Merck >98%
1-Octen-3-ol Fluka >98%
2,6-Dimethyl-6-hepten-2-ol Fluka >99.5%
3-Methyl-1-pentanol Sigma-Aldrich >97%
3-Ethoxy-1-propanol Sigma-Aldrich >97%
4-Methyl-1-pentanol Sigma-Aldrich >95%
Acetate esters Hexyl acetate Fluka >99%
Ethyl phenylacetate Aldrich ≥98%
Ethyl acetate Sigma-Aldrich >99.7%
Isoamyl acetate Riedel de Haën >98%
2-Phenylethyl acetate Fluka >99%
Ethylphenyl acetate Fluka >99%
Acids Propionic acid Fluka >99.5%
Isovaleric acid Fluka >99%
Isobutyric acid Fluka >99.5%
Valeric acid Fluka >99%
Hexanoic acid Aldrich >99.5%
Octanoic acid Aldrich >99.5%
Acetic acid Merck >98%
Decanoic acid Sigma >98%
Butyric acid Fluka >99.5%
Esters Ethyl butyrate Fluka >98%
Ethyl-2-methylbutyrate Aldrich ≥98%
Ethyl isovalerate Aldrich ≥98%
Ethyl propionate Aldrich ≥97%
Ethyl lactate Fluka >99%
Diethyl succinate Fluka >98%
Ethyl ocatanoate Fluka >98%
Ethyl hexanoate Fluka >99%
Ethyl decanoate Aldrich >99%
Ethyl-3-hydroxybutanoate Sigma-Aldrich ≥97%
Carbonyls Acetoin Fluka >97%
Diacetyl (2,3-Butanedione) Fluka >99.5%
Acetaldehyde Fluka >99.5%
Terpenes α-Terpeneol Sigma-Aldrich >99%
Citronellol Fluka >99%
Linalool oxide Aldrich >97%
Linalyl acetate Aldrich ≥97%
Geraniol Fluka >99%
Nerol Fluka >99%
Wood derived Furfuryl alcohol Aldrich >98%
Furfural Sigma-Aldrich 99%
5-Methyl-2-furfural Sigma-Aldrich 99%
Guaiacol Aldrich >98%
Whiskey lactone Aldrich >98%
Column and instrument
A J & W DB-FFAP capillary GC column (Agilent, Little Falls, Wilmington, USA), with the dimensions 60 m length by 0.32 mm internal diameter and 0.5 µm film thickness. The instrument was a Hewlett Packard 6890, gas chromatograph equipped with a flame ionization detector (GC-FID) (Agilent, Little Falls, USA)
Gas chromatographic conditions
The initial oven temperature was set at 40 ºC and held for 5 minutes, after which the temperature was increased by 8 ºCmin-1 to 225 ºC, and held for 1.2 minutes. 3 µL of the sample was injected at 200 ºC in split mode (18:1, with a split flow rate of 98.9 mL min-1).
The column flow rate was 5.5 mL min-1 and a post run of 5 minutes, with oven temperature at 240 ºC and a flow rate 6 mL min-1 cleaned off the column from any contaminants of high boiling point, making the total run time 29.3 minutes. The detector temperature was set at 250 ºC.
Sample preparation
Before the addition of wine, 2.5 grams of ammonium sulphate was added to 15 mL culture tubes (Pyrex, Milian, USA). Five milliliters of wine and 100 µL of the internal standard solution (0.5 mgL-1 4-methyl-2-pentanol and 0.1 mgL-1 2,6-dimethyl-6-hepten-2-ol in synthetic base wine) were extracted using 1 mL of dichloromethane by sonicating for one hour with shaking every 10 minutes. The extract was then centrifuged at 3000 rpm for 30 minutes and the dichloromethane layer (top layer) removed and dried on anhydrous sodium sulphate. Each wine sample was extracted in triplicate and injected into the Hewlett Packard 6890 GC-FID (Agilent, Little Falls, USA) in duplicate.
RESULTS
Calibration was performed for all the volatile compounds at the ranges stated in Table 2, with the calibration curves given in order of elution of the compounds, (Figures 1-57).
Recoveries in wine for all compounds, lowest levels of quantification and detection as well as the slope and correlation coefficient (R2) are given in Table 2.
The repeatability and intermediate repeatability, expressed in % relative standard deviation (% RSD) are given in Table 3.
Table 2. Recoveries, limits of detection and quantification, slopes, correlation coefficient and calibration ranges for aroma compounds in wine
Table 2 continued
Compound Recovery in wine LODa LOQb R2 Slope Range
(%) ± RSD (%) mg L-1 mg L-1 mg L-1
Terpenes Limonene 69.73 ± 1.34 0.04 0.11 0.999 0.125 0.1 - 1.00
Fenchone 73.65 ± 1.26 0.02 0.04 0.999 0.258 0.04 - 1.00
Linalool Oxide 73.83 ± 3.15 0.04 0.15 0.999 0.079 0.05 - 1.00
Linalool 65.02 ± 8.16 0.02 0.08 0.999 0.188 0.08 - 1.00
Linalyl acetate 85.96 ± 3.26 0.02 0.06 0.999 0.138 0.06 - 1.00
Citronellol 71.78 ± 1.29 0.02 0.06 0.999 0.193 0.01 - 1.00
α-terpeneol 81.15 ± 5.75 0.02 0.03 0.999 0.383 0.01 - 1.00
Nerol 99.52 ± 6.59 0.02 0.03 0.999 0.455 0.03 - 1.00
Geraniol 79.20 ± 1.06 0.02 0.04 1.000 0.229 0.04 - 2.00
β-Ionone 102.11 ± 1.57 0.03 0.09 1.000 0.137 0.09 - 1.00
α-Ionone 88.90 ± 1.07 0.03 0.11 0.999 0.086 0.10 - 1.00
β-Farnesol 95.22 ± 9.67 0.04 0.1 0.998 0.109 0.1 - 5.00
Acids Acetic Acid 77.68 ± 1.26 0.12 0.39 0.999 0.155 22.50 - 1000.00
Propionic Acid 115.49 ± 4.83 0.04 0.15 0.999 0.402 0.35 - 100.00
Isobutyric acid 76.36 ± 7.21 0.03 0.11 0.999 0.560 0.25 - 20.90
Butyric Acid 85.57 ± 8.47 0.03 0.1 0.999 0.566 0.25 - 21.20
Iso-Valeric Acid 100.73 ± 1.10 0.03 0.09 0.999 0.640 0.45 - 39.30
Hexanoic Acid 74.12 ± 2.21 0.02 0.08 0.999 0.731 0.38 - 29.70
Valeric Acid 97.87 ± 5.53 0.05 0.16 0.999 0.640 0.25 - 20.70
Octanoic Acid 81.19 ± 0.97 0.03 0.08 0.999 0.822 0.5 - 40.4
Decanoic Acid 101.62 ± 1.38 0.03 0.1 0.998 0.650 0.63 - 56.00
Wood derived Furfuryl alcohol 95.84 ± 7.11 0.05 0.16 0.999 1.011 0.16 - 25.00
Furfural 86.66 ± 5.07 0.01 0.03 0.999 0.943 1.00 - 100.00
5-Methyl-2-furfural 79.67 ± 9.44 0.02 0.05 0.999 0.557 0.04 - 35.00
Guaiacol 90.17 ± 4.96 0.02 0.08 0.999 0.710 0.1 - 20.00
Whiskey lactone 97.26 ± 2.50 0.03 0.09 0.999 1.120 0.10 - 20.00
Table 3 Repeatability and intermediate repeatability (expressed in% RSD) for the developed GC-FID method in white wine and synthetic wine.
Compound class Volatile compound Repeatability Intermediate repeatability White wine Synthetic White wine Synthetic Alcohols Methanol 3.97 5.70 4.17 5.98
Propanol 3.71 2.27 3.90 2.39
Isobutanol 1.46 0.76 1.53 0.79
Butanol 0.11 0.38 0.12 0.40
Isoamyl Alcohol 1.45 6.31 1.53 6.62
Pentanol 0.57 0.76 0.60 0.80
4-Methyl-1-pentanol 0.37 0.42 0.39 0.44 3-Methyl-1-pentanol 0.35 0.40 0.37 0.42
Hexanol 0.26 0.16 0.27 0.17
3-Ethoxy-1-propanol 0.62 0.34 0.65 0.36 1-Octen-3-ol 0.52 0.23 0.55 0.24 2-Phenyl Ethanol 0.94 0.53 0.99 0.55
Carbonyl compounds Acetaldehyde 2.32 2.73 2.43 2.86
Diacetyl 0.59 0.67 0.62 0.70
Ethyl-3-hydroxybutanoate 0.45 0.69 0.47 0.72 Ethyl Isovalerate 0.49 1.00 0.52 1.05
Ethyl-2-methylbutyrate 0.50 1.37 0.52 1.43 Ethyl Lactate 0.89 2.37 0.94 2.48
Ethyl-2-methylpropanoate 0.85 0.63 0.89 0.66 Ethyl Hexanoate 0.25 0.17 0.26 0.18
5-Methyl-2-furfural 0.44 0.18 0.52 0.19
Guaiacol 0.23 0.39 0.24 0.41
Whiskey Lactone 0.14 0.34 0.15 0.36
Figure 1. Calibration curve for acetaldehyde
Figure 2. Calibration curve for Methanol
Figure 3. Calibration curve for ethyl acetate
y = 0.0630x + 0.0171
Figure 4. Calibration curve for ethyl propionate
Figure 5. Calibration curve for ethyl-2-methylpropanoate
Figure 6. Calibration curve for diacetyl
y = 0.5563x ‐ 0.2014
Figure 7. Calibration curve for propanol
Figure 8. Calibration curve for ethyl butyrate
Figure 9. Calibration curve for ethyl-2-methylbutyrate y = 0.7793x + 0.3907
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01
Normalized area
Figure 10. Calibration curve for ethyl isovalerate
Figure 11. Calibration curve for ethyl isobutanol
Figure 12. Calibration curve for isoamyl acetate
y = 0.7117x ‐ 0.3788
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01
Normalized area
Concentration mg/L
Figure 13 Calibration curve for limonene
Figure 14. Calibration curve for butanol
Figure 15. Calibration curve for isoamyl alcohol
y = 0.1251x ‐ 0.0022
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01
Normalized area
Figure 16. Calibration curve for ethyl hexanoate
Figure 17. Calibration curve for pentanol
Figure 18. Calibration curve for hexyl acetate
y = 0.7047x ‐ 0.0835
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01 3.00E+01 3.50E+01
Normalized area
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01
Normalized area
Concentration mg/L
Figure 19. Calibration curve for acetoin
Figure 20. Calibration curve for 3-methyl-1-pentanol
Figure 21. Calibration curve for 4-methyl-1-pentanol
y = 0.4649x + 0.0976
Figure 22. Calibration curve for ethyl lactate
Figure 23. Calibration curve for hexanol
Figure 24. Calibration curve for 3-ethoxy-1-propanol y = 0.4613x + 0.852
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01 3.00E+01 3.50E+01
Normalized area
Figure 25. Calibration curve for fenchone
Figure 26. Calibration curve for linalyl acetate
Figure 27. Calibration curve for ethyl octanoate
y = 0.2581x + 0.0001
Figure 28. Calibration curve for 1-octen-3-ol
Figure 29. Calibration curve for acetic acid
Figure 30. Calibration curve for linalool oxide
y = 0.8512x + 0.0188
Figure 31. Calibration curve for ethyl-3-hydroxybutanoate
Figure 32. Calibration curve for propionic acid
Figure 33. Calibration curve for isobutyric acid
y = 0.5442x + 0.186
0.00E+00 2.00E+01 4.00E+01 6.00E+01 8.00E+01 1.00E+02 1.20E+02
Normalized area
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01
Normalized area
Concentration mg/L
Figure 34. Calibration curve for linalool
Figure 35. Calibration curve for furfural
Figure 36. Calibration curve for 5-methyl-2-furfural
y = 0.1876x + 0.0016
Figure 37. Calibration curve for α-terpeniol
Figure 38. Calibration curve for butyric acid
Figure 39. Calibration curve for ethyl decanoate
y = 0.3827x + 0.0002
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01
Normalized area
Figure 40. Calibration curve for citronellol
Figure 41. Calibration curve for isovaleric acid
Figure 42. Calibration curve for diethyl succinate
y = 0.1926x + 0.0003
0.00E+00 1.00E+01 2.00E+01 3.00E+01 4.00E+01 5.00E+01
Normalized area
Figure 43. Calibration curve for valeric acid
Figure 44. Calibration curve for furfuryl alcohol
Figure 45. Calibration curve for guaiacol
y = 0.6395x ‐ 0.015
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01
Normalized area
Figure 46. Calibration curve for geraniol
Figure 47. Calibration curve for ethyl phenyl acetate
Figure 48. Calibration curve for nerol
y = 0.2286x + 0.0003
Figure 49. Calibration curve for 2-phenylethyl acetate
Figure 50. Calibration curve for hexanoic acid
Figure 51. Calibration curve for α-ionone
y = 0.937x ‐ 0.0643
0.00E+00 5.00E+00 1.00E+01 1.50E+01 2.00E+01 2.50E+01 3.00E+01 3.50E+01
Normalized area
Figure 52. Calibration curve for whiskey lactone
Figure 53. Calibration curve for 2-phenyl ethanol
Figure 54. Calibration curve for β-ionone
y = 1.120x ‐ 0.02311
Figure 55. Calibration curve for octanoic acid
Figure 56. Calibration curve for decanoic acid
Figure 57. Calibration curve for β-farnesol
y = 0.8222x ‐ 0.1122
0.00E+00 1.00E+01 2.00E+01 3.00E+01 4.00E+01 5.00E+01 6.00E+01
Normalized area
CONCLUSION:
The parameters evaluated are within the acceptable limits for food analysis, as per the United States Food and Drug Administration (2001).
Please find below a list of the parameters used for method validation and a short description of each