Food Chemistry
Short communication
Microwave-assisted deuterium exchange: The convenient preparation
of isotopically labelled analogues for stable isotope dilution analysis
of volatile wine phenols
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Anna M. Crump, Mark A. Sefton, Kerry L. Wilkinson
The University of Adelaide, School of Agriculture, Food and Wine, PMB 1, Glen Osmond, SA 5064, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
This study reports the convenient, low cost, one-step synthesis of labelled analogues of six volatile
phenols, guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol, eugenol and vanillin, using micro-
wave-assisted deuterium exchange, for use as internal standards for stable isotope dilution analysis.
The current method improves on previous strategies in that it enables incorporation of deuterium atoms
on the aromatic ring, thereby ensuring retention of the isotope label during mass spectrometry fragmen-
tation. When used as standards for SIDA, these labelled volatile phenols will improve the accuracy and
reproducibility of quantitative food and beverage analysis.
Received 18 November 2013
Accepted 12 April 2014
Available online 24 April 2014
Keywords:
Volatile phenols
Microwave-assisted deuterium exchange
Isotope labelling
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2010); several volatile phenols, including guaiacol and eugenol,
have been identified as glycosidically bound constituents of
Gas chromatography–mass spectrometry (GC–MS) is a powerful
technique for the compositional characterisation of complex sam-
ples, for example the qualitative and quantitative analysis of volatile
compounds in food and beverages. Quantitative analysis typically
involves the use of internal standards to compensate for analyte
losses during sample preparation, due to evaporation or degrada-
tion. Ideally, the internal standard is a compound as similar as pos-
sible to the analyte of interest, but not already present in the sample
matrix. Stable isotope labelled compounds make excellent internal
standards (Vining, Smythe, & Long, 1981), since they possess physi-
cal and chemical properties that are virtually identical to those of the
analyte. Analytical methods using stable isotope dilution analysis
(SIDA) therefore enable accurate and reproducible quantification.
However, labelled standards are not always commercially available.
While traditional organic synthesis can be used to prepare labelled
standards via the incorporation of one or more deuterium atoms,
these methods can be complex, technically challenging, time
consuming, laborious and expensive (Vining et al., 1981);
particularly for the preparation of highly deuterated standards.
Volatile phenols have been reported as constituents of various
foods and beverages. For example, eugenol is responsible for the
distinctive aroma of cloves and also contributes to the aroma of
cinnamon (Tikunov, de Vos, González Paramás, Hall, & Bovy,
tomato and strawberry (Tikunov et al., 2010; Ubeda et al., 2012);
while vanillin, guaiacol, 4-methylguaiacol and eugenol are
oak-derived volatiles present in wine and spirits matured in oak
barrels (Pino, Tolle, Gök, & Winterhalter, 2012; Spillman, Sefton,
& Gawel, 2004). Volatile phenols have also been implicated in a
range of off-odours and taints. Guaiacol was identified as the
microbial metabolite responsible for a smoky/phenolic taint in
chocolate milk (Jensen, Varelis, & Whifield, 2001), while 4-ethyl-
phenol and 4-ethylguaiacol were found to cause off-odours in
strawberries infected with leather rot (Jelen´ , Krawczyk, Larsen,
Jarosz, & Golebniak, 2005). In wine, these last two compounds
are associated with the ‘horsy’, ‘medicinal’, ‘smoky’, ‘barnyard’
attributes indicative of Brettanomyces/Dekkera spoilage (Boidron
et al., 1988; Chatonnet et al., 1992). A range of volatile phenols,
including guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphe-
nol and o-, m- and p-cresols, have recently been identified in
grapes and wine tainted by bushfire smoke (Kennison, Gibberd,
Pollnitz, & Wilkinson, 2008; Parker et al., 2012).
The preparation of d3-labelled analogues of guaiacol, 4-meth-
ylguaiacol and vanillin have been previously reported, via syn-
thetic pathways involving methylation with deuterated methyl
iodide (Pollnitz, Pardon, Sykes, & Sefton, 2004; Spillman, Pollnitz,
Liacopoulos, Skouroumounis, & Sefton, 1997). However, the iso-
tope label is readily lost from these analogues upon cleavage of
the deuterated methoxy functional group, during mass spectrome-
try fragmentation, limiting the selection of suitable fragments for
selective ion monitoring of the standard. Incorporation of
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Corresponding author. Tel.: +61 8 8313 7360; fax: +61 8 8313 7116.
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Crump, Anna M.
