Quantitation of Linalool in Beer
J. Agric. Food Chem., Vol. 51, No. 24, 2003 7101
applied onto a glass column (20 cm × 2 cm) filled with silica. Impurities
were removed by elution with 200 mL of n-pentane followed by 70
mL of diethyl ether. [2H2]Linalool was isolated using another 130 mL
of diethyl ether as the eluent. The etheral solution was dried over
anhydrous sodium sulfate. The concentration of the target compound
was then determined by GC/flame ionization detection using methyl
octanoate as the internal standard. To consider the different detector
responses, a correction factor was determined from a mixture of known
amounts of unlabeled linalool and methyl octanoate under the same
experimental conditions; yield, 76%.
High-Resolution GC/MS (HRGC/MS). Mass spectra of linalool
and [2H2]linalool were recorded by means of a GC/MS system
consisting of a gas chromatograph 5890 series II (Hewlett-Packard,
Waldbronn, Germany) connected to a sector field mass spectrometer
type MAT 95 S (Finnigan, Bremen, Germany). MS/EI (electron
ionization) spectra were recorded at 70 eV ionization energy, and MS/
CI (chemical ionization) spectra were recorded at 115 eV ionization
energy using isobutane as the reactant gas.
volume, and headspace volume, and is also affected by aging
of the fiber and by the concentration of other, predominating
constituents present in the sample (12). Therefore, when external
calibration is used in SPME, extraction conditions must be kept
as reproducible as possible. Moreover, it has to be taken into
account that equilibrium concentrations can also drift with
increasing age of the fiber due to a loss of adsorption capacity.
Furthermore, temperature and agitation do influence the time
until equilibrium is achieved. For better reproducibility, exposure
times have, therefore, to be increased until equilibrium. How-
ever, time-consuming records of saturation curves for each
analyte have then to be measured. Another problem is that major
constituents of the sample can significantly influence the amount
of the analyte adsorbed at the fiber. These drawbacks can be
reduced, when internal calibration is used, but the results are
very much dependent on the structure of the internal standard
used.
Quantitation by Two-Dimensional HRGC/SIDA Using SPME
Isolation (Method I). Beer samples (0.5-10 mL) were placed in
headspace vials (20 mL) equipped with a stir bar and topped up to 10
mL with tap water, if necessary. An aliquot (50-100 µL) of an ethanolic
solution of [2H2]linalool (c ) 2.38 µg/mL) was added by subsurface
pipetting. After short agitation, sodium chloride (4 g) was added and
the vials were capped. After 30 min with continuous stirring, the vials
were placed into the tray of a Combi PAL autosampler (CTC Analytics,
Zwingen, Switzerland) held at 20 °C. Extraction was performed using
65 µm PDMS/DVB fibers (Supelco, Sigma-Aldrich Chemie). Com-
pounds were desorbed during 1 min into the hot injector (PPKD injector,
Thermo Finnigan, Egelsbach, Germany) of a GC (Trace GC, 2000
Series, Thermo Finnigan) and transferred onto the column in the first
dimension (DB-FFAP, WCOT Fused Silica, 30 m × 0.32 mm internal
diameter, 0.25 µm; J&W Scientific, Agilent Technologies, Waldbronn,
Germany) held at 40 °C. After 2 min, the volatiles were desorbed and
separated using a temperature gradient of 6 °C/min. At the elution time
of linalool/[2H2]linalool (∼13 min), the effluent was quantitatively
transferred to a cold trap (SGE, Darmstadt, Germany) using a moving
column stream switching system (Thermo Finnigan). After the cooling
was turned off, the trapped material was further separated using either
a DB-1701 column (WCOT Fused Silica, 30 m × 0.32 mm internal
diameter, 0.25 µm; J&W Scientific, Agilent Technologies) or a chiral
BGB-176 column (BGB Analytik, Adliswil, Switzerland) installed in
the second GC oven (CP 3800, Varian, Darmstadt, Germany). Separa-
tion on the DB-1701 started at 40 °C and was further performed at 6
°C/min, whereas for the chiral column the oven was heated from 40 to
100 °C at 10 °C/min and then to 140 °C using a gradient of 2 °C/min.
The effluent was monitored using an ion trap mass spectrometer (Saturn
2000, Varian) running in the CI mode with methanol as the reactant
gas. Elution times of linalool/[2H2]linalool were ∼13 min on column
DB-1701 and ∼19 min on column BGB-176.
It has been proven in the quantitation of many trace aroma
compounds that the most appropriate internal standard is the
isotopically labeled analogue of the analyte. In combination with
GC/MS, the analyte and internal standard can easily be differ-
entiated according to their different molecular weights (13, 14).
Combining the precision of stable isotope dilution assay (SI-
DA) with the speed of SPME isolation should, therefore, be a
very useful tool in quantitation of trace odorants. The first paper
using this method was on the analysis of caffeine in bever-
ages (15), but applications in flavor research are still rare to
date (16, 17). The purpose of the present study was, therefore,
first to develop a SIDA for the direct on-line quantitation of
both linalool enantiomers, second to compare the data obtained
by two enrichment techniques, namely, distillation/extraction
and SPME, and third to apply the SPME method to different
beers.
MATERIALS AND METHODS
Chemicals. The following chemicals were obtained from commercial
sources: ethynylmagnesium bromide (0.5 M solution in tetrahydro-
furane), (()-linalool, and 2-methyl-2-hepten-6-one were from Aldrich,
Sigma-Aldrich Chemie (Taufkirchen, Germany). Pure (R)-linalool was
purchased from Fluka, Sigma-Aldrich Chemie. Silica for flash chro-
matography (J. T. Baker, Phillipsburg, NJ) was used in column
chromatography. Lindlar catalyst (5% palladium) and methyl octanoate
were from Merck (Darmstadt, Germany) and deuterium gas (99.7%
purity) was from Messer Griesheim (Krefeld, Germany). Diethyl ether
was freshly distilled, and traces of water were removed by addition of
sodium hydride. The supernatant was decanted and used immediately.
Syntheses. 1,2-Dehydrolinalool (3,7-Dimethyl-6-octen-1-yn-3-ol). To
a solution of 2-methyl-2-hepten-6-one (2.016 g) in diethyl ether (50
mL), ethynylmagnesium bromide (40 mL of a 0.5 M solution in
tetrahydrofurane) was added, and the reaction mixture was stirred
overnight. With continuous stirring, crushed ice (50 g) was then added
and the precipitate formed was dissolved by adding a saturated solution
of ammonium chloride in water (100 mL). The organic layer was
removed, and the aqueous phase was extracted twice with diethyl ether
(total volume, 100 mL). The combined organic phases were washed
with aqueous saturated sodium hydrogensulfite (50 mL), followed by
aqueous sodium bicarbonate (0.5 mol/L; 50 mL) and finally tap water
(20 mL). The organic phase was dried over sodium sulfate. Removal
of the solvent yielded 1,2-dehydrolinalool in 84% yield. The intermedi-
ate was used for deuteration without further characterization.
Linalool concentrations were calculated from the area counts obtained
from the mass chromatograms using the following equation:
m
[2H2]linalool
Alinalool
Clinalool ) d × rf ×
×
V
A
[2H2]linalool
where C ) concentration, V ) volume of the beer analyzed, d )
dilution factor, Alinalool ) area counts for linalool, rf ) response factor,
2
2
2
A[ H ]linalool ) area counts for [ H2]linalool, and m[ H ]linalool ) amount
2
2
of [2H2]linalool added.
Quantitation by Two-Dimensional HRGC/SIDA Using Solvent
Extraction/Distillation for Volatile Isolation (Method II). The beer
sample (2-100 mL) was placed in an Erlenmeyer flask and made up
to 100 mL with tap water, if necessary. An aliquot (500 µL) of an
ethanolic solution of [2H2]linalool (c ) 2.38 µg/mL) was added, and
the mixture was stirred for 1 h. The samples were extracted three times
with diethyl ether (200 mL total volume), and the combined organic
phases were dried over anhydrous sodium sulfate. Nonvolatile com-
pounds were removed by SAFE distillation (10) at 40 °C. The distillates
were concentrated to 200 µL using a Vigreux column, and aliquots
were analyzed by HRGC/SIDA as described above.
[2H2]Linalool ([1,2-2H2]-3,7-Dimethyl-1,6-octadien-3-ol). 1,2-De-
hydrolinalool (1.5 g) and quinoline (200 mg) were dissolved in
n-heptane (20 mL) and, after addition of Lindlar catalyst (50 mg),
deuterated in an autoclave at 500 kPa for 70 min. The mixture was
washed with sulfuric acid (0.25 mol/L; 50 mL) followed by aqueous
sodium bicarbonate (0.5 mol/L; 50 mL), saturated sodium chloride
solution (50 mL), and water (20 mL) and finally dried over sodium
sulfate. For purification, the solvent was removed and the residue was