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5 20 40 60 80
Extraction time [min]
Fig. 2 Influence of extraction time to the extraction yields (n = 2); 2-AAP
solution with c = 0.275 µg/l in 15%vol ethanol; SPME fibre: 70 µm carbowax/divinylbenzene
However, for routine measurements, time is money. The
difficulty is to keep extraction time to a minimum without
sacrificing robustness. Extraction times of 5, 20, 40, 60
and 80 minutes were examined with a model solution containing
0.275 μg/l 2-AAP (see Fig. 2). The results showed
that the response of 2-AAP had reached a plateau at an
extraction time of 60 min, with an increase of 612 % over
the extraction yield at 5 minutes. Theoretically, yield rates
could be improved by about 2 % by extracting for another
20 min. However, analytical enhancement was considered
too low to invest 20 more minutes of valuable time. Therefore
60 min was considered to be the optimum extraction
time.
Addition of salts
The addition of salt to sample solutions increases the
ionic strength of water, which is called the salting-out effect
19,20) . This may lead to an increase in analyte concentration
in the gas phase which can ultimately also increase
analyte extraction by the SPME fibre. For these experiments,
sodium chloride and anhydrous sodium sulphate
were chosen. A model solution with 0.275 μg/l 2-AAP in
15 % ethanol was extracted without salt addition and
with addition of 1 g of each salt. Best results after repeat
tests were obtained for the model solution with an addition
of 1 g anhydrous dry sodium sulphate. The extraction
yields with addition of sodium sulphate were 1000 %
higher than without addition of salt and 250 % higher
than with addition of sodium chloride. A possible explanation
for the advantage of anhydrous sodium sulphate
over sodium chloride is that the dehumidifying effect of
the former increases the ionic strength of the solution and
intensifies the salting-out effect even more. Therefore an
addition of 1 g anhydrous sodium sulphate was found to
be the optimum.
Influence of pH value
According to Dollmann et al., a correlation between pH
value and extraction yield can be shown 16) . Since the model
solution of 2-AAP in 15 % vol aqueous ethanol was not
suitable for pH value experiments, a blank wine was used
for further investigations. The pH values of 4.3, 5.7, 7.2,
8.6 and 9.7 for the ready-to-measure sample solution were
compared in this study. As can be seen in Figure 3, extraction
yields changed with the hydrogen cation concentration.
Best results have been obtained with a pH value of
9.7. This pH value was achieved by an addition of 10 μl
NaOH (1.25 molar) and 0.9 ml ammonium acetate buffer
solution to 0.1 ml of the wine samples.
Stable isotope dilution analysis
Since stable isotope dilution analysis is a very accurate
method and particularly suitable for trace analysis 16) , this
method was chosen for quantification. Isotopically labelled
compounds that have nearly the same chemical and physical
properties of the analytes must be used.
For the development of a robust routine method in everyday
work, it is important to have at least two confirmation
masses for the analyte. The target ion is used for quantification
of the analyte and the qualifiers are used to rule out
interferences by checking to see if they show a fixed ratio
to the target ion. Therefore, the internal standard must be
selected and the experimental conditions have to be optimized
so that at least three different ions can be used. For
analyzing 2-AAP, the choice of the ionization mode as well
as the deuterated 2-APP standard was crucial.
Since deuterated 2-AAP was easily available, it was chosen
for the following experiments. At first, d 3 -2-AAP that was
deuterated at the acetyl group was tested as an internal
standard. The mass spectrometer was operated in the electron
ionization mode. Using this combination, only one
ion was found for each compound, m/z 135 for 2-AAP and
m/z 138 for d 3 -2-AAP. All other masses occurred in both
spectra and were not suitable as qualifier masses.
Next, the chemical ionization mode (CI) using isobutane as
a reactant gas was tried for mass spectrometry to find out
if d 3 -2-AAP could be used as an internal standard this way.
In CI mode, the two ions m/z 135 and 136 were obtained
for 2-AAP and m/z 138 and 139 were available for d 3 -2-
AAP. Apart from still insufficient confirmation, it turned
out that the m/z 136 was influenced by matrix effects. The
conclusion was that d 3 -AAP is not suitable as an internal
standard for this analytical challenge.
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4.3 5.7 7.2 8.6 9.7
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Fig. 3 Influence of pH value to extraction yields (n = 2); 2-AAP solution in
blank wine with c = 2.75 µg/l; SPME fibre: 70 µm Carbowax/Divinylbenzene;
extraction time: 60 min; addition of 1 g anhydr. NaSO 4
» November/Dezember 2008 | DLR