10.2 Automated SPME Analysis
10.2.1
Use the fiber coated with polydimethylsiloxane
100 µm (PDMS-100) and condition the fiber
before use by insertion into the GC injector as
recommended by the manufacturer.
10.2.2
Load the SPME autosampler with headspace vials
containing the prepared samples (up to a maximum
of 54 vials per tray).
10.2.3
Commence the SPME program which consists of
swirling the vial for 5 min at 50 °C, then inserting
the fiber into the head-space for 40 min at 50 °C
as the solution is swirled again, then transferring
the fiber to the injector for desorption at 250 °C
for 5 min. At the end of the program, the fiber is
transferred to the second injector (instead of the
conditioning station) for cleaning and conditioning
at 250 °C for 5 min.
10.3 GC Analysis
GC analysis is performed on a TRACE GC Ultra
™
system
with automated SPME system (Thermo Fisher Scientific,
Austin, TX USA). The GC conditions were as follows:
Column: TraceGOLD TG-5MS 5% diphenyl and
95% dimethyl polysiloxane stationary phase
(30 m, 0.25 mm ID, 0.25 µm film thickness)
Injection mode: splitless
Injection port temperature: 250 °C
Left carrier flow: 1.2 mL/min
Split flow: 50 mL/min
Splitless time: 3 min
Conditioning injector temperature: 250 °C
Right carrier flow: 0.1 mL/min
Transfer line temperature: 250 °C
Oven temperature: 60 °C hold for 1 min; to 120 °C with
15 °C/min; hold for 2 min; to 225 °C
with 30 °C/min; hold for 1 min; to
280 °C at 30 °C/min, hold for 10 min
10.4 Tandem MS/MS Detection
MS analysis is carried out using a TSQ Quantum XLS triple
quadrupole mass spectrometer (Thermo Fisher Scientific,
Austin, TX USA).
Ionization mode: electron impact (EI) positive ion at
70 eV ionization energy
Emission current: 30 µA
Ion source temperature: 250 °C
Scan type: selected reaction monitoring (SRM)
Cycle time: 0.1 s
Peak width: Q1/Q3 the full width of a peak at half its
maximum height (FWHM) of 0.70 Da
Collision gas (Ar) pressure: 1.0 mTorr
The parameters for selected reaction monitoring (SRM)
analysis for targeted compounds and internal standards
are displayed in the Table 3.
11. Calculations of Results
11.1 Identification
It is confirmed by the presence of transition ions (quantifier
and qualifier) at retention times (±0.05%) to the corre-
sponding standards. In multiple reaction monitoring
(MRM) mode the measured peak area ratios for qualifier
to quantifier ion should be in close agreement (±20%)
with those of the standards as shown in Table 3. The
quantifier and qualifier ion were selected among the
product ions produced by the fragmentation of the
selected parent ion on the basis of the intensity.
11.2 Quantification
It employs internal standardization using peak area ratios
for standards in matched matrices. Dicyclohexylmethanol is
used as the internal standard for the six flavor compounds
(thujone, menthofuran, estragole, pulegone, methyl eugenol
and
β
-asarone), and coumarin-d
4
is used as internal standard
for coumarin. Plot the calibration curves as the relative
peak areas (analyte versus the corresponding internal
standard) as a function of the compound concentration.
The flavoring concentration (c
f
) in the samples is determined
from the equation:
c
Fl
=
(
A
Fl
)
– b /a
A
IS
where,
c
Fl
– flavoring concentration in mg/kg
A
Fl
– peak area of the flavoring
A
IS
– peak area of internal standard
b
– the y-intercept
a
– the slope of calibration curve
Samples initially found to contain levels of flavoring
substances outside the linear range need to be appropriately
diluted, and the dilution factor taken into account in the
final calculations.
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