4. Principle
The method employs automated headspace solid-phase
micro-extraction (HS/SPME) for extraction of the targeted
compounds (biologically active flavorings) from very broad
types of matrices using a polydimethylsiloxane (PDMS)
SPME fiber. Samples are placed in the headspace vials,
fortified with labeled internal standards and water along
with sodium chloride (NaCl) is added. Headspace vials are
tightly sealed and after achieving equilibration headspace
partition, the headspace is sampled automatically and
analyzed by simultaneous GC-MS/MS using a Thermo
Scientific TSQ Quantum XLS gas chromatography triple
quadrupole mass spectrometer system.
5. Reagent List
Fisher Scientific
Part Number
5.1
Purified Water (obtained from
3125753
Thermo Scientific Barnstead
EASYpure II water system)
5.2
Sodium Chloride (extra pure)
194090010
5.3
Saccharose (extra pure)
S/8560/53
5.4
Ethanol (purity 99.9%)
E/0665DF/17
5.5
Methanol (purity 99.9%)
M/4058/17
6. Calibration Standards
6.1 Biologically Active Flavorings
6.1.1
Beta-asarone – purity 72% (Dr. Ehrenstorfer)
6.1.2
Coumarin (1,2-benzopyrone) – purity 99.5%
(Dr. Ehrenstorfer)
6.1.3
Estragole (1-Allyl-4-methoxybenzene) –
purity
≥
98.5% (Sigma-Aldrich)
6.1.4
Menthofuran – purity
≥
99% (Sigma-Aldrich)
6.1.5
Methyleugenol (4-Allyl-1,2-dimethoxybenzene) –
purity 99.5% (Sigma-Aldrich)
6.1.6
Pulegone – purity 98.8% (Sigma-Aldrich)
6.1.7
Thujone (alpha and beta) – purity
≥
99%
(Sigma-Aldrich)
6.2 Internal Standards
6.2.1
Coumarin – 5, 6, 7, 8 – D4, c =100 µg/mL in acetone
(Dr. Ehrenstorfer)
6.2.2
Dicyclohexylmethanol – purity 98% (Sigma-Aldrich)
7. Standards Preparation
7.1
Stock standard solutions of flavorings (1000 µg/mL):
Weigh 25.00 mg of the compound (recalculate the
amount regarding actual purity of the standard)
into volumetric flasks, dissolve in methanol and
dilute to 25 mL. Solutions can be stored at 4 °C
for at least three months.
7.2
Working standard solution of 7 flavorings
(1 respectively 10 µg/mL for coumarin): Transfer
25 µL of stock solution of thujone, menthofuran,
estragole, pulegone, methyl eugenol and
β
-asarone
(1000 µg/mL) and 250 µL of stock solution of
coumarin (1000 µg/mL) to a 25 mL volumetric flask
and dilute to marked volume with water. Solution
should be prepared fresh every time before using.
7.3
Stock standard solution of internal standard
dicyclohexylmethanol (1000 µg/mL): Weigh 25.00 mg
of the compound (recalculate the amount regarding
actual purity of the standard) into a volumetric flask,
dissolve in methanol and dilute to 25 mL. Solution
can be stored at 4 °C for at least three months.
7.4
Working standard solution of internal standard
dicyclohexylmethanol (10 µg/mL): Transfer 100 µL of
stock solution of dicyclohexylmethanol (1000 µg/mL)
to a 10 mL volumetric flask and dilute to marked
volume with water. Solution should be prepared
fresh every time before using.
8. Apparatus
Fisher Scientific
Part Number
8.1
High speed blender –
3565000
ULTRA-TURRAX
®
8.2
ULTRA-TURRAX –
1713300
Dispergation tool
8.3
ULTRA-TURRAX –
1024200
Plug-in coupling
8.4
Waring laboratory blender
68909
8.5
Fisher precision balance
XP-1500FR
8.6
Sartorius analytical balance
ME235S
8.7
SPME holder – TriPlus
™
SPME Kit 190.504.34
8.8
TSQ Quantum XLS
™
Triple Quadrupole
Mass Spectrometer – Thermo Fisher Scientific
(Austin, TX USA)
8.9
Thermo Scientific TRACE GC Ultra system with
automated SPME system – Thermo Fisher Scientific
(Austin, TX USA)
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