9.3 Clean-up
9.3.1
Condition the SPE-Cartridge with 3 mL of hexane.
9.3.2
Apply the extract to the cartridge and elute into
an evaporator tube with 5 mL of hexane.
9.3.3
Evaporate at 40 °C to dryness using a blow-down
apparatus under a gentle stream of nitrogen.
9.3.4
Reconstitute in 180 µL of cyclohexane plus 20 µL
of injection standard.
9.4 Analysis
9.4.1
GC operating conditions
GC analysis was performed on a Thermo Scientific
TRACE GC Ultra system (Thermo Fisher Scientific,
Waltham, MA USA). The GC conditions were as follows:
Column: Thermo TR-50MS 30 m, I.D.: 0.25 mm, 0.25 µm
film capillary column
Injection mode: splitless with a 5 mm injection port liner
Injection port temperature: 270 °C
Flow rate: 1.2 mL/min
Split flow: “On”, flow: 25 mL/min
Splitless time: 1 min
SSL carrier method mode: constant flow
Initial value: “On” with 1.2 mL/min
Initial time: 1 min
Gas saver flow: 15 mL/min
Gas saver time: 3 min
Vacuum compensation: “On”
Transfer line temperature: 270 °C
Oven Temperature: 60 °C for 1 min, then programmed at
12 °C/min to 210 °C, then 8 °C/min
to 340 °C with 5 min hold time
9.4.2 Mass Spectrometric Conditions
MS analysis is carried out using a TSQ Quantum XLS triple
quadrupole mass spectrometer (Thermo Fisher Scientific,
Waltham, MA USA). A satisfactory tune of the mass
spectrometer is achieved when the detector is set at
m/z
300
or less and the three FC 43 (calibration gas) ions (68, 219,
and 502) are at least half the height of their respective
windows and the ions at 502 and 503 are resolved.
The MS conditions for PAHs are as follows:
Ionization mode: EI positive ion
Ion volume: closed EI
Emission current: 50 uA
Ion source temperature: 250 °C
Scan type: Full scan in range
m/z
45-650 and SRM
Scan width: 0.15 for SRM
Scan time 0.2 s for full scan and 0.05 for SRM
Peak width: Q1, 0.7 Da; Q3, 0.7 Da FWHM
Collision gas (Ar) pressure: 0.5 mTorr
The mass spectrometer is programmed to be able
to simultaneously monitor the hydrocarbon profile in
scanning Full Scan (FS) GC-MS
and
quantify the presence
of PAHs by MS/MS within a single chromatographic run.
Eight segments are programmed each with 2 simultaneous
scan events. One scan event is used to monitor the aliphatic
hydrocarbon profile throughout the whole chromatographic
run (i.e in all segments), while SRM traces are set up for
the target PAHs in the other scan event. The program of
segments for SRM events (#1) is shown in Table 1.
Setting of scan event #2 for hydrocarbon profiling was
kept constant in all segments:
•
Scan type: FS in range 45-650
m/z
•
Scan time: 0.2 s
•
FWHM: 0.7 Da
•
Collision gas pressure: 0.5
10. Calculation of Results
10.1 Aliphatic Hydrocarbons
From the scanned GC-MS data, print a reconstructed ion
chromatogram (extracted ion chromatogram) for
m/z
57
and plot this alongside a similar
m/z
57 extracted chro-
matogram for the standard mixture of hydrocarbons. Any
detectable aliphatic hydrocarbon peaks in oysters can be
identified based on their retention times which are given in
Table 2. This is illustrated in Figure 1. Measure the specific
peak area ratios to characterize the source of hydrocarbon
contamination.
10.2 PAHs
The occurrence of one or more of any of the 16 PAHs of
food safety concern is indicated by the presence of transition
ions (quantifier and qualifier) as indicated in Table 1 at
retention times corresponding to those of the respective
standards shown in Table 1. This is illustrated in Figure 1.
Careful visual inspection of the SRM chromatograms
should be carried out to check for interferences. The
measured peak area ratios of precursor to quantifier ion
should be in close agreement with those of the standards
as shown in Table 1. If the presence of any of the 16 PAHs
is confirmed based on retention times and ion ratios then
quantification should be carried out as indicated below.
Calibration by the internal standardization is applied
for the quantification of PAHs. This calibration requires
the determination of response factors R
f
defined by the
equation below.
Calculation of the response factor:
R
f
=
A
St
×
c
[IS]
A
[IS]
×
c
St
R
f
–
the response factor determined by the analysis of
standards PAH and internal standard
A
St
–
the area of the PAH peak in the calibration standard
A
[IS]
–
the area of the internal standard peak for the
calibration standard
c
St
–
PAH concentration for the calibration standard
solution
c
[IS]
–
the internal standard concentration for the
calibration standard solution
