11. Calculation of Results
11.1 Aliphatic Hydrocarbons
Any detectable aliphatic hydrocarbon peaks in Fish can be
identified based on their retention times which are given in
Table 4. This is illustrated in Figure 4. Measure the specific
peak area ratios to characterize the source of hydrocarbon
contamination.
11.2 Identification of PAHs
The occurrence of one or more of any of the 16 PAHs of
food safety concern is indicated by the presence of transi-
tion 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.
11.3 Quantification of PAHs
Calibration by internal standardization is applied for the
quantification of PAHs. This calibration requires the
determination of response factors Rf defined by the equa-
tion below. Calibration by the internal standardization is
applied for the quantification of PAHs. This calibration
requires the determination of response factors Rf defined
by the equation below.
Calculation of the response factor:
R
f
=
A
St
×
c
[IS]
A
[IS]
×
c
St
R
f
–
response factor determined by the analysis of stan-
dards PAH and internal standard
A
St
–
area of the PAH peak in the calibration standard
A
[IS]
–
area of the internal standard peak for the calibra-
tion standard
c
St
–
PAH concentration for the calibration standard solution
c
[IS]
–
internal standard concentration for the calibration
standard solution
Calculations for each sample the absolute amount of PAH that
was extracted from the sample:
X
PAH
=
A
PAH
×
X
[IS]
A
[IS]S
×
R
f
X
PAH
–
absolute amount of PAH that was extracted from
the sample
A
PAH
–
area of PAH peak of the sample
A
[IS]S
–
area of the internal standard peak of the sample
X
[IS]
–
absolute amount of internal standard added to the
sample
The concentration of PAH in the sample (ng/g):
c (ng/g) =
X
PAH
m
c –
concentration of PAH in the sample (ng/g)
m –
sample weight in g
12. Interpretation of Results
The analytical data generated in the method requires careful
interpretation to collect convincing evidence of aliphatic
hydrocarbon contamination of fish originating from actual
crude oil sample from Gulf of Mexico and consequent
PAH contamination. The method provides a hydrocarbon
profile and quantification of PAHs which can be matched
against that of crude oil sample from Gulf of Mexico.
Although the method provides a PAH profile and simulta-
neous screening of aliphatic hydrocarbons, it should be
noted that the composition of any crude oil contamination
may change with time through biodegradation and there
may be preferential uptake by fish of individual PAHs and
aliphatic hydrocarbon eventually giving a different profile
in fish from that of the crude oil.
Subject to satisfactorily meeting the requirements for
identification of PAHs the method can be used to quantify
levels of PAHs in fish.
13. Method Performance
The method performance was established by spiking
experiments with blank oily fish with a mixture of 16 PAH
standards The method accuracy was demonstrated first by
analysis of surplus proficiency test material samples
(FAPAS smoked fish - T0642) with defined PAH values
and second by using NIST 1582 petroleum crude oil
containing certified levels of PAHs.
13.1 Recovery
Aliphatic hydrocarbons –
The method was shown to be
unsuitable for recovery of aliphatic hydrocarbons below
n
-hexadecane due to losses during concentration of the
sample extract.
PAHs –
Average recoveries of the 16 PAHs of food safety
significance ranged from 58-113%
13.2 Specificity
Aliphatic hydrocarbons –
Full scan spectra were obtained
in each case. Identification was confirmed by close agree-
ment of retention times for standards and comparison
with scanned spectra, particularly checking for evidence of
interferences. Extracted ion chromatograms using
m/z
57
were used for profiling but additional ions characteristic
of aliphatic hydrocarbons (e.g.
m/z
71) can be used as an
additional check of specificity.
PAHs –
Using Selected Reaction Monitoring (SRM) the
specificity was confirmed based on the presence of transition
ions (quantifier and qualifier) at the correct retention times
corresponding to those of the respective PAH standards.
Furthermore, the measured peak area ratios of
qualifier/quantifier ion were in close agreement with the
ion ratios of the standards as indicated in Table 1.
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