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
–
the absolute amount of PAH that was extracted
from the sample
A
PAH
–
the area of PAH peak of the sample
A
[IS]S
–
the area of the internal standard peak of the sample
X
[IS]
–
the 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 –
the concentration of PAH in the sample (ng/g)
m –
the sample weight in g
11. Interpretation of Results
The analytical data generated in the method requires careful
interpretation to collect convincing evidence of aliphatic
hydrocarbon contamination of oysters originating from an
actual crude oil sample from Gulf of Mexico and consequent
PAH contamination. The method provides a hydrocarbon
profile and PAH profile which can be matched against that
of crude oil sample from the Gulf of Mexico. The compo-
sition of crude oil from the Gulf of Mexico is given in
Table 4 indicating relatively high levels of n-hexadecane,
n-heptadecane and pristane which are characteristic.
Characteristic pristane/C-17 ratio (0.7) phytane/C-18 ratio
(0.35) were observed. The relative amounts of any combi-
nation of individual aliphatic hydrocarbons can be measured
and matched against the crude oil sample from the Gulf of
Mexico composition. As illustrated in Figure 4 which shows
both direct analysis of crude oil from the Gulf of Mexico
as well as analysis after cleanup from oysters. However,
it should be noted that the composition of the oil changes
with time and the uptake by oysters eventually may have
a different profile from the crude oil. The composition of
other samples of crude oils is illustrated in Figure 5 again
indicating differences in profile.
Similarly the pattern of PAHs found in crude oil is
very characteristic as shown in Table 4 with levels of Ant,
Phe, Flu and Chr being 100 times higher than levels of
B(a)P. Subject to satisfactorily meeting requirements for
identification of PAHs, the method gives semi-quantitative
values for the higher mass PAHs which can be used as a
good guide as to whether oysters samples are above or
below safety limits. Accurate results require confirmation
using a more refined cleanup procedure.
12. Method Performance
Method performance was established by separate spiking
experiments for blank oysters with firstly a mixture of
aliphatic hydrocarbon standards (NIST1494 – C10-C34
hydrocarbons) and secondly a mixture of 16 PAH standards.
To evaluate method performance with combined aliphatic
hydrocarbons and PAHs, spiking was carried out with
NIST 1582 petroleum crude oil.
12.1 Recovery
Aliphatic hydrocarbons –
The method was shown to be
unsuitable for recovery of aliphatic hydrocarbons below
n-pentadecane due to losses during concentration of the
sample extract. Average recoveries of n-hexadecane (C-16)
to n-tetratricontane (C-34) ranged from 52-108%.
PAHs –
Background contamination and lack of availability
of a real blank sample made it impossible to make an
accurate estimate of the recoveries of the lower mass PAHs
(Naph, Ace, Acy, Flu, Ant, Phe, Fln and Pyr). However
average recoveries of the remaining higher mass PAHs
[(B(a)P, Chr, B(b)F, B(k)F, B(k)F, B(a)P, B(g,h,i)P, and
D(a,h)A] ranged from 65-126%.
12.2 Specificity
Aliphatic hydrocarbons –
Full scan spectra were obtained in
each case. Identification was confirmed by close agreement
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 –
By SRM, 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 precursor to quantifier ion should
be in close agreement with those of the standards.
12.3 Limits of Detection
Aliphatic hydrocarbons –
LODs for aliphatic hydrocarbons
were estimated to be between 0.2 and 1 ng (on-column
injected) in full scan mode. For 1 µL of extract injected
into the GC-MS this is equivalent to 20-100 ng/g (ppb)
hydrocarbon contamination of the oysters.
PAHs –
Background contamination made it impossible to
make an accurate estimate of the LODs of the lower mass
PAHs (Naph, Ace, Acy, Flu, Ant, Phe, Fln and Pyr).
However, LODs of the remaining higher mass PAHs
[(B(a)P, Chr, B(b)F, B(k)F, B(k)F, B(a)P, B(g,h,i)P, and
D(a,h)A] were estimated to be between 0.01 and 0.07 ng
(on-column injected) in SRM mode. For 1 µL of extract
injected into the GC-MS/MS this is equivalent to 1-7 ng/g
(ppb) PAH and oil contamination of oysters.
12.4 Accuracy
The accuracy for measurement of PAHs was determined
by spiking NIST crude oil standard into oysters and
following the full extraction and cleanup procedure.
Background contamination made it impossible to make
an accurate estimate of the recoveries of the lower mass
PAHs (Naph, Ace, Acy, Flu, Ant, Phe, Fln and Pyr).
However average recoveries of (B(a)A, B(a)P, B(g,h,i)P,
and I(1,2,3-c,d)P were 124, 92, 81 and 86 % respectively
as shown in Table 3. Bearing in mind that the method is
intended as a semi-quantitative screen this accuracy was
deemed to be satisfactory.
