GC-MS Determines Hydrocarbons in Oysters Klaus Mittendorf, Laszlo Hollosi, Ebru Ates, Katerina Bousova, Thermo Fisher Scientific Food Safety Response Center, Dreieich, Germany; Eric Phillips, Hans-Joachim Huebschmann, Thermo Fisher Scientific, Austin, TX; James Chang, Thermo Fisher Scientific, San Jose, CA
Table 1: Parameters for SRM analysis of PAHs grouped according to Figure 1. Click to enlarge. | Following the recent oil spill in the Gulf of Mexico, concerns have been raised over the potential contamination of seafood with polycyclic aromatic hydrocarbons (PAHs) and aliphatic hydrocarbons. In response, a new method has been developed which can be applied to oysters to detect the presence of aliphatic hydrocarbons and PAH contamination from crude oil found in the Gulf of Mexico. Using gas chromatography coupled with tandem mass spectrometry (GC-MS/MS), this method can be used to characterize the source of contamination. Using the new method, analysts can achieve a semi-quantitative indication of whether levels of PAHs exceed safety limits for human consumption of oysters, which is essential for ensuring consumer safety.
The method uses a liquid extraction of oysters with hexane, followed by clean-up on a silica-SPE cartridge. The sample is fortified with appropriate labeled internal standards and analyzed by simultaneous GC-MS/MS using a triple quadrupole mass spectrometer system (Thermo Scientific TSQ Quantum XLS). Aliphatic hydrocarbons and PAHs of food safety significance are measured and compared with the profile from crude oil collected from the Gulf of Mexico in late May 2010.
Method
The calibration standards were obtained for sixteen PAHs. NIST injection standards were also used.
Sample Preparation
Glassware was rinsed with acetone before proceeding with the method to avoid cross contamination. A suitable amount (e.g. 250 g) of oyster meat was homogenized to give a slurry using a high speed blender (ULTRA-TURRAX, IKA).
Extraction
Table 2: Aliphatic hydrocarbons monitored in oysters spiked with NIST 1494. Click to enlarge. | The homogenized sample was accurately weighed (ca. 2 g) into a glass tube. 50 μL of PAH internal standard solution was added to the sample. The mixture was vortexed for 10 seconds and equilibration followed for approximately 10 minutes. 5 mL of hexane was added to the sample, which was put into an ultrasonic bath for 10 minutes. The supernatant hexane layer was transferred into a 50-mL round flask with a Pasteur pipette. The extraction was repeated three more times. The extracted sample was centrifuged for five minutes at 4500 rpm and 5 C and the supernatant was decanted. The sample was then evaporated to 1 mL under vacuum (220 mbar/50 C).
Clean-up
The SPE cartridge was conditioned with 3 mL of hexane before the extract was applied and eluted into an evaporator tube with 5 mL of hexane. This was evaporated at 40 C to dryness using a blow-down apparatus under a gentle stream of nitrogen and reconstituted in 180 μL of cyclohexane plus 20 μL of injection standard.
Analysis
GC analysis was performed on a Thermo Scientific TRACE GC Ultra system. 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
MS analysis was carried out a TSQ Quantum XLS triple quadrupole mass spectrometer. 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
Figure 1: Chromatogram of oyster sample spiked with aliphatic hydrocarbons plus 10 ng/g PAH mixture. Top chromatogram shows m/z 57 for hydrocarbon profiling, while lower chromatograms are SRM traces for 16 individual PAHs. Retention times for the 16 PAHs found in Table 1. Click to enlarge. | The mass spectrometer was programmed to simultaneously monitor the hydrocarbon profile in scanning full scan (FS) GC-MS as well as quantifying the presence of PAHs by MS/MS within a single chromatographic run. Eight segments were programmed each with two simultaneous scan events. One scan event was used to monitor the aliphatic hydrocarbon profile throughout the whole chromatographic run (i.e. in all segments), while selective reaction monitoring (SRM) traces were set up for the target PAHs in the other scan event. The program of segments for SRM events (#1) is shown in Table 1. The setting of scan event #2 for hydrocarbon profiling was kept constant in all segments.
Calculation of Results
From the scanned GC-MS data, a reconstructed ion chromatogram (extracted ion chromatogram) was printed for m/z 57 and plotted alongside a similar m/z 57 extracted chromatogram 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. The specific peak area ratios were measured to characterize the source of hydrocarbon contamination.
The occurrence of one or more of any of the 16 PAHs of food safety concern was 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 Rf defined by the equation below.
Calculation of the response factor:
Figure 2: Hydrocarbon profile of crude oil sample taken from the Gulf of Mexico in late May 2010 by direct analysis (top) and after 5 mg/kg spiking into oyster sample (bottom) showing m/z 57. Click to enlarge. | Rf =
ASt × c[IS]
A[IS] × cSt
Rf – the response factor determined by the analysis of standards PAH and internal standard
ASt – the area of the PAH peak in the calibration standard
A[IS] – the area of the internal standard peak for the calibration standard
cSt – PAH concentration for the calibration standard solution
c[IS] – the internal standard concentration for the calibration standard solution
Calculations for each sample the absolute amount of PAH that was extracted from the sample:
XPAH = APAH × X[IS]
A[IS]S × Rf
Figure 3: Comparison of hydrocarbon distribution of different type of oils showing m/z 57. Top: NIST1582 petroleum crude oil, middle: crude oil sample taken from the Gulf of Mexico in late May 2010, at the bottom: NIST1494 hydrocarbon standard. Click to enlarge. | XPAH – the absolute amount of PAH that was extracted from the sample
APAH – 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) =
XPAH
m
c – the concentration of PAH in the sample (ng/g)
m – the sample weight in g
Results and Discussion
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 and consequent PAH contamination. This method provides a hydrocarbon profile and PAH profile which can be matched against that of crude oil sample from the Gulf of Mexico. The composition of crude oil from the sample 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 combination of individual aliphatic hydrocarbons were measured and matched against the crude oil sample from the composition. Figure 2 shows both direct analysis of crude oil from the Gulf of Mexico and 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 3, 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 guide as to whether oyster samples are above or below safety limits. Accurate results require confirmation using a more refined cleanup procedure.
Method Performance
Table 4: Composition of crude oil from Gulf of Mexico. Characteristic pristane/C-17 ratio (0.7) phytane/C-18 ratio (0.35) were observed. Click to enlarge. | 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.
Recovery
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%. 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%.
Specificity
Full scan spectra for aliphatic hydrocarbons 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.
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.
Limits of Detection
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. 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, (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.
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. Given this assessment, the new method is a valuable tool in equipping food safety scientists to ensure that consumers are safe from the potential threat of oil contamination in oysters originating from the Gulf of Mexico.
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