Evaluation of gas chromatography columns for the analysis of the 15 + 1 EU-priority polycyclic aromatic hydrocarbons (PAHs)
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- Gómez-Ruiz, J.Á. & Wenzl, T. Anal Bioanal Chem (2009) 393: 1697. doi:10.1007/s00216-008-2585-8
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Three different stationary phases were investigated for the analysis of the 15 + 1 EU-priority polycyclic aromatic hydrocarbons (PAHs) by gas chromatography-mass spectrometry. In addition to the most commonly used 5% phenyl methylpolysiloxane, a mid-polar phase (50% phenyl methylpolysiloxane) and a recently commercialised mid-polar to polar phase (Optima® δ-6), were evaluated. Challenging groups of PAHs in terms of separation, such as the pair dibenz[a,h]anthracene-indeno[1,2,3,-cd]pyrene and the two groups benzo[b]fluoranthene-benzo[k]fluoranthene-benzo[j]fluoranthene and cyclopenta[cd]pyrene-benz[a]anthracene-chrysene, were satisfactorily separated by using the mid-polar phase. Moreover, discrimination in terms of peak height for the heaviest PAHs (caused from the strong interaction of these compounds with the stationary phase) was reduced without compromising the resolution of the other target analytes when applying the mid-polar phase in a tailor-made column geometry (20 m × 0.18 mm internal diameter and 0.14 μm film thickness) in combination with optimised chromatographic conditions. A significant enhancement of the analytical sensitivity for dibenzopyrenes is demonstrated with an almost threefold increase of the signal-to-noise (S/N) ratio for dibenzo[a,h]pyrene, the last eluting PAH. The ability of the selected column to separate potentially interfering PAHs from the target analytes in both solvent solutions and food extracts is demonstrated.
KeywordsPolycyclic aromatic hydrocarbons (PAHs)15 + 1 EU-priority PAHsGC-MSStationary phasePeak height discriminationResolution
Polycyclic aromatic hydrocarbons (PAHs) are a large group of organic compounds containing two or more fused aromatic rings constituted of carbon and hydrogen atoms. Most PAHs in the environment derives from incomplete combustion of carbon-containing materials such as oil, wood, garbage or coal. A higher amount of PAHs is formed when materials burn at low temperatures, such as in wood fires or cigarettes. High-temperature furnaces produce a lower amount of PAHs. As some PAHs (especially the lighter ones) are, to some extent, water soluble, they can be found in rivers and ground water as well .
The main source of exposure to PAHs for non-smoking humans is food. Food may be contaminated with PAHs through two different routes, either via the uptake of PAHs from the environment, as it has been shown for mussels and coastal fishes, and/or generation of PAHs during food processing such as barbecuing, frying and roasting [2, 3].
Capillary gas chromatography is most frequently used to analyse PAHs in food owing mainly to its high resolving power. Alternatively, high-performance liquid chromatography with fluorescence detection may be applied. However, the use of gas chromatography (GC) with mass spectrometry (MS) additionally offers great selectivity and good sensitivity, making GC-MS technique the most suitable for the determination of PAHs [9, 10].
The most critical point when developing a GC method is the selection of an appropriate stationary phase to separate the PAHs of interest. Isomers with identical molecular mass and similar physico-chemical properties might co-elute. However, they have to be separated for accurate quantitation. On the other hand, chromatographically overlapping peaks having different molecular weight can be resolved mass spectrometrically. Unfortunately, despite possessing different base peak ions, interferences caused by fragment ions have to be taken into account. In the group of the 15 + 1 EU-priority PAHs, the analyte pair DhA–IcP and the triplet BaA–CPP–CHR face this problem. The use of 50% phenyl methyl polysiloxane columns has solved the problems related to the separation of these critical groups of compounds [11, 12]. However, the presence of four dibenzopyrene isomers with high molecular weight (m/z 302) in the 15 + 1 EU-priority PAHs introduces some other challenges.
Among them, the most important could be related to the typical discrimination observed for compounds with high molecular weight that causes an increase of the limits of detections and quantification of these compounds.
The aim of this study was to evaluate the suitability of different capillary column stationary phases for the analysis of the 15 + 1 EU-priority PAHs. Temperature programs were optimised to the respective capillary columns to obtain best possible resolution of the 15 + 1 EU-priority PAHs and lowest discrimination of high-molecular-weight compound. Finally, the ability of the selected column to separate potentially interfering PAHs from the target analytes in both solvent solutions and food extracts was assessed.
Materials and methods
The analytes benz[a]anthracene (BaA) CAS# 56-55-3, chrysene (CHR) CAS#218-01-9, 5-methylchrysene (5-MC) CAS#3697-24-3, benzo[b]fluoranthene (BbF) CAS#205-99-2, benzo[j]fluoranthene (BjF) CAS#205-82-3, benzo[k]fluoranthene (BkF) CAS#207-08-9, benzo[a]pyrene (BaP) CAS#50-32-8, indeno[1,2,3-cd]pyrene (IcP) CAS#193-39-5, dibenz[a,h]anthracene (DhA) CAS#53-70-3, benzo[ghi]perylene (BgP) CAS#191-24-2, dibenzo[a,l]pyrene (DlP) CAS#191-30-0, dibenzo[a,e]pyrene (DeP) CAS#192-65-4, dibenzo[a,i]pyrene (DiP) CAS#189-55-9 and dibenzo[a,h]pyrene (DhP) CAS#189-64-0 were commercially available BCR certified reference materials (Institute for Reference Materials and Measurements, Geel, Belgium). Cyclopenta[cd]pyrene (CPP) CAS#27208-37-3, purity >99.0% by GC, was manufactured on request (Biochemisches Institut für Umweltkarzinogene, Großhansdorf, Germany). Benzo[c]fluorene (BcL) CAS# 205-12-9 was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The isotopically labelled compounds D12-chrysene and D12-benzo[ghi]perylene were obtained from Cambridge Isotope Labs (Andover, MA, USA). D12-benzo[a]pyrene and D12-coronene were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). 9-Fluorobenzo[k]fluoranthene, used as injection standard, was purchased from Chiron AS (Trondheim, Norway). Stock solutions of the individual PAHs were gravimetrically prepared in toluene. The different working PAH solutions containing the 15 + 1 EU-priority PAHs were prepared mixing appropriate volumes from the individual stock solution and adding toluene to achieve the desired concentrations.
Standard Reference Material® 2260a was purchased from the National Institute of Standards and Technology (NIST, Gaitherburg MD, USA).
Different oil samples were also analysed. One sample was native sunflower oil kindly provided by the European Oil and Proteinmeal Industry (FEDIOL), and a second sample was edible oil fortified with the 15 + 1 EU-priority PAHs used in an inter-laboratory comparison test organised by the Community Reference Laboratory for PAHs. In the latter sample, concentrations of the different PAHs ranged between 1.17 μg/kg for BaA and 8.70 μg/kg for IcP. Oil extracts were prepared using two consecutive steps prior to their analysis by GC-MS. First, the edible oil was mixed with cyclohexane/ethyl acetate (50:50, v/v), and 5 mL of this solution (0.18 g/mL) was analysed by gel permeation chromatography (GPC) in a GPC column filled with Bio-beads S-X3. Samples were eluted at a flow rate of 4 mL/min with cyclohexane/ethyl acetate (50:50, v/v). Then, the collected eluate from the GPC column (35−80 min) was evaporated, dissolved in 1 mL of cyclohexane and loaded into 3 mL solid-phase extraction (SPE) silica cartridges (Supelco, Bornem, Belgium). Elution of the PAHs was carried out with 8 mL of cyclohexane. The SPE eluate was evaporated under a nitrogen stream to a final volume of 200 μL and analysed by GC-MS. Prior to the evaporation, the same volume of toluene was added as a keeper.
A gas chromatograph HP 6890 from Agilent Technologies (Waldbronn, Germany) with a splitless injection port was used for the analysis of the target PAHs. The GC was coupled to an Agilent 5973 single quadrupole mass spectrometer (Agilent Technologies, Cernusco, Italy). The mass selective detector operated at 70 eV with electron ionization in single-ion monitoring (SIM) mode. Molecular ion of each compound was monitored (Table 1). Transfer line temperature and ion source temperature were maintained at 325 °C and 300 °C, respectively. A 6-mm ultra large aperture draw-out lens from Agilent Technologies (Diegem, Belgium) was used for analysis of the samples. The injection mode was pulsed splitless (pulse pressure 30 psi, pulse time 0.3 min) using a glass liner, 4 mm internal diameter (i.d.), loosely filled with silanised glass wool. The temperature of the injector was kept at 300 °C, and the injection volume was 1 μL. After 1 min, the purge valve was opened (purge flow 30.5 mL/min).
Two Optima® δ-6 columns of different column dimensions (Macherey-Nagel, Düren, Germany), 30 and 60 m, 0.25 mm i.d. and 0.25 μm film thickness. The Optima columns offer what is called by the manufacturer “autoselectivity”, meaning that the polarity of the stationary phase adjusts itself to the polarity of the analytes . Likewise, according to the manufacturer, the polarity of the column covers a range between mid-polar and polar columns.
A DB-5MS column, 60 m × 0.25 mm i.d., 0.25 μm film thickness (Agilent Technologies, Diegem, Belgium).
A DB-17MS column 60 m × 0.25 mm i.d., 0.25 μm film thickness (Agilent Technologies, Diegem, Belgium).
A tailor-made DB-17MS column 20 m × 0.18 mm i.d. and 0.14 μm film thickness (Agilent Technologies, Diegem, Belgium).
For the Optima® δ-6 30 m column: 80 °C (hold 1 min), to 216 °C at 60 °C/min, to 258 °C at 2 °C/min, to 330 °C at 120 °C/min and hold for 30 min
For the DB-5MS 60 m column: 80 °C (hold 1 min), to 200 °C at 60 °C/min, to 300 °C at 2 C/min, to 330 °C at 120 °C/min and hold for 10 min
For the DB-17MS 60 m column: 80 °C (hold 1 min), to 250 °C at 40 °C/min, to 305 °C at 25 °C/min, to 315 °C at 2 °C/min, to 330 °C at 40 C/min and hold for 35 min
For the DB-17MS 20 m column: 80 °C (hold 1 min), to 200 C at 60 °C/min, to 220 °C at 2 °C/min, to 285 °C at 3 °C/min, to 325 °C at 5 °C/min and hold for 5 min
Helium was used as carrier gas at 1.5 mL/min flow rate except for the DB-17MS 20 m column where 2 mL/min was applied.
Results and discussion
A non-polar stationary phase (DB-5MS), a mid-polar phase (DB-17MS) and a recently commercialised stationary phase (Optima® δ-6) with moderate polarity (although adjusting its polarity to that of the analyte due to its “autoselectivity” properties ) were evaluated for the separation of the 15 + 1 EU-priority PAHs. For the three columns, 60 m length was the first option selected. However, the 60-m Optima® δ-6 column was replaced by a shorter column (30 m, same stationary phase) due to the high bleeding observed in the longer column.
In the first part of the study, different temperature programs were evaluated in order to obtain best possible separation of the compounds included in the 15 + 1 EU-priority PAHs. Three groups of compounds, CPP–BaA–CHR, BbF–BjF–BkF and the pair DhA–IcP are in that respect the most challenging. Unlike DhA and IcP, CPP and BjF are not included in the 16 EPA PAH list and, therefore, only a few studies tackling these compounds are reported in the literature. Recent inter-laboratory comparison studies revealed that the separation of the three benzofluoranthenes has not yet been achieved in many laboratories . It is important to note that most of the laboratories involved in that study used stationary phases composed of 5% phenyl methylpolysiloxane. The use of this stationary phase is massively reported in the literature for the analysis of PAHs from foodstuff [15–18].
Resolution values obtained for critical pairs/triplets on three different stationary phases
DB-17MS, 60 m 0.25 mm i.d., 250 μm df
DB-5MS, 60 m 0.25 mm i.d., 250 μm df
OPTIMA 6, 30 m 0.25 mm i.d., 250 μm df
DB-17MS, 20 m 0.18 mm i.d., 180 μm df
Chromatographic separation of BbF–BjF–BkF was not achieved on all stationary phases (Fig. 1). Only the DB-17MS 60 m column was able to separate the three fluoranthenes. Acceptable resolution factors (Rs) of 1.35 for the pair BbF–BkF and 1.5 for the pair BkF–BjF were achieved on this column. The DB-5MS 60 m column allowed the separation of BkF from the other two isomers (Rs of 1.42 regarding BjF), but the pair BbF–BjF (Rs = 0.45) was not satisfactorily separated (Table 2). Finally, Optima® δ-6, 30 m could not provide separation of any of the benzofluoranthenes. Regardless the temperature program applied, a single peak containing the three isomers was obtained.
Concerning the analyte pair DhA–IcP, the optimisation of the temperature programme led to a satisfactory resolution for all three columns (Fig. 1). The highest resolution was obtained with the DB-5MS 60 m column (Rs = 1.5), while values of Rs = 1.26 and Rs = 1.41 were obtained for Optima® δ-6, 30 m and DB-17MS, 60 m columns, respectively (Table 2). Good chromatographic resolution is also needed for the pair DhA–IcP since the presence of a small fragment ion at m/z 276 from DhA could lead to biased quantification of IcP (Fig. 1).
Traditionally, little attention has been paid to the analysis of high-molecular-weight PAHs. The compound with the highest molecular mass within the group of the 16 EPA PAHs is DhA with a molecular mass of 278 g/mol. Due to the fact that most of the references found in the literature are related to the analysis of the 16 EPA PAHs, it is difficult to find analytical methods regarding the separation of heavier PAHs. However, the International Agency for Research on Cancer has classified three isomers of dibenzopyrene (DeP, DiP and DhP) as possibly carcinogenic to human beings (group 2B) and another one (DlP) as probably carcinogenic to human beings (group 2A) . Of special concern is DlP which seems to be more than ten times more potent in its effects in rats than BaP . These four compounds are included in the 15 + 1 EU-priority PAHs, and therefore, suitable analytical methods are required for their separation and accurate quantification.
A general problem in the GC analysis of PAHs is the high discrimination observed for high-molecular-weight compounds in comparison to other compounds with low and medium molecular weight. This discrimination usually refers to different behaviour of the analytes at the injection port, especially for high-molecular-weight compounds, which causes a dissimilar transfer of the analytes into the column. However, discrimination is also observed due to the strong interaction of high-molecular-weight compounds with the stationary phase which may result in broad peaks. Therefore, this causes discrimination in terms of peak height. In both cases, the main implication is a decrease of signal-to-noise (S/N) ratios and, therefore, an increase of the limits of detections of these compounds. Discrimination is already observed for the analysis of the 16 EPA PAHs [20, 21]. Therefore, even higher discrimination can be expected for the four dibenzopyrenes, which contain one additional aromatic ring moiety.
Different strategies might be applied to minimise the discrimination of high-molecular-mass compounds. In order to reduce discrimination in the injection port, injection techniques such as programmed temperature vaporization and on-column injection have been applied . When classical splitless injection is applied, as in this study, special regard has to be given to the peak shape of the late eluting compounds, which show frequently small and broad peaks with low S/N ratios. Peak distortion due to flooding effects hampers the use of high volumes in splitless injection for the enhancement of S/N ratios. However, different chromatographic parameters can be modified to obtain a better peak shape and better S/N ratios. In this study, the influence of temperature program, phase ratio (β) and column length onto the peak shape of late eluting PAHs was investigated.
The temperature program for the chromatogram shown in Fig. 2a was: 80 °C (hold 1 min), to 225 °C at 60 °C/min, to 258 °C at 2 °C/min, to 330 °C at 120 °C/min and hold for 15 min; the temperature program applied for the chromatogram in Fig. 2b is described in “Materials and methods”. The temperature profiles are printed above the chromatograms. The temperature program for the chromatogram of Fig. 2a was faster than the conditions applied for the chromatogram in Fig. 2b. At first glance, it seems that the chromatogram in Fig. 2a contains narrower and higher peaks for the dibenzopyrenes than in Fig. 2b. However, the absolute peak area, peak height and peak width were nearly identical for the four analytes in both chromatograms. Similar peak areas indicate similar mass transfer from the injector onto the column, a result that was expected since the same injection mode was used for both chromatograms. The fact that peak heights and peak areas were similar demonstrates that the steepness of the slope of the temperature program (in the tested range) has no influence on the peak shapes and S/N ratios of the four late-eluting compounds.
As expected, the elution of the dibenzopyrenes happens only at very high temperatures and is less influenced by the temperature gradient than by the end temperature. The latter is limited by the thermal stability of the respective column and should not exceed the maximum column temperature specified by the manufacturer. Hence, the temperature program has, for a given type of column and using the same injection technique, just little effect on the discrimination in terms of peak height of the dibenzopyrenes.
Figure 4 presents magnifications of the resolution obtained for the most intricate group of compounds, CPP–BaA–CHR, BbF–BjF–BkF and DhA–IcP. Resolution of the fluoranthenes on the 20-m column (Rs = 1.46 and Rs = 1.44 for the pairs BbF–BkF and BkF–BjF, respectively) was similar to that achieved on the 60-m column. The pairs BaA–CPP and CPP–CHR showed in both cases resolution values higher than 1.5, while the pair IcP–DhA presented a resolution value of 1.41 (same value as observed on the 60-m column; see Table 2). Compared to the DB-17MS 60 m column, changes in the elution order of some analytes were observed, in particular for CPP and IcP. Opposite to what was observed on the 60-m column, in the 20-m column, CPP and IcP eluted before CHR and DhA, respectively. This could be explained by the lower interaction of CPP and IcP with the stationary phase in the shorter column due to the fact that these compounds elute at lower temperature in the 20-m column than in the 60-m one. It is well known that the polarity of the stationary phase is temperature-dependent 
Absolute peak area, peak height, peak width and S/N ratio of a DhP solution (15 ng/mL) analysed in both a 20- and 60-m DB-17MS column
DB-17MS 60 m column
DB-17MS 20 m column
Absolute peak areaa
The mid-polar stationary phase (50% phenyl-methylpolysiloxane) as found in DB-17MS columns gave the best results in the chromatographic analysis of the 15 + 1 EU-priority PAHs. With this stationary phase, all target PAHs were satisfactorily separated within an analysis time of only 45 min. Discrimination in terms of peak height of the PAHs with high molecular mass (caused from the strong interaction of these compounds with the stationary phase) was minimised without compromising peak resolution by applying a tailor-made 20 m DB-17MS column with a phase ratio of 320 and optimised helium gas flow and temperature program. This reduction in peak height discrimination leads to obtaining a significant enhancement of the analytical sensitivity for dibenzopyrenes, as demonstrated by an almost threefold increase of the S/N ratio for DhP, the last eluting PAH. Suitability of the 20-m DB-17MS column to separate the target analytes (15 + 1 EU-priority PAHs) from different mixes of PAHs in both solvent solutions and food extracts was also demonstrated.
The authors would like to thank Mr. Allen Vickers from Agilent Technologies for providing tailor-made capillary columns.