Analytical and Bioanalytical Chemistry

, Volume 393, Issue 6, pp 1697–1707

Evaluation of gas chromatography columns for the analysis of the 15 + 1 EU-priority polycyclic aromatic hydrocarbons (PAHs)

Authors

  • José Ángel Gómez-Ruiz
    • European Commission, Joint Research CentreInstitute for Reference Materials and Measurements
    • European Commission, Joint Research CentreInstitute for Reference Materials and Measurements
Original Paper

DOI: 10.1007/s00216-008-2585-8

Cite this article as:
Gómez-Ruiz, J.Á. & Wenzl, T. Anal Bioanal Chem (2009) 393: 1697. doi:10.1007/s00216-008-2585-8

Abstract

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.

Keywords

Polycyclic aromatic hydrocarbons (PAHs)15 + 1 EU-priority PAHsGC-MSStationary phasePeak height discriminationResolution

Introduction

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 [1].

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].

The dietary intake of PAHs is of concern because some of them can cause cancer in humans [4]. PAHs are metabolized in the liver and other tissues by the cytochrome P450 enzyme system into reactive electrophiles. Some of these reactive electrophilic PAH species can bind to cellular macromolecules such as DNA, forming what is known as PAH–DNA adducts, and initiate mutagenic processes in the cells [5]. The Scientific Committee on Food assessed in 2002 the toxicity of 33 PAHs and concluded that 15 of them are potentially genotoxic and carcinogenic to humans [6]. In 2005, the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives identified an additional PAH (benzo[c]fluorene) as probably carcinogenic [7]. This group of PAHs is becoming recognized as the 15 + 1 EU-priority PAHs (Table 1) in opposition to the 16 EPA PAHs highlighted by the US Environmental Protection Agency (EPA) in the 1970s [8]. Among the 15 + 1 EU-priority PAHs, there are eight PAHs that are not included in the US EPA list. The presence of these PAHs has entailed new challenges for the scientific community in terms of chromatographic separation and analyte discrimination among others.
Table 1

15 + 1 EU priority PAHs under investigation (with acronyms)

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The right part of the table (in bold) shows those PAHs that are not included in the 16 EPA PAHs

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

Reference materials

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.

GC-MS analysis

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).

Five different fused-silica capillary columns were evaluated:
  • 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 [13]. 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).

The temperature programs were optimised for each column to obtain best efficiency in terms of peak resolution and sensitivity for the analysis of the 15 + 1 EU-priority PAHs. The temperature programmes were in particular:
  • 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 [13]) 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.

Resolution

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 [14]. 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 [1518].

Figure 1 shows the resolution of the critical pair/triplets of compounds on the three columns with different stationary phases. Apart from the base peak ion (m/z 228), BaA and CHR also form a fragment of low relative intensity at m/z 226, identical to the base peak ion of CPP. The co-elution of either BaA or CHR with CPP will not affect the quantification of the two first PAHs as ion m/z 228 is exclusive to them. However, the quantification of CPP would be biased due to the presence of the ions m/z 226 from BaA or CHR. Therefore, a good chromatographic resolution of CPP and the other two compounds is mandatory in order to obtain accurate quantitation of CPP.
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Fig. 1

Chromatographic separation of critical pairs/triplets on three different stationary phases. a DB-17MS column, 60 m length, 0.25 mm i.d., 0.25 μm film thickness (df), b DB-5MS column, 60 m length, 0.25 mm i.d., 0.25 μm df, c Optima® δ-6 column, 30 m length, 0.25 mm i.d., 0.25 μm df. Chromatographic conditions are described in “Materials and methods

The elution order of the triplet CPP–BaA–CHR was different in each column. For the DB-5MS 60 m column, the optimum temperature program led to longer retention times for BaA and CHR compared to CPP (Fig. 1b). On the other hand, with the intermediate polarity column (DB-17MS, 60 m), CPP showed the longest retention time among the three compounds (Fig. 1a). Finally, for the most polar stationary phase (Optima® δ-6, 30 m), CPP eluted between BaA and CHR (Fig. 1c). The compacter structure of the five-membered ring moiety in CPP and, therefore, its higher electron density could explain the stronger interaction of this compound with the two polar stationary phases resulting in a different elution order compared to that observed for the non-polar stationary phase (Fig. 1). Despite obtaining a different elution order, resolution attained with the three columns was satisfactory; in all cases, CPP was clearly resolved from BaA and CHR. Resolution values in each column for the different group of compounds are given in Table 2.
Table 2

Resolution values obtained for critical pairs/triplets on three different stationary phases

 

CHR/CPP

BaA/CPP

BbF/BkF

BkF/BjF

DhA/IcP

DB-17MS, 60 m 0.25 mm i.d., 250 μm df

1.48

>1.5

1.3

>1.5

1.41

 

CPP/BaA

BaA/CHR

BbF/BjF

BjF/BkF

IcP/DhA

DB-5MS, 60 m 0.25 mm i.d., 250 μm df

1.44

>1.5

0.45

1.42

>1.5

 

BaA/CPP

CPP/CHR

BbF/BkF

BkF/BjF

DhA/IcP

OPTIMA 6, 30 m 0.25 mm i.d., 250 μm df

1.42

1.42

No separation

No separation

1.26

 

BaA/CPP

CPP/CHR

BbF/BkF

BkF/BjF

IcP/DhA

DB-17MS, 20 m 0.18 mm i.d., 180 μm df

>1.5

>1.5

1.46

1.44

1.41

Chromatographic conditions for the different columns are described in “Materials and methods

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).

Dibenzopyrenes discrimination

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) [4]. Of special concern is DlP which seems to be more than ten times more potent in its effects in rats than BaP [19]. 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 [22]. 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.

Regarding the influence of the temperature program, Fig. 2 shows two chromatograms obtained for the 15 + 1 EU-priority PAHs on a DB-5MS column applying two different oven temperature programs. In both cases, the concentration of all the PAHs was the same (0.4 μg/mL).
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Fig. 2

Chromatograms of the 15 + 1 EU PAHs on a DB-5MS column, 60 m length, 0.25 mm i.d., 0.25 μm film thickness (df) using two different temperature programs. a 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 10 min. b 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. Vertical arrows indicate the point when maximum temperature is reached. Same concentration was used in both samples (0.4 μg/mL). Scaling was based on the highest peak in each chromatogram

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.

As it was shown before, among the tested columns, the DB-17MS 60 m column was the only one able to appropriately separate all the critical pair/triplets in the set of 15 + 1 EU-priority PAHs (Fig. 1). However, as it can be seen in Fig. 3, the four dibenzopyrenes showed strong discrimination in terms of peak height compared to the other PAHs. The first dibenzopyrene, DeP, eluted more than 20 m after reaching the final temperature (330 °C) which is already above the maximum isothermal temperature specified for this column. This indicates much stronger interaction of the dibenzopyrenes with this mid-polar stationary phase compared to the DB-5MS column. In order to reduce this interaction and, therefore, the discrimination in terms of peak height of the dibenzopyrenes, a column with the same stationary phase but shorter length and higher phase ratio was tested. The selected column was a DB-17MS column 20 m length, 0.18 mm i.d. and 0.14 μm film thickness. Phase ratio (β) of this column was 320 compared to 250 in the 60-m column.
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Fig. 3

Chromatogram of the 15 + 1 EU PAHs on a DB-17MS column, 60 m length, 0.25 mm i.d., 0.25 μm film thickness (df). Chromatographic conditions are described in “Materials and methods

Different temperature programs were evaluated, but in all cases, the resolution of the critical pair/triplets was affected when 1.5 mL/min gas flow was used. However, better results were achieved by changing the gas flow from 1.5 mL/min to 2 ml/min. Figure 4 shows the chromatographic profile of the 15 + 1 EU PAHs obtained with the DB-17MS 20 m column using the most appropriate temperature program (see “Materials and methods”). It was observed that decreasing the final temperature from 330 °C to 325 °C had no influence on the chromatography. Therefore, the lowest temperature was selected to preserve the column and prolong its life time.
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Fig. 4

Chromatogram of the 15 + 1 EU PAHs on a DB-17MS column 20 m length, 0.18 mm i.d. and 0.14 μm film thickness (df). Chromatographic conditions are described in “Materials and methods”. Peak identities are: (1) benzo[c]fluorene, (2) benz[a]anthracene (BaA), (3) cyclopenta[cd]pyrene (CPP), (4) D12-chrysene, (5) chrysene (CHR), (6) 5-methylchrysene, (7) 9-fluoro-benzo[k]fluoranthene, (8) benzo[b]fluoranthene (BbF), (9) benzo[k]fluoranthene (BkF), (10) benzo[j]fluoranthene (BjF), (11) D12-benzo[a]pyrene, (12) benzo[a]pyrene, (13) indeno[1,2,3-cd]pyrene (IcP), (14) dibenz[a,h]anthracene (DhA), (15) D12-benzo[ghi]perylene, (16) benzo[ghi]perylene, (17) dibenzo[a,l]pyrene, (18) D12-coronene, (19) dibenzo[a,e]pyrene, (20) dibenzo[a,i]pyrene and (21) dibenzo[a,h]pyrene

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 [23]

In addition to the good resolution achieved on the shorter column for the target PAHs, the four dibenzopyrenes eluted as narrow and high peaks, showing almost no discrimination in terms of peak height compared to the rest of PAHs (Fig. 4). This was confirmed by injecting a standard solution of the 15 + 1 EU-priority PAHs (15 ng/mL) onto both the 20- and 60-m DB-17MS columns and comparing performance characteristics. Table 3 shows, as an example, the absolute peak area, peak height, peak width and S/N ratio of DhP (the PAH with the longest retention time) on both columns. As expected, the peak area was very similar in both cases confirming that similar amounts of substance were transferred on the columns. However, significant differences were found for the other parameters. DhP eluted as a narrower and higher peak in the 20-m column. Furthermore, the S/N ratio of DhP in the 20-m column was almost threefold higher than in the 60-m column, demonstrating that a higher sensitivity can be achieved with the shorter column. Similar results in terms of absolute peak area, peak height, peak width and S/N ratio were also obtained for the other three dibenzopyrenes (data not shown). Therefore, the use of a 20-m column with appropriate phase ratio (β = 320) allowed us to increase the sensitivity of the heaviest molecular PAH (dibenzopyrenes) without compromising the resolution of the other PAHs achieved in a 60-m column.
Table 3

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

 

Dibenzo[a,h]pyrene (DhP)

DB-17MS 60 m column

DB-17MS 20 m column

Absolute peak areaa

20,300

21,300

Peak widthb

18

9

Peak heighta

175

409

S/N ratio

34.7

91.9

aArbitrary units

bPeak width is calculated at half height and expressed in seconds

Two solutions containing benzofluorene isomers (three) and methylchrysene isomers (five) were analysed with the selected temperature program on the DB-17MS 20 m column. Figure 5 shows that BcL and 5-MC were well resolved from their isomers, and therefore, an exact quantification can be afforded. In addition, the suitability of the DB-17MS 20 m column to analyse complex mixes of PAHs was also evaluated analysing a certified reference material, the NIST Standard Reference Material (SRM®) 2260a containing 36 different PAHs in toluene. Among the 36 PAHs, 11 belong to the 15 + 1 EU PAHs. Figure 6 shows the chromatographic resolution of these 11 PAHs and those eluting within the same retention time window. All compounds were chromatographically well resolved with exception of two pairs: chrysene–tryphenylene and coronene–dibenzo[a,e]pyrene. However, the latter pair possesses different base peak ions (m/z 300 for coronene and m/z 302 for dibenzo[a,e]pyrene) and, therefore, mass spectroscopic resolution allows appropriate quantification of both compounds. The pair chrysene–tryphenylene could not be separated regardless of the applied temperature program As it has been already described in the literature [9, 11], the use of a different stationary phase such as the non-polar DB-XLB allows an appropriate separation of these two compounds, although this column is not suitable for the quantitation of the whole set of 15 + 1 EU-priority PAHs.
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Fig. 5

Chromatographic separation of methylchrysene isomers (a) and benzofluorene isomers (b) on a DB-17MS column 20 m length, 0.18 mm i.d. and 0.14 μm film thickness (df). Chromatographic conditions are described in “Materials and methods

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Fig. 6

Chromatographic separation of different PAHs contained in SRM 2260a on a DB-17MS column 20 m length, 0.18 mm i.d. and 0.14 μm film thickness (df). Chromatographic conditions are described in “Materials and methods”. Peak identities are: (1) benzo[ghi]fluoranthene, (2) benzo[c]phenanthrene, (3) benz[a]anthracene, (4) cyclopenta[cd]pyrene, (5 + 6) chrysene and tryphenylene, (7) benzo[b]fluoranthene, (8) benzo[k]fluoranthene, (9) benzo[j]fluoranthene, (10) benzo[a]fluoranthene, (11) benzo[e]pyrene, (12) benzo[a]pyrene, (13) perylene, (14) dibenz[a,j]anthracene, (15) indeno[1,2,3-cd]pyrene, (16) dibenz[a,c]anthracene, (17) dibenz[a,h]anthracene, (18) benzo[b]chrysene, (19) picene, (20) benzo[ghi]perylene, (21) anthracene, (22) dibenzo[b,k]fluoranthene, (23 + 24) coronene and dibenzo[a,e]pyrene, (25) dibenzo[a,h]pyrene

Finally, two edible oil extracts were also analysed with the selected temperature program on the DB-17MS 20 m column. Figure 7 shows the chromatographic separation of some of the PAHs detected in an extract of sunflower oil. As it can be seen, very good resolution was achieved among PAHs that belong to the 15 + 1 EU-priority PAHs (fluoranthenes or the triplet BaA–CPP–CHR). In addition, PAHs like benzo[e]pyrene (BeP), benzo[a]fluoranthene, benzo[ghi]fluoranthene or perylene were also well resolved from the 15 + 1 EU-priority PAHs (Fig. 7). Since the analysis of this native sunflower oil revealed the almost complete absence of high-molecular-weight PAHs, it was decided to analyse a sample of edible oil fortified with the 15 + 1 EU-priority PAHs to assess the chromatographic behaviour of high-molecular-weight PAHs in the presence of food matrix. The edible oil was a sample used in an inter-laboratory comparison test organised by the Community Reference Laboratory for PAHs. Figure 8 shows the SIM traces of critical pairs/triplets and dibenzopyrenes in the edible oil extract. Besides the good resolution achieved for the different PAHs, the four dibenzopyrenes eluted as narrow and high peaks. As an example, no height discrimination was observed for DlP as compared to BaP, both compounds present in the edible oil at similar concentrations (1.73 and 1.36 μg/kg, respectively).
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Fig. 7

SIM traces corresponding to the m/z 226 and m/z 228 (a) and m/z 252 (b) of an extract of sunflower oil analysed on a DB-17MS column 20 m length, 0.18 mm i.d. and 0.14 μm film thickness (df). Chromatographic conditions are described in “Materials and methods”. BghiF benzo[ghi]fluoranthene, BcP benzo[c]phenanthrene, BaF benzo[a]fluoranthene, BeP Benzo[e]pyrene, Per Perylene

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Fig. 8

SIM traces of critical pairs/triplets and dibenzopyrenes in an edible oil extract analysed on a DB-17MS column 20 m length, 0.18 mm i.d. and 0.14 μm film thickness (df). Chromatographic conditions are described in “Materials and methods

Conclusion

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.

Acknowledgment

The authors would like to thank Mr. Allen Vickers from Agilent Technologies for providing tailor-made capillary columns.

Copyright information

© Springer-Verlag 2008