BROWSE ALL ARTICLES BY TOPIC

RELATED ITEMS

Bookmark and Share

From: Food Quality & Safety magazine, February/March 2012

Different Stationary Phases for PAH Analysis

by Claudia M. Schulz, Ansgar Ruthenschrör, Holger Fritz, Johan Kuipers, John OostdiJk, Max Erwine

Studied PAHs
click for large version
Table 1. Studied PAHs
List of studied PAHs, corresponding to the compounds on the EU list and three other important interfering PAHs.
Studied PAHs
click for large version
Figure 1: GC-MS chromatogram of a spiked salmon sample (concentration between 1 and 4 ppb) obtained on an Agilent J&W Select PAH column (15 m x 0.15 mm x 0.10 µm). Peak identification: 1 benzo[a]anthracene, 2 triphenylene, 3 chrysene, 4 5-methylchrysene, 5 1-methylchrysene, 6 benzo[b]fluoranthene, 7 benzo[k]fluoranthene, 8 benzo[j]fluoranthene, 9 benzo[e]pyrene, 10 benzo[a]pyrene, 11 perylene.
Studied PAHs
click for large version
Figure 2: GC-MS chromatogram of a spiked salmon sample (concentration between 1 and 4 ppb) obtained on an Agilent J&W Select PAH column (15 m x 0.15 mm x 0.10 µm). Peak identification: 12 indeno[1,2,3-c,d]pyrene, 13 dibenzo[a,h]anthracene, 14 benzo[b]chrysene, 15 picene, 16 benzo[g,h,i]perylene, 17 anthanthrene, 18 dibenzo[al]pyrene, 19 dibenzo[a,e]pyrene, 20 dibenzo[a,i]pyrene, 21 dibenzo[a,h]pyrene.

"The accurate quantification of PAHs in food with gas chromatography-mass spectrometry (GC-MS) requires special attention for both separation on the stationary phase and differentiation by mass ions. "

Studied PAHs
click for large version
Table 2. Method Performance of Spiked Salmon, Olive Oil Samples
Studied PAHs
click for large version
Table 3. Mean Sample Values Obtained on Agilent Select PAH Phase
Studied PAHs
click for large version
Figure 3: Separation of benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), and benzo[j]fluoranthene (BjF) on three different GC phases at m/z 252 in a mussels extract. A: Agilent Select PAH (15 m x 0.15 mm x 0.10 µm), B: VF-17ms (20 m x 0.15 mm x 0.15 µm) and C: HP-5ms (30 m x 0.25 mm x 0.25 µm).
Studied PAHs
click for large version
Figure 4: Results for benzo[k]fluoranthene in different food samples on three evaluated stationary phases (Agilent Select PAH 15 m x 0.15 mm x 0.10 µm; VF-17ms 20 m x 0.15 mm x 0.15 µm; and HP-5ms 30 m x 0.25 mm x 0.25 µm).
Studied PAHs
click for large version
Figure 5: Separation of benzo[a]anthracene (BaA), cyclopenta[c,d]pyrene (CPP), chrysene (CHR), and triphenylene (TR) on three different GC phases in a dried fish extract (left side) and a pumpkin seed oil extract (right side). A: Agilent J&W Select PAH (15 m x 0.15 mm x 0.10 µm), B: Agilent J&W VF-17ms (20 m x 0.15 mm x 0.15 µm), and C: Agilent J&W HP-5ms (30 m x 0.25 mm x 0.25 µm).
Studied PAHs
click for large version
Figure 6: Results for chrysene in different food samples on three evaluated stationary phases (Agilent J&W Select PAH 15 m x 0.15 mm x 0.10 µm; Agilent J&W VF-17ms 20 m x 0.15 mm x 0.15 µm; and Agilent J&W HP-5ms 30 m x 0.25 mm x 0.25 µm).
Studied PAHs
click for large version
Figure 7:Results for chrysene in different food samples on three evaluated stationary phases (Agilent J&W Select PAH 15 m x 0.15 mm x 0.10 µm; Agilent J&W VF-17ms 20 m x 0.15 mm x 0.15 µm; and Agilent J&W HP-5ms 30 m x 0.25 mm x 0.25 µm).
Studied PAHs
click for large version
Figure 8: Separation of dibenzo[a,l]pyrene (DlP), dibenzo[a,e]pyrene (DeP), coronene (COR), dibenzo[a,i]pyrene (DiP), and dibenzo[a,h]pyrene (DhP) on three different GC phases in a standard solution. A: Agilent J&W Select PAH (15 m x 0.15 mm x 0.10 µm), B: Agilent J&W VF-17ms (20 m x 0.15 mm x 0.15 µm), and C: Agilent J&W HP-5ms (30 m x 0.25 mm x 0.25 µm).

Polycyclic aromatic hydrocarbons (PAH) comprise a large group of more than several hundred chemical compounds containing two or more fused aromatic rings. They are produced during incomplete combustion of organic compounds. Food can be naturally contaminated with PAHs by uptake from the environment, like mussels filtering surrounding water. The main contamination sources for food, however, are processing procedures in which PAHs are generated at significant levels, such as frying, drying, smoking, grilling, or roasting.1-6

PAH exposure is a major health concern, because many are known to be carcinogenic and genotoxic. The most important lists of monitored PAHs are a group of 16 PAHs listed by the U.S. Environmental Protection Agency (EPA), and the 15+1 European Union (EU) priority PAHs (Table 1).7,8

In the past, benzo[a]pyrene was thought to be a suitable marker for the occurrence of carcinogenic PAH in food; it is, therefore, the only one currently regulated in food.9,10 In 2008, however, the European Food Safety Authority (EFSA) published an opinion on suitable indicators for the occurrence and toxicity of PAH in food.11 The EFSA’s Panel on Contaminants in the Food Chain concluded that benzo[a]pyrene is not a suitable indicator for the occurrence of carcinogenic PAH in food. Instead the sum of four PAH (PAH 4) is proposed in order to better protect consumer health. As a consequence, a European Regulation amendment is anticipated toward the use of the following four PAHs: benzo[a]pyrene, chrysene, benzo[b]fluoranthene and benzo[a]anthracene.12

One of the major techniques used for the analysis of PAH in food is gas chromatography-mass spectrometry (GC-MS). In order to obtain an accurate quantification for individual PAHs, either chromatographic separation and/or differentiation by mass ions is required.13,14 Because some PAHs exhibit identical mass ions and co-elute on the stationary phase due to similar physico-chemical properties, the choice of capillary column is one of the most critical aspects to consider when developing a suitable GC-MS method for the analysis of PAH in food.15

Here, we present the evaluation of three different stationary phases for the analysis of the EU priority PAH 15+1 in different kinds of foodstuffs. We focused on benzo[a]pyrene, chrysene, benzo[b]fluoranthene, and benzo[a]anthracene, the PAH 4, because some of these require special attention for separation on the stationary phase and mass spectrometric fragmentation.

Sample Preparation

An internal standard solution containing a mixture of deuterated PAHs was added to 1-2 g of the homogenized sample material. After the addition of 100 mL of a methanolic potassium hydroxide solution, the samples were saponified within 30 minutes at 60 degrees C in an ultrasonic bath. The PAHs were extracted several times, with a total of 150 ml cyclohexane. The combined organic extracts were dried with sodium sulphate and concentrated.

A gel permeation chromatography (GPC) cleanup was carried out using a GPC directly coupled to an evaporation unit in the same system (LCTech, Dorfen, Germany) on a GPC column (300 mm long with a 25 mm internal diameter) filled with 50 g Bio-Beads S-X3 (BioRad Laboratories, Belgium). Dichloromethane was used as eluent at a flow rate of 5 mL/min. The fraction containing the PAHs eluted between 21 and 34 min. After concentration, the extract was reconstituted in a 1:1 mixture of cyclohexane/ethyl acetate and analyzed using GC-MS.

Instrumentation

A gas chromatograph 7890A (Agilent Technologies) equipped with a UNIS 2100-PTV injection port (JAS) and a CTC Combi Pal Autosampler was used for measurement. The GC was coupled to an Agilent 5975C inert mass spectrometer operating in electron impact mode at 70 eV and selective ion monitoring mode. Ion source and transfer line temperature were maintained at 300 degrees C. Helium was used as the carrier gas, with a flow of 1.2 mL/min except for the HP-5ms, where 1.5 mL/min was applied. The injection volume was 50 µL.

For gas chromatographic separation, the following columns were used:

  • Agilent J&W Select PAH (15 m x 0.15 mm x 0.10 µm)
  • Agilent J&W VF-17ms (20 m x 0.15 mm x 0.15 µm)
  • Agilent J&W HP-5ms (30 m x 0.25 mm x 0.25 µm)

The GC temperature programs used were optimized for separation and total run time. The programs are listed here:

  • For the Select PAH: 70 degrees C (hold 0.4 min), to 180 degrees C (hold 1 min) at 70 degrees C/min, to 230 degrees C (hold 7 min) at 7 degrees C/min, to 280 degrees C (hold 7 min) at 50 degrees C/min, to 350 degrees C (hold 3.6 min) at 30 degrees C/min
  • For the VF-17ms: 70 degrees C (hold 0.7 min), to 180 degrees C at 70 degrees C/min, to 245 degrees C (hold 1 min) at 5 degrees C/min, to 270 degrees C (hold 1.5 min) at 4 degrees C/min, to 350 degrees C (hold 4 min) at 25 degrees C/min
  • For the HP-5ms: 60 degrees C (hold 1 min), to 240 degrees C at 25 degrees C/min, to 275 degrees C at 4 degrees C/min, to 315° degrees C (hold 4.6 min) at 25 degrees C/min

Results

The performance of the extraction method was tested with a salmon round robin material and an olive oil round robin material, both additionally spiked with a mixture of EU priority PAHs and other potentially interfering PAHs like triphenylene at a concentration between 1 and 4 ppb. For GC-MS measurement, a Select PAH column was used.

Figures 1 and 2 show the chromatograms obtained from the spiked salmon sample for some mass ions. Method performance results, including repeatability (n=6) and trueness for the 15+1 EU priority PAHs, as well as for some important interfering PAHs, are summarized in Table 2 for both tested matrices. Repeatability was assessed on the analysis of the same sample six times. Relative standard deviations were in the range of 2.0%-16.7% and demonstrated a good repeatability for all compounds. The trueness for all analytes was calculated from the obtained mean measured concentration and the known spiked values, ranging between 1 and 4 ppb. For most compounds, the trueness was in the range of 80%-110%. Based on these results, this method was proved suitable for the accurate measurement of PAHs in food.

Samples from different food categories known to be frequently contaminated with PAHs, including mussels, dried fish, plant oils, herbs, and spices (smoked paprika), were prepared in duplicate according to the sample preparation procedure. Three different stationary phases, a non-polar HP-5ms (5% phenyl/ 95% dimethylpolysiloxane phase), a mid-polar VF-17ms (50% phenyl/50% dimethylpolysiloxane phase), and a Select PAH were evaluated for the separation of the 15+1 EU priority PAHs, especially for the group of PAH 4. Table 3 shows an overview of the results obtained on the Select PAH column.

The first group of PAHs that have been challenging for chromatographic separation on stationary phases in the past were the three benzofluoranthenes, among them benzo[b]fluoranthene, belonging to the group of PAH 4. Because they exhibit the same base peak ion m/z 252, an optimized separation is mandatory for accurate quantification. As shown in Figure 3, the Select PAH column provided a sufficient resolution for this group.

Benzo[b]fluoranthene, belonging to the group of PAH 4, was nearly baseline separated from the two other isomers, which were separated up to 65% from each other. On the VF-17ms, a baseline separation between all three isomers was achieved. On the HP-5ms, however, only an unsatisfactory resolution of the three benzofluoranthenes was obtained. In this case, the co-eluting benzo[j]fluoranthene and benzo[k]fluoranthene were not fully separated from the earlier eluting benzo[b]fluoranthene. In recent years, functional groups (i.e., phenyl) have been incorporated into the polysiloxane backbone as arylene inclusions, as is the case for DB-5ms, increasing the thermal and oxidative resistance of the liquid phase. The arylene/phenyl functionality and percentage substitution also positively effect the separation of PAH isomers, though in some cases it is with a significantly higher total run time.14,15,16

Figure 4 shows the results obtained from the three tested stationary phases for benzo[k]fluoranthene in several food samples from different food categories. The use of a column with appropriate separation characteristics, like the Select PAH or the VF-17ms, resulted in lower amounts, whereas the value of measured benzo[k]fluoranthene is biased due to the co-elution with benzo[j]fluoranthene on the HP-5ms.

Another group of PAHs that are critical for chromatographic separation and therefore precise quantification consists of benzo[a]anthracene, cyclopenta[c,d] pyrene, chrysene, and triphenylene. Benzo[a]anthracene, chrysene, and triphenylene have the same base peak ion m/z 228 and also form a fragment ion at m/z 226, which is the base peak ion of cyclopenta[c,d]pyrene. Consequently, in order to avoid a biased quantification, an optimal stationary phase is mandatory for this group.

Application of the VF-17ms showed a good resolution of benzo[a]anthracene and cyclopenta[c,d]pyrene (Figure 5). However, co-elution was observed on this phase for the pair triphenylene and chrysene. In contrast, the Select PAH achieved a good separation for the pair chrysene and triphenylene. Although a baseline separation could not be achieved for the two compounds, the resolution in different kinds of food matrices and at different concentration levels, as well as varying ratios, was sufficient for accurate quantification.

For benzo[a]anthracene and cyclopenta[c,d]pyrene, a baseline separation was also obtained on this column. The measurement on the HP-5ms revealed an unsatisfactory separation due to co-elution of benzo[a]anthracene and cyclopenta[c,d]pyrene on the one hand and triphenylene and chrysene on the other. Benzo[a]anthracene and chrysene belong to the group of PAH 4, whereas triphenylene is a frequently occurring interfering compound in PAH-contaminated foodstuffs.

In the analyzed food samples, the application of the VF-17ms and the HP-5ms led to significantly higher amounts of chrysene due to the co-eluting triphenylene (Figure 6). In the case of the pumpkin seed oil, the created bias was more than 50%. Only the use of the Select PAH allowed an accurate quantification of the EPA and EU priority-listed chrysene.

Additionally, the quantification of cyclopenta[c,d]pyrene was biased or gave a false positive result due to insufficient separation on the HP-5ms and the interfering weak fragment ion m/z 226 produced by the benzo[a]anthracene present in the analyzed food samples (Figure 7).

The highly carcinogenic dibenzopyrenes, classified either in group 2B or group 2A by the International Agency for Research on Cancer, are of high molecular weight and therefore prone to discrimination effects in the injector as well as partial condensation effects elsewhere in the GC-MS system. Hence, special care must be taken to avoid a decrease of the signal-to-noise (S/N) ratios and limit the system peak tailing. Higher mass spectrometer interface and ion-source temperatures, as well as the use of a programmable temperature vaporization injector or an on-column injector, can limit these phenomena, resulting in increased response for the high molecular weight PAHs.17

Figure 8 demonstrates that, with the applied chromatographic parameters, a good peak shape and response for the high molecular weight PAHs were obtained on all tested columns. However, whereas on the VF-17ms and the Select PAH the four dibenzopyrenes and coronene were baseline separated, on the HP-5ms, coronene interfered with dibenzo[a,e]pyrene. Coronene and dibenzo[a,e]pyrene also produce weak fragment ions m/z 302 and m/z 300, respectively, leading to a biased quantification if both compounds are present in the analyzed sample.

The presented method proved to be suitable for the determination of the 15+1 EU priority PAHs in food samples and was applied to food samples from different kinds of food categories, including mussels, fish, plant oils, herbs, and spices. Comparison of the obtained results from the three evaluated stationary phases demonstrated that only the Agilent J&W Select PAH was able to ensure an accurate measurement of the 15+1 EU priority PAHs, especially for the group PAH 4. The use of a column with smaller dimensions (15 m x 0.15 mm x 0.10 µm) allowed the measurement of all PAHs within approximately 30 minutes’ total run time.

 


Schulz, Ruthenschrör, and Fritz are with Eurofins WEJ Contaminants in Hamburg, Germany. Kuipers, Oostdijk, and Erwine are with Agilent Technologies in Middelburg, the Netherlands.

 

References

  1. European Commission. Health and Consumer Protection Directorate-General. Scientific Committee on Food. Polycyclic aromatic hydrocarbons—occurrence in foods, dietary exposure and health effects. Available at: ec.europa.eu/food/fs/sc/scf/out154_en.pdf. Accessed Sept. 7, 2011.
  2. Larsson BK, Eriksson AT, Cervenka M. Polycyclic aromatic hydrocarbons in crude and deodorized vegetable oils. J Am Oil Chemists Soc. 1987;64(3):365-370.
  3. Jira W, Ziegenhals K, Speer K. Gas chromatography-mass spectrometry (GC-MS) method for the determination of 16 European priority polycyclic aromatic hydrocarbons in smoked meat products and edible oil. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2008;25(6):704-713.
  4. Perelló G, Martí-Cid R, Castell V, et al. Concentrations of polybrominated diphenyl ethers, hexachlorobenzene and polycyclic aromatic hydrocarbons in various foodstuffs before and after cooking. Food Chem Toxicol. 2009;47(4):709-715.
  5. Simko P. Factors affecting eliminating of polycyclic aromatic hydrocarbons from smoked meat foods and liquid smoke flavorings. Mol Nutr Food Res. 2005;49(7):637-647.
  6. Guillén MD. Polycyclic aromatic compounds: extraction and determination in food. Food Addit Contam. 1994;11(6):669-684.
  7. U.S. Environmental Protection Agency (EPA). Compendium method TO-13A. Determination of polycyclic aromatic hydrocarbons (PAHs) in ambient air using gas chromatography/mass spectrometry (GC/MS). January 1999. Available at: www.epa.gov/ttnamti1/files/ambient/airtox/to-13arr.pdf. Accessed Sept. 6, 2011.
  8. Wenzl T, Simon R, Kleiner J, et al. Analytical methods for polycyclic aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the European Union. Trends Anal Chem. 2006;25(7):716-725.
  9. European Commission. Commission Recommendation of 4 February 2005 on the further investigation into the levels of polycyclic aromatic hydrocarbons in certain foods. Off J Eur Union. 2005;48(34):43-45.
  10. European Commission, Commission Regulation (EC) No 208/2005 of 4 February 2005 amending Regulation (EC) No 466/2001 as regards polycyclic aromatic hydrocarbons. Off J Eur Union. 2005;48(34):3-5.
  11. European Commission. Commission Recommendation of 4 February 2005 on the further investigation into the levels of polycyclic aromatic hydrocarbons in certain foods. Off J Eur Union. 2005;48(34):43-45.
  12. European Commission. Commission Regulation (EC) No 208/2005 of 4 February 2005 amending Regulation (EC) No 466/2001 as regards polycyclic aromatic hydrocarbons. Off J Eur Union. 2005;48(34):3-5.
  13. Poster DL, Schantz MM, Sander LC, et al. Analysis of polycyclic aromatic hydrocarbons (PAHs) in environmental samples: a critical review of gas chromatographic (GC) methods. Anal Bioanal Chem. 2006;386(4):859-881.
  14. Ziegenhals K, Hübschmann HJ, Speer K, et al. Fast GC-HRMS to quantify the EU priority PAH. J Sep Sci. 2008;31(10):1779-1786.
  15. Bordajandi LR, Dabrio M, Ulberth F, et al. Optimisation of the GC-MS conditions for the determination of the 15 EU foodstuff priority polycyclic aromatic hydrocarbons. J Sep Sci. 2008;31(10):1769-1778.
  16. Gómez-Ruiz JA, Wenzl T. Evaluation of gas chromatography columns for the analysis of the 15+1 EU-priority polycyclic aromatic hydrocarbons (PAHs). Anal Bioanal Chem. 2009;393(6-7):1697-1707.
  17. Gómez-Ruiz JA, Cordeiro F, Lopez P, et al. Optimisation and validation of programmed temperature vaporization (PTV) injection in solvent vent mode for the analysis of the 15+1 EU-priority PAHs by GC-MS. Talanta. 2009;80(2):643-650.

Advertisement

 

Current Issue

Current Issue

August/September 2014

Site Search

Site Navigation

 

Advertisements

 

 

Advertisements