3rd meeting of young researchers at UP 1 - IJUP - Universidade do ...

Quantification of Polycyclic Aromatic Hydrocarbons in barbecued
meat by HPLC/Fluorescence
I. Mariano 1 , O. Viegas 1,2 , O. Pinho 1,2 , I.M.P.L.V.O. Ferreira 1
1 REQUIMTE- Serviço de Bromatologia, Faculdade de Farmácia da Universidade do Porto, Rua Anibal
Cunha 164, 4099-030 Porto; Portugal
2 Faculdade de Ciências da Nutrição e Alimentação da Universidade do Porto, Rua Dr. Roberto Frias,
4200-465 Porto, Portugal
Polycyclic aromatic hydrocarbons (PAHs) are a group of about 10,000 compounds, a few of
which occur in considerable amounts in the environment and food. Due to their carcinogenic
and mutagenic properties they are considered to be top of the list of the most hazardous
substances. Human beings are exposed to PAHs via air and drinking water, but mostly by
intake of food, especially, charcoal grilled and smoked meat [1].
The analysis of PAHs in food samples is hampered owing to their occurrence in food at
microtrace levels, i.e. ppb or ppt levels and the co-extraction of interfering compounds present
in the complex food matrices. Additionally, PAH are characterized by structural similarity and
many occur as isomers, which again makes identification of individual compounds extremely
difficult. Thus, the challenge for its analysts is to maximize recovery of analyte and minimize
the accompanying interferences by proper extraction and clean-up procedures.
This work concerns the optimization of a method for determination of PAHs in barbecued meat
by application of tandem solid-phase extraction (SPE) with columns filled with Extrelut
diatomaceous earth, and column chromatography on silica gel for selective isolation of the 16
PAHs highlighted by the US Environmental Protection Agency (EPA). Identification and
quantitative analysis of the individual compounds were achieved by HPLC/Fluorescence after
separation on a C18 column. Gradient elution was carried out with a mixture of three eluents:
Eluent A was water/methanol (25:75), eluent B was methanol and eluent C was ethyl acetate.
The following gradient was used: 0–18 min, 100-20% A and 0-80% B; 18-19 min 20-0% A
and 80-100% B; 19-20 min 100-90% B and 0-10% C; 20-28.5 min 90-82% B and 10-18% C;
28.5-37.5min 82-80% B and 18-20% C; returning to initial conditions at 45 min and
maintaining during 5 min., in a total run time of 50 min. The flow rate was 1.0 mL/min. These
conditions are a good alternative to the use of acetonitrile in the HPLC mobile phase [2] since
it is a less expensive solvent for monitorization of PAHs. Additionally, experiments were
carried out to determine maximum fluorescence excitation and emission wavelengths, to
improve detection limits. The multistep clean-up procedure optimized, based on tandem solidphase
extraction, in combination with the chromatographic conditions selected enabled an
accurate and reproducible separation of PAH fractions from the cooked meat samples.
References:
[1] Wenzl, T., Simon, R., Kleiner, J. and Anklam, E. (2006), Analytical methods for polycyclic
aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the
European Union. Trends in Analytical Chemistry, 25 (7), 716-725.
[2] Janoszka, B., Warzecha, L., Błaszczyk, U. and Bodzek, D. (2004), Organic compounds formed in
thermally treated high-protein food. Part I: polycyclic aromatic hydrocarbons. Acta chromatographica,
14, 115-128.
12 3 rd meeting of young researchers at UP