Determination of Polycyclic Aromatic Hydrocarbons

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Bromatology, Area of Nutrition and Bromatology, E-15782 Santiago de Compostela, Spain ... Bromatology; and University of Santiago de Compostela, Faculty of ...
GONZÁLEZ AMIGO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 1, 2002 141

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Determination of Polycyclic Aromatic Hydrocarbons from Buzzards (Buteo buteo) and Tawny Owl (Strix aluco) by Liquid Chromatography with Fluorescence Detection SUSANA GONZÁLEZ AMIGO University of Santiago de Compostela, Faculty of Pharmacy, Department of Analytical Chemistry, Nutrition and Bromatology, Area of Nutrition and Bromatology, E-15782 Santiago de Compostela, Spain MARIA ASUNCIÓN LAGE YUSTY1 University of Santiago de Compostela, Faculty of Pharmacy, Department of Analytical Chemistry, Nutrition and Bromatology; and University of Santiago de Compostela, Faculty of Pharmacy, Institute of Research and Food Analysis, Laboratory of Bromatology, E-15782 Santiago de Compostela, Spain JESÚS SIMAL LOZANO University of Santiago de Compostela, Faculty of Pharmacy, Department of Analytical Chemistry, Nutrition and Bromatology, Area of Nutrition and Bromatology, E-15782 Santiago de Compostela, Spain

Supercritical fluid extraction was applied to the determination of naturally contaminated polycyclic aromatic hydrocarbons (PAHs) in bird tissue by liquid chromatography with fluorescence detection (LC-FL). Recoveries (>90%) and relative standard deviations (£ 7.7%) were satisfactory. The levels of 10 PAHs were analyzed in 6 classes of tissues (heart, liver, intestine, muscle, lung, and kidney) of 10 buzzards and 2 tawny owls, predatory birds from the Galicia (northwest Spain). The PAHs found most abundantly were pyrene, fluoranthene, benzo[a]anthracene, and anthracene. Chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[ghi]perylene, and indeno[1,2,3-cd]pyrene were not detected. Intestine, kidney, and lung were more polluted than other tissues.

olycyclic aromatic hydrocarbons (PAHs) are a class of diverse organic compounds containing 2 or more fused aromatic rings of carbon and hydrogen atoms. They are ubiquitous pollutants formed by combustion of fossil fuels and are always found as a mixture of individual compounds. The relative concentrations of PAHs in air, water, and food are usually the same, although this can change depending on sources of pollution (1). Because of their low vapor pressures, compounds with 5 or more aromatic rings are mainly adsorbed to airborne particulate matter, such as fly ash and soot. Those with 4 or fewer rings occur both in the vapor phase and adsorbed to particles. Numerous studies refer to the occurrence of PAHs in the environment. The concentrations differ widely owing to the widely differing sampling locations and

P

Received April 10, 2001. Accepted by JS July 24, 2001. 1 Author to whom correspondence should be addressed; e-mail: [email protected].

conditions (2–12). The combination of low analyte levels and a diversity of interferences have been reported by different methods of analysis (11, 13–17), but the trend has been to the increasing use of liquid chromatography (LC) with an ultraviolet or fluorescence detector. In this work, supercritical fluid extraction (SFE) was used for the determination of PAHs in 6 classes of tissues (heart, intestine, kidney, liver, lung, and muscle) of medium-sized predators: diurnal buzzard (Buteo buteo, Order Accipitriformes, Family Accipitridae) and nocturnal tawny owl (Strix aluco, Order Strigiformes, Family Strigidae), both protected and classified species of special interest (18). At present, few data are available on the levels of PAHs in birds. Bird exposure to a mixture of these compounds in environmental situations may be applicable to humans. This study reports the determination of some PAHs commonly found in environmental matrixes. The PAHs extracted by SFE were quantitatively determined by using LC methods with a fluorescence detector. Experimental Samples A total of 68 samples of heart, intestine, kidney, liver, lung, and muscle of 10 buzzards (Buteo buteo) and 2 tawny owls (Strix aluco) were collected and dissected by Rescue Wildlife Centers of Xunta de Galicia and the Laboratory of Parasitology in the Institute of Research and Food Analysis at the University of Santiago de Compostela, Spain. Samples (number in brackets) were obtained from different zones of La Coruña [4], Lugo [1], Orense [3], and Pontevedra [4]. Ten of the birds had been shot, one was beaten, and one died by an unknown cause. We analyzed 7 males and 5 females; 11 were adults and one was immature. The 6 organs to be studied were wrapped individually in aluminium foil and frozen until analysis.

142 GONZÁLEZ AMIGO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 1, 2002 Table 1. PAH content in kidney, intestine, and lung of individual samplesa Sample

Ant

Fl

Pyr

BaA

B1

Kidney

ND

ND

5.63

ND

B2

Kidney

ND

ND

NQ

ND

B3

Kidney

ND

5.61

6.70

ND

B4

Kidney

ND

ND

3.49

3.81

B6

Kidney

ND

ND

ND

ND

B7

Kidney

ND

ND

ND

ND

B8

Kidney

ND

ND

2.70

ND

B9

Kidney

ND

ND

ND

ND

B10

Kidney

ND

ND

8.73

ND

T1

Kidney

ND

T2

Kidney

0.94

B2

Intestine

ND

B3

Intestine

ND

B4

Intestine

ND

B5

Intestine

ND

B6

Intestine

ND

NQ

ND

ND

B7

Intestine

ND

ND

ND

ND

B8

Intestine

ND

15.45

8.18

ND

B9

Intestine

ND

ND

ND

ND

B10

Intestine

ND

ND

ND

ND

T1

Intestine

ND

ND

ND

ND

T2

Intestine

ND

ND

ND

ND

B1

Lung

ND

ND

6.21

ND

B2

Lung

ND

ND

1.06

ND

B3

Lung

ND

ND

ND

ND

B4

Lung

ND

ND

NQ

2.76

B5

Lung

ND

NQ

7.79

ND

B6

Lung

ND

ND

ND

0.88

B7

Lung

ND

ND

ND

NQ

B8

Lung

ND

ND

ND

ND

B9

Lung

ND

ND

ND

ND

B10

Lung

ND

ND

5.95

ND

T1

Lung

NQ

8.48

NQ

T2

Lung

NQ

ND

ND

a

9.60

ND

ND

NQ

ND

1.19

ND

ND

ND

ND

ND

0.40

0.24

13

0.95 10.5

36.1 NQ

12.4

ND

Abbreviations: Ant = anthracene; Fl = fluoranthene; Pyr = pyrene; BaA = benzo[a]anthracene; B = buzzard; T = tawny owl; ND = not detectable; NQ = not quantitatable.

Analysis Samples were analyzed for anthracene (Ant), benzo[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), benzo[ghi]perylene (BghiP), benzo[k]fluoranthene (BkF), chrysene (Chry), fluoranthene (Fl), indeno[1,2,3-cd]pyrene (Ind), and pyrene (Pyr), following the method described by Gónzalez et al. (19). This method

involves SFE (Hewlett-Packard 7680 A) of 0.5 g dried sample (20) mixed with silicagel, 10 min static equilibration at 100° C and 256 bar, followed by 50 min extraction in the dynamic or continous flow mode at a rate of 1.5 mL/min of supercritical CO2 and a density of 0.60 g/mL, 75° C restrictor temperature, collection on octadecylsilane trap at 75° C, and elution with four 1.5 mL portions of acetonitrile. During extraction, the trap and nozzle were kept at 50° C. The acetonitrile of the 4th vial was discarded. The acetonitrile extract collected (4.5 mL) was filled to a volume of 5 mL and filtrated through 0.5 m m pore size Micro Filtration Systems-25 PTFE filters. LC analysis of the PAHs was performed with a high-pressure isocratic pump P100 from Spectra-Physics using a Sugelabor reversed-phase tracer Tr-C-160 C18 precolumn (Madrid, Spain) and Teknokroma Hypersil Green PAH column (5 mm particle size, 0.46 ´ 10 cm; Kent, United Kingdom), an FL2000 fluorescence detector, and a Datajet integrator connected by Labnet to a PC with Winner on Windows (WOW) data processing software. A 20 m L aliquot of acetonitrile solution was injected into the LC system and eluted with acetonitrile–water (85 + 15, v/v) at a constant flow rate of 0.5 mL/min. For detection and quantitation, 2 excitation (Ex) and emission (Em) wavelength programs were used: program 1 for Ant and Pyr (Ex 250 nm, Em 400 nm), BaA and Chry (Ex 270 nm, Em 390 nm), BbF (Ex 296 nm, Em 426 nm), BaP (Ex 296 nm, Em 406 nm), and Ind (Ex 304 nm, Em 470 nm); program 2 for Fl (Ex 286 nm, Em 456 nm), BkF (Ex 296 nm, Em 406 nm), and BghiP (Ex 296 nm, Em 426 nm). Quantitative analysis of the 10 PAHs was performed using external standard calibration with a reference standard solution diluted appropriately. The identity of the analytes was confirmed according to Gónzalez et al. (19) by 2 approaches: (1) By LC under the conditions described above with Ex and Em wavelength, program 1, BkF (Ex 296 nm, Em 426 nm) and BghiP (Ex 296 nm, Em 406 nm) were confirmed. By LC under the conditions described with Ex and Em wavelength, program 2, BaA and Chry (Ex 266 nm, Em 380 nm), BbF (Ex 296 nm, Em 406 nm), and BaP (Ex 296 nm, Em 426 nm) were confirmed. Under these conditions, different intense emissions bands were detected. (2) The detector was set to sweep mode, and synchronous spectra of the eluting peak were recorded and compared with those of standards. For constant-wavelength synchronous spectra, the scan type was delta, the scan length was 100 nm, and the step size was 2 nm. Because these experiments require PAH concentrations between 0.4 m g/L for Ant and 5 m g/L for BbF, the remaining solution was concentrated before being injected 10 times. Detection and quantitation limits calculated following American Chemical Society guidelines (21) as the concentration corresponding to the signals equal to the mean signal for 6 blanks plus, respectively, 3 and 10 standard deviations, were