Ld50-acenaphthene

3.1 Effects on laboratory mammals in vitro

The biological mechanisms to produce adverse effects of the majority of PAHs are not well understood yet. Except for Naph, there are only a limited number of studies available for the evaluation of acute oral toxicity. The values of oral LD50

Acenaphthene (Ace)

Acenaphtylene (Acn)

Anthanthrene (Anth)

Anthracene (An)

Benz[a]anthracene3 Benzo[£>]fluoranthene (B[a]A) B[£>]F

Benzo[/]fluoranthene Benzo[/c]fluoranthene Benzo[ g/7/']fluoranthene Benzo[g/7/']perylene B[y]F B[k]F (B[ghi]F\u) (B[ghi]P)

Dibenzo[a/7]pyrene (DB[ah]P)

Naphthacene (Naphth)

Benzo[c]phenanthrene (B[c]Phe)

Benzo[e]pyrene Chrysene

Benzo[e]pyrene Chrysene

Naphthalene (Naph)

Coronene Cyclopenta[ccf]pyrene Dibenz[a,/7]anthracene Dibenzo[ae]pyrene

Fluoranthene (Flu)

Fluorene (Fl)

Perylene (Per)

Triphenylene (TriPhen)

Perylene (Per)

Phenanthrene (Phe)

Pyrene

Triphenylene (TriPhen)

'abbreviation in the parenthesis is used in this chapter Figure 1 Structural formulae of PAHs.

Table 1 Physical and chemical properties of polycyclic aromatic hydrocarbons3

IUPAC name

(Abbreviation

CAS no.

Chemical

Crystal

Synonyms

used in this chapter)

formula, molecular weight

colourb

Acenaphthene

Ace

83-32-9

c12h10

White crystal

1,2-Dihydroacenaphthylene

154.21

Acenaphtylene

Acn

208-96-8

c^hg

No data

1,2-Dihydroacenaphthalene

152.2

Anthanthrene

Anth

191-26-4

c22h12

No data

Dibenzo[def,mono]chrysene

276.34

Anthracene

An

120-12-7

C14h10

White crystalline

Paranaphthalene

178.24

flakes, bluish-violet fluorescence

Benz[a] anthracene

B[«]A

56-55-3

c18h12

Colourless

1,2-Benzanthracene

228.3

leaflets or

2,3-Benzophenanthrene

plates

Benzo[b]fluoranthene

B[b]F

205-99-2

c20h12

Needles

2,3-Benzofluoranthene

252.32

3,4-Benzofluoranthene

Benzo[/]fluoranthene

B[j]F

205-82-3

c20h12

No data

10,11-Benzofluoranthene

252.32

Benzo-12,13-fluoranthene

Benzo[/c]fluoranthene

B[fc]F

207-08-9

c20h12

Pale yellow

11,12-Benzofluoranthene

252.32

needles

ll,12-Benzo[fc]fluoranthene

Benzo[ghi\ fluoranthene

B[ghi]Flu

203-12-3

c18h10

No data

2,13-Benzofluoranthene

226.28

Benzo[ghi\ perylene

B[ghi]F

191-24-2

c22h12

No data

1,12-Benzoperylene

276.34

Benzo[c] phenanthrene

B[c]Phe

195-19-7

c18h12

No data

3,4-Benzophenanthrene

228.3

Benzo[«]pyrene

B[a]P

50-32-8

c22h12

yellow needles

3,4-Benzopyrene

252.32

3,4-Benzpyrene

Benzol] pyrene

BMP

192-97-2

c22h22

Colourless

4,5-Benzopyrene

276.34

crystals

Melting Boiling Water Octanol- Vapor point point (°C) solubility water pressure (°C) (mg/L) (log P) (mm Hg)

93.4 279

92.5 280 264b No data 215 339.9

84 437.6

168 No data

166 No data

217 480

226.28 No data

278 >500

68 No data

176.5 311

177.5 310-312

0.00127 7.04

0.0015 5.78

0.0025 6.11

0.0008 6.11

No data No data

0.00026 6.63

0.0025 (25°C) 0.000912 (25°C) 8.75E-010 (25°C) 2.67E-006 (25°C)

No data 1E-010 (25°C) 6.66E-007 (25°C) 5.49E-009 (25°C)

Chrysene Chry 218-01-9 C1BH12

1,2,5,6-Dibenzo naphthalene 22S.3 1,2-Benzophenanthrene

Coronene Cor 191-07-1 C24H12

Hexabenzobenzene 300.36

Cyclopenta[ai]pyrene Cyclo[ai]P 2720S-37-3 C1BH12

Cyclopentano[ai]pyrene 228.29

1,2:5,6-Dibenzanthracene 27S.36

DibenzMpyrene DB[»e]P 192-65-4 C24H14

l,2:5,6-Dibenzopyrene 302.3S

3,4:S,9-Dibenzopyrene 302.3S

3,4:9,9-Dibenzopyrene 302.3S

l,2:3,4-Dibenzopyrene 302.3S

Fluoranthene Flu 206-44-0 C16H10

1.2-Benzacenaphthene 20226 Benzo[/ic]fluorene

Fluorene F1 S6-73-7 C13H10

2.3-Benzindene 166.22 2,2'-Methylenebiphenyl

Indeno[l,2,3-cd]pyrene In[ai]P 193-39-5 CEH]2

2,3-o-Phenylenpyrene 276.34

Naphthacene Naphth 92-24-0 C1BH12

2,3-Benzanthracene 22S.3 Tetracene

Naphthalene Naph 91-20-3 C10HB

128.18

Perylene Per 19S-55-0 C20H12

Pen-dinaphthalene 252.32

Phenanthrene Phe S5-01-S C14H10

178.24

Pyrene Py 129-00-0 C16H10

Benzo[ii<?/l phenanthrene 20226

Triphenylene TriPhen 217-59-4 C1BH12

9,10-Benzophenanthrene 22S.3

interactive PhysProp Database Demo. bChemFinder, http://chemfinder.cambridgesoft.com/

White crystals, orthorhombic bipyramidal plates from benzene

No data White crystals No data No data No data No data

437.3 No data 269.5 233.5 317 281.5

162.4

No data 524

No data No data

0.00014 (25°C) No data 0.00249 8.02E-005 (25°C) 7.28 3.5E-005 (25°C) 7.28

02 mmHg) No data

Coloured needles 107.8

7.64

No data 6.75

2.17E-012 (25°C) No data 1E-010 (20°C) 7.03E-011 (25°C) 6.41E-012 (25°C) 1.78E-011 (25°C) 4.8E-010 (25°C) 9.22E-006 (25°C)

White leaflets

No data No data

Colourless to brown solid No data

Colourless solid or monoclinic crystals Colourless to light yellow solid No data

163.6 536

No data No data

80.2 217.9

252.32 274

99.2 340

No data No data

Os O

reported are, B[a]P: > 1,600 mg/kg body weight in mice [50], Phe: 700,1,000 mg/kg in mice [48], Naph: 490—9,430 mg/kg in rats [51,52]. Oral toxicity of PAHs ranges from low to moderate. Adverse effects such as myelotoxicity, hemolymphatic changes and anemia were observed in the short-term studies of PAHs [53]. In long-term testing, many PAHs are known to be capable of inducing cancer in experimental animals [54].

Most studies to assess the carcinogenic potential of PAHs were carried by dermal, subcutaneous or inhalation exposure. There are only a limited number of studies that dealt with oral administration. When administrated in the diet, PAHs produced tumors in the gastrointestinal tract, liver, lungs and mammary glands of mice and rats. When administered by gavage, B[«]P induced malignant and benign forestomach tumors in mice and hamsters, and mammary tumors in female rats. Culp et al. [55] showed that papillomas and squamous cell carcinomas were observed in the forestomach of rodents. Besides these major target sites, B[«]P treatment also induced soft tissue sarcomas at various sites such as oesophagus, skin and mammary [56]. In a gavage study, B[«]A produced papillomas of the forestomach as well as lung adenomas and hepatomas in mice. When administered as an olive oil emulsion in drinking water, DB[«h]A induced alveologenic carcinomas of the lung and hemangioendotheliomas in mice [54].

As cancer is often linked to DNA damage, it is important to assess the genotoxicity of chemicals to estimate carcinogenic potency. To evaluate the genotoxicity of chemicals, a variety of short-term tests have been developed. Among them, the 'Ames test' is a well-known bacterial mutagenicity test.

Mutagenicity of selected PAHs in the Ames test are summarized in Table 2. It is apparent that most of the PAHs, especially those containing four or more aromatic rings in their chemical structure, show positive responses in the Ames test.

To examine the genotoxic profiles of PAHs, a bibliographic database, TOXNET (Toxicology Data Network), by the Environmental Mutagen Information Center (EMIC) is available. Users can search by subject terms, title words, chemical names, Chemical Abstracts Service Registry Numbers (CAS no.) and authors (EMIC) [57].

The overview of the genotoxicity of PAHs considered by the International Programme on Chemical Safety (IPCS) [58] is partly summarized in Table 3.

Among 30 PAHs listed in Table 3, 16 PAHs are genotoxically positive and finally 3 PAHs, i.e., An, Fl and Naph, are totally or probably inactive in all short-term tests. For the other PAHs, the evidence of genotoxicity is limited and based on results mainly obtained in in vitro systems. Therefore, they cannot be clearly classified as mutagenic.

In the detoxification pathway of PAHs, they are initially oxidized to arene oxides and phenols by the mixed-function oxidase, which converts the non-polar PAHs into polar hydroxy and epoxy derivatives. For example, B[«]P is initially converted to several epoxides such as 7,8-epoxide, and then hydrolyzed to the diolepoxides such as 7,8-dihydrodiol-9,10-epoxide, which are considerably more mutagenic than the parent compounds. These so-called bay-region (e.g., stereochemically hindered, cup-shaped area between carbons 10 and 11 of

Table 2 Mutagenicity of polycyclic aromatic hydrocarbons in the Ames test

TA98 TA100

Table 2 Mutagenicity of polycyclic aromatic hydrocarbons in the Ames test

TA98 TA100

+S9mix

Reference

+S9mix

Reference

Acenaphthene

[59]

[59]

Acenaphtylene

-

[59]

[59]

Anthanthrene

+

[60]

+

[61]

Anthracene

[62,63]

[62,63]

Benz[a]anthracene

+

[60,62]

+

[60,62]

Benzo[b]fluoranthene

+

[60]

+

[64]

Benzo[j]fluoranthene

No data

No data

+

[64]

Benzo[k]fluoranthene

+

[60]

+

[64]

Benzo[ghi]fluoranthene

[65,66]

+

[65]

Benzo[ghi]perylene

+

[67]

+

[68,69]

Benzo[c]phenanthrene

+

[70]

+

[69,70]

Benzo[a]pyrene

+

[62,71]

+

[62,71]

Benzo[e]pyrene

7

[60,62,63]

7

[62,72]

Chrysene

+

[59,62]

+

[60,62]

Coronene

+

[59]

No data

No data

Cyclopenta[ cd]pyrene

+

[70]

+

[70]

Dibenz[ae]anthracene

+,7

[73]

+

[73]

Dibenz[ah]anthracene

+

[60]

+

[62]

Dibenzo[flh ]pyrene

+

[74]

+

[74]

Dibenzo[ai ]pyrene

+

[60]

+

[62]

Dibenzo[flZ ]pyrene

+

[60,65]

+

[65]

Fluoranthene

+

[67,75]

+

[75]

Fluorene

+

[62]

+

[62]

Indeno[1,2,3-cd]pyrene

+

[76]

+

[77]

Naphthacene

+

[61]

+

[61]

Naphthalene

[52,63]

[62,63]

Perylene

+

[70,78]

+

[78]

Phenanthrene

[59,62]

No data

[59,62]

Pyrene

[59,62]

[59,62]

Triphenylene

+

[67,75]

+

[75]

B[«]P) diolepoxide are considered to be the ultimate mutagenic and/or carcinogenic species of PAHs [79]. It is well known that these metabolizing systems for PAHs are widely distributed in animal tissues. Among them, liver is the most active in metabolizing capacity, followed by lung, intestinal mucosa and kidneys. It is noteworthy that PAHs are generally potent inducers of mixed-function oxidases and potentiate their own toxicity. These metabolic intermediates are then converted to water-soluble conjugates with glutathione and glucuronic acid, which enable excretion to occur via the kidney. Formation of these conjugates is regarded to be a true detoxification.

Table 3 Evaluations of genotoxicity and carcinogenicity of polycyclic aromatic hydrocarbons

IUPAC name

Genotoxicitya

Carcinogenicity

IARCb

EPAc

Acenaphthene

(?)

3

Acenaphtylene

(?)

D

Anthanthrene

(+)

3

Anthracene

3

D

Benz[a ]anthracene

+

2A

B2

Benzo[b]fluoranthene

+

2B

B2

Benzo[j]fluoranthene

+

2B

Benzo[k]fluoranthene

+

2B

B2

Benzo[ghi]fluoranthene

(+)

3

D

Benzo[ghi]perylene

+

3

Benzo[c]phenanthrene

(+)

3

Benzo[a]pyrene

+

2A

B2

Benzo[e]pyrene

+

3

Chrysene

+

3

B2

Coronene

(+)

3

Cyclopenta[cd]pyrene

+

3

Dibenz[ah ]anthracene

+

2A

B2

Dibenzo[ae]pyrene

+

2B

Dibenzo[ah]pyrene

(+)

2B

Dibenzo[ai]pyrene

+

2B

Dibenzo[al]pyrene

(+)

2B

Fluoranthene

+

3

D

Fluorene

3

D

Indeno[1,2,3-cd]pyrene

+

2B

B2

Naphthacene

Naphthalene

2B

Perylene

+

3

Phenanthrene

(?)

3

D

Pyrene

(?)

3

D

Triphenylene

+

3

aRef. [78], pp. 47-96. Classification: +, positive; —, negative and ?, questionable. Parentheses, result derived from small database. Short-term tests used for the evaluation are as follows: reverse mutation test in Salmonella typhimurium (Ames test), forward mutation test in S. typhimurium strain TM677, DNA binding in mammalian cells in vitro, DNA damage/repair in bacteria, DNA damage/repair in mammalian cells, mitotic gene conversion in yeast, mitotic recombination in yeast, forward mutation in yeast, host-mediated assay (bacteria), host-mediated assay (yeast), sex-linked recessive lethals (Drosophila), somatic mutation and recombination (Drosophila), DNA repair (Drosophila), HPRT system, thymidine kinase system, ouabain resistance, diphtheria toxic resistance, chromosomal aberrations, sister chromatid exchanges, micronucleus test, DNA damage/repair in vivo (various tissues), cytogenetic effects in somatic cells, cytogenetic effects in germ cells, sperm abnormalities and dominant lethals.

bRef. [80]. Classification: Group 1, carcinogenic to humans; Group 2A, probably carcinogenic to humans; Group 2B, possibly carcinogenic to humans; Group 3, not classifiable as to its carcinogenicity to humans and Group 4, probably not carcinogenic to humans.

cEPA, US Environmental Protection Agency, Integrated Risk Information System (IRIS), Evidence for Human Carcinogenicity Weight of Evidence Characterization. Available at http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?IRIS. Classification: Group A, human carcinogen; Group B1, probable human carcinogen based on limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in animals; Group B2, probable human carcinogen based on sufficient evidence of carcinogenicity in animals; Group C, possible human carcinogen; Group D, not classifiable as to human carcinogenicity and Group E, evidence of non-carcinogenicity for humans.

3.2 Effects on humans

There is inadequate evidence for the carcinogenicity of PAHs in humans [54]. However, there are a number of occupational epidemiologic studies that show increased incidence of cancer in humans exposed to mixtures of PAHs by inhalation or dermal contact. The first one was reported by Sir Percival Pott in 1775, as has already been mentioned, who reported the increased incidence of scrotal cancer among chimneysweepers. After this pioneer study, different epidemiological studies pointed out the high incidence of tumors in workers exposed to coke oven emissions [81,82], roofing-tar emissions [83] and cigarette smoke [84,85].

As for oral exposure of PAHs, there is a little information on the influences to human health. In the Van region of eastern Turkey, upper gastrointestinal (oesophageal and gastric) cancers are endemic and dietary factors are considered to play an essential role in carcinogenesis. Turkdogan et al. [86] studied B[«]P and B[«]A levels of cooked foods and revealed that traditional foods, which are baked or cooked using animal manure or fuel oil, are highly contaminated by PAHs, and that they may constitute a serious risk factor in this region. Lopez-Abente et al. [87] reported the relationship between the oral exposure of PAHs and health effects in rural areas in Spain. In these areas, wine has been traditionally stored in leather bottles sealed with a tar-like substance, which contain PAHs. By multivariate analysis, increased risk of colorectal adenomas was more strongly associated with B[«]P intake. Sinha et al. [88] estimated dietary intake of B[«]P to test its relationship with risk of colorectal adenomas in a case-control study. They developed a food frequency questionnaire on meat-cooking methods, doneness levels and B[«]P database based on the collection and analysis of a wide range of food samples. By multivariate analysis, increased risk of colorectal adenomas was regarded to be associated with dietary intake of B[«]P. These studies provide evidence that dietary intake of B[«]P plays a role in colorectal adenoma etiology.

Evaluations on the carcinogenicity of selected PAHs by the International Agency for Research on Cancer (IARC) [80] and by the EPA are shown in Table 3. Both IARC and EPA classified PAHs into different categories according to the evidence available on their carcinogenicity. The criteria applied by each institution to classify carcinogenicity are shown in the footnote of the table.

The main problem in evaluating carcinogenicity of PAHs in food and environment is that they are not present as individual compounds but as complex mixtures. The toxic equivalence is an approach proposed to assess the carcinogenic potency of complex mixtures. This concept requires that PAH concentrations are summed and also expressed as B[«]P equivalents, their relative concentrations are weighted in relation to the carcinogenic potential of individual PAH compounds using toxic equivalency factors (TEF). Toxic equivalents (TEQs) are defined as follows:

where Q is the concentration of individual PAHs identified in a complex mixture and TEFi the relative potencies of PAHi in comparison with that of B[«]P.

The concept of TEQs was initially developed to estimate the potential toxicity of complex mixtures of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). Also in PAHs, there have been several studies on the estimation of the total carcinogenicity by TEFs of individual PAHs [89-96]. The TEFs reported are compared in Table 4. DB[«h]A appears to be equipotent or somewhat more potent than B[«]P. B[b]F, Ind[cd]P and B[«]A were about 10% and Naph, Phe and Fl were about 1% as potent as B[«]P. The greatest variation is observed for Chry.

Most studies to assess the carcinogenic potency of PAHs were carried out following skin application, pulmonary instillation and intraperitoneal injection. There are only a limited number of studies available on acute oral toxicity. The oral toxicity of PAHs, therefore, has been speculated by these studies.

There are some limitations in using the TEF approach in the risk assessment of PAHs in food. Application of the TEF approach to risk assessment is justifiable when all the chemicals in the mixture act in the same way, by the same mechanism.

Dioxins bind directly to Ah receptor without biotransformation and this binding is considered to be responsible for their toxicity. Although PAHs generally bind to the Ah receptor, this is not the only one that determines the carcinogenic effect of PAHs. PAHs cannot express their carcinogenic potential until they have been metabolized to forms that can bind to DNA. PAHs are not necessarily activated through the same metabolism. Moreover, there is no

Table 4 Estimations of carcinogenic potencies of various polycyclic aromatic hydrocarbons, relative to benzo[a]pyrene

Compound References

Table 4 Estimations of carcinogenic potencies of various polycyclic aromatic hydrocarbons, relative to benzo[a]pyrene

Compound References

[97]

[98]

[99]

[100]

[101]

Anthracene

0.28

0.01

Benz[a]anthracene

0.1

0.1

0.145

0.014

0.1

Benzo[a]pyrene

1.0

1.0

1.0

1.0

1.0

Benzo[b]fluoranthene

0.1

0.1

0.167

0.11

0.1

Benzo[e]pyrene

0

Benzo[ghi]perylene

0.012

0.01

Benzo[j]fluoranthene

0.1

0.1

0.045

Benzo[k]fluoranthene

0.1

0.1

0.020

0.037

0.1

Chrysene

0.1

0.01

0.0044

0.026

0.01

Dibenz[ah]anthracene

1

1.11

0.89

5

Dibenzo[flh]pyrene

1

10

1.2

Fluoranthene

0.001

Indeno[1,2,3-cd]pyrene

0.1

0.1

0.055

0.067

0.1

Naphthalene

0.001

Phenanthrene

0.00064

0.001

Pyrene

0

0.001

corroboration that different PAHs induce the same type of mutations in the same organs or tissues [102].

The TEFs approach essentially relies on the additive theory, in which case there is no interaction (i.e., synergistic and/or antagonistic effects) between the components in the complex mixture. Some PAHs, however, have been reported to have greater interacting effects than other PAHs. For example, B[e]P, B[ghi]P, Flu and Py show the synergistic effects to B[«]P-induced tumor incidence [55]. B[e]P, Flu and Py have weak tumor-promoting activity following initiation by B[«]P [79]. Interactions between some PAHs and B[«]P are also reported to reduce the carcinogenic activity of B[«]P in animals [79].

Krewski et al. [103] analysed the published values and derived a new set of TEF values. Applications of their TEFs to the results of bioassays with PAH mixtures [104,105] indicated that their values would be unlikely to underestimate the carcinogenic risk posed by whole mixtures [55]. Schneider et al. [106] also examined the use of the TEF derived by Brown and Mittelman [107] and concluded that the TEFs approach may underestimate the carcinogenic potencies of PAHs mixtures in most cases.

Because of some uncertainty in the TEF approach mentioned above, the Scientific Committee on Food, European Commission, does not find it necessarily appropriate to endorse the use of the TEF approach for the risk assessment of PAH in food [102].

4. ANALYTICAL METHODS

Analysis of PAHs in food samples is problematic because of their extremely low concentrations and their affinity to be in the fatty fraction of food. As food samples naturally contain large quantities of fats and lipids, it is very important to extract and eliminate these fats before instrumental analysis, without losing the PAHs in any of the steps. In the analysis of PAHs, it is also necessary to pay attention to the chemical properties, i.e., especially instability and sublimation, of these compounds at all stages to achieve accurate analysis.

Tamakawa et al. [108] showed photodecomposition of seven PAHs (i.e., An, Py, B[«]A, B[b]F, B[k]F, B[«]P and B[ghi]P) in acetonitrile under usual experimental conditions. PAHs, especially B[«]P and An, were easily decomposed within 5 h under these conditions. Takatsuki et al. [109] showed that B[«]P was decomposed by the coexistence of alkaline, light and oxygen; by peroxides in aged ethyl ether; and by oxygen when absorbed on silica gel. In the analysis of PAHs, therefore, it is recommended that samples be handled under ultraviolet (UV)-protected fluorescent lamps at all stages to achieve accurate results [110].

Moreover, low-molecular-weight PAHs, containing four or fewer aromatic rings such as An, Flu and Py, are easily sublimated during the drying-up processes in the preparation of PAHs (Figure 2). It is also critical to concentrate the extracting solvent carefully to ensure that the sample does not dry up completely.

Time (min) of N2 blowing after drying-up

—♦—Anthracene —■—Fluoranthene —A— Pyrene

—□— Benz[a]anthracene —Benzo[£>]fluoranthene —•— Benzo[a]pyrene —©— Benzo[ghi ]perylene Figure 2 Influences of N2 blowing on the recoveries of PAHs.

To correct the losses of PAHs during the analysis, the use of internal standards (surrogates) is recommended before the quantification by instrumental analysis such as high-performance liquid chromatography (HPLC) and gas chromato-graphy-mass spectrometry (GC-MS) [111-114]. Dunn and Armour [115] used tritium-labeled B[«]P as an internal standard in the HPLC determination to correct the losses of B[«]P during the purification procedure. In the United Kingdom (UK) Total Diet Study [116], 13C-labeled PAHs are also used as internal standards. Surrogates generally should be added to samples at a concentration similar to that expected for the analytes of interest [111].

A variety of analytical methods for multiresidue analysis of PAHs have been proposed since the 1970s. Typical methods include: extraction, cleanup and instrumental quantification using GC coupled with flame ionization detector (FID) or MS and HPLC with an UV detector or fluorescence detector.

Typical analytical methods for PAHs in food are shown in Tables 5 and 6.

4.1 Extraction

The PAHs extraction procedures most often used in water and beverages samples are liquid-liquid extraction (LLE) [117,118] and solid-phase extraction (SPE) [119]. These methods are applicable to food samples with comparatively low fat

Table 5 Methods used for polycyclic aromatic hydrocarbon detection: high-performance liquid chromatography (HPLC)

Analyte

Sample type Sample preparation

Quantitative HPLC

Reference

Stationary phase

Mobile phase Detection

BMP, Chry, B[e]P, B[fc]F, B[/i]F, BMP, B[g/n]P Flu, Py, BMA, Chry, B[e]P, B[fc]F, B[fc]F, BMP, DB[îi/Î]A, B[g/n]P, In[crf]P An, Py, BMA, B[fc]F, B[fc]F, BMP, B[g/n]P

Fish, shellfish

Shellfish

Daily diet

Daily diet

Naph, Ace, Fl, Phe, An, Flu, Py, BMA, Chry, B[fc]F, B[fc]F, BMP, DB[îi/Î]A, B[g/n]P, In[crf]P

Seafood

Liquid smoke flavor

Alkaline saponification with Na2S as antioxidant, hexane extraction, silica gel column purification Alkaline saponification, trimethylpentane extraction, GPC purification

Radial-Pak PAH analytical column 300 mm (L) x 15 mm (i.d.)

Vydac 201 TP54 reverse-phase CIS 150 mm (L) x 4.6 mm (i.d.)

AcCN/H20 gradient

Alkaline saponification with Na2S Radial-Pak 5PAH10 AcCN/H20

as antioxidant, hexane extraction, 100 mm (L) x 5 mm (i.d.) (80:20, v/v) Sep-Pak column purification (silica)

Alkaline saponification, hexane extraction, Sep-Pak column purification (silica)

Analytical HC-ODS AcCN/H20

Alkaline saponification,

1,1,2-trichlorotrifluoroethane extraction, SPE column purification (alumnina, silica, CIS)

Vydac 201 TP54 AcCN/H20

reverse-phase CIS gradient

Hexane extraction under alkaline condition

Separon SGX C18 AcCN/H20

Fluorescence (Ex: 370 nm, [120]

UV 254 nm, fluorescence [121]

Fluorescence An, Py, BMA [122]

(Ex: 334 nm, Em: 384 nm) B[fc]F, B[fc]F, BMP, B[g,/!,i]P (Ex: 365 nm, Em: 430 nm) Fluorescence [123]

An, Py (Ex: 334 nm, Em: 384 nm) B[fc]F, BMP, B[g,/!,i]P (Ex: 384nm,

Em: 406 nm) Fluorescence [124]

Em: 390 nm) B[fc]F, B[fc]F, BMP, DB[rt,/!]A (Ex: 290 nm, Em: 410 nm) B[g,h,i]P, In[c,rf]P (Ex: 300 nm,

Analyte Sample type Sample preparation

Phe, An, Flu, Py, Oyster B[u]A, Chry, B[fe]F, B[fc]F, BMP, B[gfa]P Naph, Fl, Phe, Smoked and An, Flu, Py, broiled fish

Phe, An, Flu, Py, BMA, Chry, BMF, BMF, B[e]P, BMP, DB[u/!]A, Pery, B[gta]P, In[ed]P, Cor, Anth

Edible oils, fats

Soxhlet extraction (methylene chloride) HPLC preparation, SPE column purification (cyanopropyl) Supercritical fluid extraction (SFE) with MeOH as modifier, SPE cleanup with bilayer mini column (alumina+silica gel), octadecyl SPE-cartridge purification (Bond Elut CI 8) On-line donor-acceptor complex chromatography (DACC) cleanup (DACC column, Chromsher PI)

Quantitative H PLC

Reference

Stationary phase Mobile phase Detection

LC-PAH column CH30H/H20 Fluorescence [126]

LiChrospher 100 RP-18 column 125 mm (L) x 4 mm (i.d.)

AcCN/H20 stepwise gradient

UV 254 nm

Chromspher 5PAH AcCN/H20

Fluorescence [128]

Naph, Ace, Fl, Smoked food Alkaline saponification, Sep-Pak Phe, An, Flu, column purification (Florisil)

Py, B[u]A, B[fc]F, B[fc]F, B[u]P, DB[u/!]A, B[g/n]P, In[e<i]P

Naph, Ace, Fl, Vegetable oils Phe, An, Flu, Py, B[u]A, Chry, B[fc]F, B[fc]F, B[e]P, B[u]P, DB[u/!]A, B[ghi]P, In[cii]P Phe, Flu, Py, Tea infusion B[u]A, B[e]P, samples

Soxhlet extraction (methylene chloride) SPE column purification (Florisil)

Extraction and preconcentration by Sep-Pak vac tC-18 cartridge

B[u]P Smoked products

Lyophilization, extraction/

sonication in hexane, SPE column purification (silica) hexane/ DMSO partition, concentration by Sep-Pak C18 plus cartridge

C18 column AcCN/H20

Fluorescence [129]

340 nm) Acn (Ex: 320 nm, Em: 533 nm) Phe (Ex: 254 nm, Em: 375 nm) An, Flu (Ex: 260 nm, Em: 420 nm) Py, B[n]A, Chry (Ex: 254 nm, Em:

390 nm) B[fa]F, BMF, BMP, DB[«,/!]A, B[g,/!,i]P (Ex: 260 nm, Em: 420 nm)

Nova-Pak CIS column AcCN/H20

Tracer PAH column AcCN/H20

Fluorescence [119]

Phe (Ex: 250 nm, Em: 365 nm) Flu (Ex: 285 nm, Em: 465 nm) Py (Ex: 270 nm, Em: 390 nm) B[n]A (Ex: 287 nm, Em: 388 nm) B[e]P (Ex: 290 nm, Em: 390 nm) B[n]P (Ex: 295 nm, Em: 405 nm) DB[«,/!]A, B[g,/!,i]P (Ex: 290 nm,

Analyte Sample type Sample preparation

B[u]A, Chry, Lipidic extracts (silica column, 250 x 4.6 mm i.d.)

Naph, Ace, Fl, Grapeseed oil Ultrasonic extraction (acetone),

Phe, An, Flu, HPLC preparation (silica column,

Py, B[u]A, 250 x 4.6 mm i.d.) B[fe]F, B[fc]F, B[u]P, DB[u/!]A, B[ghi]P, In[cii]P

B[fc]F, B[fc]F, B[u]P, Edible oils DB[u/!]A, B[g/»]P

On-line LC-LC system: (normal-phase LC -> solvent evaporator -> reversed-phase analytical LC) 1st LC: column; silica phase (250 mm x 4.6 mm) solvent; pentane,10% dichloromethane Interface: solvent evaporator

Quantitative H PLC

Reference

Stationary phase

Mobile phase Detection

Reversed-phase C-18 AcCN/H20

column gradient

2nd LC: Supelcosil LC-PAH AcCN/H20 130 mm (L) x 4.6 mm (i.d.) gradient

Phe (Ex: 250 nm, Em: 365 nm) An (Ex: 250 nm, Em: 402 nm) Flu (Ex: 240 nm, Em: 470 nm) Py (Ex: 240 nm, Em: 385 nm) BMA, Chry (Ex: 270 nm, Em: 390 nm)

B[fc]F (Ex: 260 nm, Em: 430 nm) B[fc]F, B[n]P (Ex: 255 nm, Em: 410 nm)

Fluorescence [134]

330 nm) Phe (Ex: 250 nm, Em: 365 nm) An (Ex: 250 nm, Em: 402 nm) Flu (Ex: 240 nm, Em: 470 nm) Py (Ex: 240 nm, Em: 385 nm) BMA (Ex: 270 nm, Em: 390 nm) B[fc]F, B[fc]F, BMP, diB[ii,/î]A, B[g,h,i]P, In[c,rf]P (Ex: 290 nm, Em: 410 nm) Fluorescence (Ex: 290 nm, Em: [135]

440 nm)

Naph, Ace, Phe, Toasted bread An, Flu, Py, Chry, B[e]P, B[a]P, DB[u/!]A, B[g«]P

Supercritical-fluid extraction using AcCN and C02 as modifier

Naph, Ace, Fl, Non-fatty food Extraction by sonication (ethyl Phe, An, Flu, (mashed ether-methylene chloride)

Py, B[u]A, Chry, potato, toasted B[fe]F, B[fc]F, bread, tomato)

Homogenization with cold NaOH solution, tandem SPE with Extrelut, propylsulfonic acid (PRS) column and Si02 column

Hypersil Green PAH column AcCN/H20 100 mm (L) x 4.6 mm (i.d.) gradient

Hypersil Green PAH column AcCN/H20 100 mm (L) x 4.6 mm (i.d.) gradient

ChromSpher PAH column AcCN/H20 250 mm (L) x 4.6 mm (i.d.) (84:16, v/v)

Fluorescence [136]

Naph (Ex: 270 nm, Em: 335 nm) Ace (Ex: 285 nm, Em: 330 nm) Phe (Ex: 250 nm, Em: 365 nm) An (Ex: 254 nm, Em: 402 nm) Flu (Ex: 285 nm, Em: 465 nm) Py (Ex: 270 nm, Em: 390 nm) Chry (Ex: 270 nm, Em: 384 nm) B[e]P (Ex: 290 nm, Em: 390 nm) BMP (Ex: 295 nm, Em: 405 nm) DB[ii/i]A (Ex: 290 nm, Em: 395 nm) B[g/n']P (Ex: 300 nm, Em: 420 nm) Fluorescence [137]

Phe, An (Ex: 250 nm, Em: 375 nm) Flu (Ex: 285 nm, Em: 465 nm) Py (Ex: 270 nm, Em: 390 nm) B[n]A, Chry (Ex: 270 nm, Em:

B[fc]F (Ex: 305 nm, Em: 410 nm) BMP (Ex: 290 nm, Em: 405 nm) DB[ii/i]A, B[ghi]P (Ex: 290 nm, Em: 418 nm)

In[c,rf]P (Ex: 290 nm, Em: 498 nm) Fluorescence (Ex: 360 nm, Em: [138]

460 nm)

Analyte

Sample type

Sample preparation

Quantitative HPLC

Reference

Stationary phase

Mobile phase

Detection

BMA, BMF,

Fishery product

Lyophilization, extraction through

ChromSpher PAH

ACCN/H20

Fluorescence

[139]

BMF, BMP,

chromatographic column using

100 mm (L) x 4.6 mm (i.d.)

gradient

BMA (Ex: 270 nm, Em: 390 nm)

DB[ah]A,

pentane:dichloromethane(l :1),

B[fc]F (Ex: 260 nm, Em: 430 nm)

In[crf]P

silica cartridge

BMF (Ex: 256 nm, Em: 410 nm) BMP (Ex: 256 nm,Em: 410 nm) DB[íi/í]A (Ex: 300 nm, Em: 418 nm) In[crf]P (Ex: 300 nm, Em: 418 nm)

Phe, An, Flu, Py,

Olive oils

Liquid-liquid extraction, GPC

CIS Vydac

ACCN/H20

Fluorescence (Ex: 264 nm)

[140]

11H-BMF,

purification

250 mm (L) x 2.1 mm (i.d.)

gradient

Phe (Em: 364 nm), An (Em: 402 nm),

BMA, Chry,

Flu (Em: 444 nm), Py (Em:

B[e]P, BMF,

384 nm), 11H-BMF (Em: 352 nm),

DB[nc]A, BMF,

BMA (Em: 396 nm), Chry

BMP, B[g/n']P,

(Em: 368 nm), B[e]P (Em: 384 nm),

In[crf]P

BMF (Em: 444 nm), DB[nc]A (Em: 380 nm), BMF (Em: 420 nm), BMP (Em: 404 nm), B[g/n']P (Em: 420 nm), In[crf]P (Em: 500 nm)

Flu, BMF, BMP

Coffee

SPE purification (polystyrene-

Supelcosil LC-PAH

ACCN/H20

Fluorescence

[141]

divinylbenzene)

250 mm (L) x 4.6 mm (i.d.)

(60:40, v/v)

Flu (Ex: 230 nm, Em: 410 nm) BMF, BMP (Ex: 250 nm, Em: 420 nm)

Table 6 Methods used for polycyclic aromatic hydrocarbon detection: gas chromatography (GC) and gas chromatography/mass spectrometry (GC-MS)

Analyte

Method

Sample type

Sample preparation

Column

Operating conditions

Ref.

Naph, Ace, Acn, Fl, Phe, An, GC/FID Flu, Py, BMA, Chry, B[fc]F, B[fc]F, BMP, In [erf] P, DB[íi/¡]A, B[g/n']P

GC/MSEI

Phe, An, Flu, Py, BMA, Chry/TriP, B[b;j;k]F, BMF, B[e]P, BMP, Pery, In[crf]P, DB[íi,/!;ÍI,C]A, B[g/n']P

GC/MS

Plant material (grain sorghums)

Plant material (grain sorghums)

Vegetable oils, smoked fish products, mussels,oysters, bream

Eider duck (liver)

Phe, An, 3-MePhe, 1-MePhe, GC/MSEI Flu, Py, 2-MePy, 1-MePy, B[g/n]F, CP [erf] P, BMA, Chry+TriP, B[fc]F, B[e]P, BMP, Pery, In[crf]P, B[g/n]P, Cor

3,6-DMePhe, CP[crf]P, BMA, GC/MSNICI Duck eggs B[fc]F, B[/]F, B[fc]F, B[e]P, BMP, Pery, In[crf]P, DB[íi,c]A, DB[íi;¡]A, Pic, B[ghi]P, Cor

AcCN sonication, extraction, partition into pentane, micro silic acid column cleanup

AcCN sonication, extraction, partition into pentane, micro silic acid column cleanup

(a) Vegetable oils: cyclohexane extraction

(b) Smoked meat, bacon, fish, eel, tea, herbs, vegetables:saponification, cyclohexane extraction

Back-extraction by dimethylformamide-H20, addition of Na2S04 solution, re-extraction by cyclohexane, silica gel cleanup, GPC cleanup

Freeze-dried, ground with activated sodium sulfate, Soxhlet extraction (toluene), alkaline saponification, dimethylformamide cleanup, silica column cleanup

Alkaline saponification, n-hexane extraction, water and saturated NaCl washing, dimethylformamide extraction, deactivated silica cleanup preparative TLC

DB-5 50 m (L) fused-silica column

SE-54 capillary column

Column temperature: 70°C (2 min) -> (20°C/min) -> 125°C (4°C/min) ->290°C (15 min) Column temperature: 70°C (2 min) -> (20°C/min) -> 125°C ^ (4°C/min) ->290°C (15 min) 70°C ^ (5°C/min) ->280°C

EI mode

Analyte Method Sample type

Naph, 1-MeNaph, Fl, Phe, GC/PID Ace, Acn, 2,3-DMNaph, An, Flu, Py, Chry, BMA, B[fc]F, 2-MeAn, B[fc]F, BMP Pery, 1-MePhe, In[cii]P, DB[ah]A, B[ghi]P Phe, An, 1-MePhe, Flu, Py, GC/MSEI 2-MePy, B[gfa]F, BMA, Chry+TriP, B[fc]F, B[e]P, BMP Pery, In[oi]P, B[ghi]P, Cor

Fish tissue

Eggs, chicken, duck

An, Phe, 9-MeAn, Flu, Py, GC/MSEI Chry, B[a]P, DB[ah]A (HPLC)

Roasted lamb

Phe, An, Flu, Py, BMA, Chry, B[fc]F, B[fc]F, BMP B[ghi]P, In[eii]P, DB[u/!]A

GC/MS

Naph, Ace, Acn, Fl, Phe, An, GC/MS Flu, Py, BMA, Chry, B[fc]F, (HPLC) B[fc]F, BMP, In[cii]P DB[ah]A, B[ghi]P

Sheep and goat (livers, kidneys), chicken (livers, eggs), cow and sheep (milk) Fish edible muscle

Sample preparation Column

Operating conditions Ref.

Partition into DMSO, back-partitioning into cyclohexane, filtration on silica gel+anhydrous Na2S04/ TLC on silica gel Alkaline saponification, acidification, extraction, adsorption chromatography, GPC purification

Fused-silica SPB-1 capillary Column temperature: 85°C

column

(1 min) -> (25°C/min) -> 180°C-> (7°C/min) ->300°C FID detector: 300°C

Column temperature: 50°C-> (5°C/min)->295,:,C (10 min)

Toluene extraction by shaking or SE-54 HP-1 (methyl silicone) Soxhlet extraction (toluene), saponification, centrifugation, dimethyl formamide cleanup extraction, deactivated silica gel column cleanup (100 x 10 mm)

Chloroform-MeOH extraction, CBP-1 25 m x 0.33 mm silica gel column cleanup, TLC cleanup

Alkaline saponification, silica gel column chromatography

Dichloromethane extraction, column chromatography (alumina:silica) HPLC purification (Phenogel 100 column, 250 x 22.5 mm)

DB-I

GC/MS:SE-54 30 m x 0.32 mm, splitless mode

180 °C->(5°C/min) ->270°C ion source, monitor separator temperature: 250°C EI mode, 70 eV voltage

Column temperature: 150 C-» (3°C/min) ->■ 180°C (5°C/ min)->230°C->(10,:,C/ min)->290°C (9 min) GC/MS: Column temperature: 60 °C (12°C/min) ->295°C (6 min)

2-MeNaph, 1-MeNaph, Phe, GC/MS Seafoods

Flu, Py, B[u]A, Chry, B[fc]F, B[/]F, BMP, In[cii]P, DB[ah]A, B[ghi]P

Phe, An, Flu, Py, BMA, GC/MS (HPLC) Polished rice Chry/TriP, B[fc;;;fc]F, BMF, B[e]P, BMP, Pery, In[ai]P, UB[a,h;a,c]A, B[g/n']P

Naph, 2-MeNaph, 1- GC/MS Liquid smoke flavorings

MeNaph, 2,6-DMeNaph, 1,7-DMeNaph, 1,6-DMeNaph, Fl, Phe, An, 3-MePhe, 2-MePhe,

1-MePhe, 9MeAn, DMePhe/An, Flu, Py, m-Ter, p-Ter, MeFlu/Py, B[u]A, Chry+TriP, B[fc]F, B[fc]F, BMF, B[e]P, BMP Pery, In[eii]P, DBA, B[g/!/]P

Naph, 2-MeNaph, 1- GC/MS Liquid smoke flavorings

MeNaph, 2,6-DMeNaph, 1,7-DMeNaph, 1,6-DMeNaph, Fl, Phe, An,

3-MePhe, 2-MePhe,

2-MeAn, 9-MePhe, 1-MePhe, DMePhe/An, Flu, Py, m-Ter, p-Ter,

Alkaline saponification, hexane extraction, silica gel column chromatography (elution with hexane), partition into acetonitrile Accelerated solvent extraction, sulfuric acid treatment, Florisil column cleanup

Dichloromethane sonication extraction, Soxhlet extraction, activated silica gel column chromato graphy with acetone /hexane Alkaline saponification, extraction (cyclohexane), SPE cleanup (Florisil, silica)

Cross-linked 5%

phenylmethyl siloxane column 30 m (L) x 0.25 mm (i.d.) x 0.25 |im (D) DB-35MS 30 m (L) x 0.25 mm (i.d.)

5% Phenylmethyl siloxane 60 m (L) x 0.25 mm (i.d.) x 0.25 |im (D)

Column temperature: 50°C [150]

(1 min) -> (25°C/min) -> 120°C->(10°C/min) -> 320°C (6.5 min)

Column temperature: 40°C [151]

(1 min) -> (12°C/min) -> 250°C (5°C/min) ->310 °C (3 min)

Column temperature: 100°C [152] (2 min) -> (5°C/min) -> 280°C (15 min)

EI mode, SIM mode

Column temperature: 50°C [112,113] (0.5 min) -> (8°C/min) -> 130°C (5°C/min)->290,:,C (50 min)

EI mode, 70 eV voltage

Alkaline saponification, extraction (cyclohexane), cleanup by two SPE (silica) tubes

5% Phenylmethyl siloxane 60 m (L) x 0.25 mm (i.d.) x 0.25 |im (D)

Column temperature: 50°C (0.5 min) -> (8°C/min) -> 130°C ^ (5°C/min)->290,:,C (50 min)

Table 6 (Continued)

Analyte Method Sample type

11H-BMF, MeFlu/Py, B[u]A, Chry+TriP, B[fe]F, B[fc]F, B[e]P, B[fl]P, Pery, B[gfa]P

Flu, Py, BMA, Chry, B[fc]F, B[fc]F, BMP, In[cii]P, DB[u/!]A, B[g/n]P

Naph, 2-MeNaph, 1- GC/MS Smoked cheese

MeNaph, 2,6-DMeNaph, 1,7-DMeNaph, 1,6-DMeNaph, 1,4+2,3-DMeNaph, 1,5-DMeNaph, DM/ EthylNaph, Ace, Acn, Fl, Phe, An, o-Ter, 3-MePhe, 2-MePhe, 2-MeAn, 9-MePhe, 1-MePhe,

Sample preparation Column

Operating conditions Ref.

Matrix solid-phase dispersion 5% Phenylmethyl siloxane

(mixture of Florisil and anhydrous sodium sulfate) extraction with hexane-ethyl acetate

Sonication extraction, alkaline saponification, extraction, cleanup by SPE (silica) tube

HP-5MS (5% phenylmethyl siloxane)

Column temperature: 80°C [153]

(0.5 min) -> (8°C/min) -> 230°C (5°C/min) ->280°C (17 min)

Column temperature: 50°C [154]

(0.5 min) -> (8°C/min) -> 130°C (5°C/min)->290,:,C (50 min)

DMePhe, Flu, Py, m-Ter, p-Ter, 2-MeFlu, MeFlu, 1-MeFlu+11H-B [u] F, 11H-B[fc]F, HH-B[c]F, 1-MePy, B[u]A, Chry+TriP, MeB[u]An, 3-MeChry, 2-MeChry, 1-MeChry, DMeB[u]A, B[fc]F, B[/+fc]F, B[u]F, B[e]P, B[u]P, Pery, In[cd]P, DB[fl///flc]A, B[fc]Chry, Picene, B[g/n']P, Anth, DBP

Flu, Py, B[u]A, Chry, B[fe]F, B[fc]F, B[u]P, In[e<i]P, DB[fl//]A, B[g/n]P

B[u]A, B[u]P, B[fc]F, B[gfa']P, GC/MS Primary smoke condensate

Chry, B[fc]F, DB[ah]A, In[e<i]P, B[/]F, CycloMP, DB[ue]P, DB[fl//]P, DB[fl/]P, DB[u/]P, 5-methylchrysene

Liquid-liquid extraction, cleanup by size exclusion chromatography (Bio-Beads S-X3)

Alkaline saponification, extraction (cyclohexane), cleanup by two SPE (silica) tubes

Varian FactorFour VF-5ms (5% phenyl 95% dimethyl polysiloxane) 30 m (L) x 0.25 mm (i.d.) x 0.25 nm (D) HP-5MS SV (5%

phenylmethyl siloxane) 30 m (L) x 0.25 mm (i.d.) x 0.5 |im (D)

Column temperature: 70°C (2min)->(20°C/ min) -> 120°C -> (4°C/ min)->300 °C (5 min)

Column temperature: 60 °C (1 min)->(40°C/ min) ->240°C -> (12°C/ min)->300 °C (21.5 min)

content. The analysis of samples with high fat content, e.g., fats, vegetable and mineral oils, requires the elimination of the lipids using a further purification process such as liquid-liquid partition, column chromatography, alkaline saponification/solvent extraction and gel permeation chromatography (GPC).

In the procedure of liquid-liquid partition [143,144], an edible oil sample is at first dissolved into a non-polar organic solvent such as cyclohexane or n-hexane. Following this, target PAHs in the organic solvent are extracted with a polar solvent such as DMSO or a mixture of dimethylformamide/water, while most of the lipids in samples remain in the organic phase. After dilution with water to change the coefficient of partition between the two phases, PAHs are back-extracted into cyclohexane. With this method sample mass can be reduced to 10% of the starting value.

LLE procedure is advantageous in the case of simultaneously analysing both PAHs and the other environmental contaminants such as pesticides and PCDDs, which are unstable under harsh conditions such as the alkaline digestion method mentioned later.

The Soxhlet extraction [127,130] has been widely used for the extraction of PAHs from environmental samples such as soil, sewage sludge and airborne particulates. In comparison with the liquid-liquid extraction in food analysis, Soxhlet extraction has the advantage of enabling the preparation of the extract without emulsification.

However, this method requires from 6 to 24 h for extraction, and is too time-consuming. Birkholtz et al. [157] recommended the Soxhlet extraction method with dichloromethane because alkaline digestion commonly used in food analysis is messy and cumbersome. An alternative to Soxhlet extraction is ultrasonic extraction, which has a much faster extraction time.

Alkaline saponification/solvent extraction is the most commonly used method for PAHs analysis in foods. This method is generally applied to eliminate fat, pigments and other organic contaminants [100,115], which may interfere with the further analytical determination. Alkaline digestion is adopted as an extraction procedure in the official method proposed by the United States Food and Drug Administration (FDA) [158] and the Pharmaceutical Society of Japan [159].

As mentioned in Section 2, PAHs are relatively unstable. Therefore, attention should be paid to the analytical condition in order to perform an accurate analysis, because some PAHs might be labile in the harsh saponification conditions [110,159]. Takatsuki et al. [109] proposed the used Na2S as an antioxidant during the alkaline digestion to avoid the decomposition and to obtain good recoveries. They also indicated that B[a]P was easily decomposed by the coexistence of light, peroxide in aged ethyl ether and oxygen when absorbed on silica gel. Therefore, the following precautions are recommended in the food analysis of PAHs: (1) protection from light during all steps of analysis,

(2) addition of Na2S to the alkaline digestion mixture as an antioxidant,

(3) complete removal of the peroxide in ethyl ether before use, (4) quick column chromatography on silica gel and (5) prevention of air from coming into contact with the adsorbent [105,110].

Caffeine complexation is another extraction procedure using a caffeine-PAHs complex formation. Food samples are dissolved into cyclohexane, and mixed with a caffeine-formic acid solution to form caffeine-PAHs complex. The caffeine-PAHs complex is destroyed by addition of an aqueous sodium chloride solution. PAHs are, then, back-extracted with cyclohexane [160,

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