Quantitative determination of de-conjugated chrysene metabolites in fish bile by HPLC-fluorescence...

Chemosphere 54 (2004) 1085–1097

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Quantitative determination of de-conjugatedchrysene metabolites in fish bile byHPLC-fluorescence and GC–MS

Grete Jonsson a,*, Ingrid C. Taban a, K�aare B. Jørgensen b, Rolf C. Sundt a

a RF-Akvamiljø, Mekjarvik 12, N-4070 Randaberg, Norwayb Stavanger University College, P.O. Box 8002, N-4068 Stavanger, Norway

Received 10 April 2003; received in revised form 11 September 2003; accepted 30 September 2003

Abstract

Two analytical methods have been evaluated for quantitative determination of de-conjugated chrysene metabolites

in fish bile. High performance liquid chromatography-fluorescence (HPLC-F) and gas chromatography–mass spec-

trometry (GC–MS) were compared regarding instrumental and overall limits of detection (LOD) as well as recoveries

for the following nine chrysene compounds: 1-, 2,- 3-, 4- and 6-hydroxychrysene (1-, 2-, 3-, 4- and 6-OH-chr), 1,2-

dihydroxy-1,2-dihydrochrysene (1,2-DHD-chr), 3,4-dihydroxy-3,4-dihydrochrysene (3,4-DHD-chr), 5,6-dihydroxy-5,6-

dihydrochrysene (5,6-DHD-chr) and chrysene. Instrumental LODs were comparable for the two methods whereas the

overall LOD was better for HPLC-F. Recoveries varied per chrysene compound for both HPLC-F (62–107%) and GC–

MS (48–124%). In vivo formed chrysene metabolites were studied in the bile of Atlantic cod (Gadus morhua) exposed to

chrysene (1 mg/kg) via intra-peritoneal (i.p.) and inter-muscular (i.m.) injection. Total amounts of chrysene metabolites

were three times higher in i.p. compared to i.m. exposed cod bile, but the relative distribution of determined metabolites

was very similar. 1,2-DHD-chr was the most prominent metabolite in de-conjugated bile and constituted more than

88% of the total chrysene metabolites. Additional chrysene metabolites formed were 3,4-DHD-chr and 1-, 2-, 3- and 4-

OH-chr. K-region chrysene metabolites (oxidation at carbons 5 and 6) were not detected and seem to be a less favoured

biotransformation route. The two methods were applied and evaluated for analysis of chrysene metabolites in two bile

reference materials (BCR 720 and 721) and a limited number of field exposed cods.

� 2003 Elsevier Ltd. All rights reserved.

Keywords: Chrysene metabolites; Fish bile; In vivo metabolism; HPLC-F; GC–MS

1. Introduction

Chrysene is a symmetrical polycyclic aromatic hy-

drocarbon (PAH) consisting of four condensed benzene

rings (Fig. 1). It is formed through incomplete com-

*Corresponding author. Tel.: +47-51875517; fax: +47-

51875540.

E-mail address: [email protected] (G. Jonsson).

0045-6535/$ - see front matter � 2003 Elsevier Ltd. All rights reserv

doi:10.1016/j.chemosphere.2003.09.026

bustion of organic material and is a ubiquitous envi-

ronmental contaminant (Grimmer and Pott, 1983). The

presence of chrysene in the environment is mainly due to

human activities (ATSDR, 1990). A relatively low sol-

ubility in seawater (�2 lg/l at 25 �C) will promote the

adsorption of chrysene to particulate material, followed

by sedimentation (Neff, 1979). Two- and three-ring

PAHs deposited in the sediment are quite rapidly de-

graded by bacteria, fungi and algae (Cerniglia and

Heitkamp, 1989). Larger PAHs are more resistant to

microbial attack, and their relative concentrations in the

ed.

mail to: [email protected]

OH

OH

OH

OH

OH

K-region

Bay-region

Bay-region

K-region

(a)

(b)

(c)

(d)

Fig. 1. Molecular structures of (a) chrysene, (b) 2-OH-chr,

(c) 1,2-DHD-chr and (d) 3,4-DHD-chr.

1086 G. Jonsson et al. / Chemosphere 54 (2004) 1085–1097

sediment will consequently increase with time (Cerniglia

and Heitkamp, 1989). Release of industrial waste and

refined oil products (lubricating oil, diesel oil and engine

oil) into the marine environment contributes to rela-

tively high sediment concentrations of chrysene in in-

dustrialised areas (Malins et al., 1985; Naes et al., 1995;

Witt, 1995).

Chrysene is absorbed by fish via various routes.

Uptake of water-borne chrysene is considered to be

more efficient as compared to uptake from sediment

(Beyer et al., 1998) and food (Neff, 1985; Niimi and

Dookhran, 1989). Chrysene exposure cannot be accu-

rately determined in fish by measurement of tissue levels,

due to the efficient metabolism of PAHs (Varanasi et al.,

1989). The first step in the metabolism includes an in-

troduction of oxygen by the mixed function oxygenase

(MFO) system, producing phase I metabolites such as

e.g. phenols, dihydrodiols and epoxides. Conjugation of

phase I metabolites results in highly water-soluble phase

II metabolites, such as glucuronic acid and sulphonic

acid conjugates. Excretion of the larger PAHs occurs

mainly via bile and urine following biotransformation

(Varanasi et al., 1989).

Chrysene is considered to exhibit weak carcinogenic

activity via metabolic activation to dihydrodiols (Fig. 1)

and dihydrodiol-epoxides (Wenzel-Hartung et al., 1990;

Harvey, 1991; Glatt et al., 1993). 1,2-dihydroxy-1,2-di-

hydrochrysene (1,2-DHD-chr) has been identified as a

precursor for the formation of the ultimate carcinogenic

chrysene metabolite 1,2-dihydroxy-3,4-epoxy-1,2,3,4-

tetrahydrochrysene (bay-region diol epoxide) (Grimmer

et al., 1988). Several studies have investigated the in

vitro metabolism of chrysene in various cell types, such as

e.g. human pulmonary and hepatic microsomes (Koehl

et al., 1996), rat microsomes (Jacob et al., 1996) and

hamster lung cells (Jacob et al., 1990). Oxidation at the

1,2- and 3,4-position resulting in formation of two di-

hydrodiols, 1,2-DHD-chr and 3,4-dihydroxy-3,4-dihy-

drochrysene (3,4-DHD-chr) and four phenols, 1-, 2-, 3-

and 4-hydroxychrysene (1-, 2-, 3- and 4-OH-chr) were

identified as the main metabolic pathway. Oxidation at

the 5,6-position (K-region) seems to be a less favoured

route (Grimmer et al., 1988). Pangrekar et al. (1995,

2003) and Shappell et al. (1996) have studied the in vitro

metabolism of chrysene by brown bullhead (Ameriurus

nebulosus) liver microsomes. The major metabolite

formed was 1,2-DHD-chr followed by 3,4-DHD-chr and

smaller amounts of hydroxychrysenes and 5,6-dihy-

droxy-5,6-dihydrochrysene (5,6-DHD-chr).

PAH exposure to fish has been successfully moni-

tored in feral (Krahn et al., 1986; van der Oost et al.,

1994; Aas et al., 2001) and laboratory exposed (Aas

et al., 2000a; Jonsson et al., unpublished data) fish by

measurements of their metabolites in bile. The analysis

has been performed by fixed fluorescence (FF) (Lin

et al., 1996; Aas et al., 2000b), synchronous fluorescence

scan (SFS) (Ariese et al., 1993) and high performance

fluorescence chromatography with fluorescence detec-

tion (HPLC-F) (Krahn et al., 1984; Krahn et al., 1986)

for semi-quantitative determination. Quantitative de-

termination of specific de-conjugated metabolites has

regularly been carried out by HPLC-F (Krahn et al.,

1987; van Schanke et al., 2001; Ruddock et al., 2002;

Jonsson et al., 2003) and gas chromatography with mass

spectrometry detection (GC–MS) (Solbakken et al.,

1980; Krahn et al., 1992; Jonsson et al., accepted for

publication; Jonsson et al., 2003). In a former study,

Jonsson et al. (accepted for publication) evaluated SFS

and HPLC-F as screening methods for the determina-

tion of chrysene exposure to fish. The feasibility of the

methods for chrysene metabolite identification was

G. Jonsson et al. / Chemosphere 54 (2004) 1085–1097 1087

limited due to fluorescence interference from an un-

known number of three- and four-ring aromatics.

However, both methods provided semi-quantitative con-

centrations of chrysene metabolites and distinguished

clearly between exposed and non-exposed individuals.

To our knowledge, no studies have been carried out

in order to characterize in vivo formed chrysene me-

tabolites in fish. Leonard and Hellou (2001) examined

the relative amounts of conjugated versus unchanged

chrysene in bile of orally exposed speckled trout (Salv-

elinus fontinalis) and 1-OH-chr has been used as a

monitoring parameter for PAH exposure (Ruddock

et al., 2002, 2003). Another study determined chrysene–

DNA interaction in viviparous blenny (Zoarces vivipa-

rous) and indicated its possible genotoxic potential

in fish (Noaksson et al., 1998). In the present study,

HPLC-F and GC–MS in selected ion mode (SIM) have

been evaluated for quantitative determination of chry-

sene, five OH-chrysenes and three DHD-chrysenes in

fish bile. Instrumental and overall detection limits as

well as recoveries have been established for the investi-

gated compounds. The metabolic profile of chrysene has

been identified and compared in bile of inter-muscularly

(i.m.) and intra-peritoneally (i.p.) exposed Atlantic cod

(Gadus morhua). Applicability of the two analytical

methods has been demonstrated by analyses of bile from

field exposed fish and two certified fish bile reference

materials.

2. Experimental

2.1. Chemicals

Chrysene-trans-1,2-dihydrodiol (1,2-DHD-chr),

chrysene-trans-3,4-dihydrodiol (3,4-DHD-chr), chrysene-

trans-5,6-dihydrodiol (5,6-DHD-chr) and 1-hydroxy-

chrysene (1-OH-chr) were purchased from the National

Cancer Institute’s Chemical Carcinogen Repository,

Midwest Research Institute (Kansas City, MO). 4-hy-

droxychrysene (4-OH-chr) and 6-hydroxychrysene (6-

OH-chr) were supplied by Promochem Standard Supplies

AB (Kungsbacka, Sweden) and Dr. Ehrenstorfer (Augs-

burg, Germany), respectively. 2-hydroxychrysene (2-OH-

chr) and 3-hydroxychrysene (3-OH-chr) were synthesized

by Dr. K�aare B. Jørgensen, Stavanger University College,

Norway. Chrysene, perylene and b-glucuronidase with

5% sulphatase activity (Type HP-2) were obtained from

Sigma-Aldrich (Steinheim, Germany). N-trimethylsilyl-

imidazole (TMSI) in pyridine (21%W/V) was supplied by

Varian (Morton Grove, IL, USA). Sodiumacetate,

4-chloro-1-hydroxynaphthalene (4-Cl-naphthol), anhy-

drous sodiumsulphate (pesticide grade), ammonia, ace-

tonitrile (HPLC grade) and ethyl acetate (pesticide grade)

were provided by Merck (Darmstadt, Germany).

Calibration standards for HPLC-F analysis of a

mixture of chrysene, 2-OH-chr, 4-OH-chr and three

dihydrodiols were prepared in acetonitrile at eight con-

centration levels (3–3000 ng/g). A separate set of HPLC-

F calibration standards were prepared for 1-, 3- and

6-OH-chr (0.7–1000 ng/g) to prevent chromatographic

overlap. Perylene (500 ng/g) was added as an internal

standard prior to final dilution. GC–MS calibration

standards of a mixture of all chrysene compounds were

prepared in ethyl acetate at nine concentration levels

(3–7500 ng/g). 4-Cl-naphthol (300 ng/g) was added as a

surrogate internal standard prior to derivatisation with

TMSI (100 ll), and perylene (800 ng/g) as a GC internal

standard prior to analysis.

Instrumental limits of detection (LOD), based on a

signal-to noise-ratio of 3:1 for peak areas, using diluted

standard solutions were determined for both HPLC-F

and GC–MS.

2.2. Fish bile samples

Single PAH exposure: Atlantic cod (G. morhua), 46

individuals with an average weight of 679± 135 g, were

caught in a non-polluted area outside Stavanger on the

south-west coast of Norway, and acclimatised in the

laboratory for one week prior to exposure. Exposure of

cod to single PAHs was performed in order to prepare

bile material for the present study and additional rele-

vant studies concerning determination and identification

of PAH metabolites in fish bile (Jonsson et al., unpub-

lished data; Jonsson et al., 2003). They were divided into

ten groups, n ¼ 3–7, where the number of fish in each

PAH exposure group varied with how extensive we

planned to study their respective metabolites. Eight

groups were exposed to single PAHs in a carrier (cod

liver oil) by intra-peritoneal (i.p.) injection at the fol-

lowing concentrations: Naphthalene (NPH), fluorene

(FLU), phenanthrene (PHE), dibenzothiophene (DBT),

fluoranthene (FLAN), pyrene (PYR), chrysene, benzo

[a]pyrene (BaP) at 5, 2, 2, 2, 1, 1, 1 and 1 mg/kg fish,

respectively. One group was exposed to 1 mg chrysene/

kg fish by inter-muscular (i.m.) injection between the

two major axial muscles. The remaining group was in-

jected carrier and kept in non-contaminated seawater

for preparation of ‘‘blank’’ bile. The fish were sacrificed

five days following exposure by a sharp blow to the

head, and bile from each group was pooled, homoge-

nized and stored at )80 �C until chemical analysis. The

fish were not fed during acclimatisation and exposure.

Flounder bile (Platichthys flesus), i.p. exposed to an-

thracene (ANT, 2 mg/kg fish) and benz[a]anthracene

(BaA, 1 mg/kg), was kindly received from Dr. Freek

Ariese at Vrije University in Amsterdam, the Nether-

lands. Bile from chrysene exposed cod was used for

characterization of in vivo formed metabolites. Bile

from cod and flounder exposed to the other PAHs was


used to identify possible analytical interference with

chrysene and its metabolites.

Field exposed fish: Atlantic cod (800± 450 g) were

caught with fish traps at five different sites within the

strait of Karmsundet (Krokaneset, Kopervik, Bøv�aagen,Austvik and Høgevarde) and at a reference site, Salvøy.

Høgevarde is situated close to one of the discharge-

points of the aluminium works’ gas scrubbing effluent.

The other sampling sites are less loaded with PAH-

pollution. A description of the sampling sites has been

published by Aas et al. (2001). Bile from two individual

fish at each field site was analysed.

BCR 720 and 721: The preparation of two candidate

fish bile reference materials has been described by Ariese

et al. (in press). One group of flounder (P. flesus) was

exposed to contaminated sediment (BCR 720), and one

group of plaice (Pleuronectes platessa) was exposed to

dispersed droplets of crude oil (BCR 721).

2.3. Sample preparation

Preparation of hydrolysed bile samples was per-

formed mainly as described by Jonsson et al. (2003).

Briefly, 5–50 mg of bile was treated with 3000 units of b-glucuronidase and 150 units of sulphatase activity (in

sodium acetate buffer, pH 5.0) and incubated for 2 h at

40 �C. Perylene (0.2–0.4 lg) was added to the samples as

an internal standard. Acetonitrile was added to give a

final concentration of 70%, and an overall dilution of the

bile varying from 20 to 200 times.

In the case of GC–MS analysis, 4-Cl-naphthol (0.2–

0.3 lg) was added as a surrogate standard to 20–100 mg

of fish bile prior to hydrolysis of the conjugates as de-

scribed above. Hydrolysed metabolites were extracted

with ethyl acetate (0.5 ml, 4 times) and the combined

extracts dried with anhydrous sodium sulphate before

concentration to 0.5 ml. Preparation of trimethylsilyl-

ethers (TMS-ethers) was carried out at room tempera-

ture by addition of TMSI (100 ll) and at least 15 min.

reaction time. Finally, perylene (0.5–0.6 lg) was addedas GC–MS internal standard.

Recoveries of chrysene, five OH-chrysenes and three

DHD-chrysenes by HPLC-F and GC–MS were deter-

mined for three different concentrations by adding

known amounts to ‘‘blank’’ bile. The nine chrysene

compounds had to be divided into two groups (as for

calibration standards, 2.1) for HPLC-F recovery deter-

mination, in order to prevent chromatographic overlap.

Recovery samples were carefully mixed by hand for at

least 30 s and thereafter treated as described for the

samples. For both methods, the added internal standard

corrected automatically for differences in total volume

and variations in injected volume. In addition, the sur-

rogate standard added to GC–MS samples correct for

volume changes during sample preparation, such as loss

of organic solvent during extraction. The steps assessed

by the GC–MS recovery procedure was extraction and

derivatisation efficiencies of individual chrysene com-

pounds, and compound loss during sample preparation

due to chemical instability such as oxidation. Overall

limits of detection (LOD) were determined for HPLC-F

and GC–MS by dilution of the respective recovery

samples until the signal to noise ratio was 3:1 for peak

areas.

2.4. HPLC-F analysis

The HPLC system consisted of a Waters 600E qua-

ternary pump controlled by a Waters 600 controller,

connected to a Waters 717 plus autosampler (Millipore

Corporation, Milford, MA, USA). Detection was per-

formed by a Spectra system FL 3000 fluorescence de-

tector (Thermo Separation Products, FL, USA) at the

excitation/emission (ex/em) wavelengths pairs 270/390

nm and 380/430 nm (slit widths 20 nm) for chrysene

compounds and perylene, respectively. The autosampler

injected 10 ll of standard solutions or diluted hydroly-

sed bile onto the heated (50 �C) analytical column,

Vydac 201TP52 C18 5 lm, 2.1 mm · 250 mm (Vydac,

Hesperia, CA, USA). The system was controlled by

Millenium 2010 Chromatography Manager (Millipore

Corporation, Milford, MA, USA). The HPLC mobile

phase consisted of (A) 5% acetonitrile in 0.05 M am-

monium acetate buffer (pH 4.0) and (B) 90% acetoni-

trile, containing 0.05 M ammonium acetate to keep the

ammonium acetate concentration constant throughout

the run. The elution profile was as follows: 0–3 min,

isocratic 25% B; 3–40 min, 25–100% B linear gradient;

40–42 min, isocratic 100% B; 42–45 min, 100–25% B

linear gradient; 45–65 min, isocratic 25% B. The flow

rate was 0.45 ml/min.

The optimal fluorescence wavelength pairs were de-

termined for solutions of single compounds dissolved in

60–100% acetonitrile (degassed for 10 min with nitro-

gen). Fluorescence analysis of the standard solutions

was performed immediately after preparation by a Per-

kin Elmer LS50B luminescence spectrometer. Optimi-

sation was done by a successive change of emission and

excitation scan parameters until the highest signal in-

tensity was attained. Spectra were recorded in capped

0.5 cm quartz cuvettes with slit widths set to 5 nm for

both excitation (ex) and emission (em).

2.5. GC–MS analysis

Chrysene and TMS-ethers of OH-chrysenes and

DHD-chrysenes were analysed by a GC–MS system

consisting of a HP5890 series II Gas chromatograph,

Finnigan A200S autosampler and a Finnigan MAT

SSQ7000 mass spectrometer (Thermo Finnigan, Hudd-

inge, Sweden). The system was controlled by a DEC

station 5000. Helium was used as carrier gas and the


applied column was CP-Sil 8 CB-MS, 50 m· 0.25 mm

and 0.25 lm film-thickness (Instrument Teknikk A.S.,

Oslo, Norway). Samples and calibration standards (1 ll)were injected on a split/splitless injector with splitless

mode on for 1 min. The temperatures for the injector,

transfer-line and ion source were held at 250, 300 and

240 �C, respectively, and the GC oven temperature

programme was as follows: 120 �C for 1 min., 120–250

�C at 25 �C/min, 250–320 �C at 2 �C/min and held at 320

�C for 15 min. Quantitative determination was done in

selected ion mode (SIM). The targeted mass to charge

(m=z) ratios were selected on the basis of preliminary

analysis in full scan mode (SCAN) at 70 eV in order to

identify the most abundant ions.

2.6. Calculation

Response factor curves for quantitative analysis were

fitted by least-square regression for the nine chrysene

compounds measured by both HPLC-F and GC–MS.

Pearson’s correlation coefficients, r2, calculated for

HPLC-F analysis of 1-OH-chr, 2-OH-chr, 3-OH-chr, 4-

OH-chr, 6-OH-chr, 1,2-DHD-chr, 3,4-DHD-chr, 5,6-

DHD-chr and chrysene were 0.9998, 0.9999, 0.9999,

0.9997, 0.9998, 0.9998, 1.0000, 1.0000 and 1.0000, re-

spectively. GC–MS analysis of the same order of chry-

sene compounds resulted in the following r2 values:

0.994, 0.998, 0.991, 0.996, 0.995, 0.991, 0.989, 0.997 and

0.994, respectively.

3. Results and discussion

3.1. Detection of chrysene metabolites

Maximum fluorescence signals for the investigated

chrysene compounds were achieved at excitation (ex)

and emission (em) wavelengths between 266–278 and

364–382 nm, respectively (Table 1). Acetonitrile and

distilled water in various proportions were used as sol-

Table 1

Optimal fluorescence wavelength pairs (ex/em) for chrysene compoun

Code Compound Solvent,

% acetonitrile

1 1-OH-chr 80 and 100

2 2-OH-chr 80 and 100

3 3-OH-chr 80 and 100

4 4-OH-chr 80 and 100

5 6-OH-chr 80 and 100

6 1,2-DHD-chr 60, 80 and 100

7 3,4-DHD-chr 60, 80 and 100

8 5,6-DHD-chr 60, 80 and 100

9 Chrysene 80 and 100

aWavelength pair used for HPLC-F analysis.

vents for determination of optimum wavelength pairs. A

change in solvent composition altered the signal inten-

sity, but did not lead to shift in optimum wavelength

pair. The relatively water-soluble DHD-chrysenes re-

sponded with increased signal intensity when the water

fraction increased from 0% to 40%, whereas the opposite

was observed for OH-chrysenes and chrysene (results

not shown). One of two optimum wavelength pairs for

1,2-DHD-chr, ex/em 270/390 nm, was used for HPLC-F

analysis. This selection was based on the identification

of 1,2-DHD-chr as the most abundant chrysene me-

tabolite formed in vitro by brown bullhead liver mi-

crosomes (Shappell et al., 1996; Pangrekar et al., 2003).

The use of one wavelength pair throughout the chro-

matographic run will prevent missing information due to

a turbulent baseline. This was considered to be an ad-

vantage, especially for analysis of field exposed samples,

even with a 3–42% loss of sensitivity for individual

chrysene compounds (Table 1).

The molecular ions of chrysene and OH-chrysenes (as

TMS-ethers) were found to be the most abundant ions

when analysed in full scan mode (Table 2). Other promi-

nent masses of OH-chrysenes were [M) 15]þ and

[M) 31]þ. Fragmentation of TMS-ethers of 1,2-DHD-chr

and 3,4-DHD-chr revealed m=z ¼ 191:1, [CH(OTMS)2]þ,

as the most abundant ion, whereas m=z ¼ 73:1,[Si(CH3)3]

þ, was most prominent for 5,6-DHD-chr. The

relative intensities of the molecular ions varied between

6% and 47% for DHD-chrysenes. Ions selected for

quantitative determination of chrysene compounds are

shown in Table 2. The reported fragmentation pattern

is consistent with those reported for chrysene com-

pounds (Jacob and Grimmer, 1987) and for metabo-

lites of PHE, PYR and FLAN in fish bile (Krahn et al.,

1987).

3.2. Separation and identification of chrysene metabolites

Chrysene, three DHD-chrysenes and two OH-chry-

senes were well separated by HPLC-F (Fig. 2a) when

ds

Optimal signal,

ex/em (nm/nm)

270/390 nma,

% of maximum signal

268/368 77

266/380 80

268/374 66

270/370 74

270/378 97

270/370 100

278/372 79

266/364 64

266/382 58

Table 2

Relative intensities of abundant mass fragments of chrysene compounds analysed in full scan mode

Compoundsa M(g/mol)a

Relative intensities of mass fragments (%) SIMb

M+ M) 15 M) 31 M) 90 191.1 147.1 73.1 m=z

1-OH-chr 316 100 33 20 16 316, 301

2-OH-chr 316 100 30 24 28 316, 301

3-OH-chr 316 100 31 16 23 316, 301

4-OH-chr 316 100 28 34 24 316, 301

6-OH-chr 316 100 22 14 22 316, 301

1,2-DHD-chr 406 6 5 100 15 65 406, 191

3,4-DHD-chr 406 19 6 100 15 86 406, 191

5,6-DHD-chr 406 47 32 40 60 100 406, 191

Chrysene 228 100 228

a TMS-ethers of OH-chrysenes and DHD-chrysenes.bMass to charge ratios ðm=zÞ selected for quantitative analysis in SIM mode.

Fig. 2. HPLC-F and GC–MS chromatograms of nine chrysene compounds (a) and (e), bile from chrysene (1 mg/kg i.p.) exposed fish

(b) and (f), bile from BaA (1 mg/kg i.p.) exposed fish (c) and (g), and blank bile (d) and (h). Peak identity: (1) 1-OH-chr, (2) 2-OH-chr,

(3) 3-OH-chr, (4) 4-OH-chr, (5) 6-OH-chr, (6) 1,2-DHD-chr, (7) 3,4-DHD-chr, (8) 5,6-DHD-chr and (9) chrysene.


simultaneously injected. Two pairs of OH-chrysenes, 3-

OH-chr and 2-OH-chr, as well as 4-OH-chr and 1-OH-

chr, were partly separated by HPLC-F (Fig. 2a),

whereas 2-OH-chr and 6-OH-chr suffered from total

chromatographic overlap. Bile from chrysene exposed

cod were analysed and six responding peaks (Fig. 2b)

tentatively identified as 1- and 4-OH-chr, 2-OH-chr and/

or 6-OH-chr, 3-OH-chr, 1,2-DHD-chr, 3,4-DHD-chr

and chrysene by comparison with retention times and

peak shapes of the standard mixture. The most signifi-

cant metabolite was 1,2-DHD-chr, whereas the two

corresponding diols, 1-OH-chr and 2-OH-chr, were

present as less abundant peaks. Oxidation at carbon five

or six was not revealed by the employed HPLC-F


method and the relatively limited number of available

standard compounds. This could be due to concentra-

tions below the detection limit and/or chromatographic

overlap of 6-OH-chr with 2-OH-chr. The observed oxi-

dation at carbon one to four is in agreement with the

determined regioselectivity of in vitro metabolism of

chrysene by brown bullhead liver microsomes (Pangre-

kar et al., 1995; Pangrekar et al., 2003).

Bile from environmentally exposed fish includes a

large number of PAH metabolites. Possible interferences

from metabolites of selected PAHs were evaluated by

analyses of bile from single compound exposed fish. Bile

from BaA exposed fish contained one major and one less

abundant peak responding at the employed fluorescence

wavelength pair (Fig. 2c). The major responding BaA

and chrysene metabolite (1,2-DHD-chr) were not sepa-

rated by the used HPLC-method. Metabolites originat-

ing from the other investigated two- to five-ring PAHs

did not interfere with HPLC-F determination of chry-

sene metabolites (data not shown). Background fluo-

rescence signal from ‘‘blank’’ bile is shown in Fig. 2d.

Full separation of all chrysene compounds subjected

to investigation was accomplished by GC–MS (Fig. 2e,

chrysene did not respond under the SIM conditions used

to determine its metabolites). The presence of 1-, 2-, 3-

and 4-OH-chr, 1,2-DHD-chr and chrysene in bile from

chrysene exposed cod was confirmed by GC–MS (Fig.

2f), but a relatively high overall LOD (Table 3) com-

plicated the confirmation of 3,4-DHD-chr. Further-

more, 5,6-DHD-chr and 6-OH-chr was not present at

detectable levels in the bile of chrysene exposed cod,

although the overall LODs for these two compounds

were relatively low (Table 3). BaA metabolites did not

interfere with GC–MS determination of 1,2-DHD-chr,

but to a large extent with 1-OH-chr. Less pronounced

interference was observed between BaA metabolites and

Table 3

Instrumental and overall limits of detection (LOD) for chrysene com

Compound Instrumental LODa

HPLC-F GC–MS SIM

pg injected pg injected

1-OH-chr 12 5

2-OH-chr 2.0 12

3-OH-chr 1.0 3

4-OH-chr 1.8 2

6-OH-chr 0.5 2

1,2-DHD-chr 5.0 15

3,4-DHD-chr 0.8 5

5,6-DHD-chr 1.5 1

Chrysene 0.8 3

a Signal to noise ratio was 3:1.b TMS-ethers of OH-chrysenes and DHD-chrysenes.c Spiked ‘‘blank’’ bile.

2- and 3-OH-chr (Fig. 2g). MS selectivity prevented any

interference from PAH metabolites with dissimilar

masses.

3.3. Limits of detection and recoveries

Instrumental LODs were comparable for the two

analytical techniques, HPLC-F and GC–MS, regarding

the analysis of nine chrysene compounds (Table 3).

However, better LODs were achieved for seven out of

nine compounds when analysed by HPLC-F. The posi-

tion of the oxidised carbon in the chrysene molecule

seems to have an influence on the LOD. Oxidation at

carbon three to six results in better LOD as compared to

oxidation at carbon one and two. 1-OH-chr and 5,6-

DHD-chr yielded improved sensitivity by GC–MS

analysis. Overall LOD was generally better by HPLC-F

analysis of all chrysene compounds as compared to GC–

MS (Table 3). Three compounds, 1-OH-chr, 4-OH-chr

and 5,6-DHD-chr, displayed comparable overall LOD

for both methods. The loss of GC–MS sensitivity when

analysing chrysene compounds in spiked bile reflects a

labour demanding sample preparation procedure. The

slowest eluting compounds (1- and 2-OH-chr and 1,2-

DHD-chr) result in broader chromatographic peaks and

hence a decrease in sensitivity (Jonsson et al., 2003).

HPLC-F recoveries of OH-chrysenes and DHD-

chrysenes were >85%, at all tested concentrations (Table

4). Added concentrations were at least four times the

overall LOD. HPLC-F recovery of chrysene was poor

(62–75%) as compared to the other chrysene compounds

(Table 4). Chrysene is less water soluble than OH-

chrysenes and DHD-chrysenes and will therefore pre-

cipitate more easily with particulate material during

sample preparation and centrifugation. Decreasing re-

coveries with increasing concentrations was observed for

pounds

Overall LODa in 30 ll bileb HPLC-F GC–MS SIMb

ng/g bilec ng/g bilec

60 120

18 300

10 90

24 60

5 50

50 500

12 150

14 50

6 80

Table 4

Recoveries of chrysene compounds analysed by HPLC-F and GC–MS SIM at varying concentration levels

Compound HPLC-F GC–MS SIM

Added

(ng/g bile)

Recovery, n ¼ 5%

(±1 SD)

Added

(ng/g bile)

Recovery, n ¼ 5%

(±1 SD)

1-OH-chr 1731 99 (3) 2852 111 (13)

5309 93 (3) 8938 124 (15)

18 290 88 (2) 30 665 109 (13)

2-OH-chr 115 99 (7) 4926 109 (6)

348 91 (4) 15 441 112 (9)

1036 92 (4) 52 976 99 (13)

3-OH-chr 82 101 (7) 2491 99 (8)

249 95 (3) 7808 102 (10)

742 94 (3) 26 787 107 (10)

4-OH-chr 106 100 (9) 1351 102 (7)

325 93 (6) 4235 105 (13)

1121 85 (5) 14 531 121 (10)

6-OH-chr 67 104 (7) 1147 99 (4)

207 95 (4) 3593 96 (12)

712 86 (3) 12 328 93 (8)

1,2-DHD-chr 4324 107 (5) 8007 58 (6)

13 262 100 (4) 25 097 47 (5)

45 690 95 (5) 86 105 48 (7)

3,4-DHD-chr 192 100 (4) 3011 63 (6)

588 94 (1) 9439 52 (6)

2027 91 (1) 32 384 52 (6)

5,6-DHD-chr 182 99 (7) 667 84 (6)

558 92 (1) 2091 73 (4)

1921 90 (2) 7175 72 (6)

Chrysene 69 75 (4) 1767 100 (9)

213 69 (2) 5540 91 (6)

733 62 (2) 19 008 92 (5)


all chrysene compounds. These findings may indicate

saturation of the solvent phase (70% acetonitrile) and

equilibrium between dissolved and precipitated com-

pounds. GC–MS recoveries of chrysene were increased

as compared to HPLC-F (Table 4), and four repetitive

extractions with ethyl acetate explain the improved re-

covery. Average recoveries for 1-, 2-, 3- and 4-OH-chr

exceeded 100% and might partly result from decompo-

sition of 1,2- and 3,4-DHD-chr (Table 4) in the injector,

as reported for TMS-derivatives of BaP-dihydrodiols

(Takahashi et al., 1979). The relatively low recoveries

(47–63%) of these two DHD-chrysenes support this as-

sumption. GC–MS recovery of the third DHD-chrysene

(5,6-DHD-chr) was better (72–84%), and might be a

result of less decomposition as indicated by the recovery

of 6-OH-chr (<100%). Better results for 5,6-OH-chr can

also be explained by the fact that this molecule is more

compact and volatile than the two other dihydrodiol-

isomers, 1,2- and 3,4-DHD-chr, and will therefore elute

at a shorter retention time as a narrower chromato-

graphic peak (Fig. 2e). The generally low recovery of the

DHD-chrysenes could partly be a result of decomposi-

tion, as indicated above, although TMS-derivatives of

DHD-chrysenes are less vulnerable to decomposition

than the free dihydrodiols (Krahn et al., 1987). A re-

duced extraction efficiency of the relatively polar DHD-

chrysenes as compared to chrysene and OH-chrysenes

could also explain the low recoveries for these com-

pounds.

3.4. Metabolic profile of chrysene exposed Atlantic cod

Quantification of individual metabolites in chrysene

exposed cod was accomplished by use of HPLC-F, after

confirmation of the compound identity by GC–MS

analysis. One prominent metabolite, 1,2-DHD-chr

(>88%), was excreted in the bile of chrysene exposed cod

(Table 5). Minor concentrations, <6%, of 3,4-DHD-chr

Table 5

Chrysene metabolites determined by HPLC-F in the bile of intra-peritoneal (i.p.) and inter-muscularly (i.m.) exposed Atlantic cod

(G. morhua)

Route Concentration, ng/g bile (±SD), n ¼ 5

Sum 1,2-DHD-chr 3,4-DHD-chr 1- and 4-OH-chra 2-OH-chr 3-OH-chr Chrysene

i.p. 12 581 (317) 11 160 (389) 176 (19) 281 (19) 720 (65) 131 (3) 113 (12)

i.m. 4263 (181) 3814 (174) 53 (8) 76 (12) 209 (22) 51 (2) 60 (9)

aNot separated.

HPLC - FFluoresence270 / 390 nm

Time (min)

(a)

(b)

(c)

7

450

7

7

6

6

6

34+1

34+1

34+1

is

is

is

Fig. 3. HPLC-F of field exposed (Austvik) fish bile (a), BCR

720 (b) and BCR 721 (c). Peak identity: (1) 1-OH-chr, (3) 3-OH-

chr, (4) 4-OH-chr, (6) 1,2-DHD-chr, and (7) 3,4-DHD-chr.


and 1-, 2-, 3- and 4-OH-chr were determined as well. K-

region oxidised metabolites were not detectable by any

of the two analytical techniques, and seem to be a less

favoured route for biotransformation of chrysene by

Atlantic cod. The relative distribution of chrysene me-

tabolites in bile was nearly identical for i.m. and i.p.

injected cod (Fig. 4b). Total amount of chrysene me-

tabolites were approximately three times higher in i.p. as

compared to i.m. injected cod (Table 5, Fig 4a). This

may indicate a rapid uptake of i.p. administered chry-

sene into the systemic circulation. A delayed entrance

into the bloodstream and hence a delay in excretion

might explain lower levels of bile metabolites in i.m.

injected cod. However, we cannot draw any firm con-

clusions regarding distribution and excretion of i.p.

versus i.m. administered chrysene due to the limited

number of samples and only one single sampling point.

The HPLC-F profile of chrysene metabolites in cod bile

displayed at least four peaks that could not be identified

with the available standards (Fig 2b, i.p. injection).

Chromatographic retention times indicate decreased

water solubility for these unknown compounds as

compared to DHD-chrysenes. Possible identities might

be quinones and/or epoxides of chrysene. A similar se-

lectivity for formation of DHD metabolites with a bay-

region double bond by fish have been observed for PHE

(1,2-DHD-PHE) (Solbakken et al., 1983; Goksøyr et al.,

1986) and BaP (7,8-DHD-BaP) (Varanasi and Gmur,

1981; Kennedy et al., 1989; van Schanke et al., 2000).

3.5. Chrysene metabolites in bile of fish exposed to

complex PAH mixtures

The applicability of HPLC-F for determination of

chrysene metabolites in bile of fish exposed to complex

mixtures of xenobiotics was evaluated by analysis of two

reference materials, BCR 720 and BCR 721, and bile

from field exposed fish (Fig 3). HPLC-F analysis re-

vealed responding peaks at the retention times of two

diols, 1,2- and 3,4-DHD-chr, and three hydroxychryse-

nes, 3- and 1- + 4-OH-chr, (Fig. 3a–c). GC–MS analysis

confirmed the presence of the most abundant metabo-

lite, 1,2-DHD-chr. The other peaks were identified by

HPLC-F analysis of the respective bile samples spiked

with low concentrations of standard compounds due

insufficient concentration for GC–MS analysis. Indica-

tive values of total and relative amounts of determined

chrysene metabolites are presented in Fig. 4. All com-

pound concentrations, except for 1,2-DHD-chr which

was quantified by GC–MS, were determined by HPLC-

F and corrected with the average recovery for each

compound (Table 4). Despite the variation in total

chrysene metabolite concentration (Fig. 4a), relative

amounts of individual metabolites were similar for 1,2-

DHD-chr (Fig. 4b). However, a relatively large differ-

ence in the levels of 3,4-DHD-chr and hydroxychrysenes

was observed between field samples and laboratory in-

jected samples. HPLC-F of bile from fish exposed to

combustion type PAHs (Fig. 3a and b) or oil PAHs

(Fig. 3c) displayed a large number of eluting peaks.

Chromatographic overlap of e.g. BaA metabolites or

alkylated PAH metabolites complicated the accurate

quantitative determination of chrysene metabolites.

However, additional analysis by GC–MS enabled con-

firmation and determination of the most abundant

Fig. 4. Total concentrations of chrysene compounds (chrysene, 1,2-DHD-chr, 3,4-DHD-chr, 1-OH-chr, 3-OH-chr and 4-OH-chr) in

analysed fish bile (a) and the relative distribution of individual chrysene metabolites (b). All compounds were determined by HPLC-F,

except for GC–MS determination of 1,2-DHD-chr in BCR 720 and 721, and field exposed samples.


chrysene metabolites. Analysed reference materials and

field exposed bile represent two additional exposure

routes to i.p. and i.m. injection. The prominent 1,2-

DHD-chr in all analysed samples (Fig. 4b) indicates a

similar regioselective metabolism, independent of expo-

sure route. The relative proportions of individual me-

tabolites vary more pronounced for the less abundant

metabolites and may be a result of variation in exposure

route or chromatographic interference from unknown

peaks in fish bile from complex exposures.

1,2-DHD-chr (carcinogenic precursor) was the major

abundant metabolite in studied fish bile, independent of

exposure source and route. The absolute configuration

of the metabolite plays an important role for the car-

cinogenic activity (Thakker et al., 1985), and the [R,R]

configuration of 1,2-DHD-chr is far more potent than

the [S,S] isomer. In vitro metabolism of chrysene by

brown bullhead liver microsomes yields 70–78% of the

most potent stereoisomer (Pangrekar et al., 2003). The

absolute configuration of in vivo formed 1,2-DHD-chr

has to the authors’ knowledge not yet been described.

Chrysene, BaA and their alkylated homologues are

constituents in fossil PAH sources continuously released

into the marine environment (Neff, 1979; Utvik, 1999).

Acquiring knowledge about the potential adverse effects

of specific PAHs to marine biota has been an important

topic for many years. Understanding the stereoselective

in vivo metabolism and effects of PAHs still remains as a

future challenge.

4. Conclusions

HPLC-F and GC–MS have been evaluated for

quantitative analysis of chrysene, five OH-chrysenes and

three DHD-chrysenes in fish bile. Instrumental LODs

were comparable for the two employed methods, but

slightly better for seven out of nine compounds when

analysed by HPLC-F. The overall LOD was better for

all nine chrysene compounds when analysed by HPLC-F

as compared to GC–MS. HPLC-F recoveries were

>85% for OH-chr and DHD-chr and between 62% and

65% for chrysene. GC–MS recoveries for chrysene

increased to >92%, whereas the recoveries for DHD-

chrysenes decreased. OH-chrysene recoveries, except 6-

OH-chr, were exceeding 100% and may be a result from

degradation of 1,2- and 3,4-DHD-chr in the injector.


In vivo formed chrysene metabolites have been

characterized in cod bile by HPLC-F and GC–MS

analysis. Oxidation occurs at carbon 1–4 in the chrysene

molecule, with 1,2-DHD-chr as the most prominent

metabolite following i.p. and i.m. administration. 1,2-

DHD-chr is a precursor of the carcinogenic 1,2-DHD-

3,4-epoxide, and thus an important indication of the

carcinogenic activity of chrysene.

The combination of HPLC-F and GC–MS allowed

characterization of chrysene and its metabolites in bile

of fish exposed to pure chrysene. Determination of

chrysene metabolites in two reference bile samples and

field exposed fish bile were more challenging, but could

be accomplished for the most abundant compounds by

combining the two analytical techniques.

Acknowledgements

The authors thank Dr. Endre Aas for providing us

with field exposed samples. Dr. Jonny Beyer is ac-

knowledged for assistance with laboratory exposures

and for reviewing the manuscript. We thank Norsk Agip

in Stavanger, a Norwegian subsidiary of the Italian

EniGroup, for financial support. This work was per-

formed at the RF-Akvamiljø facilities in Stavanger,

Norway.

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Quantitative determination of de-conjugated chrysene metabolites in fish bile by HPLC-fluorescence...

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