Jou nal o Ch oma og aphy A, 907 (2001) 291–299 www.else ie .com/loca e/ch oma
Lase -induced luo escence de ec ion a 266 nm in capilla y
elec opho esis
Polycyclic a oma ic hyd oca bon me aboli es in bio a
ab cba
´
J. Kuij , C. Ga cıa-Ruiz , G.J. S oombe g , M.L. Ma ina , F. A iese ,
aa,
*
U.A.Th. B inkman , C. Gooije
a
V ije Uni e si ei
,
Depa men o Analy ical Chemis y and Applied Spec oscopy
,
De Boelelaan
1083, 1081
HV Ams e dam
,
The Ne he lands
b
´´´´
Uni e sidad de Alcala
,
Depa amen o de Quımica Analı ica
,
Facul ad de Quımica
,
C a
.
Mad id–Ba celona
,
Km
. 33,600,
´
E-
28871
Alcala de Hena es
,
Mad id
,
Spain
c
V ije Uni e si ei
,
Ins i u e o En i onmen al S udies
,
De Boelelaan
1115, 1081
HV Ams e dam
,
The Ne he lands
Recei ed 30 June 2000; ecei ed in e ised o m 29 Sep embe 2000; accep ed 13 Oc obe 2000
Abs ac
The sepa a ion o i e phenolic polycyclic a oma ic hyd oca bon me aboli es (hyd oxy-PAHs) has been pe o med by
cyclodex in-modi ied micella elec okine ic ch oma og aphy (CD-MEKC) using a 30 mMbo a e bu e (pH 9.0)
con aining 60 mMsodium dodecyl sul a e and a ying concen a ions o g-cyclodex in (g-CD). A concen a ion o 12.5
mMg-CD was ound o p o ide a baseline sepa a ion o he i e hyd oxy-PAHs. We applied con en ional luo escence and
lase -induced luo escence (LIF) de ec ion, using a new, small-size, quad upled Nd–YAG lase emi ing a 266 nm. The bes
limi s o de ec ion, in he low ng/ml ange, we e achie ed using LIF de ec ion. Fo all analy es, linea i y was obse ed up o
ca. 100 ng/ml. As an applica ion, conjuga ed py ene me aboli es in hepa opanc eas samples om he e es ial isopods
Oniscus asellus and Po cellio scabe we e sepa a ed and de ec ed. Finally, la ish bile samples om indi iduals exposed o
pollu ed sedimen o c ude oil, which we e pa o an in e labo a o y s udy, we e analyzed by CD-MEKC wi h con en ional
luo escence and LIF de ec ion o de e mine he 1-hyd oxypy ene concen a ions. 2001 Else ie Science B.V. All igh s
ese ed.
Keywo ds
:
Lase -induced luo escence de ec ion; De ec ion, elec opho esis; Fish; Polynuclea a oma ic hyd oca bons
1. In oduc ion elec opho esis (CE), mainly because LIF de ec ion
using exci a ion in he deep UV ange is no s aigh -
A p esen , lase -induced luo escence (LIF) de- o wa d. Un il now, such exci a ion equi ed he use
ec ion o analy es exhibi ing na i e luo escence, o la ge ame a gon ion lase s o excime lase -
ollowing exci a ion in he deep UV ange, has been pumped dye lase sys ems [1,2]. Un o una ely, hese
ha dly exploi ed o de ec ion pu poses in capilla y lase sys ems a e expensi e and imp ac ical in
analy ical applica ions because o hei la ge dimen-
sions.
*Co esponding au ho . Fax: 131-20-4447-543.
E-mail add ess
:
[email p o ec ed] (C. Gooije ). In his s udy, he pe o mance in LIF de ec ion o
0021-9673/01/$ – see on ma e 2001 Else ie Science B.V. All igh s ese ed.
PII: S0021-9673(00)01040-2
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a new, small-size, quad upled Nd–YAG lase emi - was used o p epa e bu e s and analy e s anda d
ing 266-nm adia ion a a high pulse equency was solu ions. Bu e s we e adjus ed o he equi ed pH
in es iga ed. Such a lase sys em will be o special using 1 MNaOH. Bo ic acid and asco bic acid we e
in e es in bioanaly ical s udies since he 266-nm ob ained om J.T. Bake (De en e , The Ne he -
ou pu is ully compa ible wi h he exci a ion o lands), absolu e e hanol (analy ical- eagen g ade)
¨
na i ely- luo escen amino acids ( y osine, om Riedel-de Haen (Seelze, Ge many) and sodium
yp ophan and phenylalanine). In he p esen s udy, dodecyl sul a e (SDS) om Fluka (Buchs, Swi ze -
howe e , i s app op ia eness o de ec ion o polynu- land). 1,5-Naph halenedisul onic acid and g-cyclo-
clea a oma ic hyd oca bon (PAH) me aboli es is dex in hyd a e we e pu chased om Ald ich (S .
es ed. Louis, MO, USA). b-Glucu onidase/a yl sul a ase
Because he me abolism o PAHs a e PAH ( om Helix poma ia, EC 3.2.1.31 and EC 3.1.6.1, 30
exposu e is e y as o mos o ganisms, he con- and 60 U/ml, espec i ely) was ob ained om
cen a ions o PAH me aboli es in bio ic ma ices a e Me ck (Da ms ad , Ge many). Bu e solu ions we e
gene ally much highe han hose o he pa en il e ed o e 0.45-mm sy inge il e s (Schleiche &
PAHs. I is he e o e ad an ageous o ocus a en ion Schuell, Dassel, Ge many) be o e use.
on he de e mina ion o PAH me aboli es, ins ead o
on hei pa en s. In he las decade he quan i ica ion
2.2.
S anda d solu ions and samples
o PAH me aboli es in exc e a (u ine, bile and eces)
has become an impo an me hod o he biomoni o - The hyd oxy-PAHs used in his s udy a e 1-hy-
ing o PAH exposu e in a ious e es ial and d oxypy ene, 3-hyd oxybenzo[a]py ene, 1-hyd oxy-
aqueous ecosys ems [3–7], as well as o moni o ing phenan h ene, 1-hyd oxych ysene and 2-hyd oxy-
human PAH exposu e [8–13]. naph halene. S ock solu ions con aining hese i e
Speci ically, 1-hyd oxypy ene has ex ensi ely hyd oxy-PAHs (10 mg/ml) as well as he ish bile
been used as a bioma ke o PAH exposu e. The samples we e dis ibu ed as pa o a Eu opean
de e mina ion o 1-hyd oxypy ene and o he hy- Union (EU)- unded in e labo a o y s udy (SMT 4-
d oxy-PAHs is pe o med a e enzyma ic hyd olysis CT 98-2250) [17]. The s ock solu ions had been
o phase II conjuga es such as 1-hyd oxypy ene p epa ed wi h e hanol–wa e (80:20, / ), con aining
sul a e and 1-hyd oxypy ene glucu onide. Al hough 5 mg/ml asco bic acid o a oid oxida ion. F om he
s anda ds a e no ye comme cially a ailable, he s ock solu ions, 1 mg/ml solu ions we e p epa ed
po en ial o di ec moni o ing o phase II me aboli es using e hanol–wa e (80:20, / ) wi h 5 mg/ml
has also been explo ed [5,9,10,13]. asco bic acid. In o de o p epa e calib an solu ions,
Phenolic and conjuga ed PAH me aboli es a e u he dilu ions we e made by mixing wi h e hanol–
usually analyzed by liquid ch oma og aphy (LC) wa e (80:20, / ) wi hou asco bic acid.
combined wi h luo escence de ec ion [3–13]. Few Hepa opanc eas samples om he isopods Po cel-
s udies epo on he sepa a ion o hyd oxy-PAHs by lio scabe and Oniscus asellus, which con ained
CE [14–16], bu un il now CE has no been applied conjuga ed py ene me aboli es, we e ob ained as
o he de ec ion o hese compounds in eal (bio ic) desc ibed by S oombe g e al. [5]. B ie ly, he
samples. In he p esen s udy he sepa a ion and isopods we e s a ed o 3 days and indi idually
de ec ion o i e hyd oxy-PAHs, and o py ene and placed in scin illa ion ials o 48 h, oge he wi h
i s me aboli es, in biological ma ices ( ish bile and 100 mg o g ound lea es spiked wi h py ene a a
isopod hepa opanc eas) is s udied. le el o 10% (w/w) d y mass. A e dissec ion,
hepa opanc eases o 10 animals we e pooled in 100
ml o T is bu e (pH 9.0) and 20 ml o p o ease K
2. Ma e ials and me hods was added. P o eolysis was pe o med o 18 h a
378C. Nex , an equal olume o e hanol was added
2.1.
Chemicals and ma e ials and he emaining deb is was p ecipi a ed by 3-min
cen i uga ion a 5000 pm. Indi idual me aboli e
Wa e demine alized and dis illed in he labo a o y solu ions we e ob ained by ac iona ion o he
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samples by means o e e sed-phase LC, wi h a Meylan, F ance), emi ing a 266 nm wi h an a e age
g adien o ace oni ile and ammonium ace a e bu e ou pu powe o 5.4 mW, a pulse wid h o 0.34 ns
(10 mM, pH 5.0) [5]. Fo p olonged use, all s an- and a epe i ion a e as high as 7.8 kHz, was ocused
da ds and samples we e s o ed in he e ige a o a in o he liquid co e o he capilla y by a qua z lens
2208C. Be o e use, he solu ions we e sonica ed o wi h a ocal dis ance o 3 cm. A 253, N.A. 0.4
5 min. e lec i e objec i e (Ealing, Hollis on, MA, USA)
was used o collec he luo escence emission. The
2.3.
Ins umen al se -up 9558 QA pho omul iplie ube (EMI, Middlesex,
UK) used o de ec ion was connec ed o a 456H
An LS-50B luo escence spec ome e (Pe kin- powe supply (O ec, B acknell, UK) ope a ed a
Elme , Beacons ield, UK) was used o eco d luo- 1000 V. Neu al densi y il e s we e used in o de o
escence exci a ion and emission spec a. Elec- dec ease he i adiance o he capilla y de ec ion
ophe og ams we e ob ained using a P ince high- window. A UG-1 bandpass il e and BG-14 o
ol age/injec ion sys em (Laue labs, Emmen, The GG-13 cu -o il e s (Scho , Mainz, Ge many) we e
Ne he lands) in combina ion wi h a con en ional FP- used o collec all emission and disc imina e agains
920 luo escence de ec o (Jasco, Tokyo, Japan) o a sca e ed exci a ion ligh .
labo a o y-buil se -up o LIF de ec ion. CE sepa-
a ions using he con en ional luo escence de ec o
we e pe o med a a ol age o 20 kV wi h a 90 3. Resul s and discussion
cm375 mm I.D. uncoa ed used-silica capilla y
(BGB Analy ik, Anwil, Swi ze land) wi h an e ec- This sec ion i s discusses he CE sepa a ion and
i e leng h o 60.5 cm. Fo he LIF expe imen s a luo escence de ec ion o i e hyd oxy-PAHs which
104 cm capilla y wi h an iden ical e ec i e leng h would be he a ge analy es in he ish bile samples.
was used; he sepa a ion was un a 23.3 kV o The pa en PAHs a e usually no obse ed in such
main ain he same ield s eng h (221 V/cm). The samples. The e o e, i is no necessa y o in ol e he
injec ion p essu e was 25 mba and he injec ion ime pa en compounds in he sepa a ion. Emphasis is on
0.1 min. Fo da a collec ion a compu e equipped he pe o mances o he new 266-nm LIF me hod
wi h CLASS-VP da a-acquisi ion so wa e was used. compa ed wi h con en ional luo escence de ec ion.
The LIF se -up is shown in Fig. 1. The ligh om Subsequen ly, he de e mina ion o py ene me abo-
a quad upled Nd–YAG NanoUV lase (Uniphase, li es in isopods and ish bile samples will be dis-
cussed. Fo such complex ma ices he LIF me hod
will, no doub , su e om he disad an age ha i
o e s no selec i i y in exci a ion se ings: only one,
single, wa eleng h is a ailable, i.e., 266 nm. Com-
pa ison wi h con en ional luo escence de ec ion is,
he e o e, o dis inc in e es .
3.1.
CE o hyd oxy-PAHs
To pe o m con en ional luo escence de ec ion
unde op imum condi ions, he exci a ion and emis-
sion luo escence spec a o he hyd oxy-PAHs we e
eco ded in he selec ed bu e sys em (see below). I
mo e han one maximum was p esen , as is he case
o he exci a ion spec a, he maximum gi ing he
Fig. 1. De ec ion sys em o LIF de ec ion. UVL: 266-nm lase ; highes signal- o-noise a io (S/N) in he elec-
F1: neu al densi y il e ; L: ocusing lens; C: capilla y ( ocusing ophe og ams was chosen. The maxima used o
poin ); O: e lec ing objec i e; M: mi o ; F2: bandpass o cu -o
il e ; PMT: pho omul iplie ube. de ec ion in CE a e gi en in Table 1. The con en-
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Table 1
a
Limi s o de ec ion (ng/ml) o i e hyd oxy-PAHs using CE wi h LIF and con en ional luo escence (CF) de ec ion
Compound CF LIF, UG-1 LIF, BG-14
3-Hyd oxybenzo[a]py ene 2 (383/435) 25 2
2-Hyd oxynaph halene 40 (275/344) 3 4
1-Hyd oxyphenan h ene 7 (306/361) 3 2
1-Hyd oxych ysene 35 (307/370) 10 6
1-Hyd oxypy ene 1 (347/387) 4 2
a
In pa en heses (CF): exci a ion/emission wa eleng hs used o de ec ion.
ional luo escence de ec o has a ixed exci a ion sli jec ions close o he de ec ion limi . As shown in
o 10 nm, bu he emission sli can be a ied (10, 18 Table 1, he LODs we e in he low ng/ml ange o
o 40 nm bandwid h). An emission bandwid h o 40 h ee o he es analy es, bu abou 10- old highe
nm ga e he bes esul s in e ms o S/N. o 2-hyd oxynaph halene and 1-hyd oxych ysene.
As a as he CE sepa a ion is conce ned, micella Using peak a eas, linea anges om 10 o 500
elec okine ic ch oma og aphy (MEKC) as such does ng/ml we e ob ained o 3-hyd oxybenzo[a]py ene
2
no su ice: using a 30 mMbo a e, 60 mMSDS and 1-hyd oxypy ene (wi h R alues o 0.9997 and
bu e (pH 9.0), all analy es we e ound o mig a e 0.9993, espec i ely; i e da a poin s in duplica e o
simul aneously. This is no su p ising because all bo h). Fo peak heigh s, linea i y was obse ed om
2
a ge compounds a e a he hyd ophobic and he e- 10 o 100 ng/ml wi h R alues o 0.9992 and
o e ha e simila a ini y o he micelles [18]. In 0.9969 ( ou da a poin s in duplica e) o 3-hyd oxy-
o de o gene a e selec i i y, g-cyclodex in (g-CD) benzo[a]py ene and 1-hyd oxypy ene, espec i ely.
was added o he bu e , an app oach p o en o be Fo 1-hyd oxyphenan h ene, linea i y was obse ed
aluable in he sepa a ion o a di e en se o om 25 o 200 ng/ml, using ei he peak a eas o
2
hyd oxy-PAHs [14]. The selec i i y ha can be peak heigh s (R50.9940; ou da a poin s in dupli-
2
achie ed wi h a g-CD–SDS bu e sys em is based ca e, and R50.9952; i e da a poin s in duplica e,
on he di e en pa i ioning o he analy es be ween espec i ely). Because he LODs o 2-hyd oxy-
he micella , aqueous and g-CD phases. The a ini y naph halene and 1-hyd oxych ysene we e a he
o a pa icula analy e o a CD molecule s ongly high, no linea anges we e de e mined o hese
depends on i s i in he CD ca i y. Al hough b-CDs compounds.
a e gene ally conside ed o be mo e e icien com- In gene al, he linea anges a e sho e when
plexing agen s han g-CDs, he la e a e p e e ed in using peak heigh s, which can be eadily explained
CD-MEKC because he b-CD molecules a e no able by he peak b oadening occu ing a le els abo e ca.
o bind analy es s ongly in micella bu e sys ems. 100 ng/ml. This peak b oadening is p esumably
I has been sugges ed ha his can be asc ibed o he caused by o e loading o he micella and g-CD
co-inclusion o indi idual SDS molecules which phases. The co-inclusion o indi idual SDS mole-
educes he ac ual ca i y size, especially in he case cules in o CD ca i ies, as men ioned abo e, implies
o b-CDs [19,20]. ha he e ec i e micella concen a ions a e lowe
In his wo k we used a 30 mMbo a e, 60 mM han expec ed om he o al SDS concen a ion and
SDS, pH 9.0 bu e wi h a ying concen a ions o he c i ical micella concen a ion. Also, he con-
g-CD (10, 12.5, 15 and 20 mM) o achie e he cen a ion o g-CD molecules wi h a ca i y la ge
sepa a ion o he i e hyd oxy-PAHs. A concen- enough o s ong inclusion o hyd oxy-PAHs will be
a ion o 12.5 mMwas ound adequa e o sepa a e educed. These assump ions a e suppo ed by he
all hyd oxy-PAHs. obse a ion ha he peak o 3-hyd oxy-
The limi s o de ec ion (LODs) o he hyd oxy- benzo[a]py ene, which shows he highes a ini y o
PAHs ob ained wi h con en ional luo escence de- he g-CD molecules, shows ailing a high con-
ec ion we e de e mined om h ee eplica e in- cen a ions, which indica es o e loading o he g-CD
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phase. On he o he hand, he on ing obse ed o il e wi h a ansmission (T) o 6.3%, he capilla ies
he 1-hyd oxypy ene peak a highe concen a ions could be used o a leas se e al weeks, while
sugges s o e loading o he micella phase. main aining adequa e de ec ion sensi i i y. A ypical
Wi h he con en ional luo escence de ec o op- LIF-de ec ed elec ophe og am o a s anda d mix u e
imum de ec ion sensi i i y o all compounds e- o he i e hyd oxy-PAHs is shown in Fig. 2. High
qui es p og ammed adjus men o he exci a ion and e iciency peaks we e ob ained wi h pla e numbe s o
emission wa eleng hs. This p o ed o be qui e 128 000, 199 000, 241 000, 191 000 and 214 000
di icul , because he mig a ion imes we e ound o (peak numbe s 2–6, espec i ely).
show sligh un- o- un a ia ions and he compounds The luo escence exci a ion spec a (no shown) o
a e mig a ing closely oge he . Mo eo e , he LODs he hyd oxy-PAHs sugges ha only 2-hyd oxy-
o 2-hyd oxynaph halene and 1-hyd oxych ysene naph halene and 1-hyd oxypy ene can be exci ed
will o en be inadequa e o he de e mina ion o e icien ly a 266 nm. Fo he o he me aboli es he
hese compounds in eal samples. The e o e, i is ex inc ion coe icien s a 266 nm a e a he low.
ce ainly wo hwhile o explo e he possibili ies o In e es ingly, hese di e ences a e no e lec ed in
LIF de ec ion. he LIF esul s: LODs in he low ng/ml ange we e
The LIF se -up (Fig. 1) was op imized unde achie ed o all compounds s udied (Table 1).
low-d i en condi ions (p essu e5100 mba ), using a P obably, measu emen s we e pe o med unde sa u-
26
solu ion o 5?10 M1,5-naph halenedisul onic acid. a ion condi ions: despi e he low mola abso p-
The backg ound le el was checked a e e e y i i ies a 266 nm, maximum exci a ion is eached
adjus men o a oid collec ion o s ay ligh om he due o he high peak powe . When using he BG-14
capilla y co e and wall. This was done by adjus ing cu -o il e , he LODs o 1-hyd oxypy ene and
he p essu e o ze o: hen, in he absence o low, he 3-hyd oxybenzo[a]py ene we e be e han wi h he
luo escence signal almos ins an aneously collapses UG-1 bandpass il e because he UG-1 il e , while
due o pho odecomposi ion. con enien ly excluding ambien ligh , is ha dly ans-
Fi s ly, he capilla y holde posi ion was op imized pa en abo e 400 nm. Compa ed wi h he esul s
so ha he ocal poin on he capilla y was exac ly in ob ained wi h con en ional luo escence de ec ion,
he cen e o he e lec i e objec i e. Then, he he LODs we e signi ican ly be e o 2-hyd oxy-
ocusing in o he capilla y liquid co e was op imized naph halene and 1-hyd oxych ysene.
by adjus ing he posi ion o he ocusing lens.
Subsequen ly, he dis ance o he e lec i e objec i e
o he illumina ed poin o he capilla y was op i-
mized o achie e op imum luo escence collec ion
and sca e ed ligh ejec ion. Finally, he posi ion and
angles o he mi o p ojec ing he luo escence
image on he pho omul iplie ube we e a ied o
op imum luo escence in ensi y. P ope alignmen o
he LIF se -up could easily be main ained o e
se e al mon hs, wi hou any e-alignmen . Also,
excellen long- e m ou pu powe s abili y was ob-
se ed, as was appa en om he s able and la
baselines ob ained.
I should be ealized ha he peak powe o he
lase used he e is s ill as high as 1.8 kW, despi e i s
modes a e age powe o 5.4 mW. I was obse ed Fig. 2. Elec ophe og am o a mix u e o i e hyd oxy-PAHs (20
ha such a high peak powe damages he capilla y ng/ml) using 266-nm LIF de ec ion. Emission il e : BG-14. O he
wi hin a ew hou s. Also, ai bubbles we e o med condi ions: see ex . EOF: elec oosmo ic low; 15asco bic acid;
wi hin he capilla y, p obably due o hea ing o he 253-hyd oxybenzo[a]py ene; 352-hyd oxynaph halene; 451-hy-
bu e . Fo una ely, when using a neu al densi y d oxyphenan h ene; 551-hyd oxych ysene; 651-hyd oxypy ene.

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To summa ize his sec ion on s anda d solu ions, i
can be concluded ha he 266-nm LIF de ec ion
me hod in es iga ed he e is gene ally applicable o
analy es exhibi ing na i e luo escence, despi e he
ixed exci a ion wa eleng h. The de ec ion limi s a e
sligh ly be e han hose ob ained wi h con en ional
luo escence de ec ion. A quan i a i e compa ison
wi h o he LIF de ec ion me hods is di icul o make
since di e ences in de ec ion con igu a ion play a
ole. In an MEKC sepa a ion o hyd oxy-PAHs,
Smi h e al. [14] achie ed LODs in he 0.08–3 mM
ange, alues dis inc ly less a o able han he da a
in Table 1. Howe e , hey used a capilla y wi h an
inne diame e o 50 mm and made use o ibe
op ics. In gene al i can be s a ed ha con inuous Fig. 3. Elec ophe og ams o py ene me aboli es isola ed om he
wa e a gon ion lase s pe o m be e in e ms o hepa opanc eas o (a) Oniscus asellus and (b) Po cellio scabe
achie able de ec ion limi s. In p elimina y expe i- using 266-nm LIF de ec ion. Emission il e : BG-14. Bu e : 30
men s on naph halene sul ona es, using 266-nm LIF mMbo a e, 60 mMSDS, 10 mMg-CD, pH 9.0. 151-Hyd oxy-
py ene sul a e; 25unknown py ene me aboli e; 351-hyd oxy-
de ec ion, LODs in he low nM ange we e ob ained py ene; 45py ene; 551-hyd oxypy ene glucoside.
[21], which is abou one o de o magni ude highe
han ob ainable using exci a ion by a con inuous
wa e a gon ion lase [1,2]. Such a di e ence in men ioned abo e, eco ded wi h LIF de ec ion using
pe o mance should be expec ed in iew o he he BG-14 cu -o il e , a e shown in Fig. 3.
pulsed na u e o he p esen lase (du y cycle: 3?Theo e ical pla e numbe s a e ypically 241 000,
26
10 ). 93 000, 137 000, 97 000 and 91 000 o peak num-
be s 1–5. The conjuga ed PAH me aboli es could be
3.2.
Py ene me aboli es in isopods well sepa a ed by CE using a 30 mMbo a e, 60 mM
SDS, 10 mMg-CD, pH 9.0 bu e . Howe e , py ene
In a p e ious s udy i was concluded ha he and 1-hyd oxypy ene glucoside showed pa ial o e -
py ene me aboli e concen a ions in isopods, spe- lap (R: 0.40) wi h his bu e composi ion. Thei
s
ci ically, Oniscus asellus and Po cellio scabe , can esolu ion equi ed 12.5 mMg-CD in he bu e .
be used as a bioma ke o PAH exposu e in he Un o una ely, unde hese condi ions he 1-hyd oxy-
e es ial en i onmen [5]. Moni o ing he conju- py ene sul a e peak o e lapped wi h a compound
ga ed py ene me aboli es di ec ly, ins ead o 1-hy- p esen in he sample ma ix. Ne e heless, when
d oxypy ene a e enzyma ic hyd olysis, can be using bo h bu e s sys ems all a ge analy es can be
ad an ageous because he analy e de ec abili y is moni o ed.
3–5- imes highe in ha case [9,10,13]. Mo eo e ,
de ec ion o he conjuga ed PAH me aboli es ins ead
3.3. 1
-Hyd oxypy ene in ish bile
o he hyd oxy-PAHs can gi e impo an in o ma ion
ega ding phase II me abolic pa hways. He e, he As a second applica ion, wo ish bile samples
applicabili y o CE–LIF was es ed o he de ec ion we e analyzed, bile om plaice (Pleu onec es pla es-
o py ene, 1-hyd oxypy ene and he h ee conjuga ed sa) exposed o c ude oil, and om lounde (Pla ich-
py ene me aboli es, 1-hyd oxypy ene sul a e, 1-hy- hys lesus) exposed o con amina ed sedimen . These
d oxypy ene glucoside and an unknown phase II samples had been dis ibu ed as pa o an in e -
py ene me aboli e. Since he conjuga es a e no labo a o y s udy [17]. The me hods used in his s udy
comme cially a ailable, quan i ica ion was no pos- by o he labo a o ies we e LC wi h con en ional
sible. luo escence de ec ion and gas ch oma og aphy–
Elec ophe og ams o he wo isopod samples mass spec ome y (GC–MS).
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297
In his applica ion he quan i a i e aspec s o CE
wi h con en ional luo escence and LIF de ec ion,
we e es ed, he a ge analy e being 1-hyd oxy-
py ene. In iew o he da a assembled in Table 1, o
his pa icula me aboli e con en ional luo escence
de ec ion will p obably be he me hod o choice:
compa ed o 266-nm LIF i pe o ms equally in
e ms o LODs bu i p o ides addi ional exci a ion
selec i i y. The ish bile samples (50 ml) we e
weighed and hyd olysed enzyma ically o 2 h a
378C a e he addi ion o 20 ml o enzyme (b-
glucu onidase/a yl sul a ase) and 180 ml o wa e .
A e incuba ion, 1000 ml o absolu e e hanol was
added and he sample was cen i uged o 5 min a
7000 pm; nex , he supe na an was aken o
analysis.
As was al eady indica ed abo e, i was no su p is-
ing ha analyzing he ish bile samples by CE wi h
266-nm LIF de ec ion and using he BG-14 cu -o
il e , ga e ise o a la ge numbe o peaks in he
elec ophe og ams, especially wi h he oil-exposed Fig. 4. Elec ophe og ams o ish bile samples a e enzyma ic
hyd olysis using 266-nm LIF de ec ion. Emission il e : GG-13.
ish bile. The in e e ing peaks inhibi ed eliable O he condi ions: see ex . (a) Oil-exposed plaice bile, (b) sedi-
ecogni ion o mos o he hyd oxy-PAHs discussed men -exposed lounde bile. The 1-hyd oxypy ene peaks a e
in Sec ion 3.1 by means o spiking. Only 1-hyd oxy- indica ed by a ows.
py ene, which was ound o be p esen a a much
highe concen a ion han he o he hyd oxy-PAHs,
could be unambiguously iden i ied. In o de o sen ed in Table 2. I is in e es ing o compa e he
imp o e he selec i i y o LIF de ec ion, a GG-13 esul s wi h hose ob ained in he in e labo a o y
cu -o il e , which emo es all adia ion below ca. s udy [17]. Fo he sedimen -exposed lounde bile
395 nm, was used ins ead. This caused a subs an ial he concen a ions as de e mined independen ly by
educ ion o he numbe o peaks o he sedimen - se en labo a o ies we e ound o be in he 3.061.9
exposed ish bile, bu no o he oil-exposed ish bile mg/g ange (n57, 95% con idence in e al), while
(Fig. 4). o he oil-exposed plaice bile he ange was 1.260.4
I was expec ed ha he use o con en ional (n56). The esul s p esen ed in Table 2 a e ully in
luo escence de ec ion would u he imp o e he line wi h hese da a. The small di e ences be ween
selec i i y, since he exci a ion and emission maxima he LIF and CF da a migh be sys ema ic, bu he
o 1-hyd oxypy ene a e a a he high wa eleng hs ange o 1-hyd oxypy ene concen a ions de e mined
(c . Table 1). Indeed, he elec ophe og ams now by he o he labo a o ies is o b oad o a de ini e
became much cleane (Fig. 5), wi h he elec- conclusion.
ophe og am o he lounde bile exhibi ing only one
la ge peak.
Quan i ica ion was pe o med by using he ex e - 4. Conclusions
nal s anda d me hod wi h calib a ion cu es in he
ange om 16 o 100 ng/ml ( ou da a poin s in Compa ed o o he a ailable deep UV lase sys-
iplica e). Be o e injec ion he oil- and sedimen - ems he quad upled Nd–YAG lase used in his
exposed ish bile samples we e dilu ed wi h e hanol– s udy has se e al ad an ages. I is compac , inexpen-
wa e (80:20, / ) o gi e a o al dilu ion o 503si e and ugged (expec ed ope a ing li e ime: 10 000
and 1003, espec i ely. Rele an esul s a e p e- h). Nex , he alignmen o he LIF se -up is easily
298 J
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907 (2001) 291
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il e is used o educe he inciden powe . The las
cha ac e is ic implies ha he 266-nm CE–LIF de ec-
o can be gene ally used o analy es exhibi ing
na i e luo escence; e en when he mola abso p-
i i y a 266 nm is poo . This is illus a ed by he
LODs ob ained o he i e hyd oxy-PAHs s udied:
simila LODs (2–6 ng/ml) a e ob ained o all o
hem al hough h ee o hem ha e low abso p i i ies
a 266 nm.
O cou se, i eal samples ha e o be analyzed, he
inhe en lack in exci a ion selec i i y can be dis-
ad an ageous. This is clea ly illus a ed by he
elec ophe og ams o he oil-exposed ish bile.
Ne e heless, he 1-hyd oxypy ene concen a ions
de e mined in bo h bile samples using 266-nm LIF
de ec ion a e compa able o hose ob ained wi h
con en ional luo escence, and wi hin he ange o
concen a ions ound by o he labo a o ies using LC–
luo escence de ec ion.
Fig. 5. Elec ophe og ams o ish bile samples a e enzyma ic Finally, i is expec ed ha 266-nm LIF will ha e
hyd olysis using con en ional luo escence moni o ing wi h
l
ex
pe spec i e o de ec ion in CE, especially when
347 nm and
l
387 nm. O he condi ions: see ex . (a) Oil-
em
a en ion is ocused on molecules like y osine,
exposed plaice bile, (b) sedimen -exposed lounde bile. The yp ophan and phenylalanine, because he 266-nm
1-hyd oxypy ene peaks a e indica ed by a ows. lase line is close o he luo escence exci a ion
maxima o hese amino acids.
main ained o e se e al mon hs and he lase shows
excellen long- e m ou pu powe s abili y p o iding
s able baselines and eliable de ec ion. Finally, i s
ou pu powe is high enough o sa u a e he abso p- Acknowledgemen s
ion ansi ions e en when a 6.3% Tneu al densi y
The au ho s wish o hank he Du ch Founda ion
Table 2 o he Ad ancemen o Science (NOW) o inancial
1-Hyd oxypy ene concen a ions in PAH-exposed ish bile and suppo and equipmen (g an No. 344-006). Also,
calib a ion plo cha ac e is ics, using CE wi h LIF and con en ion-
a
he echnical assis ance o M . J. Buijs is much
al luo escence (CF) de ec ion app ecia ed.
Concen a ion 1-hyd oxy-
py ene (mg/g)
LIF CF
Plaice bile (oil-exposed) 1.8 (0.07) 1.5 (0.04) Re e ences
1.7 (n51) 1.3 (n52)
[1] S.J. Kok, I.C.K. Isbe g, C. Gooije , U.A.Th. B inkman, N.H.
Flounde bile (sedimen -exposed) 4.5 (0.07) 4.0 (0.28) Vel ho s , Anal. Chim. Ac a 360 (1998) 109.
4.1 (n52) 3.8 (n52) [2] S.J. Kok, G.P. Hoo nweg, T. de Ridde , U.A.Th. B inkman,
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a
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