Luminescence 2002;17:191–291 ABSTRACTS

Extended abstracts from Xth International

Symposium on Luminescence Spectrometry, Granada,
Spain, 4±7 June 2002
Chemiluminescence systems to study the differentiate the CL produced by neutrophils when this
role of Fcg and complement receptors in response was mediated by FcgR (IgG-IC) from that
stimulating the oxidative burst in mediated by cooperation of FcgR and CR, as well as from
neutrophils from patients with systemic that mediated only by CR [(Fab')2-IC/NHS]. Moreover,
lupus erythematosus the results also show that the luminol–CL system can be
sensitive to measure the CL production by neutrophils in
C. M. O. S. Alves,1* C. M. Marzocchi-Machado,2 diseases such as SLE, which can present quantitative and/
I. F. Carvalho3 and Y. M. Lucisano Valim2 or functional abnormalities of FcgR and/or CR. In the
1
Depto. BioquõÂmica e ImunologõÂa da Faculdade de Medicina de lucigenin–CL system, we found similar results, as shown
RibeiraÄo Preto. E-mail: ifdcarva@fmrp.usp.br in Fig. 2. The lucigenin–CL produced by the neutrophils
2
Depto. FõÂsica e QuõÂmica da Faculdade de CieÏncias of SLE patients was lower than that produced by the
FarmaceÏuticas de RibeiraÄo Preto, Universidade de SaÄo Paulo,
neutrophils of healthy controls. For both stimuli, IgG-IC
RibeiraÄo Preto, SP, Brazil 14040-903
3
Depto. ClõÂnica MeÂdica da Faculdade de Medicina de RibeiraÄo
and IgG-IC/NHS, the lucigenin system was sensitive to
Preto. E-mail: yaluva@usp.br differentiate the CL responses of neutrophils when
mediated by FcgR only and mediated by FcgR/CR. With
regard to the lucigenin–CL system, only the superoxide
We have established chemiluminescence (CL) systems to anion, the first of the reactive oxygen species (ROS), can
investigate the role of the receptors for immunoglobulin be measured. The activation of NADPH oxidase
G (IgG receptors or FcgR) and the receptors for com- generates the other ROS from the superoxide anion. In
plement proteins (CR) in mediating the oxidative burst in contrast, the luminol–CL system measures all of the
neutrophils. Immune complexes (IC) containing IgG and/ ROS. Then, the CL systems, described in the present
or complement bind to neutrophils and stimulate the study, can be important tools to evaluate the involvement
NADPH oxidase complex, which leads to the oxidative
burst. The IC–neutrophils interaction is mediated by
FcgR and/or CR and might be impaired in autoimmune
diseases such as systemic lupus erythematosus (SLE) (1).
Using CL systems, we have been able to evaluate the
involvement of these receptors in the immunopatho-
genesis of SLE, which is mediated by deposition of IC in
a vulnerable organ and massive complement activation
and influx of neutrophils. In the luminol–CL system, we
prepared IC containing anti-BSA IgG/BSA, as described
previously (2), to stimulate the neutrophils via FcgR, and
opsonized these complexes with complement from
normal human serum (NHS) to also stimulate the cells
via CR. In an attempt to stimulate the neutrophils via CR
only, we prepared the fragment F(ab')2 of the anti-BSA
IgG and opsonized it with NHS. In the lucigenin–CL
system, we used anti-OVA IgG/OVA, opsonized or not
with NHS, as complexes. The opsonization of complexes
was performed with NHS diluted 1:2 in veronal-buffered Figure 1. Luminol-dependent chemiluminescence of neutro-
saline, containing CaCl2 and MgCl2, pH 7.2, and phils mediated by Fcg and/or complement receptors. Cells
(2  106) were stimulated with 60 mg immune complexes (IC)
incubation at 37°C for 30 min. Neutrophils were isolated containing IgG (IgG-IC), IgG-IC opsonized with comple-
from blood samples of SLE patients, diagnosed at the ment from normal human serum (IgG-IC/NHS) or with
local University Hospital according the American F(ab')2-IC/NHS. The reaction was kept at 37°C for 15 min in
College of Rheumatology criteria for SLE (3), and from the presence of 10 4 mol/L luminol and recorded in mV. Cells
healthy controls. The CL production by neutrophils and luminol in the absence of IC were used as background
controls. All tests were run in duplicate. These results are
stimulated with IC was accompanied in a luminometer presented as the values of the areas under the curves of the
(Bio-Orbit 1250) in the presence of lucigenin or luminol. chemiluminescence profiles and are representative of three
Fig. 1 shows that the luminol system was able to experiments.

Copyright  2002 John Wiley & Sons, Ltd.

192 ABSTRACTS

to an inadequacy of a two-exponential model and a

shortage of other proposals that have been seen for a few
decades in this field. The main cause of that inadequacy
lies in a discrepancy between the beginning phase of the
two-exponential model and a sigmoidal form of the
ascending phase of chemiluminescence, therefore con-
struction of a new model was founded on a superposition
of infinitesimal forms of logistic, CLasc (t) = D[1 ‡ exp
(a ‡ bt)] 1, and exponential, CLdes (t) = Dexp( kt),
functions, describing the ascending or descending phases
of the time course, respectively. A resulting velocity of
this two-component process is described by the following
differential equation:
Figure 2. Lucigenin-dependent chemiluminescence of
neutrophils mediated by Fcg and/or complement receptors.
Cells (4  106) were stimulated with 80 mg immune complexes
dCL…t† Db exp…a ‡ bt†
containing IgG (IgG-IC) or with IgG-IC opsonized with ˆ kCL…t† …1†
complement from normal human serum (IgG-IC/NHS). The dt ‰1 ‡ exp…a ‡ bt†Š2
reaction was kept at 37°C for 10 min in presence of 10 3 mol/L
lucigenin and recorded in mV. Cells and lucigenin in the
absence of IC were used as background controls. All tests were
run in duplicate. These results are presented as the values of the at the CLdes(t)  CL(t) assumption. An integral
areas under the curves of the chemiluminescence profiles and R exp‰a ‡ …b ‡ k†tŠ
are representative of three experiments. dt (which is not an elementary
‰1 ‡ exp…a ‡ bt†Š2
function, in general) comprised in the solution of
Equation 1, having the form of CL(t) =
R exp‰a ‡ …b ‡ k†tŠ
bD exp… kt† dt was approximated
‰1 ‡ exp…a ‡ bt†Š2
of the FcgR and CR in the production of specific ROS
during the oxidative burst of neutrophils. These methods
can be applied in monitoring the functions of these
receptors in the immunopathogenesis of SLE.

REFERENCES
1. Davies KA. Complement, immune complexes and systemic lupus
erythematosus. Br. J. Rheum. 1996; 35: 5–23.
2. Marzocchi-Machado CM, Polizello ACM, Azzolini AECS, Luci-
sano-Valim YM. The influence of antibody functional affinity on the
effector functions involved in the clearance of circulating immune
complexes anti-BSA IgG/BSA. Immunol. Invest. 1999; 28: 89–101.
3. Tan EM, Cohen AS, Fries JF et al. The 1982 revised criteria for the
classification of systemic lupus erythematosus. Arthritis Rheum.
1982; 25: 1271–1277.

Logistic±exponential model for a time

course of chemiluminescence: theory and
applications
Bonawentura Kochel
Wrocøaw University of Medicine, Department of Toxicology,
57/59 Traugutta Street, PL-50417 Wrocøaw, Poland. E-mail:
kochel@pnet.pl
A logistic–exponential (LE) model for a time course of
chemiluminescence (Fig. 1) was constructed in response
Figure 1. The time course of chemiluminescence of human
neutrophils stimulated with fMLP. The LE model parameters
and characteristics of this process were found to be as follows:
D  SD(D) = 39757  1139 cps; a  SD(a) = 3.085  0.059;
b  SD(b) = 0.426  0.024/s; k  SD(k) = 0.036  0.011/s;
tm  SD(tm) = 12.8  1.0 s, CLm  SD(CLm) = 22930  3359
cps; ti1  SD(ti1) = 6.5  0.5 s; ti2  SD(ti2) = 19.2  1.6 s.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

 ABSTRACTS 193

exp…kt† (Equation 3):

by k 1 exp…kt†. This approximation is
1 ‡ exp…a ‡ bt†    
effective for sufficiently large values of time. 1 k
tm ˆ
ln a ;
Indeed, at a > 0, b < 0 and k > 0 (the experimental b b‡k
reality conditions), the following holds:     
  …b ‡ k†D k k
exp…kt† CLm ˆ
exp ln a ti1=i2 ˆ
lim ˆ exp…kt†. Consequently, b b b‡k
t!1 1 ‡ exp…a ‡ bt†
" p! #
CL(t) = D{[1 ‡ exp(a ‡ bt)] 1 1} ‡ Cexp ( kt) as a 1 b2 2k…b ‡ k†  b b2 4k…b ‡ k†
solution of Equation 1 is valid precisely for large ln a
values of t, where C = D[1 ‡ exp ( a)] 1 is an inte- b 2…b ‡ k†2
gration constant.
 The final result can be rewritten as …3†
exp… kt† ‡ exp…bt†fexp…a†‰exp… kt† 1Š 1g
CL…t† ˆ D ,
‰1 ‡ exp…a ‡ bt†Š‰1 ‡ exp… a†Š Corresponding standard deviations of the LE model’s
where lim …exp…bt†fexp…a†‰exp… kt† 1Š 1g† ˆ 0, parameters and above-mentioned main characteristics
t!1
which results in the following simple formula useful were determined. Moreover, a set of reactions underlying
in applications: the time course described by the LE model was found and
shown to constitute a second-order dynamic system.

D
exp… kt†
CL…t†  …2†
1 ‡ exp…a ‡ bt†
at the exp ( a)  0 assumption, justified by either
sufficiently large values of the a parameter or the
CL(t) measurement error. A relative error of the CL(t)
evaluation (daCL) produced by exp( a)  0 assump-
tion takes the form of aCL: = 100[1 ‡ exp (a)] 1
(%). In practical applications of the LE model (eg
native and perturbed neutrophils or erythrocytes,
intoxicated yeast cells, ferrous ion-treated bull sper-
matozoa cells, autoxidizing L-DOPA, or luminol
oxidized in the Fenton reaction), it was found by
the author that 2  a  10, therefore 12%  aCL 
0.004%. Thus means that in the majority of
(bio)chemiluminescence measurements the daCL error
may be left out of account. R exp…kt†
An approximation of dt by
1 ‡ exp…a ‡ bt†
F…t† ˆ b 1 ‰a ‡ bt ln…1 ‡ exp…a ‡ bt††Š at small values
of t allowed one to determine a relative error of the CL(t)
evaluation given by the LE model (Equation 2):

t CL…t†
t CL…t† :ˆ
100…%†, where DtCL(t): = CL(t)
CL…t†  regulation has not been fully understood.
1
CLcorr(t) and CLcorr …t† ˆ D ‡ exp… kt†
  1
 ‡ exp…a ‡ bt†
k 1 ‡ exp…a ‡ bt† 1 PATIENTS AND METHODS
bt ‡ ln . dtCL(t)
b 1 ‡ exp…a† 1 ‡ exp…a†
takes positive/negative values if the CL(t) value is
overestimated/underestimated by the LE model.
There are three main characteristics describing the
chemiluminescence time course in the LE model: a time
moment, when the CL(t) function reaches its maximal or
peak value tm, the peak value CLm, and two time
moments corresponding to two inflection points ti1, ti2
Regulation of the ischemia reperfusion
syndrome in liver ischemia by transcription
factorsÐa clinical trial using
chemiluminometric methods
S. Kersting,1 T. Zimmermann,1 G. v. Gagern,1
D. Ratayczak,1 S. Albrecht,2 D. Ockert1 and
H. D. Saeger1
1
Department of Surgery, Technical University Dresden,
Fetscherstrasse 74, D-01307 Dresden, Germany. E-mail:
zialblcl@rcs.urz.tu-dresden.de
2
Department of Gynaecology, Technical University Dresden,
Fetscherstrasse 74, D-01307 Dresden, Germany
Transcription factors such as NFkB regulate the release
of numerous mediators, eg immediate early gene
products, which seem to play an important role in
ischaemia and reperfusion damage occurring after liver
resections. Earlier findings suggest that disturbance of the
intracellular redox equilibrium by reactive oxygen
species, normally maintained by seleno-enzymes
PH-Gpx and thioredoxin-reductase, lead to activation of
NFkB with a simultaneous rise of the inactivating factor
of NFkB, IkBa. Until now the detailed mechanism of
In order to evaluate the factors of the redox equilibrium
in vivo, the authors investigated these processes on a
molecular level in 40 patients undergoing liver resec-
tions, comparing operations under ischaemic conditions
(by clamping of the hepatoduodenal ligament) and
operations without interruption of the blood supply.
Blood samples were take pre-operatively, intra-opera-
tively from the portal vein and vena cava) and directly

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

194 ABSTRACTS

Figure 1. Activity of NO.

post-operatively, and at 6 h, 24 h, 48 h and 72 h there- RESULTS

after.
Reactive oxygen species (ROS) were determined by
means of chemiluminescence, the binding activity of the
subunits of NFkB p50 and p65 by EMSA. Additionally,
mRNA of TNFa, tissue factor, IkBa and MIF was traced
by RT–PCR.
Comparing both groups, we find a significantly higher
activity of nitric oxide (NO) in the clamping group, with a
simultaneous relative overexpression of p50 over p65,
leading to a lower exprimation of the immediate early
gene products (Figs 1 and 2).

Figure 2. Expression of p50/p65.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291


DISCUSSION
NO seems to play a crucial role in the reperfusion
injury—on the one hand, NO is known for vasodilatation,
the inhibition of platelet aggregation and adhesion and
for protective effects on hepatocytes by reduction of the
metabolic activity in the cells. It ‘scavenges’ radicals and
thereby acts a cytoprotective. However, in the presence
of oxygen-derived free radicals, NO reacts immediately
with them to peroxynitrite, which has deleterious
cytotoxic effect. On the other hand, since these effects
of nitric oxide seem depending on its redox state, the
maintenance of the redox balance seem to be of major
importance for the reduction of the cell damage.
Promising substances for the restoration of the redox
balance could be antioxidants, corticoids or NO-releasing
substances.
Intra-operative chemiluminometric assay
during surgery for primary
hyperparathyroidism
H. Bergert, D. Ockert, T. Zimmermann, S. Albrecht,
U. Kessler and H. D. Saeger
Department of Visceral, Thoracic and Vascular Surgery, Dresden
University Hospital, D-01307 Dresden, Germany. E-mail:
zialblcl@rcs.urz.tu-dresden.de
Hyperparathyroidism is no longer considered an un-
conunon endocrinopathy. With widespread use of multi-
channel analysers for serum calcium determination as a
part of general medical examination, about one case of
primary hyperparathyroidism/1000 hospital patients/year
may be detected. That is why fewer patients with
advanced skeletal, urologic and hypercalcemic syn-
dromes are referred for surgical treatment compared
with the numbers reported in the literature more than 30
years ago. About 60% of our treated patients were in an
asymptomatic state and the operation is increasingly
becoming a prophylactic procedure.
PATIENTS AND METHODS
Between January 1995 and December 2001 a total of 194
patients underwent neck exploration for hyperpara-
thyroidism in our department for visceral, thoracic and
vascular surgery at Dresden University Hospital: 98
operative interventions were done for primary hyperpara-
thyroidism and 96 for secondary hyperparathyroidism; 5
operations were performed on patients who had pre-
viously undergone neck exploration. 3 in primary and 2
in secondary hyperparathyroidism (Fig. 1). The average
age of our patients with primary hyperparathyroidism
was 43.4 years with a male: female ratio of 1:3.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 195
The sensitivity of the preoperative localization diag-
nostic methods is 65–85%, but the sensitivity of the
surgical neck exploration itself is 90–97%.
The operative strategy for hyperparathyroidism is well
known and widely accepted. It is:
. To identify all four parathyroid glands.
. If there is only one gland enlarged, then this
parathyroid gland should be removed.
. An obvious macroscopically normal gland should be
taken and sent for perioperative frozen section as well.
Although the succes rate of parathyroid surgery is
90–97% there are some intraoperative problems, namely:
. The small size of adenomatous or hyperplastic glands.
. Atypically located glands.
. The occurence of abnormal hyperfunctioning super-
numerary glands. That may be expected in about 6% of
the cases.
To reduce the operation time as well as the failure rate
that accompanies the incomplete excision of hypersecret-
ing parathyroid tissue, we perform an intraoperative
monitoring of parathyroid hormone.
In early 1990 an immunochemiluminometric assay
with an incubation time of about 2 h was developed. We
have modified this assay by reducing the incubation time,
in a first step to 20 min and now to 7 min. With this
modification it was possible to perform the assay in the
operating theatre and to obtain hormone levels within
10 min.
Intact parathyroid hormone is a single-chain polypep-
tide of 84 amino acids and has a short half-life of
approximately 5 min, which confirms a rapid rate of
disappearence of the molecule from the peripheral
circulation. This method utilizes two antibodies which
bind to separate portions of intact parathyroid molecule
and a chemiluminescent label as the detection system.
Blood samples are incubated with chemiluminescent
acridinium ester-labelled polyclonal PTH (1–34) anti-
Figure 1. Operations in hyperparathyroidism (HPT), Depart-
ment of Surgery, University Hospital Dresden, Germany.
Luminescence 2002;17:191–291
196 ABSTRACTS
Figure 2. Method of chemiluminometric assay for PTH
monitoring.
body. In the same step the sample is incubated with a
polyclonal (44–68) PTH antibody, which is covalently
bonded on a polysterene bead. In this way the parathyroid
hormone is sampled in form of this sandwich complex
(Fig. 2). The tubes are decanted and after a repeat wash
the bound luminescence in the assay tubes after injection
of a trigger solution are measured using a luminometer.
The concentration of intact parathyroid hormone is
directly proportional to the amount of chemilumi-
nescence.
RESULTS
For monitoring purposes, blood samples were taken from
a peripheral vein at following times: preoperatively just
before induction of anesthesia, after identifying the
suspicious parathyroid gland during the neck exploration,
first at the moment of the excision and then at two 10 min
intervals after excision.
Initial intraoperative concentrations of PTH were in
the range 79–1380 pg/mL. The maximum values are
attributed to manipulation of the adenomatous glands
during exploration; 10 min after excision, PTH serum
values declined to an averaged value of 89 pg/mL. In all
cases of successful parathyroidectomy the PTH values
declined to under 20% of the initial intraoperative PTH
values. In one case of operation failure the PTH value did
not decrease to the baseline value of 20%, which
indicated that we had not found the hypersecreting gland,
although we had removed four glands already. This
female patient had a fifth supernumerary gland in the
mediastinum but she rejected an extended operation with
sternotomy. Our results demonstrate that intraoperative
parathyroid hormone monitoring is a cost-effective
method of controlling the effect of surgical treatment.
We use it as our “biochemical frozen section”. A
decrease of the PTH concentration to under 20% percent
of the baseline values predicts the success of parathyroid
surgery.
Copyright  2002 John Wiley & Sons, Ltd.
Chemiluminometric detection of CTX and
osteocalcin in cases of postmenopausal
women using the Elecsys analyser
S. Albrecht,1 T. Zimmermann,3 K. Scheunert,1
V. Konnegen,1 G. Wunderlich2 and W. Distler1
1
Department of Gynaecology and Obstetrics at Dresden
University of Technology, Fetscherstrasse 74, D-01307 Dresden,
Germany. E-mail: zialblcl@rcs.urz.tu-dresden.de
2
Department of Nuclear Medicine at Dresden University of
Technology, Fetscherstrasse 74, D-01307 Dresden, Germany
3
Department of Visceral, Thoracic and Vascular Surgery,
Fetscherstrasse 74, D-01307 Dresden, Germany
It is known that bone turnover increases both perimeno-
pausally and during early menopause (1). During these
stages cell activity on the bone surface intensifies, with
primary stimulation of osteoclasts. At a later point in
time, osteoblast activity also rises but with reduced
efficiency. This effect may be caused by insufficient
function of the ovaries, because the osteoblasts already
have receptors for oestrogen. This finally results
predominantly in bone resorption, with perforation of
the bone trabeculae (2).
Bone resorption may be assessed by measuring bone
mineral density (BMD) and biochemical markers of bone
metabolism. Type I collagen is a helical protein that is
cross-linked at the N- and C-terminal ends of the
molecule. During renewal of skeleton, type I collagen
is degraded and small peptide fragments are excreted into
the bloodstream. The C-telopeptides (CTx) of the
a1-chains of the collagen triple helix then can be
determined (3, 4). Osteocalcin (OC) is a vitamin
K-dependent, calcium-binding and the most abundant
non-collagenous bone protein. It is a specific product of
the osteoblasts and is thus a marker for bone formation. A
fraction of a newly synthesized molecule is released in
the blood, where it can be detected by a radioimmuno-
assay (RIA) or electrochemiluminescence immunoassay
(ECLIA). Besides the intact molecule, proteolytic frag-
ments such as the N-terminal mid-fragment can also be
measured.
One aim of the study was to test the behaviour of OC
and CTx at the beginning of the menopause as well as a
comparison of two different assays for the determination
of these two markers within clinical practice.
MATERIALS AND METHODS
Subjects
176 Postmenopausal, ambulatory women, mean age
60.4  6.1 years (range 41–77 years) were studied over
a 2 year period. Blood samples were taken by veno-
puncture and stored at 20°C. For the determination of
bone loss, BMD of the distal radius was measured using a
Stratec CT-Bone Scanner (SCT 900). On the basis of
Luminescence 2002;17:191–291
 ABSTRACTS 197

BMD values and hormone replacement therapy (HRT) all

subjects were subdivided into four groups:
1. Women (mean age 59.3 years, n = 73) with normal
BMD and without HRT.
2. Women (mean age 59.9 years, n = 43) with normal
BMD and with HRT.
3. Women (mean age 64.3 years, n = 40) with BMD > 2
SD below the mean for a particular age and without
HRT.
4. Women (mean age 58.7 years, n = 18) with BMD > 2
SD below the mean for a particular age and with HRT.

Markers of bone formation and resorption

Intact OC was detected in serum by RIA (OSCAtest1,
Brahms, Berlin, Germany). In addition, the N-terminal
mid-fragment was determined by electrochemilumi-
nescence immunoassay (ECLIA, Roche Elecsys System
1010). The Elecsys analyser is a fully automatic multi-
batch analytic system for the determination of immuno-
logical tests using the chemiluminometric procedure.
Serum CTx was assessed using a commercially
available enzyme-linked immunosorbent assay (Serum
CrossLaps One Step ELISA, Osteometer Bio Tech, IBL,
Hamburg, Germany) and ECLIA (Roche Elecsys System
1010). All analyses were performed according to the Figure 1. Results of biochemical parameters.
manufacturer’s instructions.

RESULTS
The results are shown in Figs 1–6 and in Table 1. Com-
pared with healthy postmenopausal women, untreated
osteoporotic women had significantly higher mean
concentrations of OC in both measurements ( p = 0.011
for RIA, p = 0.022 for ECLIA), as shown in Figs 1 and 2.
Furthermore, HRT results in a significantly ( p = 0.004 for
RIA, p = 0.007 for ECLIA) drop of OC values, which was
only tested for persons with reduced BMD. Intact OC was
highly correlated (r = 0.878) with the N-terminal mid-
fragment (Fig. 5).
The mean concentrations of CTx were not significantly
different ( p = 0.514 for ELISA, p = 0.130 for ECLIA)
between women with normal and low BMD without
HRT, as shown in Figs 3 and 4. In women with HRT and
BMD > 2 SD below the mean for a particular age, the
mean values of CTx decreased significantly ( p = 0.016
for ELISA, p = 0.028 for ECLIA) in comparison to those
without any HRT. Fig. 6 shows that ELISA and ECLIA
obtained similar results with a high correlation of both
methods (r = 0.801).

DISCUSSION
Postmenopausal women with an amplified loss of bone
mass show significantly higher levels for OC than those Figure 2. Results of biochemical parameters.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

198 ABSTRACTS

Figure 3. Results of biochemical parameters.

Figure 5. Results of biochemical parameters.

Figure 4. Results of biochemical parameters. Figure 6. Results of biochemical parameters.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291


Table 1. Biochemical markers related to bone mineral density and HRT
Markers of bone turnover Group 1
Intact OC (ng/mL) 13.09  6.35
N-terminal mid-fragment (ng/mL) 25.25  10.14
CTx (ELISA) (pmol/L) 3189  1376
CTx (ECLIA) (ng/mL) 0.286  0.136
Data are shown as mean  1 SD.
with normal BMD. This finding of increased OC in
women with low bone mass agrees with previous studies.
By contrast Seibel et al. did not establish a significant
difference between menopausal healthy and osteoporotic
women. The suppression of OC during HRT was also
found in earlier studies. Considering the presented out-
comes, it follows that intact OC and the N-terminal mid-
fragment are equivalent for assessing bone formation.
CTx were unchanged in women with decreased BMD.
No significantly difference between normal women and
those with reduced BMD could be found. In contrast,
oestrogen therapy causes a reduction of the intensified
bone turnover in terms of a significant drop of CTx (4).
Compared with conventional assays, the fully automatic,
simple to handle analysis system mass similar results.
In conclusion, intact OC and N-terminal mid-fragment
are probably helpful in assessing bone loss and therapy
effects, while CTx is only useful for therapy monitoring.
The ELISA and ECLIA can equally be used for detecting
OC and CTx.
REFERENCES
1. Ravn P, Fledelius C, Rosenquist C, Overgaard K, Christiansen C.
High bone turnover is associated with low bone mass in both pre-
and postmenopausal women. Bone 1996; 19: 291–298.
2. Kanis JA. Osteoporose. Blackwell Wissenschaftsverlag: Berlin and
Vienna, 1995; 33–38.
3. Pedersen BJ, Ravn P, Bonde M. Type I collagen C-telopeptide
degradation products as bone resorption markers. J. Clin. Ligand
Assay 1998; 21: 118–127.
4. Rosenquist C, Fledelius C, Christgau S et al. Serum CrossLaps One
Step ELISA. First application of monoclonal antibodies for
measurement in serum of bone-related degradation products from
C-terminal telopeptides of type I collagen. Clin. Chem. 1998; 44:
2281–2289.
A new experimental strategy in the
application of the peroxyoxalate
chemiluminescence system in ¯ow injection
analysis
L. GaÂmiz-Gracia,1 A. M. GarcõÂa-CampanÄa,1
F. AleÂs Barrero,1 M. A. Dorato,2 M. RomaÂn Ceba1 and
W. R. G. Baeyens3
1
Department of Analytical Chemistry, Faculty of Sciences,
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 199
Group 2 Group 3 Group 4
9.52  3.78 16.72  8.44 10.34  4.11
15.59  6.11 32.18  15.27 18.75  4.57
1697  1243 3387  1752 2237  1278
0.140  0.070 0.355  0.220 0.178  0.105
University of Granada, Av Fuentenueva s/n, E-18071 Granada,
Spain. E-mail: lgamiz@ugr.es
2
National University of CoÂrdoba, Argentina
3
Department of Pharmaceutical Analysis, Faculty of
Pharmaceutical Sciences, Ghent University, Harelbekestraat 72,
B-9000 Ghent, Belgium
Peroxyoxalate (PO) chemiluminescence (CL) reaction is
one of the most efficient system applied in analytical
chemistry for the determination of hydrogen peroxide,
some fluorescent molecules and compounds that are not
directly fluorescent but can be easily derivatized with
fluorescent labels (1). Furthermore, imidazole has been
known for a long time as the most efficient catalyst in the
PO-CL reaction, showing a complex concentration
dependence on chemiluminescence intensity and reaction
kinetics (2).
The use of POs as precursors in these chemilumi-
nescence reactions presents some drawbacks, the most
important ones being those arising from the of use
organic solvents. POs are very unstable in the presence of
water, and must be dissolved in acetonitrile in order to
avoid their degradation. Thus, the systems described in
the literature employ special pumps or special pump-
tubes when using POs in flow injection (FI) systems. By
contrast, we propose a cheaper and easier FI configura-
tion, based on the use of a two-injection valve system for
the introduction of both PO and a fluorophore (or a
derivatized analyte) solution into the flow system,
avoiding the problems arising from the use of acetoni-
trile, as neither special tubes nor special pumps are
required. Furthermore, the use of a micellar medium as
carrier avoids the rapid degradation of POs in water
(3, 4).
EXPERIMENTAL
Chemical
Human albumin (fraction V), glycine, o-phthalaldehyde
(OPA), fluorescamine (FR), fluoresceine, imidazole,
polyoxyethylene 23 lauryl ether (Brij 35), cetyltrimethyl-
ammonium bromide (CTAB), and acetone were pur-
chased from Sigma. Hydrogen peroxide, acetonitrile and
sodium dihydrogen phosphate were obtained from
Panreac. Sodium hydrogen carbonate and rhodamine B
were purchased from Merck. Sodium dodecyl sulphate
Luminescence 2002;17:191–291
200 ABSTRACTS
Figure 1. Proposed manifold. PMT, photomultiplier tube; PP, peristaltic pump; TCPO, bis(2,4,6-trichlorophenyl) oxalate.
(SDS) and Triton X-100 were obtained from Riedel De
Haën, and bis(2,4,6-trichlorophenyl) oxalate (TCPO)
from Wako Pure Chemical Industries Ltd. All the
reagents were of analytical reagent grade. Deionized
water was used for the experimental work.
Apparatus
CL measurements were carried out on a Jasco CL 1525
detector, equipped with a PTFE spiral detection cell, data
control and acquisition programme. Two Gilson Mini-
pulse-3 peristaltic pumps, two Rheodyne 5020 manual
injection valves, and Omnifit tubing and connections
were used for constructing the FI manifold.
Proposed FI manifold
The conditions (not optimized) employed for carrying out
those experiments were as follows: surfactant solutions
(10 mmol/L) were prepared by dissolving them in
phosphate buffer (0.1 mol/L, pH 7.0) and used as the
carrier solution. Hydrogen peroxide (100 mmol/L, pre-
pared daily) and imidazole (10 mmol/L) were prepared in
surfactant solution and used as oxidant and catalyst,
respectively. Three different streams introduced the
solutions into the FI system at a flow rate of 2 ml/min.
TCPO (1 mmol/L) was prepared in acetonitrile and
introduced into the carrier stream by means of a manual
injection value (500 mL loop). The fluorophore (fluor-
esceine or rhodamine B) or the labelled analyte (glycine
labelled with OPA or albumin labelled with FR) was
Table 1. CL intensities for different CL determinations applying the proposed FI manifold in
presence of different surfactants as carriers using the PO-CL system
Fluorophore Analyte
Rhodamine B None
Fluoresceine None
o-Phthalaldialehyde Glycine
Fluorescamine Albumin
* Results expressed as average CL intensity in arbitrary units (n = 3).
Copyright  2002 John Wiley & Sons, Ltd.
subsequently introduced into the FI system 3 s later by
means of a second injection valve (100 mL loop). The
content of the two injection valves were mixed in a
reaction coil (50 cm length, 0.5 mm i.d.) placed just after
the second injection valve and before reaching the
detector. The chemiluminescence intensity was instanta-
neously measured by the PMT placed in front of the
detection cell.
RESULTS AND DISCUSSION
The proposed FI-manifold (Fig. 1) was checked coupled
to a CL detector, making use of TCPO as CL precursor,
hydrogen peroxide as oxidant and imidazole as catalyst in
both direct CL (using two different fluorophores such as
fluoresceine and rhodamine B as analytes) and indirect
CL systems, considering non-fluorescent analytes, such
as glycine and albumin, and employing two different
fluorescent derivatizing agents of the CL reaction, viz.
OPA (a label of amino acids and proteins tested with
glycine) and FR (a label of amino groups which provided
good sensitivity and selectivity, tested with albumin).
Different surfactants were tested with this system as
carriers and solvents, viz. SDS, anionic; CTAB, cationic;
and Triton X-100 and Brij 35, non-ionic. The concentra-
tions of all the surfactants were upon the critical micellar
concentration.
The results obtained in terms of intensity of chemi-
luminescence for different CL systems are showed in
Table 1. The best results were obtained when CTAB and
SDS were used as carriers and solvents, offering an
Surfactant*
CTAB SDS TRITON X-100 BRIJ 35
28 26 15 15
337 36 13 11
249 70 81 36
– 941 784 748
Luminescence 2002;17:191–291

interesting alternative in the use of the PO-CL reaction as
a detection technique in continuous analysis systems,
avoiding the use of organic solvents in continuous
analysis systems. Further results will be obtained in the
optimization of some of the proposed determinations.
Acknowledgement
The International Olympic Committee (IOC) (ref. 1767/
2000) supported this work.
REFERENCES
1. Stigbrand M, Jonsson T, Pontén E, Irgum K, Bos R In Chemilumi-
nescence in Analytical Chemistry, Garcı́a-Campaña AM, Baeyens
WRG (eds). Marcel Dekker: New York, 2001; 141–173.
2. Emteborg M. Theory and Applications of Imidazole-Mediated
Peroxyoxalate Chemiluminescence. PhD Thesis, Department of
Analytical Chemistry, Umeå University, Sweden, 1997.
3. Dan N, Ling Lau M, Grayeski, ML. Anal. Chem. 1991; 63: 1766–
1771.
4. Steijger OM, Van Mastbergen HM, Holthuis JJM. Anal. Chim. Acta
1989; 217: 229–237.
Optimization of the chemiluminescent
determination of protein in micellar
medium by ¯ow injection analysis using
experimental design
L. GaÂmiz-Gracia,1 A. M. GarcõÂa-CampanÄa,1 F. AleÂs
Barrero,1 L. Cuadros RodrõÂguez,1 M. Schiavone2 and
W. R. G. Baeyens3
1 phosphate were obtained from Panreac. Sodium hydro-
Department of Analytical Chemistry, Faculty of Sciences,
University of Granada, Av. Fuentenueva, s/n, E-18071 Granada,
Spain. E-mail: lgamiz@ugr.es
2
University of Bologna, Italy
3 from Wako Pure Chemical Industries Ltd. All the
Department of Pharmaceutical Analysis, Faculty of
Pharmaceutical Sciences, Ghent University, Harelbekestraat 72,
B-9000 Ghent, Belgium
Figure 1. Pareto chart for screening design.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 201
Peroxyoxalate chemiluminescence (PO-CL) is an in-
direct or sensitised type of chemiluminescence in which a
peroxyoxalate is oxidized in presence of H2O2 and an
intermediate from the primary reaction transfers its
energy to an energy-accepting fluorophore, which
becomes electronically excited and subsequently emits
light. The usefulness of this reaction is based on the
possibility of detecting native fluorophores or compounds
derivatized with fluorescent labels (1).
In this contribution we propose the use of an original
flow injection analysis (FIA) configuration for the CL
determination of proteins based on the use of a two-
injection valve system for the introduction of both PO
and derivatized analyte solutions in the flow system,
avoiding the problems arising from the use of acetonitrile
as solvent, as neither special tubes nor special pumps are
required by using micellar medium as carrier, which also
avoids the rapid degradation of POs in water. Chemical
and instrumental variables affecting CL emission have
been optimized, using an experimental methodology
based in the use of both univariate optimization and a
sequential experimental design, viz. 27 4 screening
design Face-centred Draper–Lin Small Composite
Design (scarcely used in analytical chemistry) and a
23 ‡ star Face-centred Central Composite Design.
EXPERIMENTAL
Chemical
Human albumin (fraction V), fluorescamine (FR),
imidazole and acetone were purchased from Sigma.
Hydrogen peroxide, acetonitrile and sodium dihydrogen
gen carbonate was purchased from Merck. Sodium
dodecyl sulphate (SDS) was obtained from Riedel De
Haën, and bis(2,4,6-trichlorophenyl) oxalate (TCPO)
reagents were of analytical reagent grade. Deionized
water was used for the experimental work.
Luminescence 2002;17:191–291
202 ABSTRACTS

Table 1. Estimated effects for CL response in the screening Procedure

design
For the CL determination of albumin using the proposed
Average 284.13  68.32
CL system and the new configuration, SDS (10 mmol/L)
A: Peroxide ‡ BD ‡ CE ‡ FG 301.61  160.22 was prepared in phosphate buffer (0.1 mol/L, pH 7.0) and
B: Imidazole ‡ AD ‡ CF ‡ EG 49.04  160.22 used as carrier solution in the first stream at a flow rate of
C: TCPO ‡ AE ‡ BF ‡ DG 296.64  160.22
D: pH ‡ AB ‡ CG ‡ EF 23.29  160.22 3 mL/min. Hydrogen peroxide (400 mmol/L, prepared
E: Imidazole flow rate ‡ AC ‡ BG ‡ DF 417.49  160.22 daily), incorporated in one of the streams at a flow rate of
F: Peroxide flow rate ‡ AG ‡ BC ‡ DE 19.56  160.22 2.1 mL/min, and imidazole (4 mmol/L) delivered in the
G: Dummy ‡ AF ‡ BE ‡ CD 30.71  160.22 third stream at a flow rate of 0.2 mL/min, were prepared
in surfactant solution and used as oxidant and catalyst (3),
respectively. TCPO (1 mmol/L) was prepared in aceto-
nitrile and introduced into the carrier stream by means of
Apparatus a manual injection valve (50 mL loop). Albumin labelled
with FR was finally introduced into the FI carrier stream
CL measurements were carried out on a Jasco CL 1525 by means of a second injection valve (20 mL loop). The
detector, equipped with a PTFE spiral detection cell, data content of both injection valves is mixed in a reaction coil
control and acquisition programme. Two Gilson Mini- (50 cm length, 0.5 mm i.d.) placed just after the second
pulse-3 peristaltic pumps, two Rheodyne 5020 manual injection valve. The CL reaction takes place in the
injection valves, and Omnifit tubing and connections detection cell and the CL intensity is registered
were used for constructing the FI manifold, described instantaneously by the PMT situated just in front of the
previously (2). cell.

Figure 2. Response surfaces for Face-centred Draper–Lin Small Composite Design.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

 ABSTRACTS 203

The labelling of the albumin with FR was carried out Table 3. Performance characteristics of the method
off-line as follows: 1 mL albumin in buffer (NaHCO3
Work range 1.56–12 ppm
6.5 mmol/L, pH 11.5) was mixed with 1 mL FR Slope 28.87 c.u./ppm
(4.5 mmol/L in acetone) and placed in an ultrasound Intercept 80.64 c.u.
bath for 1 min at room temperature. SR,c 17.14 c.u./ppm
R2 98.15%
Linearity 96.03%
Analytical sensitivity 28.87 c.u./ppm
RESULTS AND DISCUSSION Analytical resolution 0.59
Optimization of the experimental variables Precision (a) RSD, 4.27% (n = 13)
(b) Reproducibility limit, 12.1 c.u.
The variables (both chemical and FI variables) involved Detection limit (a) IUPAC, 0.31 ppm (9.3  10 14 mol)
in the proposed CL-FIA system (viz. concentration of (b) ISO, 0.38 ppm (1.1  10 13 mol)
Quantification limit 1.02 ppm
SDS, peroxide, imidazole and TCPO, pH, flow rate of Upper limit 12 ppm
SDS, imidazole and peroxide, injection volume of TCPO Linear range 1.87
and labelled albumin, reactor length, and labelling
reaction) were optimized by means of both univariate
and multivariate methods.
Once the flow rate of the carrier has been selected on
the basis of previous studies, a 27 4 screening fractional was optimised by means of a 23 ‡ star Face-centred
factorial design was carried out in order to evaluate the Central Composite Design, studying the following
significance of the other variables involved in the system. variables: concentration of FR and buffer, and pH. Once
The results are summarized in Fig. 1, which represents the chemical variables had been optimized, a 32 design
the Pareto chart for the studied variables, and in Table 1, was chosen for the optimization of temperature and
which gives the estimated effects. All those variables mixing time.
whose effect was lower than that of the dummy were All selected optimum values are summarized in
considered as non-significant. Table 2.
A Face-centred Draper–Lin Small Composite Design
(4) was then carried out for the optimization of the four
significant variables, viz. concentration of TCPO, Calibration and performance characteristics
peroxide and imidazole, and flow rate of imidazole. This The method provides good stability and repeatability
design allows us the optimization of a relatively high (RSD, 4.27% of the CL signal for a concentration of
number of variables with a low number of experiments. 6 ppm albumin; n = 13) and a linear range of 0.31–
The obtained response surfaces are shown in Fig. 2. 12 ppm albumin (ie 0.47–1.80 fmol albumin), with a
Other variables involved in the design (as the volume detection limit (IUPAC) of 0.31 ppm, which corresponds
of the injection valves and length of the reactors) were to 0.93 fmol albumin. All the performance characteristics
optimized by the univariate method. are shown in Table 3.
Finally, the labelling reaction between FR and albumin The proposed method is being applied to the deter-
mination of albumin in pharmaceutical products and
biological fluids. Further results will be available soon.
Table 2. Optimized values
FIA system Acknowledgement
[SDS] 10 mmol/L in buffer NaH2PO4
0.1 mol/L, pH 7.0 The International Olympic Committee (IOC) (Ref. 1767/
[H2O2] 400 mmol/L (in SDS) 2000) supported this work.
[imidazole] 4 mmol/L (in SDS)
[TCPO] 1 mmol/L in ACN
SDS flow rate 3 mL/min
H2O2 flow rate 2.1 mL/min REFERENCES
Imidazole flow rate 0.2 mL/min
Valve 1 (TCPO) 50 mL 1. Stigbrand M, Jonsson T, Pontén E, Irgum K, Bos R. In Chemilumi-
Valve 2 (FR ‡ albumin) 20 mL nescence in Analytical Chemistry, Garcı́a-Campaña AM, Baeyens
Reactor 50 cm, 0.5 mm i.d. WRG (eds). Marcel Dekker: New York, 2001; 141–173.
2. Gámiz-Gracia L, Garcı́a-Campaña AM, Alés Barrero F, Dorato MA,
Labelling reaction Román Ceba M, Baeyens WRG. Luminescence (this issue)
[FR] 4.5 mmol/L in acetone (1 mL) 3. Emteborg M. Theory and Applications of Imidazole-mediated
Buffer NaHCO3 6.5, pH 11.5 (1 mL) Peroxyoxalate Chemiluminescence. PhD Thesis, Department of
Ultrasounds 1 min Analytical Chemistry, Umeå University, Sweden, 1997.
4. Draper NR, Lin DKJ. Technometrics 1990; 32: 187–194.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

204 ABSTRACTS
Determination of oxymetazoline
hydrochloride by ¯ow injection analysis
with inhibited chemiluminescent detection
M. P. Bueno Vargas,1 A. M. GarcõÂa-CampanÄa,1
J. M. Bosque Sendra1 and X. Zhang2
1
Analytical Chemistry, Faculty of Sciences, University of
Granada, Av. Fuentenueva s/n, E-18071 Granada, Spain
2
Department of Chemistry, Tsinghua University, 10084 Beijing,
People's Republic of China
Oxymetazoline hydrochoride is an imidazoline derivate
that is usually found as a decongestant in different
medications used in the treatment of eye irritation and
nasal congestion caused by colds, rhinosinusitis and/or
allergic symptoms. In this paper, we have studied the
potential of flow injection analysis (FIA) combined with
chemiluminescence (CL) detection for the analysis of this
intranasal drug, to be used in quality control. For this
purpose, a study of the effect of oxymetazoline hydro-
chloride on the signal of different chemiluminescence
systems using some chemiluminescent precursors or
involving different oxidants in different media and
applying a typical flow injection manifold has been
carried out.
EXPERIMENTAL
Chemical
Oxymetazoline hydrochloride and 3-aminophthal-
hydrazide (luminol) were purchased from Sigma. Potas-
sium permanganate was obtained from Panreac. Sodium
hydrogen carbonate and sodium hydroxide were pur-
chased from Merck. All the reagents were of analytical
reagent grade. Deionized water was used for the
experimental work.
Apparatus
CL measurements were carried out on a Jasco CL 1525
detector, equipped with a PTFE spiral detection cell, data
control and acquisition programme. Two Gilson Mini-
pulse-3 peristaltic pumps, a Rheodyne 5020 manual
injection valve and Omnifit tubing and connections were
used for constructing the FI manifold.
RESULTS AND DISCUSSION
FI manifold and optimization of experimental
variables
From the screening of several chemiluminescence
systems, we found the presence of oxymetazoline
produced a strong inhibition of the chemiluminescence
intensity from the oxidation of luminol using permanga-
nate in basic medium. By considering that permanganate
Copyright  2002 John Wiley & Sons, Ltd.
Figure 1. Proposed FI-CL manifold.
is an oxidant applicable in a luminol-based CL system
(1), we observed a negative CL peak from the injection of
standard or sample solution of oxymetazoline, since it
can be oxidized by permanganate in this medium, which
implies a decrease of the background CL signal. In this
sense, it is possible to establish a flow injection (FI)
method for the determination of oxymetazoline hydro-
chloride based on its inhibition on the CL intensity of the
luminol–permanganate system. First, different FIA con-
figurations were examined, selecting from this study a
three-channel FIA manifold from which better signal:
noise ratios and good reproducibility were obtained. A
schematic diagram of the flow system is shown in Fig. 1.
Then, the effect of both experimental (concentrations of
permanganate and luminol and pH) and instrumental
variables (injected volume of analyte and flow rates)
were studied in order to achieve the highest decrease in
the CL signal proportional to the analyte concentration.
Considering the optimized conditions, the proposed
FIA assembly consisted of three different streams
containing buffer solution (pH 11.5), ie the carrier
channel, a second one for permanganate solution
(3.5 mmol/L), both at flow rates of 2.2 ml min, and the
last one with luminol (100 mmol/L), prepared in buffer
solution, at a flow rate of 4.5 mL/min. Standard and
sample solutions are injected directly into the buffer
stream. An injection volume of 20 mL was selected
because it gave the best analyte:background signal ratio
and faster chemiluminescence signals were acquired.
Calibration and performance characteristics
Using these experimental conditions, a standard calibra-
tion curve was established, obtaining a good linear
relationship between the oxymetazoline concentration
(C) logarithm and the CL intensity (ICL), the regression
equation being, ICL = 383.60 ‡ 537.14 log C (Fig. 2).
The performance characteristics were calculated, show-
ing a linear dynamic linear range of 1.88–200 ppb, a
IUPAC limit of detection of 1.21 ppb, a linearity on-line
of 99.01% and a relative standard deviation of 1.60%
(50 ppb oxymetazoline).
This method is very simple and sensitive and offers an
easy alternative for the determination of oxymetazoline
hydrochloride. Pharmaceutical compounds are being
Luminescence 2002;17:191–291

Figure 2. Variation in CL intensity with the change of oxy-
metazoline concentration. Concentrations of oxymetazoline
injected were (repeated three times for each): blank, 25, 50,
100, 150 and 200 ppb.
analysed to test the usefulness of the proposed method in
real samples.
REFERENCE
1. Easwaramoorthy D, Yueh-Chuan Yu, Hsuan-Jung Huang. Anal.
Chim. Acta 2001; 439: 95.
Enzymatic determination of sugars at trace
level in homogeneous phase using
multisyringe ¯ow injection analysis (MSFIA)
and chemiluminescence detection
Nicolau PizaÁ, Manuel MiroÂ, Jose Manuel Estela and
VõÂctor CerdaÁ
Department of Chemistry, University of the Balearic Islands,
E-07071-Palma de Mallorca, Spain. E-mail: vcerda@p01.uib.es
Organic analytes are commonly analysed by chemilumi-
nogenic reactions in the liquid phase. In this context,
extensive reviews have been recently published by
Fletcher et al. (1), Palilis and Calokerinos (2) and
Garcı́a-Campaña and Baeyens (3), wherein the evolution
of chemiluminescence (CL) through time is overviewed
and some mechanisms of CL reactions are discussed in
detail. Although direct determinations have been used in
biomedical chemistry to analyse compounds of pharma-
ceutical interest in biological fluids, the most widely
extended CL applications are related to indirect determi-
nations based on either the quenching of a certain CL
reaction (4) or the enzymatic conversion of the target
compound into an ingredient of the CL reaction. In the
latter case, when the indirect CL determination is carried
out by flowing stream methods, particularly by flow
injection analysis (FIA), the most commonly employed
strategy relies upon the implementation of packed-bed
reactors with the enzyme immobilized by covalently
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 205
bonding on controlled-pore glass into the flow assembly
(5) in order to achieve a better enzyme stability and a low
reagent consumption. Hence, scarce flow methodologies
use the enzyme in homogeneous phase, wherein unlike
immobilization-based systems the enzymatic activity
may be improved by proper adjustment of pH of reaction
medium (pH <6).
In this communication, a novel multisyringe flow
injection analysis (MSFIA) set-up for the indirect CL
determination of sugars (viz. glucose) in real samples is
reported. This novel technique (6) assembles some
features of the precedent FIA and sequential injection
analysis (SIA) methodologies that make it especially
adapted to implement CL-enzymatic procedures in
homogeneous phase. The automated manifold (see Fig.
1) comprises a multisyringe burette with four syringes
connected in block to the same step-by-step motor,
allowing the simultaneous propulsion of sample/reagents
to the flow system at the required time, and the selection
of the proper flow-rate for each operation of the
procedure. A greater versatility, demonstrated by the
simplicity of sample aspiration and choice of enzyme
volume, is achieved with three-way commutation valves,
as used in the multicommutation approach. The metho-
dology is based on the on-line oxidation of the substrate
(b-glucose) by the glucose oxidase (GOD) flavoprotein to
gluconolactone and hydrogen peroxide in the presence of
molecular oxygen. Subsequently, the generated oxidant
merges downstream with an alkaline slug of 3-amino-
phthalhydrazide (luminol) and a metal catalyst zone, viz.
Co (II) at a total flow rate as high as 72 mL/min in order
to develop the CL reaction into a laboratory-made spiral-
shape flow-through cell placed in front of a metal case-
type photomultiplier.
Optimization of flow parameters, performance of
reactor, buffer and reagent concentrations and set-up
configuration with the aim of achieving maximum
glucose to hydrogen peroxide conversion rate and
Figure 1. Schematic diagram of the multisyringe flow injec-
tion system for glucose determination at trace levels. MSB,
multisyringe burette; SV, solenoid valve; HC, holding coil; KR,
knotted reactor (130 cm); RC, reaction coil (4 cm); D, detector
(photomultiplier tube); W, waste. Carrier, 2 mmol/L acetic/
acetate buffer, pH 5.1; GOD, glucose oxidase (0.24 mg
enzyme/mL); luminol (80 mmol/L in 30 mmol/L NaOH) and
catalyst [1 mg/L Co (II)].
Luminescence 2002;17:191–291
206 ABSTRACTS
maximum light collection is discussed in detail. The
feasibility of implementing stopped-flow strategies is
also assessed. A conversion yield of 77% is attained by
coupling a knotted reactor, and thus, the enzyme–
substrate mixing is improved owing to the toroidal flows,
and the axial dispersion of hydrogen peroxide released is
minimized. This value is slightly higher than b-anomer
ratio at the mutarrotation equilibrium as a consequence of
applying a stopped-flow modality for 150 s. The inter-
ference from other aldohexoses, cetohexoses and di-
saccharides in the enzymatic reaction in thoroughly
studied.
Under the optimum conditions, a glucose concentra-
tion as low as 90 mg/L may be easily detected by merging
simultaneously a 200 mL sample plug with a 200 mL zone
of GOD (viz. 17 enzyme units/injection) at a 1000-fold
photomultiplier tube gain. A linear log–log regression
equation between light emission and glucose concentra-
tion is found over the range from 90 mg/L to 2.7 mg/L
albeit a maximum concentration of 180 mg/L glucose is
able to be analysed by suitable gain selection without
requiring manifold reconfiguration. An injection through-
put of 20/h, a repeatability lower than 2.5% (n = 8) at the
1 mg/L level and a detection limit of 72 mg/L at the 3s
level are the figures of merit of the developed analyser for
determination of ultratraces of glucose. The MSFIA
methodology is applied to low glucose content pharma-
ceutical preparations and dietetic drinks with satisfactory
results.
REFERENCES
1. Fletcher P, Andrew KN, Calokerinos AC, Forbes S, Worsfold PJ.
Luminescence 2001; 16: 1–23.
2. Palilis LP, Calokerinos AC. Anal. Chim. Acta 2000; 413: 175–186.
3. Garcı́a-Campaña AM, Baeyens WRG. Analusis 2000; 28: 686–698.
4. Gregorio-Alapont A, Lahuerta-Zamora L, Martı́nez-Calatayud J.
J. Pharm. Biomed. Anal. 1999; 21: 311–317.
5. Nielsen J, Nikolajsen K, Benthin S, Villadsen J. Anal. Chim. Acta
1990; 237: 165–175.
6. Cerdà V, Estela JM, Forteza R. et al. Talanta 1999; 50: 695–705.
Bioluminometric tumorchemosensitivity-
assay (TCA) in cases of gynaecology
malignanciesÐuseful as pretherapeutical
factor?
S. Albrecht, T. Zimmermann, T. KoÈppen and W. Distler
1
Department of Gynaecology and Obstetrics of the Technical
University. E-mail: zialblcl@rcs.urz.tu-dresden.de
2
Department of Visceral-, Thoracic- and Vascular Surgery,
Fetscherstrasse 74, D-01307 Dresden, Germany
Despite the improved survival rates associated with
adjuvant therapy, the survival patients with breast cancer
Copyright  2002 John Wiley & Sons, Ltd.
and ovarian cancer remains poor. Systemic treatment of
breast and ovarian cancer is largely empirically derived,
based on data obtained from clinical trials (1). However,
patients with the same tumour pathology may not
respond to the same drug treatment. Tumour hetero-
geneity is well known in breast cancer.
Drug discovery and toxicological safety testing share a
need for dependable in vitro cellular toxicity tests.
Ideally, such tests should be objective, quantitative,
reproducible and able to lend themselves to automation
(2).
A number of assays fulfil these criteria well, but
recently we have developed a short-term cell culture
assay based on the detection of adenosine triphosphate
(ATP) by the luciferin–luciferase reaction, which shows
considerable promise as an in vitro toxicity assay as well
as a tumour chemosensitivity assay. This microplate ATP
assay requires relatively few cells and is therefore
suitable for the assessment of toxicity using tissue-
derived cells as well as cell lines.
The TCA is commercially available based on the rapid
loss of ATP from dead cells. It is an ex vivo microplate
assay requiring only 20,000 cells into each well. Four to
six drugs or drug combinations can be tested in each plate
at six different concentrations. In this study we have used
the results of 12 breast carcinomas and 28 ovarian
carcinomas. We examined the efficacy of three anti-
cancer-drugs (Carboplatin, Taxol and Novantron) for
ovarian cancers and Taxol, Epirubicin and 5-fluorouracil
for breast cancer.
MATERIAL AND METHODS
Patients
Small surgical biopsies from ovarian cancer (n = 28) and
breast cancer (n = 11) patients were obtained on a
Figure 1. Schedule of the ATP-TCA.
Luminescence 2002;17:191–291

sequential basis during surgery in Dresden prior to
palliative or neo-adjuvant chemotherapy.
Data analysis
The reaction of the cells to the chemotherapy was defined
at the beginning of the evaluation. We prefer the
sensitivity index (SI). A tumour growth inhibition
(TGI) >70% at 25% test concentration was defined as
sensitive, while inhibition 50%    70% was defined
as partially sensitive and inhibition 50% as resistant.
ATP-TCA
The ATP-TCA was performed according to the manu-
facturer’s instructions and as previously published. The
Figure 2. Two examples of TC assays in breast cancer patients.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 207
tumours were dissociated by enzymes to obtain the
constituent cells, which were then cultured on micro-
plates for 6–7 days in the presence of varying concentra-
tions of cytotoxic agents. At the end of the culture period,
the ATP content of surviving cells was measured by a
sensitive luminescence assay.
The results from cells cultured with drugs were
compared with no-drug controls (M0) and wells to which
a maximum inhibitor of cell growth has been added (MI)
to derive a percentage inhibition for each drug concen-
tration tested (Fig. 1)
The degree of inhibition of ATP is expressed as a
percentage of M0, subtracting the MI values as follows:
% inhibition ˆ 1 …test M1†=…M0 M1†
Luminescence 2002;17:191–291
208 ABSTRACTS
RESULTS
Taxol seems to be a very sensitive drug for breast cancer
patients (Fig. 2). It foretells a TGI of 90, 12% at 25% test
concentration. Epirubicin and 5-FU are resistant (TGI
32,48% and 28.31% respectively). The second patient
shows, for all drugs, anticancer-drug resistance. Epi-
rubicin predics 35.82%, Taxol 19.95% and 5-FU foretells
17.85%.
Of a total of 40 patients from both groups tested with
the ATP-TCA, all had evaluable results, giving a 100%
assay evaluability rate. There are only a small number of,
sensitive tumours. For ovarian cancer, 27 tumours
showed resistance for Taxol and only one shows nearly
the same sensitivity for Carboplatin. Only one tumour
predicted partial sensitivity and 28 resistances. Novan-
tron influenced the tissues better than the other one. Only
two were resistant. For breast cancer no tumour showed
sensitivity for 5-FU but seven specimens showed
resistance. Taxol and Epirubicin predicted nine and eight
resistant tumours, respectively.
DISCUSSION
Previous work has shown the ATP-based assay to be a
reproducible and reliable assay of cell viability, useful for
molecular studies as well as the characterization of drug
effects on cell lines and tumour samples. In this paper, we
show its ability to measure the effects of cytotoxic agents
on human cells derived breast cancer and ovarian cancer.
The ATP-based assay has recently been shown to be
useful for the design of new regimens.
Several TCA methods are in widespread use in the
pharmaceutical industry for the evaluation of new anti-
cancer drugs.
Dose-dependency of responsiveness is evident in many
of the tumours we have studied, a phenomenon that is
now being exploited clinically by dose intensification
regimens. Chemosensitivity testing could help to identify
patients who might benefit from such regimens and aid in
the choice of an appropriate regimen, since several agents
known to be suitable for this therapeutic approach can be
tested on the same tumour sample. It is also possible that
the assay could be used to indicate the dose of the agent.
Therefore, at present, cisplatin or carboplatin and
paclitaxel are the main agents for treatment of ovarian
cancer. In this study Novantron for ovarian cancer proved
to have a significantly higher in vitro efficacy at 25% test
concentration as Taxol or Carboplatin. It is suggesting
that it would be worthwile to test this agent in a
randomized trial.
Taxol, Epirubicin and 5-FU did not show a significant
sensitivity for breast cancer. Most of the tissues predicted
resistance. The ATP assay was able to predict resistance
more reliably than drug sensitivity.
The use of ex vivo chemosensitivity testing in clinical
Copyright  2002 John Wiley & Sons, Ltd.
practice is controversial but could be of great benefit to
patient care. The number of drugs with proven efficacy in
breast and ovarian cancer is rapidly increasing beyond
the capaticity of clinical trials to test potential regimens.
Tumour heterogeneity makes the choice of the best
agents for individual patients ever more difficult and
some method of individualizing chemotherapy is ur-
gently required.
This report shows that the new generation ATP-based
assays have the ability to predict solid tumour chemo-
sensitivity. This technology is now ready for much wider
evaluation and could be introduced to clinical practice
within a few years.
REFERENCES
1. Bellamy WT. Prediction of response to drug therapy of cancer. A
review of in vitro assays. Drugs, 1992; 44: 690–708.
2. Cree IA et al. Correlation of the clinical response to chemotherapy
in cases of in vivo cellular toxicity tests. Anti-Cancer Drugs. 1996;
7: 630–635.
Investigation of ¯uorescein as a biolabelling
agent for a crustacean species by
¯ow-injection analysis and high
performance liquid chromatography
W. R. G. Baeyens,1 G. Van der Weken,1 J. Mertens2
and A. M. GarcõÂa-CampanÄa3
1
Laboratory of Drug Quality Control, Department of
Pharmaceutical Analysis, Ghent University, Harelbekestraat 72,
B-9000 Ghent, Belgium. E-mail: willy.baeyens@rug.ac.be
2
Laboratory of Animal Ecology, Department of Morphology,
Systematics and Ecology, Ghent University, K. L.
Ledeganckstraat 35, B-9000 Ghent, Belgium
3
Department of Analytical Chemistry, University of Granada,
Avda. Fuentenueva s/n, E-18071 Granada, Spain
Branchipus schaefferi is a freshwater fairy shrimp
(Anostraca, Crustacea). It is a filter-feeder that mostly
lives in temporary pools. In view of a general study on the
biological food-chain, a fluorogenic labelling reaction
was envisaged using sodium fluorescein (Fig. 1). The
analytical elaboration was performed by means of a semi-
quantitative estimation of this spiked crustacean species,
based on a mark–recapture estimate of population size, so
as to follow the links in natural food chains (1). Semi-
quantitative determination by visual control under UV
light was verified by an HPLC determination employing
fluorescence detection, based on the high sensitivity and
specificity offered by this technique.
Besides the well-known influence of pH upon the
fluorescence emission of the labelling reagent in a
phosphate-buffered medium, various organic modifiers
and potential fluorescence enhancers were controlled in
Luminescence 2002;17:191–291

Figure 1. Sodium fluorescein.
off-line and on-line modes in order to establish a mobile
phase compatible with HPLC conditions and to introduce
a column-switching technique at the same time.
EXPERIMENTAL
In order to eliminate differences in results originating—
amongst other factors—from variations in size, only
males were labelled, by placing them for 30 min in a bath
containing 20 mg sodium fluorescein together with
200 mg charcoal in a 1 L medium. For controlling
different extraction parameters, the same labelled batch
of Branchipus schaefferi was used in each case. Different
parameters for reaching maximum extraction and for
sample preparation were investigated.
The HPLC set-up consisted of a column switching
system of two pre-columns and two Valco valves to
enable a flexible system for purification and preconcen-
tration. The samples were injected on a guard column
LiChroCART 4-4 (LiChrospher 100 RP-18; 5 mm) in a
mobile phase consisting of a phosphate buffer (0.01
md/L, pH 7.5) at a flow-rate of 0.7 mL/min (mobile phase
1). After about 1 min the 4-4 guard column was back-
flushed (pump 2, flow 0.7 mL/min) with 10% (v/v)
acetonitrile added to the same buffer and the separation
of fluorescein and 2',7'-dichlorofluorescein (internal
standard) was carried out on a guard cartridge 30 
4.6 mm, 5 mm (Bio-Sil C 18 HL 90-5). Fluorescence
signals were detected on an RF-551 fluorescence HPLC
monitor (Shimadzu) equipped with a 2 mL cell set at
lexc = 492 nm and lem = 508 nm (values obtained on-
line). Retention time of fluorescein and the internal
standard were, respectively, 2.9 and 9.8 min, an impurity
of the latter eluted at 4.6 min.
RESULTS AND DISCUSSION
Mobile phase parameters
The pH is the most important parameter influencing the
fluorescence emission of fluorescein. In the off-line mode
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 209
(Shimadzu Spectrofluorophotometer RF-5001PC) the
intensity was evaluated in 0.05 mol/L phosphate buffer,
pH range 5–9. A ten-fold emission increase was noted
from pH 5 to 8, whereafter signal flattening occurred. In
flow injection analysis (FIA) applying a transporting fluid
of 10% (v/v) acetonitrile in 0.05 mol/L phosphate buffer
and a flow of 0.7 mL/min, an increase of about 10% of
peak height was noted at pH 7.75 compared with pH 6.5.
As reversed phases have a limit of use around about pH 8,
the pH of the phosphate buffer for eluting fluorescein was
kept at pH 7.5, phosphate proving to be the most suited
buffer.
As no differences in fluorescence intensity of fluor-
escein were observed when methanol or acetonitrile were
added in concentrations lower than 20% (v/v), the latter
solvent was applied as organic modifier in the HPLC set-
up, keeping the concentration of organic modifier as low
as possible. Only when concentrations above 50% (v/v)
were applied were lower intensities noted, especially
when utilizing acetonitrile.
The influence of buffer molarity was controlled. When
applying 0.2 mol/L instead of 0.01 mol/L, the k ' value of
fluorescein increased from 2.4 to 4.4. For the internal
standard, the k ' value increased from 7 to 30. Testing the
influence of molarity upon the intensity of fluorescein in
the FIA mode, a slight increase (3%) was noticed when
utilizing the above phosphate buffers.
When applying commonly used fluorescence en-
hancers (Brij 35, octanesulphonic acid sodium salt)
added in a concentation of 10 mmol/L to the phosphate
buffer 0.01 mol/L, pH 7.5, ‡ 10% (v/v) acetonitrile,
fluorescein and the internal standard eluted at the dead
volume of the column.
Internal standard
In the proposed determination of fluorescein in extracts
of B. schaefferi, the introduction of an internal standard is
neccesary. The purity of different fluorescence deriva-
tives tested as internal standard was of high importance,
as most of them showed multiple peaks in the chroma-
togram obtained with the proposed mobile phase. Both
columns were serially coupled and the mobile phase
consisted of 10% (v/v) acetonitrile in 0.01 mol/L
phosphate buffer, pH 7.5. From the fluorescing com-
pounds 5-carboxyfluorescein dibromofluorescein, eosine,
malachite green, 4',5'-dichlorofluorescein and 2',7'-di-
chlorofluorescein, all within a retention time of 10 min.,
the latter fluorophore was chosen as an internal standard.
Retention was also tested in function of pH (Fig. 2).
Performance characteristics
The linearity of fluorescein emission was tested in the
concentration range 1–40 mg/mL, applying a 10 mL
injection volume, and a linear regression coefficient of
Luminescence 2002;17:191–291
210 ABSTRACTS
Figure 2. Influence of mobile phase pH on the retention of
fluorescein and the internal standard; flow of 0.7 mL/min.
r = 0.9999 was obtained. Using the ALAMIN program
(2, 3) employing the experimental calibration data set,
the performance analytical characteristics were calcu-
lated: analytical sensitivity (0.2314 mg/mL), limit of
detection (0.601 mg/mL), limit of quantification
(2.004 mg/mL) and linearity (99.25%).
In the suggested HPLC set-up, injections up to 500 mL
were possible. When injecting low concentration fluor-
escein solutions, problems arose due to bleeding of the
HPLC system (4). Irregular signals occurred with the
same retention as fluorescein due to valve turning,
solvent injection and ‘memory effects’. A 25 pg injection
of fluorescein was considered the detection limit,
although the performance limit of the detector was not
yet reached. When substituting the 2 mL cell by a 12 mL
cell, no signal amelioration was obtained. The ‘memory
effects’ occurred also in the FIA set-up, where 1 pg was
considered the detection limit.
Sample preparation
Preliminary extraction of marked B. schaefferi showed
that acceptable disintegration of this organism was
obtained after a treatment with sodium hydroxide
solutions instead of employing an ultrasonic bath,
phosphate buffers or different acidic solutions.
Volume (range 1–5 mL) and molarity (0.1–0.5 mol/L)
of sodium hydroxide solutions in combination with time
(30–120 min) and temperature (5–85°C) were investi-
gated for maximum extraction of fluorescein from the
labelled species. Each parameter was controlled at the
same day and on the same batch of labelled animals. A
buffer molarity of 5 mol/L could not be applied due to the
high viscosity obtained. As fluorescein solutions are
stable at 100°C (5), a temperature of 85°C, where
maximum extraction is obtained, can be used. After
neutralization (phosphoric acid solution), the internal
Copyright  2002 John Wiley & Sons, Ltd.
standard was added (2',7'-dichlorofluorescein solution in
0.01 mol/L phosphate buffer, pH 7.5) and the solutions
filtered through a disposable syringe filter (regenerated
cellulose, 0.2 mm, 25 mm; Chromacol).
CONCLUSIONS
The values obtained for maximum extraction of 10 B.
schaefferi specimens were obtained using 5.0 mLNaOH
(0.5 mol/L or 1 mol/L) for a period of 30 min–1 h at a
temperature of 85°C. Prior to injection in the HPLC
column, the solution is to be neutralized with a phosphate
buffer and the organic material needs to be eliminated.
Besides a cleaning-up step, retaining the analytes and
washing away foreign matter, the 4-4 guard column
provided the advantage of a pre-concentration effect.
Multiple injections or injection volumes up to 500 mL are
possible. Diluted or low-concentration solutions may be
used and, even when lowering the mobile phase flow-rate
(for miniaturization purposes) for the 30 mm column,
higher response signals can be obtained, thus expanding
the analytical potentials of the proposed method. More-
over, besides high sensitivity, the system offers the
advantage of chromatographic flexibility.
Acknowledgements
The authors wish to thank C. Dewaele (BIO-RAD RSL,
Begoniastraat 5, B-9810 Nazareth, Belgium) for the gift
of the guard cartridges (30  4.6 mm, 5 mm Bio-Sil C 18
HL 90-5).
REFERENCES
1. Breeuwer P, Drocourt J-P, Bunschoten N, Zwietering MH,
Rombouts FM, Abee T. Appl. Environ. Microbiol. 1995; 4: 1614–
1619.
2. Garcı́a Campaña AM, Cuadros Rodrı́guez L, Alés Barrero F, Román
Ceba M, Sierra Fernández JL. Trends Anal. Chem. 1997; 16: 381–
385.
3. Garcı́a-Campaña AM, Baeyens WR, Van der Weken G, Cuadros
Rodrı́guez L, Alés Barrero F. Biomed. Chromatogr. 1998; 12: 177–
178.
4. Fink W, Auch J. Deutsch. Lebensm. Rundschau. 1993; 89: 246–251.
5. Lancaster FE, Lawrence JF. J. Chromatogr. 1987; 388: 248–252.
Fluorimetric determination of acyclovir in a
pharmaceutical preparation applying HPLC
with an acidic micellar mobile phase
W. R. G. Baeyens,1 G. Van Der Weken,1 Y. Zhao1 and
A. M. GarcõÂa-CampanÄa2
1
Laboratory of Drug Quality Control, Faculty of Pharmaceutical
Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent,
Belgium. E-mail: willy.baeyens@rug.ac.be
Luminescence 2002;17:191–291

2
Department of Analytical Chemistry, Faculty of Sciences,
University of Granada, Avda Fuentenueva s/n, E-18071
Granada, Spain
Acyclovir (9-[(2-hydroxyethoxy)methyl]guanine) (ACV)
(Fig. 1), an acyclic analogue of guanosine, demonstrates
strong and selective activity against Herpes simplex and
Varicella zoster viruses. Determination of ACV in bio-
logical samples has been achieved by radioimmunoassay,
ELISA, and by chromatographic methods, e.g octadecyl-
silica or polymer-based reversed phase HPLC techniques
applying photometric detection at about 250 nm. The
fluorescence (FL) of ACV in acidic media is made
possible by the protonation of the molecule. Recently, a
micelle-enhanced fluorimetric detection method coupled
with HPLC (straight phase) was successfully applied for
ACV determination in biological fluids (1).
In preliminary work the effects of various surfactants
in an acidic mobile phase upon ACV fluorescence by
means of off-line measurements were investigated (2). A
study of the fluorescence behaviour of ACV mixed with
sodium dodecyl sulphate (SDS) was conducted in terms
of matrix pH, buffer concentration and type (eg phos-
phate) as well as of SDS concentration in the matrix
media. Off-line measurements clearly indicate that ACV
fluorescence occurs exclusively under acidic conditions
and is enhanced with increasing SDS content, while the
buffer concentration only exerts a slight influence upon
the cited fluorescence intensity.
The present paper describes the quantitative determi-
nation of ACV in a pharmaceutical preparation by a
newly developed reversed-phase HPLC–FL technique.
An FIA set-up was applied to investigate the parameters
in the mobile phase in order to optimize the area
(fluorescence intensity) independently from retention on
the RP column.
EXPERIMENTAL
Chemicals
SDS and tetramethylammonium bromide (TMAB) were
obtained from Janssen Chimica (Beerse, Belgium) and
Figure 1. Structure of acyclovir.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 211
ACV from Alpha Pharma (Zwevegem, Belgium).
Adenosine was purchased from Aldrich (Milwaukee,
WI, USA). All reagents were of analytical reagent grade.
SDS was dissolved in the phosphate-containing mobile
phase by gentle heating, then kept at room temperature.
Deionized water was used throughout.
Instrumentation
Chromatography was performed with an LC-9A pump
using a Valco (C6W) injector with a 20 mL (HPLC) or
10 mL loop (FIA). Detection was controlled by a
fluorescence HPLC monitor RF-551 (2 mL flow cell)
equipped with a C-R6A integrator. All instruments were
from Shimadzu (Shimadzu, ‘s Hertogenbosch, The
Netherlands).
Experimental conditions
The separation was performed on a narrow-bore column
EcoCART 125-3 with a guard column 4-4, both filled
with LiChrospher 100 RP-18, 5 mm (Merck Eurolab,
Darmstadt, Germany). The mobile phase consisted of an
aqueous solution containing SDS 40 mmol/L, TMAB
5 mmol/L and phosphoric acid 52 mmol/L, brought to pH
2.2 with sodium hydroxide (20% g/v). The mobile phase
was pumped at a flow rate of 0.6 mL/min and chroma-
tography was carried out at 35°C. The column eluate was
monitored for 8.5 min at a setting of lexc 262 nm, lem
376 nm, low sensitivity, and thereafter automatically
switched to lexc 282 nm, lem 376 nm, high sensitivity.
Standard and sample solution preparation
Calibration solutions (0.1–0.5 mg/mL ACV) and con-
taining 0.4 mg/mL adenosine as an internal standard were
prepared in diluent (6.0 g/L phosphoric acid brought to
pH 2.2 with sodium hydroxide (20% g/v). For sample
solution preparation, a portion of the creme was weighed
so as to provide a theoretical concentration of
0.25 mg/mL ACV, the internal standard was added
(0.4 mg/mL) and brought to volume with diluent, then
the mixture was slightly warmed and placed in an
ultrasonic bath for 5 min. After cooling at room tem-
perature the solution was filtered through a regenerated
cellulose syringe filter 0.45 mm  25 mm (Chromacol
Ltd.)
RESULTS AND DISCUSSION
Enhanced ¯uorescence
Previous off-line tests indicated an increase of native
fluorescence of about 450% for ACV (strongly pH-
dependent) and of about 150% for adenosine in the
presence of 50 mmol/L SDS. The temperature (25–85°C)
Luminescence 2002;17:191–291
212 ABSTRACTS
had no effect on the fluorescence emission of ACV, but
for adenosine a 10% decrease was noted at 85°C.
Phosphoric acid content (25–75 mmol/L) had no effect
upon either the fluorescence intensity or the maxima of
excitation and emission. A concentration higher than
6 mmol/L TMAB (tested from 0–10 mmol/L) decreased
the fluorescence intensities of both compounds by about
10%. The increase of SDS 30–50 mmol/L increased the
fluorescence intensity of ACV by 40%, and by 15% for
adenosine. Changing the pH from 2.0 to 3.2 also
decreased the fluorescence intensity of ACV by about
40%. The pH had no effect upon the adenosine
fluorescence intensity.
Mobile phase
Separational resolution between ACV and adenosine
increased slightly with temperature till 35°C, after which
stabilization occurred. Strong differences were observed
with the phosphoric acid content: 26 mmol/L, Rs 2.9;
52 mol/L, Rs 4.6; and 78 mol/L, Rs 3.7. Increasing
TMAB content (0–10 mol/L) slightly improved the
resolution but on the other hand a decrease in retention
was noted. TMAB had a positive effect on the peak
width.
The increase of SDS concentrations (25–75 mol/L) had
a negative effect on the resolution together with lower
retention for both compounds. Higher pH values
improved the resolution and decreased the retention
capacity of ACV. However, the latter was also noted by
adding methanol without resolution changes A 10% v/v
methanol content of the mobile phase also decreased the
fluorescence intensity by about 20%. Decreasing fluor-
escence intensity was also observed when adding other
organic modifiers (acetonitrile, isopropanol).
Internal standard
The method enables a separation of ACV (RT = 8 min),
guanosine (RRT = 0.88), adenosine (RRT = 1.29), gua-
nine (RRT = 1.52) and adenine (RRT = 2.23). Due to the
obtained chromatographic separation of ACV, and taking
into account the fluorescence intensity of adenosine, the
latter was used as an internal standard. However, the
detector clearly needs time between the signals of both
components to switch the excitation wavelength, together
with its sensitivity settings, in order to obtain an adequate
integration (Fig. 2).
Stability
The purity of ACV solutions was controlled by the
injection of a 0.58 mg/mL ACV solution. No signals were
observed exhibiting an area above 0.02% of the mean
peak. Under the influence of light (daylight or UV) or
heat (35 °C, 1 day) no degradation could be noticed; even
Copyright  2002 John Wiley & Sons, Ltd.
by heating until boiling for 5 min, no degradation was
observed. After heating until dry and subsequent dis-
solution in diluent, no ACV was found (only guanine).
Upon forced degradation (1 day) with an oxidant (H2O2,
2% v/v), a loss of ACV content of 6–9% was noticed.
Linearity
A standard solution (0.502 mg/mL ACV) was injected
(n = 10) for intra-day determinations and a relative
standard deviation of 1.04% was calculated by area
determination, 0.54% by peak height, respectively. The
recovery of the internal standard was controlled (n = 4), a
value of 100.2% (rsd 1.0%) was found based on
calculation with area and 100.1% (rsd 0.4%) based on
peak height calculations. The highest correlation was
obtained (n = 5, r = 0.9998, rsd = 1.9%) when calculating
with peak area ratios. Although a lower correlation was
obtained (n = 5, r = 0.9992, rsd = 1.4%) when calculation
was done without internal standard, the rsd value of 1.4%
clearly indicates that the determination definitely may be
performed. The highest correlation was obtained with
calculations based on peak area ratios. The results
indicate that the determination can also be performed
with peak height ratios and without internal standard. The
limit of detection was 2 mg ACV/mL, calculation based
on three times noise, the limit of quantitation being
situated at 10 mg/mL.
Pharmaceutical preparation
Filtration through the regenerated cellulose filter did not
alter the ACV concentration (n = 3, recovery 100.5%, rsd
1.5%), respectively the internal standard values (n = 8,
recovery 100.3%, rsd 1.6%). Five batches of a prepara-
tion with a theoretical value of 50 mg ACV/g gel were
Figure 2. Typical chromatogram of a standard solution:
acyclovir (RT = 7.8 min) and adenosine (RT = 10.8 min) at a
flow of 0.6 mL/min.
Luminescence 2002;17:191–291

controlled. Of the marked theoretical content, 96.0–
99.8% ACV was found with an rsd (n = 10) of 1.6–3.5%
(calculations: peak area ratios).
CONCLUSION
Adding SDS to the mobile phase (pH 2.2) of the proposed
chromatographic set-up increases the fluorescence in-
tensity of ACV with a factor of 3.9. TMAB inclusion
decreases the enhancement with about 6% but peak shape
improved, resulting in higher Rs-values employing the
internal standard. The determination of ACV applying
adenosine as the internal standard also demonstrated the
applicability of fluorescence detection in the HPLC set-
up, when appropriately applying wavelength program-
mation in between ACV and adenosine peaks. The
application of a micellar environment for the fluorimetric
determination of ACV was thus established and vali-
dated. Only when the micellar condition is applied in
acidic aqueous media SDS was found a suitable
fluorescence enhancer resulting in a more sensitive
detection compared with conventional UV detection.
REFERENCES
1. Macka M, Borák J, Seménková L, Popl M, Mikes V. Determination
of acyclovir in blood serum and plasma by micellar liquid
chromatography with fluorimetric detection. J. Liqu. Chromatogr.
1993; 16(11): 2359–2386.
2. Zhao Y, Baeyens WRG, Van der Weken G, Garcı́a-Campaña AM.
Fluorimetric study of acyclovir in acidic micellar media. Biomed.
Chromatogr. 1999; 13: 143–144.
Determination of benzo[a]pyrene
metabolites by human cytochrome P450
using column-switching HPLC with
¯uorescence detection
Akira Toriba, Eriko Kaji, Thaneeya Chetiyanukornkul,
Ryoichi Kizu, Kazuichi Hayakawa
Graduate School of Natural Science and Technology, Kanazawa
University, 13-1, Takara-machi, Kanazawa, Ishikawa 920-0934,
Japan. E-mail: toriba@mail.p.kanazawa-u.ac.jp
Benzo[a]pyrene (BaP) is an ubiquitous environmental
pollutant produced during combustion processes. BaP is
bioactivated by enzymes such as cytochrome P450
(P450) to acquire its mutagenic and carcinogenic proper-
ties. Recently, it has been reported that hydroxylated
metabolites of BaP, monohydroxybenzo[a]pyrenes
(OHBaPs), exhibited oestrogenic activity using a yeast
two-hybrid assay and a oestrogen receptor reporter gene
assay (1, 2). The major metabolite in the 12 kinds of
OHBaPs included the 3-OH, 7-OH and 9-OH species,
which result from hydroxylase activity shown to be
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 213
mediated through the P450 1A, 1B and 3A families.
However, 3-OHBaP, which particularly showed the
highest affinity for the oestrogen receptor, has been
quantified as the sum of the other metabolites because
several OHBaPs were co-eluted from an ODS column.
To estimate the activation of BaP to oestrogenic metab-
olites, a determination method of OHBaP isomers using
column-switching HPLC with fluorescence detection was
developed. The block diagram for the present HPLC
system is shown in Fig. 1. Fluorescence detection was
Figure 1. Block diagram for the determination of OHBaP
isomers. I1, injector; P1, P2 and P3, pumps; D1 and D2,
fluorescence detectors; GC, guard column; C1, alkylamide type
column; C2, trapping column (ODS column); C3, b-cyclo-
dextrin bonded column; MP1, MP2 and MP3, mobile phases;
V1, switching valve.
performed at 432 nm with excitation at 265 nm. OHBaPs
except for 6-OHBaP in 12 kinds of the isomers were
separated on an alkylamide type reversed phase column
(C1) and, via column-switching, on a b-cyclodextrin
bonded silica gel column (C3). The metabolism of BaP
with recombinant P450 1A1, 1A2, 1B1, 2C8, 2E1 and
3A4 was examined by the measurement of OHBaPs as
the metabolite. Reaction mixtures contained 100 mmol/L
potassium phosphate (pH 7.4), 5 mmol/L MgCl2,
40 mmol/L BaP, 5 pmol P450 and 250 mg/mL epoxide
hydrolase. After the addition of an NADPH generation
system, the reactions were done at 37°C for 20 min.
Resulting metabolic products were subjected to the
column-switching HPLC system. As an example, separa-
tion of BaP metabolites by P450 1A1 analysed by the
HPLC system is shown in Fig. 2. 9-OHBaP metabolite
was separated from the other OHBaPs on the first
separation column (C1), while 1-, 3- and/or 12-OHBaPs
were co-eluted as shown in the chromatogram A. 1-, 3-
and 12-OHBaPs were separated clearly on the second
separation column (C3) after column-switching and 1-
and 3-OHBaPs were detected as the metabolites. P450
1A1 was the most active of the P450s tested here for the
metabolism to OHBaP.
Luminescence 2002;17:191–291
214 ABSTRACTS
Figure 2. Representative chromatograms of BaP metabolites by human P-450 1A1 analysed by the
proposed HPLC system. Chromatogram B was obtained by applying the black part of chromatogram
A with the column-switching. The number on the chromatograms indicates the position of hydroxyl
group to BaP.
REFERENCES
1. Hirose T, Morito K, Kizu R, et al. J. Health Sci. 2001; 47: 552–558.
2. Charles GD, Bartels MJ, Zacharewski TR, Gollapudi BB, Freshour
NL and Carney EW. Toxicol. Sci. 2000; 55: 320–326.
On-line screening method for antioxidants
by liquid chromatography based upon
indirect chemiluminescence detection
Toshimasa Toyo'oka, Tomoaki Kashiwazaki and
Masaru Kato
Department of Analytical Chemistry, School of Pharmaceutical
Sciences, University of Shizuoka, 52-1 Yada, Shizuoka
422-8526, Japan. E-mail: toyooka@ys2.u-shizuoka-ken.ac.jp
Antioxidants have been efficiently resolved by electron
spin resonance (ESR) using spin-trapping agents such as
DEMPO and TEMPO (1). Although the identification of
radical species is possible by the method, the antioxidants
in complex matrices, such as biological and food
samples, are difficult to determine. Thus, the antioxidant
potential of individual substances is actively investigated
and several methods including the use of chemilumi-
nescence (CL) reaction have been developed as sensitive
assay (2–4). However, it is difficult to carry out a quan-
Copyright  2002 John Wiley & Sons, Ltd.
titative assay of antioxidant activity, owing to the short
lifetime of these radicals. Furthermore, it is unknown
what kinds of radicals are eliminated by the substance
having antioxidant activity.
In this paper, we present novel assay systems for anti-
oxidants, which are mainly based on eliminating super-
.
oxide anion radical (O2 ) and hydrogen peroxide (H2O2)
by HPLC with luminol-based chemiluminescence. The
proposed methods are also applied to the determination
of antioxidants in tea products.
MATERIALS AND METHODS
On-line HPLC±CL detection of antioxidants
The mobile phase and the reagent solutions were con-
tinuously flowed by three HPLC pumps. A Shimadzu
CLD-10A CL monitor equipped with an 80 mL flow cell
was used for the detection of the antioxidants. The signals
obtained from CL detector were recorded on a Hitachi
833A Data Processor. The mobile phase and the reagent
solutions were degassed by sonication, prior to use. The
negative peaks obtained from the CL detector were
recorded as the positive peaks with phase exchange.
The separation of an antioxidant mixture (C, CG, GC,
GCG, EC, ECG, EGC and EGCG) (see Fig. 1) was
carried out by J’sphere ODS-H80 (150  4.6 mm i.d.,
Luminescence 2002;17:191–291

Figure 1. Structures of catechins and flavones tested.
5 mm) with methanol: water (28:72) containing 1%
H3PO4. The eluent was neutralized with sodium hy-
droxide solution at the outlet of the column (via) a mixing
device.
Sample pretreatment and the analysis of tea
products
Green tea leaves (Thea sinensis L.) dried (1.0 g) was
extracted with 100 mL hot water at 80°C for 10 min.
After cooling at around room temperature, the leaves
were filtered out using filter paper. The filtrate was
centrifuged at 3000 rpm for 15 min and then the cloudy
solution was passed through a 0.45 mm membrane filter
under reduced pressure. The solution was diluted to
appropriate concentration with water. The sample pre-
treatments were performed throughout just before
analysis. The diluted tea solutions were separated by
HPLC and determined using UV and CL detectors.
Green tea and oolong tea products on the market were
also filtered through the membrane, diluted with water,
and analysed by HPLC–CL as for the procedures for
green tea leaves.
RESULTS AND DISCUSSION
Novel screening methods for antioxidants based upon on-
line CL by HPLC were developed. The luminescence was
constantly produced from the chemical reaction of
luminol and H2O2. The CL intensity was dependent
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 215
upon the concentrations of luminol and H2O2. The CL
reaction was optimized using flow injection analysis
(FIA) without an analytical column. From the results of
optimization, 4.16 mmol/L luminol and 4.0 mmol/L H2O2
were selected for the screening system of H2O2 elimi-
nation.
Luminol also emits light from the chemical reaction
.
with O2 generated from enzyme reaction of hypox-
anthine (HX) and xanthine oxidase (XOD). The final
concentrations recommended for luminol, HX and XOD
were 4.16 mmol/L, 3.12 mmol/L and 3.58 units/L,
respectively. Excess amounts of catalase (15000 units/L)
were added to XOD solution to remove the effect of
H2O2, because luminol also emits light in the presence of
H2O2. When the sample contains antioxidants eliminat-
.
ing O2 or H2O2, the down-peaks corresponding to each
antioxidant are observed on the chromatogram.
The antioxidant activities of catechins and flavones
reported as a series of antioxidants were determined with
the proposed HPLC–CL systems (Fig. 1). The down
peaks were observed with all the compounds tested by
FIA. The order of scavenging potential of catechins
toward H2O2 was EGCG > GCG > ECG > CG > GC >
EGC > C > EC. Judging by the results, gallate and
pyrogallol groups in the catechin structure seem to be
important to the elimination of H2O2. However, the effect
of stereostructures was negligible for the elimination
activity from the comparison of the activity between C
and EC or GCG and EGCG. In the case of flavones,
7,8-dihydroxyflavone scavenged H2O2 most strongly
Luminescence 2002;17:191–291
216 ABSTRACTS

among the other flavones. Kaempferol, quercetin, morin REFERENCES

and myricetin, which have phenolic hydroxyl groups at
positions 3, 5, 7 in the structures, also scavenged H2O2 1. Thornalley PJ, Bannister JV. The Spin Trapping of Superoxide
Radicals, Greenwald RA (ed.). CRC Handbook of Methods for
efficiently. Oxygen Research. CRC Press; Boca Raton, FL, 1985; 133.
.
With respect to the scavenge activity of O2 , GC and 2. Frei B, Yamamoto Y, Niclas D, Ames B. Anal. Biochem., 1988;
EGC possessing pyrogallol structure were strong, as 175: 120.
3. Hirayama O, Takagi M, Fukumoto K, Katoh S. Anal. Biochem.,
compared with the gallate-bearing catechins, such as CG 1997; 247: 237.
and ECG. The activities of GCG and EGCG, having both 4. Visioli F, Galli C. Anal. Biochem., 1997; 249: 244.
gallate and pyrogallol groups in the structure, were
stronger than those of C and EC but lower than those of
GC and EGC. Hence, the scavenger effect seems to
depend upon the substituents binding on positions 2 and
3. On the other hand, 7,8-dihydroxyflavone, myricetin,
fisetin and kaempferol provide a strong effect, and the Derivative synchronous spectro¯uorimetric
scavenge potential was in this order. These flavones determination of organochlorinated
possess catechol and pyrogallol groups in the structure. compounds after their microwave micellar
Thus, the scavenger ability seems to be due to the number extraction
of phenolic hydroxyl groups and their position in the
structure. C. PadroÂn Sanz, C. Mahugo Santana, J. R. Betancor
The mixture of catechins was separated by reversed- RodrõÂguez, Z. Sosa Ferrera and J. J. Santana RodrõÂguez
phase liquid chromatography using an ODS column. Department of Chemistry, Faculty of Marine Sciences, University
of Las Palmas de Gran Canaria, E-35017 Las Palmas de Gran
Since catechins were relatively instable in neutral and
Canaria, Spain. E-mail: jjsr@cicei.ulpgc.es
alkaline solutions, an acidic mobile phase, methanol:
water (28:72) containing 1% H3PO4 was used for the
separation. After the separation, the eluent was neutra-
lized with sodium hydroxide solution at the outlet of the INTRODUCTION
analytical column, because the CL reaction proceeds in
Polychlorinated biphenyls and polychlorinated dibenzo-
neutral to slight alkaline medium. Although the separa-
furans (PCBs, PCDFs) constitute two families of pollu-
tion between C and EGC was poor under the elution
tants that are environmentally persistent. They are highly
condition, the other catechins separated well.
lipophilic, toxic (1) and tend to bioaccumulate in fatty
The proposed HPLC–CL method was applied to the
. tissues along food chains.
determination of antioxidants scavenging O2 or H2O2 in
Combination of synchronous and derivative fluores-
green tea extract and tea products. Since various com-
cence spectrometry has proved to have enormous
pounds appeared on the chromatogram utilizing UV
. application in the resolution of mixtures, due to the
detection at 265 nm, only antioxidants eliminating O2
posibility of a greater discrimination of spectra. Thus,
and H2O2 were identified on the chromatograms with the
second derivative synchronous fluorescence has been
indirect CL detection. The results suggest that the
applied to the analysis of mixtures of different organic
proposed methods are selective for the antioxidants.
compounds (2). In this work we use second derivative
The green tea and oolong tea solutions in PET bottle were
synchronous fluorescence to the resolution of a mixture
also analysed using the HPLC–CL methods. From the
containing two of the most important PCBs and PCDFs,
chromatographic comparison, the green tea on the market
3,3',4,4'-tetrachlorobiphenyl (TTCB) and 2,3,7,8-tetra-
contained C together with the other catechins, whereas
chlorodibenzofuran (TTCDF), respectively. These com-
only three catechins, GC, GCG and EGCG, were detected
pounds are determined after their microwave-assisted
in oolong tea solution.
micellar extraction (MAME) from marine sediment
In conclusion, the proposed HPLC–CL methods seem
samples, using polyoxyethylene 10 lauryl ether (POLE)
to provide new assay systems for antioxidants possessing
. micellar medium as extractant.
scavenging effect of O2 and H2O2. An on-line screening
system for the compounds scavenging hydroxy radicals is
currently being investigated in our laboratory. RESULTS AND DISCUSSION
Simultaneous determination of TTCB and TTCDF
Acknowledgements
The excitation, emission and synchronous spectra of
The authors thank Central Research Laboratories, Tokyo TTCB and TTCDF in the presence of POLE are over-
Food Techno Co., for the generous gift of catechins. This lapped. However, the use of second derivative synchro-
work was supported in part by a Goto Research Grant nous fluorescence spectra allows the simultaneous
from the University of Shizuoka. determination of both compounds in different concentra-

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291


tion ratios. The characteristics of second derivative
synchronous fluorescence spectrum of TTCB and
TTCDF have been previously established and reported
by Santana Rodrı́guez et al. (3).
Optimization of the extraction
1 g sediment sample was spiked with 200 ppb each
analyte and 10 mL NaCl (3.5%, w/v) were added. It was
allowed to equilibrate for 1 h in a rotatory system. After
its centrifugation the supernatant was discarded. The
sample was introduced in the microwave system (300 W)
with different salt and surfactant concentrations, and then
irradiated at several radiation times in order to optimize
the extraction process. The extract, previously filtered,
was directly measured in the fluorescence spectro-
photometer.
The results show how TTCDF recovery increases with
POLE concentration (1–8%, w/v) up to 5%, after which it
is stabilized. For TTCB no influence is observed. When
NaCl is added (0–8%, w/v) the best recovery for TTCB
was obtained at 1%; however, a decrease is observed for
the TTCDF with the salt concentration increase. A range
of microwave radiation times between 1 and 15 min was
studied. The results show a soft decrease in the recoveries
of both compounds when the exposure time is increased.
After these studies we conclude that the optimal
parameters for the extraction of both compounds are:
radiation time 1 min, POLE concentration 5% (w/v) and
salt concentration 1% (w/v). Fig. 1 shows the second
derivative synchronous fluorescence spectrum of a
mixture of TTCB and TTCDF extracted from a marine
sediment sample. Under these conditions and after
applying two extraction steps, recoveries of 73  2%
Figure 1. Second derivative spectra synchronous fluores-
cence of TTCB and TTCDF extracted from a spiked marine
sediment sample, 200 ppb for each analyte, in the presence of
POLE solution (5%, w/v).
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 217
and 84  7% (n = 6) were obtained for TTCB and
TTCDF, respectively. According to these results, the
proposed methodology, after its validation, could be
applied to determine these compounds in different kinds
of marine sediment samples.
REFERENCES
1. Waid JS. PCBs and the Environment. CRC Press: Boca Raton, FL,
1986.
2. Konstantianos DG, Ioannou PC. Analyst 1996; 121: 909.
3. Santana Rodrı́guez JJ, Sosa Ferrera Z, Hernández Garcı́a J, Bermejo
Martı́n-Lázaro AJ. Analyst 1994; 119: 2241.
Use of partial least square method for the
sensitive resolution of mixtures of
chlorophenoxyacid herbicides by micellar
enhanced photochemically induced
¯uorescence
E. M. Almansa-Lopez,1 A. M. GarcõÂa-CampanÄa,1
J. J. Aaron2 and L. Cuadros-Rodriguez1
1
School of Qualimetrics, Department of Analytical Chemistry,
University of Granada, E-18071 Granada, Spain. E-mail:
ealmansa@urg.es
2
Interfaces, Traitments, Organization et Dynamique des
SysteÁmes de l'Universite Paris 7 `Denis Diderot,' CNRS, UPRES-A
7086, 1 Rue Guy de la Brosse, 75005 Paris, France
Chlorophenoxyacid herbicides do not show native
fluorescence but, as well as other aromatic pesticides,
they can be photolysed into strongly fluorescent photo-
products. This fact has permitted the establishment of a
new method for their quantitative analysis, based in a
simple technique called ‘room temperature photochemi-
cally-induced fluorescence’ (RT-PIF), in which quantifi-
cation is based on the obtained PIF signals, reproducible
and directly proportional to the concentration of the non-
fluorescent analyte. The method for the analysis of total
chlorophenoxyacid herbicides was carried out in a
mixture of methanol and pH 5 buffer (50/50 v/v), using
irradiation times of 15 min (1). Recently, micellar media
have been incorporated in the establishment of new
methods, considering the photochemically-induced fluor-
escence properties of the analytes. The methodology has
been named ‘micellar-enhanced photo-induced fluores-
cence’ (MEPIF) and it is based on the enhancement of the
fluorescence intensity provided by organized media,
increasing the sensitivity and reducing the need to use
organic solvent. In a previous work, we have studied the
influence of the presence of several surfactants in
aqueous buffer solutions on the PIF properties of the
Luminescence 2002;17:191–291
218 ABSTRACTS

chlorophenoxyacid herbicides in batch solutions (2). This RESULTS AND DISCUSSION

study allowed the use of PIF detection for the sensitive
Selection of the optimum number of factors
determination of 2,4-dichlorophenoxyacetic acid (2,4-D)
and Mecroprop (MCPP) in micellar medium, and we also In order to select the optimum number of factors, in the
proposed the determination of both herbicides using a PLS algorithm, to model the system without overfitting
FIA system with MEPIF detection in cationic micellar the concentration data, a cross-validation method leaving
medium (3). out one sample at a time, was used. The concentration of
In this study we have carried out a simultaneous each sample was then predicted and compared with the
determination of binary mixtures of MCPP and 2,4-D known concentration of this reference sample.
applying multivariate calibration methodology by using The prediction errors sum of squares (PRESS) is a
the partial least square algorithm (PLS-1) (4), which is a measure of how well the training set is predicting the
typical full-spectrum calibration method in which multi- concentration for each number of factors. The F-ratio
ple instrumental measurements are obtained from probability is used to determine the significance of
different specimens at each of a number of wavelengths, PRESS value greater than the minimum. As the
the simultaneous determination of different analytes in a difference between the minimum PRESS and other
sample being possible by means of the decomposition PRESS values become smaller, the probability that each
and regression for only one component at a time. In order additional factor is significant becomes smaller. Empiri-
to select the multivariate calibration matrix to apply cally, a number of factors for the PRESS value showing
PLS-1, a central composed design based on a orthogonal an F-ratio probability (6) below 0.75 and is a good
star and three central points was applied to select the choice, because using the number of factors that yields a
experimental points (11 binary mixtures of the MCPP and minimum PRESS usually leads to some overfitting (Fig.
2,4-D; the concentrations were varied in the range 1). For the mixture MCPP ‡ 2,4-D, seven and six factors
0.78–4 mg/L MCPP and 2,4-D). The spectral region were used as optima for MCPP and 2,4-D prediction,
between 280 and 330 nm was selected for the analysis, respectively.
with 51 points/spectrum, and for this training set the PIF
spectra (5) were recorded.
Statistical parameters
The values of the root mean squares (RMSD), which is an
indication of the average error in the analysis, and the
EXPERIMENTAL
square of the correlation coefficient (R 2), which is a
Fluorimetric measurements were carried out on a indication of the quality of fit of all the data to a straight
Kontron SFM-25 spectrofluorimeter (Zurich, Switzer- line, were calculated. The predictive ability of each
land), equipped with a data control and acquisition method and for each component can be also described in
program. For the photodegradation reaction in the batch terms of the relative error root of prediction (REP), which
procedure, a 200 W HBO Osram high-pressure mercury is the square root of the mean square of the error in
lamp with an Oriel model 8500 power supply was used
and a standard Hellma 1 cm2 quartz reaction cuvette was
placed on an optical bench at 45 cm from the lamp.
Standard stock solutions of chlorophenoxyacid herbi-
cides (25 mg/L) were prepared from the corresponding
compounds by dissolving them in methanol. Standard
stock solutions of CTAC (2%) was prepared by dilution
with distilled water. Working solutions were prepared by
transferring 4 mL 50/50 v/v mixture of methanol and pH
5 buffer solution, 2 mL CTAC aqueous solution and
0–1 mL aliquots of the herbicide standard solution into a
10 mL volumetric flask and adjusting to the marker with
distilled water. The solutions were then shaken lightly
before irradiation and analytical measurements.
The photolysis reaction was performed by irradiating
for 10 min with UV light a 3 mL volume of dilute
herbicide solution which was magnetically stirred at
room temperature in the cuvette. The emission spectra
(281–330 nm) were registered at constant excitation Figure 1. PRESS plot and optimum number of factors
(270 nm) wavelength for all herbicide photoproduct generated from prediction of MCPP-2,4-D by the PLS-1
mixtures. method.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

 ABSTRACTS 219

prediction, expressed as a percentage of the mean of the REFERENCES

true concentration.
s Pn 1. Eremin SA, Laasis B, Aaron J-J. Talanta 1996; 43: 259–301.
…^ci ci †2 2. Garcı́a-Campaña AM, Aaron J-J. Luminescence 2000; 15: 130.
1X n
2 iˆ1 3. Garcı́a-Campaña AM, Aaron J-J, Bosque-Sendra JM. Talanta 2001;
RMSD ˆ …^ci ci † R ˆP
2
n 55: 531–539.
N iˆ1
…ci ci †2 4. Martens H, Naes ST. Multivariate Calibration. Wiley: New York,
iˆ1 1989.
s 5. Sanchez-Peña M, Muñoz de la Peña A, Salinas F, Mahedero MC,
100 1 X n Aaron J-J. Analyst 1994; 119: 1177–1181.
6. Haaland DM, Thomas EV. Anal. Chem. 1988; 60: 1193.
REP ˆ …^ci ci †2
c N iˆ1

In this expression, ci and ĉ are the true concentration

and the estimated concentration of the analyte in sample
i, c is the mean of the true concentration in the prediction
set, and N is the total number of samples used in the A computational method for ¯uorescence
prediction set. These values found are summarized in determination of organochlorinated
Table 1. compounds of environmental interest
J. R. Betancort RodrõÂguez,1 P. GarcõÂa Baez,2
Table 1. Statistical parameters of the PLS-1 method, using J. J. Santana RodrõÂguez1 and C. P. SuaÂrez Araujo3
the PIF spectral data set 1
Department of Chemistry, Faculty of Marine Sciences,
University of Las Palmas de G.C. E-35017 Las Palmas de G.C.,
Number Spain. E-mail: jjsr@cicei.ulpgc.es
Component of factors RMSD R 2 (%) REP (%) 2
Department of Statistics, Operating Research and
MCPP 7 0.036 99.99 1.46 Computation, University of La Laguna, E-38071 La Laguna,
2,4-D 6 0.058 99.98 2.33 Spain
3
Department of Computer Sciences and Systems, University of
Las Palmas de G.C., E-35017 Las Palmas de G.C., Spain
Table 2. Recoveries obtained from PLS-1 method, using the
PIF spectral data set One of the most important environmental pollutants are
the polychlorinated biphenyls (PCBs). They are ubiqui-
MCPP 2,4-D tous toxic contaminants, due their bioaccumulative
Mixture Added (ppm) %R Added (ppm) %R capacity and persistence and specific physicochemical
properties. They are generally found in the environment
1 1.0 116.29 2.5 89.63
2 4.0 86.53 2.5 113.72
as complex mixtures, their presence often reflecting local
3 2.5 82.45 1.0 128.58 anthropogenic impacts.
4 2.5 96.54 4.0 97.32 Fluorescence spectrometry combined with the use of
5 2.5 81.78 2.5 113.45 micellar systems has proved to be a sensitive technique
for the determination of a great variety of organic com-
pounds, including hazardous pollutants such as PCBs.
However, the similar structures of PCBs (1) produce
APPLICATION
overlapping in fluorescence spectra, which leads to
The proposed PLS-1 method allowed us the resolution of limited selectivity.
binary mixtures of the herbicides. The composition of the We propose an alternative and complementary method
binary mixtures analysed is shown in Table 2. Recoveries of facing these problems, the neural computation method,
were 81–128%. A t-test, which compares the average which is a distributed, parallel and self-programming
recoveries, was carried out in order to check whether this computational approach. Its principal structure for
value was significantly different
pfrom 100%. The calculated information processing is the artificial neural network,
statistics, t = (Rm 100) n=sR (where Rm = pooled re- which has been biologically inspired. This method is very
covery for each component, sR = recovery standard devia- suitable for the specific tasks of fluorescence spectro-
tion, n = number of analyses) was obtained for comparison metry, essentially analysing complex data, which can
of the means of paired samples. The null hypothesis (the involve no explicit knowledge of the problem, no linear
recovery is close to 100) is accepted for a significance level processes, overlapping information or noise (2).
greater than 5%. In both cases, the pooled recovery
(92.72% and 108.54% for MCPP and 2,4-D, respectively)
RESULTS AND DISCUSSION
was not significantly different from 100% (32.2% and
28.0% p value for MCPP and 2,4-D, respectively). The excitation, emission and synchronous fluorescence

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

220 ABSTRACTS
Figure 1. Diagram of the artificial neural net.
spectra of seven congeners of PCBs were collected using
a Perkin-Elmer LS-50 spectrophotometer. Four working
solutions with concentrations of 5  10 7–10 
10 6 mol/L were prepared by dilution in deionized water.
A surfactant, polyoxyethylene (10) lauryl ether (POLE),
with a final concentration of 2.5  10 2 mol/L was added
in order to enhance the fluorescence signal.
To simplify the data input to the neutral network,
spectrofluorimetic characteristics were fixed for all
PCBs. The excitation and emission spectra were recorded
using a wavelength of 325 (lem) and 252 (lexc) nm,
respectively. Synchronous fluorescence spectra were
recorded using Dl = 74 nm.
The solution to face the determination of PCBs
mixtures is based on a hierarchical unsupervised modular
adaptive neural network (HUMANN) (3), which consists
on a set of neural layers working with independent
unsupervised learning (Fig. 1). This set consists of a
kohonen, tolerance, and labelling layers. The kohonen
layer generates a topological map, which represents the
different kinds of analysed compounds; The tolerance
layer performs a discrimination process in order to
evaluate which difference matrix data can be considered
representative; and the labelling layer will identify the
different analytes (PCBs) present in the mixture by the
firing of a labelling neuron.
Copyright  2002 John Wiley & Sons, Ltd.
The proposed system has also a pre-processing stage
that performs a feature extraction task. It is based on a
radial basis function approximation network which, using
Gaussian mixtures, adjusts and compares fluorescence
spectra of single substances and complex mixtures (4). In
our case we use excitation, emission and synchronous
fluorescence spectra data with four concentrations. These
spectra are characterized by the great similarity of the
seven PCBs, making more complex the discrimination
between them. The determination of different analytes
(PCBs) present in the mixtures is indicated by the firing
neurons in the labelling layer and by the activation level
of these neurons. This layer has a dynamic dimension and
an open behaviour to different topological structures
present in the processing data.
REFERENCES
1. Garcı́a Báez P, Suárez Araujo CP, Santana Rodrı́guez JJ, Hernández
Garcı́a J. Biomed. Chromatogr. 1999; 13: 181–183.
2. Suárez Araujo CP. Biomed. Chromatogr. 1999; 13: 187–188.
3. Garcı́a P, Suárez CP, Rodrı́guez J, Rodrı́guez M. Neurosci. Methods
1988; 82: 59–73.
4. Garcı́a Báez P, Suárez Araujo CP, Fernández López P. Systems
Analysis Modelling and Simulation (in press, 2002).
Fundamentals and some applications of a
new approach for quanti®cation purposes
in luminescence analysis: method of the
slope ratio (MOSR)
L. Cuadros-RodrõÂguez, A. M. GarcõÂa-CampanÄa and
E. M. Almansa-LoÂpez
School of Qualimetrics, Department of Analytical Chemistry,
Faculty of Sciences, University of Granada, E-18071 Granada,
Spain. E-mail: ealmansa@ugr.es
In instrumental chemical analysis it is very common to
use the external standard calibration, which is established
from measurements of standard analyte solutions in
working solvent. The corresponding equation for linear
fit is:
R ˆ a ‡ b
CA …1†
where R is the analytical response, a and b are the
calibration coefficients (intercept and slope, respectively)
and CA is the analyte concentration added to solvent.
When a sample is measured, the analytical response is
then given by:
R ˆ a ‡ b
CS …2†
where CS indicates the ‘fictitious’ analyte concentration
Luminescence 2002;17:191–291

that the measurement system measures and which is
attributed to the analyte content in the sample. Obviously,
CS is agree with the actual analyte content when no
matrix effect is disturbing the measurement.
Therefore, when the analyte is added into a sample
solution, the analytical response depends on both added
analyte concentration and initial analyte concentration in
sample, and Equations 1 and 2 can be generalized:
R ˆ a ‡ b
…CA ‡ CS † …3†
If a matrix effect is disturbing, CS term can be decom-
posed in:
CS ˆ X
S ‡ K ‡ P
…CA ‡ X
S†
S …4†
where X
S represents the true analyte concentration in
the measuring solution (X is the true analyte content in
the sample and S is the sample concentration), K is a
component of constant matrix systematic error, and
P
(CA ‡ X
S)
S represents a component of proportional
matrix systematic error (CA is the added analyte
concentration and P is a proportionality factor). Evi-
dently, if K and P are null, there are no matrix effects and
the results obtained from a external standard calibration
are free of matrix systematic error.
By combining Equations 3 and 4 and grouping terms, a
general equation is obtained:
R ˆ …a ‡ K
b† ‡ b
CA ‡ b
X
S
…1 ‡ P
S†
‡ P
b
CA
S …5†
This equation indicates the dependence of the analytical
response with the analytical variables: added analyte
concentration; true analyte content in sample; and sample
concentration in solution; and it represents a mathema-
tical model that describes the metrological behaviour of a
general analytical system when there is analyte and
matrix interaction.
When the measurement of analytical signals are
disturbed from matrix effects, four calibration methodol-
ogies would be applied in analytical chemistry in order to
screen and correct the possible deviation between the
found result of the analysis and the true value (or what is
conventionally considered as the true value (1). These
considered calibrations are formally named as follows,
external standard calibration (EC); matrix-matched
calibration (MC); standard-added calibration (AC); and
Youden calibration (YC). By applying different con-
straints on Equation 5, it is possible to find the equations
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 221
corresponding to several calibration methodologies (2):
S ˆ 0 …K ˆ 0 and P ˆ 0†; CA variable
EC function ) R ˆ ‰aŠ ‡ ‰bŠ
CA …6†
S ˆ constant, X ˆ 0; CA variable
MC function ) R ˆ ‰a ‡ K
bŠ‡‰b
…1 ‡ P
S†Š
CA
…7†
S ˆ constant, X 6ˆ 0; CA variable
AC function ) R ˆ ‰a ‡ K
b ‡ b
X
S

…1 ‡ P
S†Š ‡ ‰b
…1 ‡ P
S†Š
CA
…8†
S ˆ variable, CA ˆ 0
YC function ) R ˆ ‰a ‡ K
bŠ ‡
‰b
X
…1 ‡ P
S†Š
S …9†
If the EC curve is a straight line, the MC and AC curves
are also rectilinear and all the terms between brackets
indicate the corresponding independent term (intercept)
and linear coefficient (slope) of the different calibration
curves, which can be represented by aE, bE, aM, bM, aA
and bA, respectively.
It is necessary to consider that, when a proportional
matrix systematic error contributes to the analytical
response, the YC curve is not linear and a parabolic curve
is obtained:
YC function ) R ˆ ‰a ‡ K
bŠ ‡ ‰b
X Š
S ‡
‰b
X
PŠ
S 2 …9†
where the three terms between brackets indicate the
intercept, aY, the linear coefficient, bY, and the quadratic
coefficient, byc, respectively.
For this reason, when a YC curve is established, it is
possible to screen the presence of matrix effects. In
effect, if aY ≠ aE there is a component of constant
systematic matrix error (K ≠ 0), while if bY is not
constant (YC is not linear) then there is a component of
proportional systematic matrix error (P ≠ 0).
Traditionally, the actual analyte content is estimated
from AC curve applying the expression:
aA 1
X ˆ
…10†
bA S
however, as it can easily checked, this expression is
erroneous because of it presupposes that K is always
null. The correct way for calculating the (true) X values
Luminescence 2002;17:191–291
222 ABSTRACTS

is (3–5): Table 1. Analyte content (X) and its uncertainty [s(X)],

relative standard deviation (RSD) and recovery R found by
aA aY 1 applying MOSR in the determination of 2,4-D, 2,4,5-T,
X ˆ
…10† MCPP, MCPA and from A- and C-data analysis
bA S
Analyte
which implies that it is always necessary to establish a content s(X)* RSD
YC when the method of standard additions (MOSA) is Analyte (matrix) X (mg/g) (mg/g) (%) R (%)
being applied. 2,4-D (tap water) 6.97 0.19 2.7 96.9
An equivalent alternative way to find a matrix free- 2,4-D (river water) 21.58 0.66 3.0 99.9
error analytical result is to calculate X from the quotient 2,4,5-T (tap water) 14.11 0.40 2.8 97.3
between the linear coefficients of both YC and EC 2,4,5-T (river water) 17.59 0.39 2.2 100.5
MCPP (tap water) 9.78 0.16 1.6 97.9
curves: MCPP (river water) 32.79 1.82 5.5 96.4
MCPA (tap water) 17.42 0.38 2.2 96.8
bY MCPA (river water) 15.28 0.42 2.8 95.5
X ˆ …11† Molybdenum (cabbage) 0.37 0.01 3.3 102.7**
bE
Oxalate (urine) 3537 265 7.5 105.5**
Equation 11 constitutes the basis of what we term the * Calculated by application of Fieller’s theorem.
“method of the slope ratio” (MOSR), which is proposed ** These recoveries are calculated using the analyte content declared
in this communication. MOSR presents two important in the original paper.
advantages on MOSA:
1. When the (constant or proportional) matrix effect is
disturbing, it is possible to estimate free-error
for each application, the standard deviation of the analyte
analytical results only from a YC curve for each
content, s(X), the relative standard deviation, RSD, and
sample and a general EC curve, while the MOSA
the recovery, R, are calculated. The found RSD and
application always requires the establishment of both
recovery values show that the proposed quantification
YC and AC curves for each sample.
strategy is accurate.
2. For all cases, the uncertainty associated with the
analyte content can be easily calculated from the
standard deviation of YC and EC slopes by applying
REFERENCES
Fieller’s theorem (6).
To test the applicability of this quantification strategy 1. Garcı́a-Campaña AM, Cuadros Rodrı́guez L, Aybar Muñoz J et al.
Biomed. Chromatogr. 1999; 13: 151–154.
for routine analysis, it was applied to different data sets 2. Cuadros Rodrı́guez L, Gámiz Gracia L, Almansa López EM,
obtained from the spectrofluorimetric molybdenum Bosque Sendrà JM. Trends Anal. Chem. 2001; 11: 620–635.
determination in cabbage leaves with alizarin S after 3. Cuadros Rodrı́guez L, Garcı́a-Campaña AM, Alés Barrero F,
Jiménez Linares C, Román Ceba M. J. AOAC Int. 1995; 78: 471–
Cuadros Rodrı́guez et al. (C-data) (3), from the micellar 476.
spectrofluorimetric oxalate determination in urine with 4. Garcı́a-Campaña AM, Cuadros Rodrı́guez L, Aybar Muñoz J, Alés
Alizarin Red S and Zr(IV) after Garcı́a-Campaña et al. Barrero F. J. AOAC Int. 1997; 80: 657–664.
5. Castells RC, Castillo MA. Anal. Chim. Acta 2000; 423: 179–185.
(A-data) (4), and finally from the determination of four 6. Fieller EC. J. R. Stat. Soc. 1940; 7: 1–64.
chlorophenoyacid herbicides: 2,4-dichlorophenoxyacetic 7. Garcı́a-Campaña AM, Aaron J-J, Bosque Sendra JM. Talanta 2001;
acid (2,4-D), 2-(2-methyl-4-chorophenoxy) propionic 55: 531–539.
acid (MCPP), 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T) and 2-methyl-4-chloro-phenoxyacetic acid
(MCPA) in tap water and river water, carried out for
this study.
In this last case, three calibration curves have been Determination of the ¯uoxetine
established for each herbicide: a general EC curve and enantiomers, derivatized with
two YC curves, one for each kind of matrix. Known 4-¯uoro-7-nitro-2,1,3-benzoxadiazole
concentrations of the four analytes were spiked into the (NBD-F) by HPLC with ¯uorimetric detection
two different matrixes (tap water and river water) and
were analysed using the room temperature photochemi- Tineke Vankeirsbilck,1 Ann Vercauteren,1
cally-induced fluorescence (RTPIF) technique to convert Guido Van der Weken,1 Willy R. G. Baeyens,1
them in strongly fluorescent photoproducts after direct Takeshi Fukushima2 and Kazuhiro Imai2
irradiation with ultraviolet light (7). 1
Department of Pharmaceutical Analysis, Faculty of
The analyte contents (X, calculated applying MOSR) Pharmaceutical Sciences, Ghent University, Harelbekestraat 72,
obtained from each analysis are shown in Table 1. Also, B-9000 Ghent, Belgium. E-mail: willy.baeyens@rug.ac.be

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291


2
Laboratory of Bio-Analytical Chemistry, Graduate School of
Pharmaceutical Sciences, University, of Tokyo, 3-1, 7-Chome,
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
A substantial number of pharmaceuticals contain an
asymmetric centre. These drugs are generally adminis-
tered as racemates. As the literature illustrates, the
individual enantiomers often differ in pharmacological
action. It is thus important that separation methods are
developed to determine both enantiomers in racemic
mixtures. Fluoxetine, ()-N-methyl-g-[4-(trifluoro-
methyl)phenoxy]benzene-propanamine, is administered
as a racemic mixture and is an important antidepressant
drug that enhances serotoninergic neurotransmission
through potent and selective inhibition of presynaptic
serotonin re-uptake. In this work, a relatively simple and
sensitive HPLC method with fluorimetric detection is
described for the determination of the fluoxetine
enantiomers based on the coupling with the fluorogenic
label NBD-F.
EXPERIMENTAL
HPLC conditions
Isocratic elution was applied and all separations were
performed at ambient temperature. Optimization of the
derivatization procedure was done on a 12.5 cm  4 mm
i.d. Lichrospher 100 RP-18 column (Merck, Darmstadt,
Germany) with a mobile phase consisting of 30:70 (%
v/v) phosphate buffer, pH 4, 50 mmol/L acetonitrile at a
flow-rate of 1 mL/min. Enantiomeric chromatography
was performed on a 15 cm  4.6 mm i.d. Chiralcel OJ-R
column (Daicel Co., Tokyo, Japan). Mobile phases
consisting of different mixtures of acetonitrile and
NaClO4-buffers with varying molarity and pH values
were tested. The excitation and emission wavelengths of
the detector were set at 470 and 530 nm, respectively.
Figure 1. Reaction of fluoxetine with NBD-F.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 223
Derivatization procedure
The stock standard solutions of fluoxetine (1.00 mg/
25 mL) were prepared by dissolving appropriate amounts
of the compound in a 0.2 mol/L borate buffer (pH 8.0)
containing 4 mmol/L EDTA. 50 mmol/L NBD-F solu-
tions were prepared by dissolving NBD-F in acetonitrile.
Both solutions were kept in amber-coloured bottles and
renewed every week. 10 mL fluoxetine solution and 30 mL
NBD-F solution were mixed and 960 mL 1% (v/v) acetic
acid in methanol was added to stop the reaction. 10 mL
final solution was injected onto the HPLC system (1, 2).
RESULTS AND DISCUSSION
As fluoxetine has only weak native fluorescence,
chemical derivatization with NBD-F was selected with
this benzoxadiazole reagent (Fig. 1) because of its high
reactivity with amines. It was found a suitable reagent
because of its rapid derivatization reaction under mild
conditions. In a first stage, the optimal derivatization
conditions were investigated. The effects of temperature
and reaction time were controlled by varying the reaction
time (1, 5, 10, 20 and 30 min), at 20, 40 and 60°C. The
reaction was most rapid at 60°C; however, derivatization
for 5 min at room temperature was preferred because of
the fluoxetine thermolability.
According to Pichini et al. (3), the starting mobile
phase that was applied consisted of 0.3 mol/L NaClO4,
CH3CN and triethylamine (TEA) in v/v concentrations of
170, 330 and 2.5 respectively, brought to pH 2,5.
Different mobile phase constituents were investigated.
TEA was not found essential to obtain an enantiomeric
separation and pH changes of mobile phase did not
explicitely affect the chromatogram. When the perchlo-
rate buffer was changed to a phosphate buffer, no
separation was noticed. The molarity of the perchlorate
buffer was varied between 0.1 and 0.5 mol/L, but no
Luminescence 2002;17:191–291
224 ABSTRACTS
Figure 2. Chromatogram of fluoxetine derivatized with NBD-F. Reaction and chromatographic conditions as
given in the text. The peaks at 16 and 17 min are the fluoxetine enantiomers.
significant change in resolution could be observed. The
percentage of acetonitrile was varied from 50% to 80% as
this solvent seemed to be the most important factor in the
separation process. Base-line separation was obtained
using 60% (v/v) of acetonitrile. The final optimal mobile
phase consisted of 40% (v/v) 0.3 mol/L NaClO4 and 60%
(v/v) CH3CN brought to pH 3, with a flow-rate of 0.6 mL/
min. The derivatives were well-separated within 20 min
(Fig. 2). The specific peaks have not been identified so far
as pure enantiomers were not available (further work in
this respect is in progress). The limit of detection was
somewhat better than 100 pg using the proposed method.
The intraday variation was 1.53% for the first peak and
1.62% for the second peak (n = 7). The interday variation
was 4.17% and 4.84% for the first and second peaks,
respectively (n = 5).
CONCLUSION
A sensitive HPLC method has been developed for the
determination of both enantiomers of the fluoxetine
antidepressant. The method is based on the pre-column
coupling reaction between fluoxetine and the fluorogenic
tag NBD-F. The reaction takes place at room temperature
within 5 min and is terminated with 1% (v/v) acetic acid.
The resultant solution was injected onto a Chiralcel OJ-R
column. The mobile phase consisted of 40% (v/v)
0.3 mol/L NaClO4 and 60% (v/v) CH3CN at pH 3.0.
The peaks of the derivatized enantiomers are separated
with base-line resolution within 20 min.
Copyright  2002 John Wiley & Sons, Ltd.
REFERENCES
1. Fukushima T, Kato M, Santa T, Imai K. Enantiomeric separation
and sensitive determination of D,L-amino acids derivatized with
fluorogenic benzofurazan reagents on Pirkle type stationary phases.
Biomed. Chromatogr. 1995; 9(4): 10–17.
2. Fukushima T, Kato M, Santa T, Imai K. Enantiomeric separation
and spectrofluorometric detection of the racemic drugs ()-1-(2,6-
dimethylphenoxy)-2-propamine (Mexiletine) and (3 RS)-4-amino-
3-hydroxybutanoic acid (GABOB), derivatized with 4-fluoro-7-
nitro-2,1,3-benzoxadiazole on a phenylcarbamylated cyclodextrin
bonded stationary phase. Analyst 1995; 120(2): 381–383.
3. Pichini S, Pacifici R, Altieri I, Pellegrini M, Zuccaro P. Stereo-
selective determination of fluoxetine and norfluoxetine enantiomers
in plasma samples by high-performance liquid chromatography. J.
Liq. Chromatogr. Relat. Technol. 1996; 19: 1927–1935.
Methodology for different resolution
degrees of LAS homologues and isomers in
commercial formulations by HPLC-FD
A. Garballo,1 M. del Olmo,1 A. Gonzalez-Casado,1
A. NavaloÂn,1 J. L. VõÂlchez,1 I. LoÂpez,2 J. A. de Ferrer,2
A. Moreno2 and J. L. Berna2
1
Department of Analytical Chemistry, University of Granada,
Avda. Fuentenueva S/N, E-18071 Granada, Spain. E-mail:
mdolmo@ugr.es
2
PetroquõÂmica EspanÄola S.A., E-28042-Madrid, Spain
Linear alkylbenzenesulphonates (LAS) are applied
extensively as surfactants in consumer formulations as
Luminescence 2002;17:191–291

complex mixtures of homologues and isomers. The alkyl
chain length of the homologues usually varies from 10 to
13 carbon atoms and the phenyl group may be attached to
the alkyl chain at any carbon except at the end position.
The determination of the total LAS content is sufficient
for general assessment of the pollution of waste water and
surface water. Homologous and isomeric separation of
LAS are important in industrial and environmental
samples in order to explain their behaviour. Furthermore,
the isomeric distribution gives information about the
manufacturing process and can be considered as a
fingerprint of the product.
To deal with the resolution of homologous and isomers
of LAS by HPLC and fluorimetric detection (FD), the
resolution parameter P = A/B shown in Fig. 1 (A, distance
between the top of the minor peak and the valley; B,
height of the minor peak) was used to optimize each of
the experimental variables tested in this study.
First, we used the isoeluotropic model (1) for the
selection of the mobile phase composition. Acetonitrile,
tetrahydrofuran, methanol and water were tested in
binary, ternary and quaternary mixtures established
according to mixture design. The maximum resolution
was achieved with acetonitrile/water, while methanol/
water showed the poorest resolution.
Figure 1. Relationship between the SDS concentration in mobile phase and the resolution
parameter P calculated for 31 and 41 isomers of LAS homologue C11 using a 250 mm
octadecylsilica column and acetonitrile/water as the mobile phase.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 225
Next, sodium perchlorate, triethylamine/acetic acid,
ammonium acetate and non-ionic, cationic and anionic
surfactants were checked as modifiers. The solvophobic
association of LAS with SDS (2) produced better results
in both cases. Fig. 1 shows the relationship between the
SDS concentration in mobile phase and the resolution
parameter P calculated for 31 and 41 isomers of LAS
homologue C11, using a 250 mm octadecylsilica column
and acetonitrile/water. The P parameter is maximum for
a SDS concentration of 5 mmol/L, decreasing markedly
when SDS concentration increases.
Column temperature and pH of the mobile phase were
also tested as experimental variables. While pH affected
neither resolution, retention time nor sensitivity, an
increase of the work temperature caused shorter retention
time and therefore poorer resolution. We established
40°C as the optimum column temperature because lower
values increased the viscosity of the mobile phase.
Consequently, the homologues resolution was
achieved by using a 125 mm octylsilica column,
methanol/water as mobile phase and 30 mmol/L SDS.
The initial composition of the mobile phase was 55% of
methanol and 45% 30 mmol/L SDS in water, increasing
the methanol content to 70% over a period of 15 min
using linear gradient elution, as shown in Fig. 2A. For the
Luminescence 2002;17:191–291
226 ABSTRACTS

Figure 2. (A) Chromatogram corresponding to LAS homologues. (B) Chromatogram corresponding to LAS isomers.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

 ABSTRACTS 227

isomeric resolution of LAS, a 250 mm octadecylsilica

column, with acetonitrile/water as mobile phase and
5 mmol/L SDS was used. In this case, the initial com-
position of the mobile phase was 20% acetonitrile and
80% 5 mmol/L SDS in water, increasing the acetonitrile
content to 40% over a period of 155 min using linear
gradient elution, as shown in Figure 2B. The column
effluent in both cases was monitored using fluorescence
detection at 230 nm for excitation and 290 nm for
emission.
Figure 1. The structures of the NBD compounds (1) and
Acknowledgement ABD compounds (2) used in this study.

The authors wish to express their thanks to Petroquı́mica

Española S.A. for their financial support, for providing
the LAS homologues standards and their valuable con-
tributions to this project.
REFERENCES
1. Nyiredy SZ, Meier B, Erdelmeier CAJ, Sticher O. J. High Resol.
Chromatogr. 1985; 8: 186–188.
2. Heinig K, Vogt C, Werner G. Analyst 1998; 123: 349–353.
The method for predicting the ¯uorescence
quantum yields (F) of the benzofurazan
compounds and the design of a new
photoinduced electron transfer (PET)
reagent
Maki Onoda, Seiichi Uchiyama, Tomofumi Santa and
Kazuhiro Imai
Graduate School of Pharmaceutical Sciences, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
E-mail: ff16008@mail.ecc.u-tokyo.ac.jp
Recently, the reagents utilizing PET (1), called ‘PET
reagents’ (2), have been developed as a new type of
fluorescent ‘on–off’ reagent. In general, the PET reagents
consist of a fluorophore and a reacting group as an
electron donor (D) and an electron acceptor (A) in one
molecule. They are non-fluorescent or weakly fluores-
cent, since the electron transfer from D to A accelerates
radiationless relaxation. After the reaction with analytes,
the electron transfer is suppressed and the fluorescence of
the fluorophore is restored. In this research, we intended
to establish a method for predicting the F values of the
benzofurazan compounds to develop new PET reagents.
First, we investigated the effect of the spacer length on
the F values, using nitrobenzoxadiazole (NBD) com-
pounds (1, see Fig. 1; n = 0, 2–12, R = NMe2) (3). They
have -NMe2 moiety and NBD fluorophore, as D and A,
respectively. The F values were reduced as the
methylene chain length became shorter (n e 4).
Next, we investigated the effect of the aliphatic sub-
stituent group (R) on the F values, using NBD com-
pounds (1, see Fig. 1; n = 2, R = NEt2, NMe2, NHMe,
NH2, NHAc, NMeAc, OH, OMe, OAc) and aminosul-
phonylbenzoxadiazole (ABD) compounds (2, see Figure
1; R = NHAc, OH) (4). The F values were reduced in the
compounds bearing an aliphatic substituent group (R)
which had a high electron-donating ability (e.g. R = NEt2,
NMe2, NHMe): the ability was estimated by a HOMO
energy (the semi-empirical PM3/COSMO calculation)
and a K value (the Stern–Volmer plotting).
These results enable us to predict the F values of the
benzofurazan compounds bearing an aliphatic substituent
group for designing new PET reagents. The following
criteria seem to be necessary for designing the PET
reagents: i.e. (a) D and A are linked with a short spacer;
(b) a reacting moiety has a high electron-donating ability
(the reagents must be non-fluorescent or weakly fluor-
escent); and (c) the reacted moiety has a low electron-
donating ability (the derivatives must be strongly
fluorescent).
Accordingly, we have designed new PET reagent for
peroxyacetic acid (PAA), 4-ethylthioacetylamino-7-
phenylsulphonyl-2,1,3-benzoxadiazole (EPB, 3; Fig. 2),
using the established method for predicting the F values.
As estimated, EPB (3, F = 0.0014) was non-fluorescent
and its derivative with PAA, EPBO (4, F = 0.34), was
strongly fluorescent in acetonitrile. EPB (3) and EPBO
Figure 2. The structures of a new PET reagent for
peroxyacetic acid, EPB (3) and its derivative, EPBO (4).

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

228 ABSTRACTS
(4) were separated by reversed phase HPLC and
fluorometrically detected at 479 nm with excitation at
362 nm. The attained detection limit for PAA was 105
fmol (s/n = 3) and the cross-reactivity toward hydrogen
peroxide was very low, indicating that EPB (3) is a highly
sensitive and selective reagent for PAA.
REFERENCES
1. de Silva AP, Gunaratne HQN, Gunnlaugsson T et al. Chem. Rev.
1997; 97: 1515–1566.
2. de Silva AP, Gunaratne HQN, Gunnlaugsson T. Tetrahedron Lett.
1998; 39: 5077–5080.
3. Onoda M, Uchiyama S, Santa T, Imai K. Luminescence 2002; 17:
11–14.
4. Onoda M, Uchiyama S, Santa T, Imai K (submitted).
A new ¯uorogenic derivatization reagent
for thiols: 4-(N-acetylaminosulphonyl)-7-
¯uoro-2,1,3-benzoxadiazole
Kohki Okabe, Reiko Wada, Seiichi Uchiyama,
Kenichi Ohno, Tomofumi Santa and Kazuhiro Imai
Graduate School of Pharmaceutical Sciences, University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
E-mail: kimai@mol.f.u-tokyo.ac.jp
High-performance liquid chromatography (HPLC) with
fluorometric detection is the most popular method for the
determination of thiol compounds. Thus, many fluoro-
genic derivatization reagents with thiols for HPLC have
been developed (1).
We have already developed fluorogenic derivatization
reagents with benzofurazan structure for thiol compounds
(2), ie ammonium 7-fluoro-2,1,3-benzoxadiazole-4-
sulphonate (SBD-F) (3); 4-(aminosulphonyl)-7-fluoro-
2,1,3-benzoxadiazole (ABD-F) (4); and 4-(N,N-dimethyl-
aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-
F) (5). Among them, SBD-F is most popularly used,
since the generated derivatives were sensitively detected
and highly water-soluble. However, SBD-F requires a
long reaction period of about 60 min for the derivatiza-
tion of thiols, and therefore more reactive reagent is still
required.
Here we report a new fluorogenic reagent, 4-(N-
Figure 1. Chemical structure of AcABD-F.
Copyright  2002 John Wiley & Sons, Ltd.
acetylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole
(AcABD-F; Fig. 1) for thiols. AcABD-F reacted with
thiols within 10 min at 50°C. The fluorescence intensities
of the generated derivatives were as strong as those of
SBD thiols. Furthermore, the acetylaminosulphonyl
group ionized at above pH 5, suggesting that AcABD
thiols are highly water-soluble at above pH 5. Finally, the
derivatives of thiols such as cysteine, homocysteine,
glutachione and N-acetylcysteine with AcABD-F were
separated on the reversed-phase HPLC and detected with
detection limits at femtomol range.
REFERENCES
1. Shimada K, Mitamura K. J. Chromatogr. B 1994; 659: 227–241.
2. Uchiyama S, Snata T, Okiyama N, Fukushima T, Imai K. Biomed.
Chromatogr. 2001; 15: 295–318.
3. Imai K, Toyo’oka T, Watanabe Y. Anal. Biochem. 1983; 128: 417–
473.
4. Toyo’oka T, Imai K. Anal. Chem. 1984; 56: 2461–2464.
5. Toyo’oka T, Suzuki T, Saito Y, Uzu S, Imai K. Analyst 1989; 114:
413–419.
Study of the interaction of b-cyclodextrins
with gem®brozil using a spectro¯uorimetric
method
Laura FernaÂndez, Cristina MartõÂnez-OhaÂrriz, Miguel
SaÂnchez, Carmen MartõÂn, Arantza Zornoza and Itziar
VeÂlaz
Departamento de QuõÂmica y Edafologia (SeccioÂn de QuõÂmica
FõÂsica), Facultad de Ciencias, Universidad de Navarra, E-31080
Pamplona, Spain. E-mail: misango@unav.es
Gemfibrozil (5-(2,5-dimethylphenoxy)-2,2-dimethyl-
pentanoic acid; Fig. 1) is an oral lipid regulation agent.
This drug exhibits intrinsic fluorescence that may be
derived from substituents on the phenoxy ring. This
property was used for the determination of gemfibrozil in
plasma by HPLC with fluorescence detection (1).
b-cyclodextrins (b-CD) display a doughnut-shaped
structure with a hydrophobic inside and two hydrophilic
rims, and gemfibrozil can lodge into the b-CD cavity,
forming reversible non-covalent complexes.
Figure 1. Chemical structure of gemfibrozil.
Luminescence 2002;17:191–291

In this work, the inclusion complexes of gemfibrozil
with b-CD have been studied by fluorescence spectro-
scopy, taking into account the drug ionization. Gem-
fibrozil is a weak acid ( pKa 4.7); consequently, the
binding constants at different temperatures (range
15–45°C), have been determined at pH 2.8 and 7.0,
assuming a 1:1 stoichiometry. The corresponding
changes in the standard enthalpy and entropy upon bind-
ing have been also evaluated. The same procedure has
been carried out with methyl-b-CD, in order to test the
influence of the methylated rims on the complex stability.
The fluorescence emission of gemfibrozil is remark-
ably enhanced when b-CD concentration is increased;
this fact proves the inclusion complexation and allows to
obtain the binding constants. This increase is higher with
methyl-b-CD than with unmodified b-CD. The values
obtained for the binding constants, K1:1 at 25°C, were:
(3.7  0.3) 103 mol/L with methyl-b-CD and (2.5 
0.3) 103 mol/L with b-CD. This difference in binding
can be related with the hydrophobic interactions when the
methyl groups are present in the cyclodextrin rims (2).
It has been found that the stability constants at pH 7.0
are lower than those at pH 2.8, showing the influence of
the ionization in the drug–CD interactions. In the same
way, those at pH 2.8, the binding constants of gemfibrozil
with methyl-b-CD, at 25°C (K1:1 = (7.5  0.4) 102
mol/L), are higher than those with the natural b-CD
(K1:1 = (6.8  0.4) 102 mol/L).
Acknowledgement
We gratefully acknowledge financial support from the
Gobierno de Navarra and the Ministerio de Educación y
Cultura (Fund No. BJU2000-0264).
REFERENCES
1. Nakagawa A, Shigeta A, Iwabuchi H, Horiguchi M, Nakamura K,
Takahagi H. Biomed. Chromatogr. 1991; 5: 68–73.
2. Duchěne D. New Trends in Cyclodextrins and Derivatives. Editions
de Santé: Paris, 1991.
Spectro¯uorimetric study of pindolol and
other monosubstituted indole derivatives
C. Gazpio, M. SaÂnchez, A. Zornoza, C. MartõÂn,
C. MartõÂnez-OhaÂrriz and I. VeÂlaz
Departamento de QuõÂmica y EdafologõÂa (SeccioÂn de
QuõÂmica-FõÂsica), Facultad de Ciencias, Universidad de Navarra,
E-31080 Pamplona, Spain. E-mail: misango@unav.es
Pindolol [1-(1H-indol-4-yloxy)-3-[(1-methylethyl)ami-
no]-2-propanol] is a non-cardioselective b-blocker com-
monly used in the treatment of cardiovascular disorders.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 229
Figure 1. Chemical structure of pindolol and indole deriva-
tives.
This drug enhances the clinical effects of antidepressant
drugs (1).
Spectrofluorimetric characteristics of pindolol have
been investigated with the aim of using this technique for
analytical determinations. Other monosubstituted indole
derivatives, 4-methoxy and 5-methoxyindole, have been
also studied for comparative purposes (Fig. 1). Corrected
excitation and emission wavelengths in different solvents
are reported. The emission maxima suffer a batho-
chromic shift as the solvent polarity increases. The effect
of the solvent on the Stokes’ shift of these compounds has
been analysed using the Lippert equation, it seems that
there is an increase in the dipolar moments upon
excitation. In addition, the solvatochromic plot of the
fluorescence spectrum of pindolol in 1,4-dioxane–water
mixtures shows a marked red-shift due to the hydrogen
bonding which arises for H2O mole fractions above 0.8.
Fluorescence quantum yields have been determined in
different solvents at 25°C (Table 1). In all the cases, it has
been found that quantum yields in water and dichloro-
methane are considerably lower than those in other
solvents. In dichloromethane, quenching takes place
probably due to the donation of an electron from the
fluorophore to the solvent. It is observed that 5-
methoxyindole is markedly more fluorescent than the
4-methoxy derivatives in all the solvents studied, it could
be attributed to the higher electronic delocalization
through the p-system in that position. With respect to
the sensitivity of this technique, low detection limits have
been obtained for pindolol in different solvents.
Table 1. Fluorescence quantum yields of pindolol and indole
derivatives at 25°C in different solvents
4- 5-
Pindolol Methoxyindole Methoxyindole
Water 0.013 0.010 0.27
Acetonitrile 0.14 0.16 0.58
Methanol 0.17 0.23 0.61
Ethanol 0.23 – 0.36
Dichloromethane 0.027 0.027 –
Luminescence 2002;17:191–291
230 ABSTRACTS
Figure 2. Stability constants of pindolol with different cyclodextrins. Non-linear
adjustment of the experimental data. [Pindolol] = 1.21  10 5 mol/L; lexc = 264 nm;
lem = 319 nm; slits = 4.0 nm.
Pindolol is a weak base, pKa = 9.46 (2), and its
fluorescence is pH-dependent, the drug is more fluor-
escent in acid solutions and the intensity decreases in
alkaline media.
Finally, the complexation of pindolol with different
cyclodextrins has been investigated in pH 12.1 buffer
solution. The emission wavelength of pindolol presents a
slight hypsochromic shift in presence of increasing
concentrations of b-cyclodextrin, probably related to
the inclusion of the chromophore in its apolar cavity. In
addition, with b-cyclodextrin, a four-fold fluorescence
enhancement has been observed, this increase is even
higher with methyl (ten-fold) and hydroxypropyl-b-
cyclodextrin (six-fold). This fact is less marked with
g-cyclodextrin and negligible with a-cyclodextrin.
Previously, we studied the complexation of this drug
with different cyclodextrins in pH 5.5 aqueous solution
(3) and it led to a lower increase of fluorescence which
evidences the role of ionisation in the formation of these
inclusion complexes. Complexation with b-cyclodextrins
can be applied to improve the sensitivity of pindolol in
water. The stability constants of the complexes (pH 12.1)
have been determined at 25°C (Fig. 2).
Acknowledgement
Authors acknowledge the Gobierno de Navarra, the
Ministerio de Educación y Cultura (Grant No. BJU2000-
0264) for financial support.
Copyright  2002 John Wiley & Sons, Ltd.
REFERENCES
1. Blier P, Bergeron R. J. Clin. Psychiat. 1998; 59 (5): 16–23.
2. Laxer M, Capomacchia AC, Hardee GE. Talanta 1981; 28: 973–
976.
3. Gazpio C, Sánchez M, Zornoza A, Martı́n C, Martı́nez-Ohárriz C,
Vélaz I. 9th European Conference on the spectroscopy of biological
molecules, September 8–13 2001, Prague, Czech Republic.
Excited-state reaction between ¯uorescein
and N-acetyl-D,L-aspartic acid
L. Crovetto, A. Orte, E. M. Talavera and
J. M. AÂlvarez-Pez
Department of Physical Chemistry, University of Granada,
Cartuja Campus, E-18071 Granada, Spain. E-mail:
jalvarez@platon.ugr.es
In the course of our studies with fluorescein-labelled
proteins, we have observed complex nanosecond emis-
sion kinetics which indicate the excited state relaxation
processes. Since fluorescein is possibly the most
frequently used fluorophore in biology, and we found
that in presence of a suitable proton donor-acceptor such
as phosphate buffer at 1 mol/L concentration, the excited
state monoanion-dianion proton transfer reaction occurs
very efficiently (1, 2), we have tried to establish whether
amino acids can act as a proton donor/acceptor. In this
Luminescence 2002;17:191–291

work we investigate the excited state reaction between
fluorescein and N-acetyl-D,L-aspartic acid (3, 4) as a
model of amino acid residue in proteins.
RESULTS AND DISCUSSION
N-acetyl-D,L-aspartic acid did not significantly perturb
the fluorescein absorption spectrum. Hence, it does not
form ground state complexes. Steady state fluorescence
from aqueous fluorescein solutions at pH 5.9 showed that
as the N-acetyl-D,L-aspartic acid concentration increased
there was a pronounced increase in the intensity of the
emission peak at 515 nm (lex = 440 nm). At this pH the
monoanion: dianion ratio in the ground state is >1, and
the 440 nm wavelength preferentially excites the less
fluorescent monoanion. Under these conditions the
excited monoanion is converted to the dianion during
its lifetime and this conversion is detected experimentally
as a fluorescence intensity increase due to the higher
dianion fluorescence efficiency. To quantify the reaction,
we have recorded titration graphs of fluorescence inten-
sity, normalized by absorbance, vs. pH for fluorescein
solutions with N-acetyl-D,L-aspartic acid buffer 1 mol/L
pH 4.6–6.8 (lex = 440; lem = 515), as shown in Fig. 1. For
this model we can express the normalized fluorescence
intensity by:
M D
Iˆ pK †
‡ …1†
1 ‡ 10…pH 1 ‡ 10…pK pH†
The experimental results were fitted to the theoretical
expression. We obtained very good fit parameters and a
value of pK* = 6.40  0.02 in accordance with the value
obtained from another reaction system (1, 2). The
statistical parameters show that the excited reaction is
Figure 1. Normalized fluorescence intensity vs. pH of
fluorescein solutions and N-acetyl-D,L-aspartic acid at 1 mol/L
concentration.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 231
Figure 2. Fluorescence decay obtained by the expression:
I(t) = A ‡ a1e t/1 ‡ a2e t/2 vs. time/10 9 s, with the values
obtained from a solution of fluorescein 5  10 5 mol/L and
N-acetyl-D,L-aspartic acid 1 mol/L at pH 5.5.
sufficiently fast to achieve equilibrium during the life-
times of the excited states.
We also recorded nanosecond emission graphs with
preferential monoanion excitation (lex = 440; lem = 515)
at low (0.005 mol/L) and high (1 mol/L) buffer concen-
tration in the pH range 4.6–6.5. Decays were bi-
exponential. At a low buffer concentration, the two
lifetimes were pH-independent and correspond to the
lifetimes of monoanion and dianion form in the absence
of excited-state proton exchange. By contrast, at a high
buffer concentration they were pH-dependent and the
pre-exponential of the short lifetime was negative. Fig. 2
shows the representation of the expression I(t) =
A ‡ a1e t/t1 ‡ a2e t/t2 vs. time/10 9 s with the values
obtained from fluorescein 5  10 5 mol/L. The decay
shape is characteristic of excited state reactions.
These results are indicative of excited-state reactions
and support the hypothesis that there has been a proton
exchange reaction between fluorescein and N-acetyl-D,L-
aspartic acid.
Acknowledgement
This work was supported by Grant PB98-1285 from the
Spanish Ministry of Education and Culture.
REFERENCES
1. Yguerabide J, Talavera E, Alvarez JM, Quintero B. Steady-state
fluorescence method for evaluating excited state proton reactions:
application to fluorescein. Photochem. Photobiol. 1994; 60: 435–
441.
2. Alvarez-Pez JM, Ballesteros L, Talavera EM, Yguerabide J.
Fluorescein excited-State proton exchange reactions: nanosecond
Luminescence 2002;17:191–291
232 ABSTRACTS
kinetics and correlation with steady-state fluorescence intensity. J.
Phys. Chem. A 2001; 105: 6320–6332.
3. Costa Pesoa J, Gadja T, Gillard R et al. Oxovanadium (IV) com-
plexes of the dipeptides glycyl-L-aspartic acid, L-aspartylglycine
and related ligands; a spectroscopic and potentiometric study. J.
Chem. Soc. Dalton Trans. 1998; 3587–3600.
4. Kushwaha PS, Mishra PC. Stability of the normal, zwitterionic
neutral and anionic forms of aspartic acid in gas phase and aqueous
media. J. Mol. Struct. Teochem. 2001; 549: 229–242.
Determination of ground-state dissociation
constant from the system ¯uorescein/
2-mercaptoethanol
L. Crovetto, A. Orte, E. M. Talavera and
J. M. Alvarez-Pez
Department of Physical Chemistry, University of Granada,
Cartuja Campus, E-18071 Granada, Spain. E-mail:
jalvarez@platon.ugr.es
The widespread use of fluorescein as a protein label has
encouraged us to study its interaction with amino acid
residues containing side chains with charged groups that
can act as a proton donor–acceptor. The complex-
forming reaction between 2-mercaptoethanol, as a model
of cysteine, and fluorescein was investigated using
absorption, steady-state and time-resolved fluorescence
techniques.
RESULTS AND DISCUSSION
Visible absorption and emission spectra of fluorescein
were recorded at constant pH. Absorption spectra slightly
decreased with increasing concentrations of 2-mercapto-
ethanol. Absorption and emission spectra were also
shifted slightly to red, although isosbestic or isoemissive
points were not found in the wavelength region 350–
650 nm. These spectral features suggest that the spectral
changes are caused by the formation of an association
between 2-mercaptoethanol and fluorescein or a medium
effect of concentration (ionic strength). The influence of
concentration was excluded by recording absorption and
emission spectra from solutions of different sodium
perchlorate concentrations (0–2 mol/L). Therefore, we
postulate an association according to the general kinetic
model of Scheme 1.
In this scheme, species 1 represents the free form of
fluorescein, while species 2 represents the corresponding
bound form. Scheme 1 assumes a 1:1 stoichiometry
between species 1 and X. Only species 1 and 2, which are
in chemical equilibrium with each other, absorb light at
the excitation wavelength. Excitation by light creates the
excited-state species 1* and 2*, which can decay by
fluorescence and non-radiative processes. The composite
rate constants for those processes are denoted by k01 and
k02, respectively. The second-order rate constant describ-
Copyright  2002 John Wiley & Sons, Ltd.
ing the association is represented by k21. The first-order
rate constant for the dissociation of 2* is denoted by k12.
Although the absorption spectra do not show enough
differences to quantify the interaction and to calculate the
ground state dissociation constant, fluorescence spectro-
scopy can be used to evaluate these constants if the
association–dissociation leads to a change in the
measured steady-state fluorescence signal (1). Steady-
state fluorescence spectra of fluorescein as a function of
2-mercaptoethanol concentration (lex = 420, 440 and
460 nm) show that the emission maximum at 515 nm,
characteristic of fluorescein, gradually decreases with
increasing concentrations of 2-mercaptoethanol, with a
concomitant shift to red.
To avoid distortion of the fluorimetric titrations, the
possible association reaction in the excited state must be
sufficiently slow, as established by Kowalczyk et al. (2).
Hence, a relatively simple experimental test based on
time-resolved fluorescence measurements was per-
formed. The decays of fluorescein at different concentra-
tions of 2-mercaptoethanol (range 0–2 mol/L) were
recorded at two different excitation wavelengths (420
and 440 nm) and one emission wavelength (515 nm). The
decays from solutions with identical 2-mercaptoethanol
concentrations were analysed globally as bi-exponential
functions. Each simultaneous bi-exponential analysis
contained six decay curves. Global bi-exponential
analysis gave good fits. With increasing [2-mercapto-
ethanol], two concentration-independent lifetimes were
consistently obtained (t1 = 4.21  0.03 ns and t2 =
2.24  0.04 ns). Therefore, there is no interference from
the excited-state complex-forming reaction (k2  [X] and
k12 are small enough).
When the above conditions are applied, the kinetic
equations derived from Scheme 1 can be rearranged in
the form of a Hill plot (2, 3).
F Fmin
log ˆ a  log‰X Š log KD
Fmax F
where Fmin and Fmax are the fluorescence signals at [X] →
Scheme 1. Kinetic model for a system of free fluorescein (1)
and its reversible association with 2-mercaptoethanol (X) to
give corresponding bound form, 2, and their excited-state
species (1*, 2*).
Luminescence 2002;17:191–291

0 and [X] → ?, respectively. The validity of the equation
is restricted to the [X] range where the decay times
remain constant, and Fmin and Fmax should be measured
at the [X] extremes of this region.
To determine the ground-state dissociation constant,
KD, we recorded the fluorimetric titration curve at pH 7.
Fig. 1 shows the plot of the left side of the equation vs.
Figure 1. Hill plot of fluorescein 2-mercaptoethanol system
measured at lex 420 nm and lem 515 nm in aqueous solution at
room temperature and pH 7.5.
log [X]. The straight line obtained intersects the abscissa
at the value corresponding to log KD. From this value we
obtain KD = 0.277  0.025. The slope, a, of the plot
indicates that 2-mercaptoethanol binds to fluorescein
with a 2:1 stoichiometry, according to the predominant
ionic charge of fluorescein at physiological pH.
Acknowledgement
This work was supported by Grant PB98-1285 from the
Spanish Ministry of Education and Culture.
REFERENCES
1. Kowalczyk N, Boens N, Van den Bergh V, De Schryver FC.
Determination of the ground-state dissociation constant by fluori-
metric titration. J. Phys. Chem. 1994; 98: 8585–8590.
2. Kowalczyk N, Boens N, Ameloot M. Determination of ground-state
dissociation constant by fluorescence spectroscopy. Methods
Enzymol. 1997; 278: 94–113.
3. Kowalczyk N, Boens N, Meuwis K, Ameloot M. Potential mis-
evaluation of the ground-state from fluorimetric titrations: applica-
tion to the ion indicators SFBI, PBFI and fura-2. Anal. Biochem.
1997; 245: 28–37.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 233
2',7'-Di¯uoro¯uorescein excited-state
proton exchange reactions:
monoanion±dianion equilibrium
A. Orte,1 L. Crovetto,1 R. Bermejo,2 E. M. Talavera1 and
J. M. AÂlvarez-Pez1
1
Department of Physical Chemistry, University of Granada,
Cartuja Campus, E-18071 Granada, Spain. E-mail:
jalvarez@platon.ugr.es
2
Department of Analytical Chemistry and Physical Chemistry,
EUP of Linares, E-23700 Linares, Spain
Fluorescein is a well-known fluorophore, widely used as
a fluorescent label. However, it undergoes photobleach-
ing reactions, which limit its use. For this reason,
fluorescein derivatives with higher resistance to photo-
degradation have been synthesized (1). One of these
derivatives with high photostability and quantum yield is
2',7'-difluorofluorescein (Oregon Green 4881). It dis-
plays four prototropic forms: cation (C), neutral (N),
monoanion (M) and dianion (D), of which the dianionic
form shows much higher fluorescence than the others.
Two fluorine atoms give higher polarity to the molecule,
lessening pK values, as in chlorinated fluoresceins (2).
This is favourable in biochemical applications since, at
biological pH, the prevalent prototropic form is dianion.
Like fluorescein (3, 4), Oregon Green 488 shows
proton exchange reactions during its excited state when
a suitable proton donor–acceptor is present at sufficiently
high concentration. These reactions might influence some
experimental results, and it is therefore necessary to
characterize them for a correct interpretation of the
fluorescence experimental data when the dye is used in
protein labelling. In our experience, the acetic acid–
acetate buffer operates as a proton donor–acceptor, and
could be considered as a model of an aspartic acid protein
residue.
RESULTS AND DISCUSSION
Excited-state proton reactions of Oregon Green 488 are
clearly demonstrated by measurements of emission
spectra as a function of buffer concentration at constant
pH and preferential monoanion excitation (lexc =
440 nm). With increasing buffer concentration the
fluorescence intensity at lem = 515 nm increases and
some changes in spectral shape are observed. These
effects saturate above 1 mol/L concentration and can be
explained as follows: with preferential excitation of the
monoanion, the concentration of directly excited mono-
anions is higher than expected in equilibrium and the
excited monoanions will thus tend to become dianions to
establish equilibrium. Such relaxation does not occur
significantly at low buffer concentration but does occur at
high acetate buffer concentrations, as demonstrated by
the increase in dianion emission with increasing buffer
Luminescence 2002;17:191–291
234 ABSTRACTS

concentration. The buffer concentration does not appre-

ciably modify the ground state equilibria of the proto-
tropic forms, since only slight ionic strength effects are
observed in absortion spectra.
Measurements of fluorescence decays using a time-
correlated single-photon-counting technique have pro-
vided more evidence for this excited-state reaction. With
no buffer and at a pH in which only monoanion and
dianion forms are present, two pH-independent lifetimes
were obtained. The mean values from global analysis of
fluorescence decays, fitted to a bi-exponential function,
were t1 = 5.1  0.4 ns and t2 = 3.8  0.5 ns. Both pre-
exponentials were positive. This is in agreement with a
model of two uncoupled species decaying independently
of each other. Hence, the absence of excited-state proton
transfer reactions must be considered. At high buffer
concentration, fluorescence decays showed two pH- Figure 2. Fluorescence intensity vs. pH curve fitting. (~)
lex = 420 nm, lem = 520 nm. (&) lex = 440 nm, lem = 520 nm.
dependent lifetimes: a short lifetime, whose pre-expo- Dlex = Dlem = 1.5 nm.
nential is negative, representing excited state species
concentrations rearrangement before equilibrium is
reached; and a long lifetime, which represents both
prototropic forms decaying in equilibrium. Fig. 1 shows
d-impulse decays in both the absence of buffer and at fluorescent intensity normalized by the absorbance
high buffer concentration. (IT/AT), is based on an excited-state equilibrium between
A steady-state fluorescence method to quantitatively three excited forms: neutral (N*), monoanion (M*) and
evaluate excited-state proton transfer reactions induced dianion (D*), N* , M* , D*, promoted by the proton
by an effective proton donor-acceptor (3) has been donor–acceptor, with two constants involved ( pKN* and
applied to 2',7'-difluorofluorescein. Equation 1, for the pKM*). R N, R M and R D are adjustable parameters, related
to N, M and D quantum yields, respectively.

IT RN
ˆ pKN pKN pKM
‡
AT 1 ‡ 10pH ‡ 102
pH
RM

pKM
‡
1‡ 10 pKN pH ‡ 10pH
RD
  
1‡ 10 pKM pH ‡ 10 pKN ‡pKM 2
pH

Excited-state equilibria were quantitatively evaluated

through global non-linear least squares curve fitting of
experimental data (Fig. 2), obtaining pKN* = 3.9  0.1
and pKM* = 5.08  0.04.

Acknowledgement
This work was supported by Grant PB98-1285 from the
Spanish Ministry of Education and Culture.

Figure 1. d-impulses from fluorescence decays fitted to a
biexponential function: I(t) = A ‡ b1e t/1 ‡ b2e t/2. (—) No
buffer concentration, pH = 4.3, 1 = 3.5  0.1 ns, 2 = 5.1 
0.2 ns; (




) Buffer concentration 1 mol/L, pH = 4.3,
1 = 0.82  0.03 ns (with b1 < 0), 2 = 5.1  0.2 ns.
REFERENCES
1. Sun WC, Gee KR, Klaubert DH, Haugland RP. Synthesis of
fluorinated fluoresceins. J. Org. Chem. 1997; 62: 6469–6475.
2. Leonhardt H, Gordon L, Livingston R. Acid–base equilibria of
fluorescein and 2',7'-dichlorofluorescein. J. Phys. Chem. 1971; 75:
245–249.
3. Yguerabide J, Talavera EM, Alvarez JM, Quintero B. Steady-state

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291


fluorescence method for evaluating excited state proton reactions:
application to fluorescein. Photochem. Photobiol. 1994; 60: 435–
441.
4. Alvarez-Pez JM, Ballesteros L, Talavera EM, Yguerabide J.
Fluorescein excited-state proton exchange reactions: nanosecond
emission kinetics and correlation with steady-state fluorescence
intensity. J. Phys. Chem. A 2001; 105: 6320–6332.
Lipoprotein oxidation with chrysin and
copper by ¯uorescence, IR and UV
spectroscopies
J. A. Gallego-Nicasio,1 M. R. MartõÂnez-MunÄoz,1
G. LoÂpez-RodrõÂguez,1 P. Carmona2 and M. V. Fraile1
1 F2Ca windows.
Universidad San Pablo CEU, Facultad de Ciencias
Experimentales y TeÂcnicas, Departamento de Ciencias BaÂsicas,
Ctra. Boadilla del Monte Km. 5.300, E-28668 Madrid, Spain.
E-mail: vfradot@ceu.es
2 by fluorescence spectroscopy with those measured by
Instituto de Estructura de la Materia (CSIC), E-28006 Madrid,
Spain
LDL oxidation is considered to be an important model of
functional tissue degeneration in diseases such as
diabetes mellitus (1–3). The atherosclerosis increase in
diabetes seems to a large extent to be a result of LDL
oxidation, which involves accumulation of products
stemming from lipid peroxidation. Oxidated LDL
themselves are taken by ‘scavenger’ receptors present
in macrophage cells and start the formation of atherome
plaque. The structural changes produced during the
oxidation of LDL proteins seem to be caused by the
.
presence of OH groups, although the course of the
oxidative reactions is determined by the availability of
. .
O2, O2 or H2O . The structural changes resulting from
LDL oxidation can be studied by Fourier transform
infrared spectroscopy (4), while the lipid oxidation
process can be monitored by fluorescence and UV
spectroscopies. The main aim of this investigation is
focused on the knowledge of the action mechanism of
chrysin associated with LDL in the course of the
oxidation of these lipoprotein particles.
This work has been carried out on previous isolation
and purification of LDL lipoproteins from EDTA-con-
taining plasma of healthy human beings. Briefly, these
lipoproteins were isolated by density gradient ultracen-
trifugation at 90,000 rpm using a NVTi rotor. LDL
proteins were then dialysed against NaCl solution for
12 h. Just before monitoring oxidation in the presence or
absence of chrysin, EDTA and unassociated chrysin were
removed through filtration with a Sephacryl-400 column.
Chrysin was incorporated into the LDL particles by
means of phospholipid vesicles consisting of dipalmito-
tylphosphatidylcholine (DPPC), using chrysin concentra-
tions in the 0.3–0.5 mmol/L range.
The fluorescence spectra were measured using a
spectrofluorimeter Perkin-Elmer LS-50B, using a cell
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 235
of 1 cm pathlength at 37°C. Methods have been applied
to determine the protein concentrations, which were in
the range of 10 mg/mL. The excitation wavelength was
280 nm, which generated emission spectra of 200–
600 nm.
Ultraviolet spectra were scanned at 37°C with a
Perkin-Elmer Lambda 14 spectrophotometer, assisted
by a personal computer, and thermostated cells of 0.5 cm
pathlength. The protein concentrations analysed were the
same as those used for the fluorescence study. Absor-
bance at 234 nm, generated by conjugated dienes, was
monitored at 10 min intervals. The infrared spectra were
recorded with 4/cm resolution, as solid films, by means of
a Perkin-Elmer 2000 FTIR spectrophotometer and using
The results obtained with the above spectroscopic
techniques can be summarized as follows. There seems to
be correlation between oxidation latency times measured
ultraviolet spectroscopy, either in the presence or the
absence of chrysin (Fig. 1). This correlation suggests that
the structural changes revealed by fluorescence spectro-
scopy are influenced by formation of conjugated dienes
or other concomitant oxidation products. In this connec-
tion, it has been reported that oxidizing lipids cause
alterations in both function and structure of proteins (5).
In particular aldehydes, which are lipid oxidation
products, have been described to cross-react with pro-
teins, eg through Schiff base formation between aldehyde
carbonyl group and lysine side chain in proteins.
Accordingly, it seems reasonable to attribute the ob-
served fluorescence spectral changes either to alterations
in protein tertiary structure resulting from the above
oxidation reactions or environment changes of aromatic
protein residues.
As to the chrysin effects on LDL oxidation, this
compound causes LDL to have longer oxidation latency
times as compared with chrysin-free LDL, this delaying
effect being observed in both the presence and absence of
copper ions (Fig. 2). This result is in agreement with the
antioxidant character found for chrysin in previous
Figure 1. Correlation between fluorescence and UV oxida-
tion latency times.
Luminescence 2002;17:191–291
236 ABSTRACTS
Figure 2. Normalized intensities of the LDL emission
fluorescence band in the 200–600 nm range vs. time. (&)
LDL ‡ chrysin ‡ Cu2‡; (*) LDL ‡ chrysin; (r) LDL ‡
Cu2‡; (&) LDL.
works, where it was reported that this compound, by
virtue of its antioxidant action, has significant therapeutic
use as an anti-inflammatory agent (6). Comparison of the
fluorescence spectra of pure LDL and this lipoprotein
containing chrysin shows that this compound reduces the
fluorescence intensity, which means that there is quench-
ing resulting from interaction of the flavonoid with
aromatic residues of LDL proteins. This effect can be
unambiguously attributable to chrysin interacting with
LDL particles, either inside them or on their surfaces,
since free chrysin was removed through gel filtration.
The infrared spectra have provided the following
results. After removing free chrysin, it was noticed that
this compound affects not only protein structure but also
the carbonyl components. In other words, the protein:
carbonyl components ratio is greater when chrysin is
present. This is in agreement with the antioxidant
character of chrysin, which reduces the formation of oxi-
dation products. On the other hand, the band component
(about 1625 cm) corresponding to the b-structure shows
higher intensity in pure LDL than in LDL incubated with
chrysin. In addition, turns, appearing near 1650 cm, are
more abundant in LDL associated with chrysin. These are
the main protein structural effects that chrysin produces
upon association with LDL. No significant structural
influence was observed for lipids in the 3000–2800 cm
range, with the exception of the carbon–hydrogen bond
cleavage involved in lipid oxidation.
REFERENCES
1. Esterbauer H, Dieber-Rotheneder M, Waeg G, Striegl G, Jürgens D.
Biochemical, structural and functional properties of oxidized low-
density lipoprotein. Chem. Res. Toxicol. 1990; 3: 77–92.
2. Jessup W. Cellular modification of low-density lipoproteins.
Biochem. Soc. Trans. 1993; 21: 321–325.
Copyright  2002 John Wiley & Sons, Ltd.
3. Mazière C, Auclair M, Rose-Robert F, Leflon P, Mazière JC.
Glucose-enriched medium enhances cell-mediated low density
lipoprotein peroxidation. FEBS Lett. 1995; 363: 277–279.
4. Herzyk E, Lee DC, Dunn RC, Bruckdorfer KR, Chapman D.
Changes in the secondary structure of apoprotein B-100 after Cu‡2
catalysed oxidation of human low-density lipoproteins monitored by
Fourier transform infrared spectroscopy. Biochim. Biophys. Acta
1987; 922: 145–154.
5. Singh S, Suri R, Agrawal GCh. Fluorescence properties of oxidized
human plasma low-density lipoproteins. Biochim. Biophys. Acta
1995; 1254: 135–139.
6. Ali RM, Houghton PJ, Raman A, Hoult JRS. Antimicrobial and
antiinflammatory activities of extracts and constituents of Oroxylum
indicum. Phytomedicine 1998; 5: 375–381.
b-Phycoerythrin as an invited guest of
reverse micelles: a ¯uorescence study
Ruperto Bermejo,1 Diego J. Tobaruela,1
Eva M. Talavera,2 Angel Orte2 and
Jose M. Alvarez-Pez2
1
Department of Physical and Analytical Chemistry, Jean
University, E.U.P. of Linares, E-23700 Linares, Spain. E-mail:
rbermejo@ujaen.es
2
Department of Physical Chemistry, Granada University, Cartuja
Campus, E-18071 Granada, Spain. E-mail: jalvarez@ugr.es
INTRODUCTION
At present, there is a general tendency towards the use of
natural rather than synthetic dyes in foods and cosmetics.
Many synthetic pigments are being excluded from food
and cosmetics use on the grounds that they are toxic,
carcinogenic or otherwise unsafe. The consequent search
for natural colourants might be resolved with natural
dyes, such as biliproteins from algae. Biliproteins are a
family of proteins derived from cyanobacteria and
eukaryotic algae. These proteins contain covalently
linked tetrapyrrole groups, and have evolved to maximize
both absorption and fluorescence and to minimize the
quenching caused either by internal energy transfer or by
external factors, such as changes in pH or ionic
composition (1). b-phycoerythrin (b-PE) is the most
valuable of the biliproteins due to its large absorption
coefficient and great fluorescence properties, such as the
high quantum yield and high Stokes’ shift (2). Bilipro-
teins are highly water-soluble and reasonably stable
proteins. However, some applications require their
solubilization in apolar solvents (3). In this paper we
studied the inclusion of this protein in reverse micelles, a
microheterogeneous system that mimics the water-
membrane interface well.
RESULTS AND DISCUSSION
The fluorescence properties of b-PE are investigated in
Luminescence 2002;17:191–291
 ABSTRACTS 237

be expected. Nevertheless, at w0  35 the water pool is

Figure 1. (*) Fluorescence intensity at 580 nm and (*)
anisotropy, of b-PE as a function of water:surfactant molar
ratio (w0). (*) lex = 540 nm, D lem = D lem = 5 nm. (*)
lex = 540 nm, lem = 580 nm, D lex = 3 nm, D lem = 10 nm for
w0 range 15–28 and D lem = D lex = 3 nm for w0 range 35–55.
Biliprotein concentration was 10 6 mol/L.
large enough and the protein must be completely
confined in the water pool. Thus, the unchanging value
of fluorescence intensity means that the protein micro-
surroundings are also unchanging. Moreover, the sharp
decreases in anisotropy means that the mobility of the
protein returns, when included in micelles, to almost the
same values as in the homogeneous solution and it is not
limited by strong interactions of the protein amino acids
with the AOT sulphonate groups or the structured
interfacial water layer.
Fig. 2 shows steady-state excitation fluorescence
anisotropy spectra of b-PE in 20 mmol/L sodium phos-
phate, pH 7.0 (hexameric b-PE), b-PE in 20 mmol/L
sodium phosphate, pH 7.0, with addition of sodium
perchlorate 1 mol/L (dissociated b-PE) (4), and b-PE
incorporated in reverse micelles with water content
w0 = 40. The anisotropy values shown in Fig. 2 were
slightly higher than those corresponding to hexameric
b-PE and considerably lower than those corresponding to
dissociated b-PE.
The fluorescence emission spectra are very similar in
both aqueous media (hexameric and dissociated b-PE)
showing the same fluorescence emission maximum at
575 nm and the same bandwith (37 nm), although the

aerosol-OT [AOT, sodium bis-(2-ethylhexyl) sulpho-

succinate]/water/i-octane reverse micelles. Highly pure
b-PE was obtained from cells of Porphyridium cruentum,
as described elsewhere (4).
Steady-state fluorescence from b-PE was studied by
following its behaviour as a function of the water:
surfactant molar ratio (w0) in the range 15–55. Fig. 1
shows that fluorescence intensity at the wavelength of
emission maximum (580 nm) increased continuously
with increasing water content. This effect remained
practically constant at w0 > 35 and saturated at w0  40.
Anisotropy also increases initially with the water content
rise up to w0 = 28, and then decreases sharply, staying
practically constant at w0 > 35.
The above spectral effects can be explained as follows:
the b-PE structure is a disc formed by two (ab)3,
assembled face-to-face with the g-subunit located in a
central hole with a thickness of 60 Å and a diameter of
110 Å (5). On the other hand, the inner-core radius of
reverse micelles in the AOT-iso-octane system (at room
temperature) is around 50 Å (6). Consequently, the
hexameric b-PE is longer than the micelles when the
water pool is relatively small (w0 < 30). In this phase, the Figure 2. Steady-state excitation anisotropy spectra of b-PE.
hydrophilic amino acid side chains of the protein interact (*) b-PE in 20 mmol/L sodium phosphate buffer, pH 7.0
with the AOT sulphonate headgroups, whereas the (lem = 575 nm). (*) b-PE in 1 mol/L sodium perchlorate,
20 mmol/L sodium phosphate buffer, pH 7.0 (lem = 575 nm).
hydrophobic parts of the protein remain partially exposed (!) b-PE in reverse micelles (w0 = 40) (lem = 580 nm). The
to the hydrocarbon solvent. Thus, both decreases in anisotropy values shown in this figure are the means of five
fluorescence efficiency and large anisotropy values might values and were calculated for each 5 nm.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

238 ABSTRACTS
fluorescence of hexamers is larger than that of dissociated
b-PE. The steady-state emission spectrum of micelled
b-PE had a characteristic shape, almost identical to that
of its spectrum in aqueous solution (hexameric state).
However, the fluorescence emission maximum shifted
5 nm (580) and its bandwidth was 41 nm. The fluores-
cence intensity of hexameric B-PE was slightly larger
than b-PE in reverse micelles.
From fluorescence analysis the stability of micelled
b-PE with time was higher than that of the b-PE in
aqueous solution. Likewise, the thermal stability for b-PE
in reverse micelles was higher than in aqueous solution.
These results indicate that the protein chromophores are
better shielded than when they are in aqueous solution,
and can be explained in terms of their inclusion in the
inner core of reverse AOT micelles. Moreover, there are
no important interactions between the amino acid
residues neighbouring the bilin chromophores and the
AOT sulphonate headgroups.
REFERENCES
1. Glazer AN. Phycobiliproteins. In Chemicals from Microalgae,
Cohen Z (ed.). Taylor and Francis: London, 1999; 261–280.
2. Bermejo R, Fernández E, Alvarez-Pez JM, Talavera E. Labeling of
cytosine residues with biliproteins for use as fluorescent DNA
probes. J. Lumin. 2002 (in press).
3. Bermejo R, Talavera EM, del Valle C, Alvarez-Pez JM. C-phyco-
cyanin incorporated into reverse micelles: a fluorescence study.
Colloids Surface B, 2000; 18: 51–59.
4. Bermejo R, Talavera EM, Alvarez-Pez JM. Chromatographic
purification and characterization of b-phycoeryhrin from Porphy-
ridium cruentum. Semipreparative high-performance liquid chro-
matographic separation and characterization of its subunits. J.
Chromatogr. A 2001; 917: 135–145.
5. Ficner R, Lobeck K, Schmidt G, Huber R. Isolation, crystallization,
crystal structure analysis and refinement of b-phycoerytrin from the
red alga Porphyridium sordidum at 2.2 Å resolution. J. Mol. Biol.
1992; 228: 935–950.
6. Zulauf M, Eicke HF. Inverted micelles and microemulsions in the
ternary system H2O/aerosol-OT/isooctane as studied by photon
correlation spectroscopy. J. Phys. Chem. 1979; 83: 480–486.
Comparative study of determination
methods of DNA intercalator binding
constants
Eva M. Talavera, Rosa M. Gonzalez and
Jose M. Alvarez-Pez
Department of Physical Chemistry, University of Granada,
Cartuja Campus, E-18071 Granada, Spain. E-mail:
etalaver@platon.ugr.es or jalvavez@ ugr.es
Ethidium bromide (EB) belongs to a class of agents
known as intercalating drugs that are able to bind to
double-stranded nucleic acids. The intercalation within
DNA shields the EB molecules from quenching by the
aqueous solvent, thus increasing its visible fluorescent
Copyright  2002 John Wiley & Sons, Ltd.
emission manifold. The EB also interacts with DNA in a
second lower-affinity site, due to electrostatic interac-
tions between the positive charge on EB and the
negatives on DNA phosphate groups. In a recent study,
using time-resolved fluorescence spectroscopy, we were
able to separate the fluorescence corresponding to each
binding site from the total fluorescence (1). With the
normalized weighting coefficients from triexponential
fitting of nanosecond decay graphs, we can quantify the
concentrations of free EB, those intercalated within base
pairs and those bound to the secondary sites. Moreover,
we have demonstrated that the interaction in both sites is
independent of ionic concentration and that the saturation
of the intercalative sites is not necessary in order for EB
molecules to bind at secondary sites, as was thought until
now.
The analysis of binding data has so far been carried out
using the easier classical Scatchard treatment (2) or by
the neighbour exclusion model (3). The first is applicable
to the case of identical, independent and discrete binding
sites on the lattice and implies a linear plot of n/L vs. n
(where L is the concentration of free ligand in mol/L of
ligand and n represents the binding density in mol bound
ligand mol/L total nucleotide bases). The second is more
suitable and corresponds to the case of overlapping, and
non-cooperative binding sites. Each model requires the
evaluation of best fitting values for two parameters, viz.
K, the affinity constant for binding, and n, the number of
nucleotide base-pairs per binding site. The existence of at
least two kinds of binding for EB in DNA is supported by
the break in the Scatchard plots.
Figure 1. Graphs of [EB]B/([DNA]0 [EB]F) vs. [EB]B/
[DNA]0 for DNA–EB solutions in 20 mmol/L Tris–HCl,
1 mmol/L EDTA, pH 7.5, 0.4 mol/L NaCl (*). (– – –), Graph
fitted to Scatchard model. (—), Graph fitted to McGhee–von
Hipple model. This fitting yielded the intrinsic binding
constants and sites number values for high-affinity binding
shown in Table 1.
Luminescence 2002;17:191–291

Figure 2. Graphs of [EB]B/([DNA]0 [EB]F) vs. [EB]B/
[DNA]0 for DNA-EB solutions in 20 mmol/L Tris–HCl,
1 mmol/L EDTA, pH 7.5, 0.05 mol/L NaCl (*). (– – –), Graph
fitted to Scatchard model. (—), Graph fitted to McGhee–von
Hipple model. This fitting yielded the intrinsic binding
constants and sites number values for high-affinity binding
shown in Table 1.
In this work we compare the binding parameters, at salt
concentrations of 0.05 and 0.4 mol/L, obtained with the
classical expression and those from the McGhee–von
Hipple theory, for binding of EB to E. coli DNA. Figs 1
and 2 show both fittings for the intercalation in high
affinity sites for 0.4 mol/L NaCl and 0.05 mol/L NaCl,
respectively. The binding isotherms are not linear, even
when there is only a single class of binding site. The
small slope of the isotherm at large values of [EB]B/
[DNA]0, implying a smaller apparent binding constant,
results from the large reduction in the number of ways of
achieving a given degree of binding as saturation is
approached. The best fitting values of binding constant
and sites number for the two interaction places at
different salt concentrations are shown in Table 1. The
discrepancy in the values obtained for K and n may be
explained as follows: consider a DNA molecule which
has N nucleotide residues and which at saturation con EB
Table 1. Values of binding constants and sites number from fits of free and binding (at secondary and high-affinity sites) ligand
concentrations to Scatchard and McGhee–von Hippel models, at two NaCl concentrations
Model [NaCl] (mol/L) K1
103 (mol/L)
Scatchard 0.4 71.35  6.00
0.05 132.36  26.0
McGhee–von Hippel 0.4 30.19  1.71
0.05 75.28  1.35
Subscript 1 refers to intercalative binding sites and subscript 2 refers to low-af®nity binding sites.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 239
displays n nucleotide residues per binding and a total of
N/n binding sites. If the binding sites do not overlap
(binding sites are discrete and regularly spaced every n
residues, as assumed in the Scatchard model), then for
fractional saturation less than 1, the number of free sites
is equal to the total number of sites minus the number of
sites that are occupied. However, if the binding sites
overlap, the latter statement is not true and the number of
free binding sites on the bare DNA is actually
(N n ‡ 2)/2. Classical theory for non-overlapping sites
thus underestimates the number of intercalator-free
binding sites for fractional saturation less than 1. A
consequence of this phenomenon is that the value of
association constants determined by analysis of experi-
mental intercalator–DNA binding data with the classical
expression may be as much as n times greater than the
more correct value obtained by analysis with the
McGhee–von Hipple expression.
In conclusion, at any degree of binding saturation, the
number of free ligand binding sites depends not only on
the number of ligands already bound, but also on the
distribution of these bound ligands on the lattice. The
result is a curved Scatchard plot, where the curvature
arises as a consequence of the overlap of potential ligand
sites and cannot be attributed to binding site hetero-
geneity or ligand binding cooperativity.
Acknowledgement
This work was supported in part by Grant PB98-1285
from the Spanish Ministry of Education and Culture.
REFERENCES
1. Talavera EM, Guerrero P, Ocana F, Alvarez-Pez JM. Photophysic
and direct determination of binding constants of ethidium bromide
complexed to E. coli DNA. Appl. Spectrosc. 2002 (in press).
2. Scatchard G. The attraction of proteins for small molecules and ions.
Ann. N. Y. Acad. Sci. 1949; 51: 660–672.
3. McGhee JD, von Hippel PH. Theoretical aspects of DNA–protein
interactions: cooperative and non-cooperative binding of large
ligands to one dimensional homogeneous lattice. J. Mol. Biol. 1974;
86: 469–489.
n1 r2 K2
103 (mol/L) n2 r2
3.1  0.09 0.97 10.31  1.35 6.4  0.61 0.92
3.7  0.16 0.84 20.07  4.76 8.1  1.06 0.78
2.6  0.06 1.00 1.66  0.09 4.1  0.40 0.99
3.3  0.11 0.98 2.81  0.40 5.6  0.78 0.96
Luminescence 2002;17:191–291
240 ABSTRACTS
Spectral study of molecular `light switch'
complexes and their applications in the
determination of DNA
F. Chen and Z. K. He
Department of Chemistry, Wuhan University, Wuhan 430072,
People's Republic of China. E-mail: zhkhe@whu.edu.cn
A molecular ‘light switch’ is a kind of complex that is not
photoluminescent in water but does emit in non-aqueous
solvents or in the presence of DNA (1). The emission
spectra of Ru(bipy)2(dppx)2‡ [bipy = 2,2'-bipyridine,
dppx = 7,8-dimethyl-dipyrido[3,2-a:2',3' c]phenazine,
synthesized according to reference (1) ] in different
solvents has been studied using a Shimadzu RF-5301
spectrofluorometer. It was found that the solvent polarity
and the ability of donating and transferring protons are
the important factors in predicting luminescence intensity
in different systems. The increasing concentrations of
water in solutions of Ru(bipy)2(dppx)2‡ dissolved in
organic solvents leads to decrease in emission intensity
that follows the Perrin sphere of quenching model (Fig.
1). This model assumes that the emitting molecules are
quenched completely if the quencher molecules are
within a sphere of radius r, and any quenchers outside this
sphere do not quench the emission at all (2). The Perrin
equation is ln(F0/F) = N0V[Q], where F0 and F are the
relative quantum yield of emission in the absence and the
presence of quencher, respectively; N0 is Avogadro’s
number, V is the volume of the quenching sphere, and [Q]
is the quencher concentration. The radius of quenching
sphere in CH3CN, C2H5OH and DMF are 0.609 nm,
0.652 nm and 0.709 nm, respectively. The radius of the
Figure 1. Perrin plots for the quenching of Ru(bipy)2
(dppx)2‡ (1.0  10 6mol/L) in CH3CN (a), CH3CH2OH (b)
and DMF (c).
Copyright  2002 John Wiley & Sons, Ltd.
quenching sphere shows that the water within 0.6–0.7 nm
will quench the fluorescence of Ru(bipy)2(dppx)2‡
completely. As water is a good quencher of
Ru(bipy)2(dppx)2‡, Ru(bipy)2(dppx)2‡ may be used to
probe low-level water in organic solvent.
The effect of DNA on the absorption spectra of
Ru(bipy)2(dppx)2‡ was also studied (Fig. 2), by use of a
Shimadzu UV-1601 spectrophotometer. The absorption
spectra of Ru(bipy)2(dppx)2‡ with and without DNA
clearly show that there exists strong hypochromism and
red shift in the presence of DNA, which revealed that
intercalation exists between Ru(bipy)2(dppx)2‡ and
DNA. The apparent binding constant (K) of four
molecular ‘light switch’ complexes, Ru(bipy)2(dppz)2‡,
Ru(bipy)2(dppx)2‡. Ru(phen)2(dppz)2‡, Ru(phen)2-
(dppx)2‡ (phen = 1, 10-phenanthroline, dppz = dipyrido
[3,2-a:2',3' c]phenazine) and DNA were calculated
according to their absorption spectra (3). The apparent
binding constants are 1.90  104 L/mol, 2.63  104
L/mol, 2.80  104 L/mol and 4.86  104 L/mol, respec-
tively. The methods for the determination of DNA by use
of four molecular ‘light switch’ complexes have been
developed. Under optimum conditions, the detection
limits of calf thymus DNA are 0.76 ng/mL, 0.33 ng/mL,
0.54 ng/mL, 0.19 ng/mL with Ru(bipy)2(dppz)2‡,
Ru(bipy)2(dppx)2‡, Ru(phen)2(dppz)2‡ and Ru(phen)2-
(dppx)2‡, respectively. Ru(phen)2(dppx)2‡ is the most
sensitive probe for the systemically determination of
DNA (see Table 1). The present method can be
successfully applied to the determination of nucleic acids
in synthetic samples. When 1.0  10 3 mol/L NaCl,
5.0  10 5 mol/L MgSO4, 5.0  10 5 mol/L CaCl2,
1.0  10 4 mol/L K2HPO4, 1.65  10 5 mol/L guanine,
Figure 2. Ultraviolet visible absorbance spectra of
Ru(bipy)2(dppx)2‡ (1.0  10 5mol/L) in the presence of
DNA (top to bottom 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL) in
0.01 mol/L boric buffer, pH 7.5.
Luminescence 2002;17:191–291

Table 1. Analytical parameters of determination of CT-DNA by use of molecular ‘light switch’ complexes (4.0  10 7L/mol) in
0.01 mol/L boric buffer, pH 7.5
Linear regression equation Limit of detection
‘Light switch’ complex (C, mg/mL) (3s, ng/mL)
Ru(bipy)2(dppz) 2‡
4.333 ‡ 416.50C
Ru(bipy)2(dppx)2‡ 4.430 ‡ 678.03C
Ru(phen)2(dppz)2‡ 4.320 ‡ 326.73C
Ru(phen)2(dppx)2‡ 7.879 ‡ 1175.39C
1.0  10 5 mol/L uracil, 1.9  10 5 mol/L thymine were
added, respectively, the change of fluorescence intensity
was lower than 2.0% in this system. The change of
fluorescence intensity is lower than 5.0% when
1.65  10 5 mol/L adenine, 4.0 mg/mL BSA or
8.0.0 mg/mL RNA was added. This reveals clearly that
the proposed method is reliable, sensitive, simple and
practicable.
REFERENCES
1. Friedman AE, Chambron J-C, Sauvage JP, Turro NJ, Barton JK. J.
Am. Chem. Soc. 1990; 112: 4960.
2. Turro NJ. Modern Molecular Photochemistry. Benjamin Cum-
mings: Menlo Park, CA; 1978.
3. Wolfe A, Shimer GH Jr, Meehan T. Biochemistry 1987; 26: 6392.
Effect of polymyxin B on the organization
of Escherichia coli lipid membranes
AdriaÁ Clausell, Montserrat Pujol, M. AsuncioÂn Alsina
and Yolanda Cajal
Department of Physical Chemistry, Faculty of Pharmacy,
University of Barcelona, Avn. Joan XXIII, s/n E-08028 Barcelona,
Spain
Cationic peptide antibiotics are ubiquitous in nature (1).
Polymyxin B (PxB), as a prototypical example, is a non-
ribosomally synthesized peptide produced by the Gram-
positive Bacillus polymyxa, and is rather selective and
potent against Gram-negative organisms. Even though
PxB has been in clinical use for several decades, there is
no indication of antibiotic resistance in clinical isolates.
PxB acts on membranes, but the mechanism of antibiotic
action has not yet been established. Studies with model
membranes of synthetic phospholipids show that PxB
forms vesicle–vesicle contacts through which anionic
phospholipids exchange (2). This phenomenon has been
proposed as the basis for its antibiotic action by
promoting the scrambling of lipids between inner and
outer membranes of Gram-negatives. It has recently been
established that PxB induces a highly selective cellular
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 241
Correlation coefficient
(r) Line range (ng/mL)
0.76 0.9997 0.76–400
0.33 0.9995 0.33–300
0.54 0.9990 0.54–300
0.19 0.9997 0.19–300
stress that interferes with hyperosmotic response and
leads to stasis and cell death (3). To gain more insight
into the molecular basis for the antibacterial stress caused
by PxB, biophysical studies have been conducted with
vesicles prepared with membrane lipids extracted from
the Gram-negative Escherichia coli. The effect of
different concentrations of PxB in the mobility of the
phospholipids and the gel-to-liquid crystal phase transi-
tion has been studied by steady-state anisotropy, using
three types of probes located at different positions in the
membrane: 1,6-diphenylhexa-1,3,5-triene (DPH), a
hydrophobic molecule that reports on the order of the
lipid fatty acyl chains in the core region of the bilayer;
1-[4-(trimethylammonium) phenyl]-6-phenyl-1,3,5-
hexatriene (TMA-DPH), anchored at the water–lipid
interface; and N-(7-nitro-2-1,3-benzoxadiazol-4-yl)
dioleoyl phosphatidyl ethanolamine (NBD-PE), an indi-
cator of the mobility of the lipid headgroup region of the
membranes. E. coli lipid vesicles incorporating the
probes were submitted to heating and cooling cycles
from 5 to 45°C, and in all cases anisotropy decreased with
temperature due to the increase in lipid mobility that
characterizes the gel-to-liquid crystal phase transition
(Fig. 1). At low temperatures, anisotropies were higher
for DPH and TMA-DPH as compared to NBD, thus
indicating that the maximal mobility of the lipids occurs
at the headgroup region. Upon melting, when the
membrane is in the more fluid liquid crystal phase,
anisotropy was highest for TMA-DPH, indicating that the
interfacial region of the membrane, close to the glycerol
backbone, remains more ordered than the hydrophobic
core or the headgroup regions. After a heating cycle,
samples were cooled to the initial temperature and
anisotropy values were the same, ie there was no
hysteresis in the liquid crystal-to-gel transition. Results
in the presence of PxB show that the antibiotic adopts two
different forms in the membrane, with different activities.
At concentrations below 2 mol%, PxB had no effect on
the ordering of the phospholipids in either the gel or
liquid-state phase of the lipid bilayer. Only a small
increase in order was observed at the external part of the
membrane, were PxB binds. At this concentration range,
PxB forms vesicle–vesicle contacts and induces selective
lipid exchange. This is consistent with the hypothesis that
Luminescence 2002;17:191–291
242 ABSTRACTS

Figure 1. Temperature dependence of the steady-state fluorescence anisotropy of E. coli lipid vesicles labelled with NBD (left),
TMA-DPH (centre, or DPH (right), in the presence of PxB: 0 mol% (open circles and line), 0.5 mol% (open triangles); 1 mol% (open
squares); 2 mol% (closed circles). Vesicles (133 mmol/L) were cooled in 10 mmol/L Tris, pH 8.0, to the starting temperature, and then
anisotropies were measured at the desired temperatures. Excitation, 365 nm; emission, 425 nm. The data are representative of at least
three independent experiments (0.002).

PxB in the intervesicle contacts is accessible through the were obtained for the other probes (not shown). In
aqueous phase and does not alter the bilayer structure. At conclusion, data presented here are strong evidence that
concentrations of PxB in the membrane of 2 mol% and the molecular contacts formed by PxB between vesicles
higher, PxB inserts deeply in the membrane, and modifies of E. coli lipid extract do not modify membrane lipid
lipid dynamics. Lipid fluidity decreases significantly both order and phase properties.
in the polar and the water/lipid interfacial regions of the
bilayer, as deduced by the increases in NBD and TMA-
DPH anisotropies, respectively (Fig. 1, left and centre).
At the hydrophobic core region, determined by the acyl
chains of the phospholipids, lipid order also decreases,
but changes are smaller, suggesting that PxB remains
close to the interface (Fig. 1, right). At these concentra-
tions, above 2 mol%, PxB has a membrane-perturbing
effects, increasing the permeability of the vesicles and
destroying the bilayer organization of the lipids, e.g.
experiments of lipid accessibility using vesicles contain-
ing NBD-PE and adding ditionite as a reduction reagent,
showed that below 2 mol% of PxB, vesicles remained
intact, but above this concentration the vesicles became
permeable. The absence of hysteresis in the cooling
cycles of anisotropy is a clear indication that the bound
peptide does not leave the interface when the lipids are in
the more fluid crystalline state, above the transition
temperature (Tm). To define more precisely the concen-
tration of PxB that induces changes in lipid order,
experiments were conducted at a constant temperature.
Figure 2. Steady-state fluorescence anisotropy of THM-DPH
As shown in Fig. 2 for TMA-DPH, PxB does not affect in E. coli extract lipid vesicles (133 mmol/L) as a function of the
lipid dynamics below 2 mol%, either in the gel or in the concentration of PxB, at constant temperature: below (open
liquid crystal states of the membrane. The same results circles) and above (closed circles) the Tm for the lipid.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

 ABSTRACTS 243

REFERENCES PHYCOERYTHRIN FLUORESCENCE-BASED ASSAY

1. Hancock REW, Chapple DS. Antimicrob. Agents Chemother. 1999; In the phycoerythrin assay, gallic acid protects phyco-
43: 1317–1323. erythrin from damage by peroxyl radicals, and hence
2. Cajal Y, Rogers J, Berg OG, Jain MK. Biochemistry 1996; 35: 299– inhibits the quenching of its characteristic fluorescence.
308.
3. Oh JT, Van Dyk TK, Cajal Y, Dhurjati PS, Sasser M, Jain MK. 2,2'-Azobis(2-amidinoropane) dihydrochloride (AAPH),
Biochem. Biophys. Res. Commun. 1998; 246: 619–623. which thermally decomposes to yield peroxyl radicals on
being heated to 37°C, is used as a constant source of
peroxyl radicals. B-phycoerythrin was used as the
detector agent; it is normally highly fluorescent (excita-
tion 545 nm; emission 575 nm), but loses fluorescence
intensity when damaged by peroxyl radicals. Mixing
phycoerythrin with AAPH results in a slow and
progressive loss of phycoerythrin fluorescence over a
Antioxidant activity of gallic acid by a period of time (Fig. 2). Addition of an antioxidant
¯uorimetric assay solution of gallic acid inhibits the loss of fluorescence
intensity, this inhibition being proportional to the anti-
M. LoÂpez-VeÂlez, F. MartõÂnez-MartõÂnez and oxidant capacity of the added sample. Phosphate buffer
C. del Valle-Ribes was used as a blank and 1 mmol/L Trolox (6-hydroxy-
Department of Physical Chemistry, Faculty of Pharmacy, 2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-
University of Granada, E-18071 Granada, Spain. E-mail:
soluble a-tocopherol analogue, was used as a standard.
msvelez@ugr.es
The reaction was started by the addition of AAPH. The
antioxidant activity value refers to the net protection area
Antioxidants are of great interest to the food industry
under the quenching curve of B-PE in the presence of
because they prevent rancidity. Antioxidants are also of
gallic acid. The net areas corresponding to the different
interest in biological processes because they may help to
concentrations of gallic acid were 21.69 and 33.19,
protect the human body against damage by reactive
respectively, and the calculated antioxidant activity
species.
values were 1.53 and 2.74. Antioxidant activity increased
Gallic acid (3,4,5-trihydroxybenzoic acid) is a low
molecular phenolic acid widely distributed in the plant
kingdom. It has been reported to exert potential health-
promoting effects as an antioxidant agent and is included
in the List of Existing Food Additives as natural
antioxidants in some countries.
The purpose of this work was to study the antioxidant
effect of this phenolic compound, present in red wines
(1, 2), using the fluorimetric method previously described
by Cao et al., (3). This assay measures the effect of
antioxidant compounds on the decline in B-phycoerythrin
(B-PE) fluorescence induced by a peroxyl radical
generator, 2,2'-azobis(2-amidinoropane) dihydrochloride
(AAPH). Furthermore, other fluorescence properties of
this compound are studied using steady-state fluores-
cence.

CHANGE IN FLUORESCENCE WITH THE

CONCENTRATION OF GALLIC ACID
The method utilized here was the direct method in which
the natural or intrinsic fluorescence of the analyte
molecule itself is monitored. Gallic acid shows fluores-
cence at 370 when excited in the intense band region at
280 nm. The fluorescence of various solutions (0.1 
10 5 to 7  10 5 mol/L) was measured and a calibration
graph for this compound was constructed (Fig. 1). Good Figure 1. (A) Fluorescence spectra of gallic acid in methanol
linearity (r2 > 0.99) was obtained for the concentration at different concentrations (lexcitation, 280 nm). (B) Change of
range 3.5  10 5 mol/L. fluorescence with the concentration of gallic acid.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

244 ABSTRACTS
Figure 2. Principle of the fluorimetric assay with B-PE as a target for free radical action
and AAPH as a peroxyl radical generator.
with increasing gallic acid concentration. The antioxidant
activity of this phenolic acid depends on the number of
hydroxyl groups in the molecule. In this way, the
antioxidant activity of gallic acid corresponding to the
three available hydroxyl groups.
REFERENCES
1. López M, Martı́nez F, Del Valle C, Orte C, Miró M. J. Chromatogr.
A 2001; 922: 359–363.
2. López-Vélez M, Martı́nez-Martı́nez F, del Valle-Ribes C. Crit. Rev.
Food Sci. Nutrit. 2002 (in press).
3. Cao G, Prior RL. Methods Enzymol. 1999; 299: 50–62.
Fluorescence analysis of the interaction of
two peptide sequences of hepatitis G virus
with model membranes
Montse MunÄoz,1 Conxita Mestres,1 Isabel Haro,2
Nuria Rojo2 and Victoria Girona1
1
Physical Chemistry Department, Faculty of Pharmacy, Avgda
Joan XXIII, s/n E-08028 Barcelona, Spain. E-mail:
mmunoz@farmacia.far.ub.es
2 a heating and a cooling cycle was performed.
Department of Peptide and Protein Chemistry, IIQAB.CSIC,
Jordi Girona Salgado 18, E-08034 Barcelona, Spain
The present study was undertaken to examine, by means
Copyright  2002 John Wiley & Sons, Ltd.
of fluorescence techniques, the interaction with lipo-
somes of two peptide sequences corresponding to the
fragments 39–53 and 32–53 of the structural E2 protein
of hepatitis G virus. The peptides with a sequence of
GNVTLLCDCPNGPWV and GERVWDRGNVTLLCD
CPNGPWV were synthesised manually, purified and
characterized as described elsewhere. Small unilamelar
vesicles (SUVs) of 1,2-dimyristoyl-sn-glycero-3-phos-
phocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-
3-[phospho- rac-(1-glycerol)] (DMPG), alone or with
the fluorescent probes 1,2-dioleoyl-sn-glycero-3-phos-
phoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)
(NBD-PE) or 1-(4-trimethyl ammonium phenyl)-6-phe-
nyl-1,3-5-hexantriene-p-toluenesulphonate (TMA-DPH)
were prepared by sonication in a bath-type sonicator (Lab
Supplies, Hickesville, NY) above the gel–fluid transition
temperature until a clear dispersion was obtained. SUVs
had a mean diameter of 40 nm, and a narrow size
distribution (polydispersity <0.1). The intrinsic
fluorescence of the peptides resulting from the presence
of a tryptophan (Trp) residue in the molecule was
analysed upon peptide incubation with DMPC or
DMPG SUVs (lexcitation = 285 nm). Steady-state fluor-
escence anisotropy of the samples with and without
peptides was measured automatically. For each sample,
Excitation and emission wavelengths were 365/425
for TMA-DPH and 460/534 nm for NBD-PE. Fluores-
cence measurements were conducted in an AB-2
Luminescence 2002;17:191–291
 ABSTRACTS 245

spectrofluorimeter (SLM-Aminco) in the temperature

range 20–60°C.
The presence of DMPC or DMPG SUVs did not
modify either the Trp quantum yield or the maximum
emission wavelength (350 nm) of the peptides, thus
indicating that, in presence of lipids, the peptides remain
in an exposed environment (1).
For steady-state fluorescence studies, we have used
two probes that locate at different depth in the bilayer.
TMA-DPH is anchored at the water-lipid interface with
the hydrophobic fraction, DPH orientated parallel to the
lipid fatty acid chains and the trimethylamonium group
located close to the lipid headgroup. On the other hand,
NBD-PE tend to sense the lipid–water interface region of
membranes instead of the hydrophobic interior, therefore
it is an indicator of the mobility of the headgroup region
of the membranes (2).
The probe that reports on the anisotropy closest to the
glycerol backbone region (TMA–DPH) did not show any
change in chain mobility both in the DMPC or DMPG
SUVs (not shown). This indicates that the peptides had no
effect in this region of the membrane. In the case of
Figure 2. Temperature dependence of the steady-state
NBD-PE-labelled SUVs, phospholipid composition was fluorescence anisotropy of NBD-PE-labelled SUVs of DMPG
determinant in the anisotropy values. While DPPC (closed symbols) alone or in presence of 1.26 mmol/L E2
samples were not affected by peptide presence, the (32–53) peptide (open symbols).

anisotropy of DPPG liposomes changed in a different

degree, depending on the peptide.
Fig. 1 shows the heating curves of SUVs with and
without E2 (39–53) incubated with NBD-PE containing
SUVs. Although the changes are not important, peptide
addition seems to play a slight rigidifying effect in the gel
state (r  0.28) if compared to the control (r  0.22). The
curve shapes are superimposed at higher temperatures,
thus indicating no effect in the fluid state. The longer
peptide E2 (39–53) decreased the mobility at the
headgroup region for DMPG-SUVs, both below and
above the Tm (23°C), as seen by the increase of the
anisotropy of the NBD-PE groups (Fig. 2). The results
suggest a surface interaction between the peptides and
DMPG vesicles having peptide length, as well as charge a
role on membrane structure. Further studies are being
performed to gain insight on peptide structure influence
on lipid mono- and bilayers.

Figure 1. Temperature dependence of the steady-state
fluorescence anisotropy of NBD-PE labelled SUVs of DMPG
(closed symbols) alone or in presence of 1.26 mmol/L E2
(39–53) peptide (open symbols).
REFERENCES
1. Sospedra P, Mestres C, Haro I, Muñoz M, Busquets MA. Effect of
amino acid sequence on peptide–membrane interaction. Langmuir
2002; 18: 1231–1237.
2. Cajal Y, Busquets MA, Carvajal H, Girona V, Alsina MA. Effects of
fungal lipase on membrane organization evaluated by fluorescence
polarization. J. Mol. Cat. B. (in press).

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

246 ABSTRACTS
Spectro¯uorimetric determination of
carbetamide in water samples applied to
adsorption studies in soils
G. MunÄoz-Soto, F. Carricondo, M. del Olmo,
A. Gonzalez-Casado, A. NavaloÂn and J. L. VõÂlchez
Department of Analytical Chemistry, University of Granada,
Avda. Fuentenueva s/n, E-18071-Granada, Spain. E-mail:
mdolmo@ugr.es
Pesticide contamination of soil and groundwater con-
tinues to be a problem in spite of the higher quality of
modern pesticides. This work is centered on the study of
carbetamide [(R)-1-(ethylcarbamoyl)ethyl carbanilate], a
selective herbicide (1) which belongs to the family of
amides and is used primarily as a pre-emergence
herbicide that kills plants by interfering with cell division
and plant growth.
In aqueous medium, carbetamide does not show
natural fluorescence but the aniline, obtained by alkaline
hydrolysis with NaOH, is fluorescent. The selection of
the main experimental variables (temperature, time and
volume of NaOH solution) on the hydrolysis reaction of
carbetamide were studied by using a 24 factorial screen-
ing design (2). In order to optimize these variables, a
‘Face Central Cube Design’ was selected, since it
presents better representation of the experimental
Figure 1. Estimated total fluorescence function (response surface) for: (A) temperature reaction and
hydrolysis time, (B) hydrolysis time and NaOH volume and (C) hydrolysis time and NaOH volume.
Copyright  2002 John Wiley & Sons, Ltd.
domain. Fig. 1 shows the obtained response surfaces.
The application of Lagrange’s criterion indicates the
presence of a maximum which corresponds to a
temperature of 80°C, 30 min for hydrolysis time and
a volume of 0.5 mL 6 mol/L sodium hydroxide
solution.
The fluorescence intensity (at 20.0  0.5°C) is always
measured at lem = 345 nm and lex = 280 nm, after acidi-
fication of the sample solutions. The calibration graph
obtained with fluorescent signals is linear for the con-
centration range 2.5–25.0 mg/L carbetamide.
The proposed method was applied to study the adsorp-
tion capacity of soil for carbetamide, by a calcareous silt
loam soil located in a fertile area, above one of the most
important aquifers in Andalucia. The mobility of the
pesticide through the soil profile was established by the
study of the adsorption kinetics and isotherms (3) carried
out in soil samples taken at three different soil depths
(0–25 cm, 50–75 cm and 125–150 cm) of the unsaturated
zone. Kinetics shown in Figure 2A represent the amount
of pesticide adsorbed vs. the contact time between the
pesticide solution and the soil sample. The adsorption
isotherms, shown in Figure 2B, represent the amount of
pesticide adsorbed as a function of the equilibrium
concentration of the adsorbing species in solution. The
Langmuir isotherm was used to obtain the distribution
coefficients in every case.
Luminescence 2002;17:191–291

Figure 2. (A) Adsorption kinetics of carbetamide and (B)
adsorption isotherms of carbetamide (^, 0–25 cm; &, 50–
75 cm; ~ 125–150 cm).
The carbetamide molecule has a hydrophilic character
(water solubility 3.5 g/L at 20°C) and shows a low
adsorption, consequently it is necessary to control its use
to avoid contamination of the aquifer.
Acknowledgement
This work was supported by the Spanish Interministerial
Commission of Science and Technology (CICYT)
(Project No. AMB-97-1222).
REFERENCES
1. Tomlin C. The Pesticide Manual: A World Compendium, 10th edn.
British Crop Protection Council: Croydon, 1994.
2. Box GEP, Hunter WG, Hunter JS. Estadı́stica para Investigadores.
Introduction al Diseño de Experimentos, Análisis de Datos y
Construcción de Modelos. Reverté: Barcelona, 1989.
3. Matallo M, Romero E, Sanchez-Rasero F, Peña A, Dios G. J.
Environ. Sci. Health 1998; B33: 51–66.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 247
Fluorimetric determination of neopterin and
thiobarbituric acid reactive substances in
elderly patients
Dagmar SolichovaÂ,1 BozÏena JurasÏkovaÂ,1
Bohuslav Melichar,2 MilosÏ Klejna,3 Lucie KraÂlovskaÂ,4
VladimõÂr BlaÂha,1 Petr ZÏd'aÂnskyÂ1 and ZdeneÏk ZadaÂk1
1
Department of Metabolic Care and Gerontology, Teaching
Hospital, CZ-500 05 Hradec KraÂloveÂ, Czech Republic. E-mail:
solich@lfhk.cuni.cz
2
Department of Oncology and Radiotherapy, Teaching Hospital,
Hradec KraÂloveÂ, Czech Republic
3
Hospital for the Chronically Ill, Hradec KraÂloveÂ, Czech Republic
4
Faculty of Chemical Technology, University of Pardubice, Czech
Republic
With ageing, the population becomes more hetero-
geneous with regard to health status, due to the increasing
prevalence of chronic illnesses and disability. Deteriora-
tion in health and functional status often induces
important modifications in several biological parameters,
including lipids. Among biochemical parameters usually
used to assess the existence and severity of specific
diseases, the lipid metabolites, e.g. cholesterol, fatty
acids and lipid peroxidation, have long been recognized
as indicators of general health and nutrition and/or
increased morbidity and mortality, but also with an
increased rate of complications (especially infections),
clear biochemical evidence of malnutrition, and higher
duration and cost of hospitalization in the elderly. The
aim of the present study was the bioanalysis of urinary
neopterin, an indicator of systemic immune activation
(1), and serum thiobarbituric acid-reactive substances
(TBARS), a marker of lipid peroxidation in the elderly
patients, and also to evaluate the association of these
biochemical parameters and health status.
SUBJECTS AND METHODS
Twelve self-sustaining nonagenarians, 10 women and
two men, aged 94  3 years, and eight institutionalized
nonagenarians, eight women, aged 91  1 year as well as
11 control subjects, seven women and four men aged
84  5 years, entered the study. Urinary neopterin, an
indicator of systemic immune activation (1), and serum
thiobarbituric acid reactive substances (TBARS), a
marker of lipoperoxidation, were determined initially,
and collection of the blood and urine samples was
repeated at 3 month intervals. Neopterin was measured
by reversed-phase high performance liquid chromatogra-
phy. A C18 reversed-phase column 3.3  150 mm, 5 mm
diameter packing Separon SGX was used. Potassium
phosphate buffer (15 mmol/L, pH 6.4) at flow rate of
0.8 mL/min was used as the mobile phase. Neopterin was
identified by its native fluorescence (353 nm excitation,
438 nm emission). The concentration of neopterin in
Luminescence 2002;17:191–291
248 ABSTRACTS
urine was expressed as neopterin:creatinine ratio
(mmol/mol creatinine). TBARS were determinated spec-
trofluorometrically (excitation wavelength 528 nm, emis-
sion wavelength 558 nm) after extraction with n-butanol
treatment with thiobarbituric acid. The significance of
differences between the nonagenarians and the control
group was examined by ANOVA—Kruskal–Wallis tests,
using statistical software NCSS 6.0.21 (Kaysville, UT,
1996). The decision on significance was based on
p = 0.05.
RESULTS AND CONCLUSION
Urinary neopterin was significantly higher in institu-
tionalized compared to self-sustaining subjects and
controls (625  565 vs. 203  63 mmol/mol creatinine,
and 198  128 mmol/mol creatinine, respectively;
p = 0.006). The serum TBARS were higher in both
groups of nonagenarians (3.23  1,16 mmol/L and
2.69  0.39 mmol/L vs. 2.12  0.83 mmol/L for the self-
sustaining, institutionalized and controls, respectively;
p = 0.023). We conclude that the fluorimetric determina-
tions of urinary neopterin and serum TBARS can be
useful for monitoring health status in elderly patients.
Acknowledgement
Supported by grants of Ministry of Health Czech
Republic, No. NG/6770-3, NC/6171-3 and NB/6822-3.
REFERENCE
1. Solichová D, Melichar B, Svobodová I, Bláha V, Zadák Z.
Fluorescence analysis of antioxidant vitamins and neopterin in
nonagenarians. Biomed. Chromatogr. 1999; 13: 117–119.
Synthetic peptides belonging to structural
and non-structural proteins of GB virus C: a
spectroscopic and calorimetric study of its
interaction with model lipid membranes
N. Rojo,1 M. J. GoÂmara,1 M. A. Busquets,2
M. A. Alsina2 and I. Haro1
1
Department of Peptide and Protein Chemistry, IIQAB-CSIC,
Jordi Girona 18-26, E-08034 Barcelona, Spain. E-mail:
alsina@farmacia.far.ub.es
2 with increasing amounts of peptides was considerable.
Physical Chemistry Department, Faculty of Pharmacy, Avgda
Joan XXIII, s/n E-08028 Barcelona, Spain
GB virus C (GBV-C) and hepatitis G virus (HGV) are
strain variants of a recently discovered enveloped RNA
virus belonging to the Family Flaviviridae, which is
Copyright  2002 John Wiley & Sons, Ltd.
transmitted by contaminated blood and/or blood pro-
ducts, intravenous drug use, from mother to child, and
sexually. The natural history of GBV-C/HGV infection is
at present not fully understood and its potential to cause
hepatitis in humans is questionable (1).
Having in mind the potential use of synthetic peptides
as antiviral therapies and attempting to better understand
the molecular interaction of envelope E2 protein of
GBV-C with lipid bilayers as model membranes, a
potential epitope of GBV-C located at the 99–118 region
(VSWFASTGGRDSKIDVWSLV) of this structural
protein and a peptide belonging to the non-structural
protein NS3 of the same virus (sequence 440-460:
AIAYYRGKDSSIIKDGDLVVC) were selected. The
main aim of the present study was to get insight into
the interaction of the above-described synthetic peptides
with lipid bilayers.
Peptides were synthesized manually following proce-
dures previously described (2, 3). Crude peptides were
purified by preparative high performance liquid chroma-
tography (HPLC) on a Shimadzu chromatograph
equipped with a C8-silica column. Purified peptides were
characterized by analytical HPLC, amino acid analysis
and electrospray mass spectrometry.
Differential scanning calorimetry (DSC) experiments
of multilamellar vesicles composed of dipalmitoylphos-
phatidylcholine (DPPC MLVs) were performed using a
DSC 821E Mettler Toledo calorimeter. Differences in the
heat capacity between the sample and the reference cell
were obtained by raising the temperature at a constant
rate of 5°C/min over the range 0–60°C. All samples were
submitted to three heating/cooling cycles. Data from the
first scan were always discarded to avoid false results
coming to the possible lipid–peptide mixing in the
sample under heating. DSC runs were carried out within
the same day of liposomes preparation. The thermotropic
phase behaviour of zwitterionic DPPC vesicles was
studied in the absence and presence of increasing
concentrations of GBV-C peptides. At high lipid:peptide
ratios, the chain melting transition of DPPC was not
affected by the NS3 peptide. Contrarily for the E2-
peptide, low quantities clearly induced a peak reduction
(Fig. 1). As previously obtained for other synthetic
peptides (4) the addition of low concentrations of the
peptides caused the complete disappearance of the DPPC
pretransition and also the enthalpy of the main transition
peak was considerably diminished. Moreover although
E2 and NS3 peptides did not show a significant dis-
placement of the phase transition mid-point of the DPPC,
the broadening of the transition profile of DPPC bilayers
The higher the percentage of peptide into the bilayer, the
greater the broading of the transition.
Fluorescence experiments were carried out on a
Perkin-Elmer spectrofluorimeter LS 50. NS3 (that con-
tains Tyr) and E2 (that contains Trp) peptides were
Luminescence 2002;17:191–291
 ABSTRACTS 249

respectively excited at 280 nm and 285 nm. Emission

spectra were recorded from 310 to 340 nm. The inter-
action of GBV-C peptides with DPPC was investigated,
by measuring the fluorescence quantum yield and the
wavelength of the emission maximum, in the absence and
in the presence of different concentrations of large
unilamellar liposomes (LUVs). To study the effect of
membrane fluidity on lipid/peptide interaction, the
experiments were carried out both below (25°C) and
above (55°C) the gel/liquid crystalline phase transition
temperature for the DPPC. Fluorescence spectra showed
that in the presence of lipids there was a blue shift in the
emission maximum of E2(99–118) peptide and an
increase in the fluorescence quantum yield. Besides, for
NS3(440–460) a highly moderate fluorescence and no
blue shift were detected for the phenol ring of Tyr
(Fig. 2).
Our results obtained by DSC and fluorescence spectro-
scopy measurements suggest that the selected GBV-C
synthetic peptides (mainly the structural one) interact
with vesicles composed of DPPC. This report represents

Figure 2. Fluorescence uncorrected spectra of (a) E2(99-118)

and (b) NS3(440-460) GBV-C peptides in presence of in-
creasing amounts (from a lipid-peptide ratio of 0 (&), 25 (~),
50 (*), 100 (&), 200 (~) and 300 (*) of DPPC LUVs.

the first step toward establishing a database to gain

insights into the binding of structural and non-structural
GB virus C peptides to lipid membranes.

Acknowledgements
The excellent technical assistance of Josep Carilla and
Amelia López (Laboratori d’Anàlisi Tèrmica IIQAB-
CSIC, Barcelona) is greatly acknowledged. Grant
BQU2000-0793-CO2-02 from the Ministerio de Ciencia
y Tecnologı́a, Spain, funded this work.

Figure 1. DSC heating endotherms of DPPC-MLVs in the
presence of increasing amounts of (a) E2(99-118) (b) NS3(440-
460) GBV-C peptides. The curves refer to the second scan.
REFERENCES
1. Halasz R, Weiland O, Sallberg M. Scand. J. Infect. Dis. 2001; 33:
572–580.
2. Pérez JA, González-Dankaart JF, Reig F, Pintó RM, Bosch A, Haro
I. Biomed. Peptides Proteins Nucleic Acids 1995; 1: 93–100.
3. Haro I, Pérez JA, Reig F, Mestres C, Egea MA, Alsina MA.
Langmuir 1996; 12: 5120–5125.
4. Sospedra P, Mestres C, Haro I, Muñoz M, Busquets MA. Langmuir
2002; 18: 1231–1237.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

250 ABSTRACTS

An improved ¯uorimetric method for the Chemicals

quantitative analysis of antitumoural
11-methylbenzophenothiazine in aqueous
solutions of b-cyclodextrins. Analytical
application in urine
Mame Diabou Gaye Seye,1 Christelle Parrain,1
Jean-Jacques Aaron1 and Noboru Motohashi2
1
Interfaces, Traitement, Organisation et Dynamique des
SysteÁmes (ITODYS), Universite Paris 7ÐDenis Diderot, AssocieÂ
au CNRS (UMR 7086), 1 Rue Guy de la Brosse, F-75005 Paris,
France. E-mail: aaron@paris7.jussieu.fr
2
Department of Medicinal Chemistry, Meiji College of
Pharmacy, I-22-1 Yato-Gho, Tanashi-Shi, Tokyo 188, Japan
11-Me BPHT was synthesized in our laboratories, by
means of a previously described method (2). b-CD and
HP-b-CD were purchased from Aldrich and used as
received. Deionized water and analytical-grade ethanol
were utilized for the preparation of solutions. The
concentration ranges of the cyclodextrins under study
were 0–10 2 mol/L for b-CD and 0–10 1 mol/L for
HP-b-CD. For all fluorescence spectral measurements, a
11-Me-BPHT concentration of 10 5 mol/L was em-
ployed. For the determination of 11-Me-BPHT in urine,
the standard addition procedure was applied.

RESULTS AND DISCUSSION

Fluorescence spectra of 11-Me-BPHT and
INTRODUCTION cyclodextrin effects
11-methylbenzophenothiazine (11-MeBPHT) belongs to Fluorescence excitation and emission spectra of
a new family of benzophenothiazines (BPHTs), with 11-Me-BPHT were recorded at 20°C in pure ethanol,
important antiviral and antitumoural properties (1, 2). water:ethanol (99:1 v/v) (WE) mixture, 10 2 mol/L
Although BPHTs have been the object of recent b-CD and 2.5  10 2 mol/L HP-b-CD WE solutions
photochemical and photophysical studies (3–5), their (Fig. 1). As can be seen, the emission spectra (obtained
strong hydrophobicity makes them, in general, poorly with excitation at lex = 338 nm) were characterized in all
soluble and unstable in aqueous solutions, the preferred media by a single, structureless, broad peak with a
medium for biomedical applications. In order to solve maximum located at 500 nm in ethanol and red-shifted to
this problem it is of interest to use b-cyclodextrins 525 nm in the WE and b-CD solutions and 517 nm in the
(b-CDs), which can include selectively BPHTs and other HP-b-CD solution. The fluorescence signal was signifi-
phenothiazine molecules, in their apolar cavity, forming cantly increased in the presence of HP-b-CD and b-CD
inclusion complexes (4–6). Moreover, the interactions relative to the WE solution. Upon progressively increas-
with b-CDs can produce an increase in the fluorescence ing the b-CD and HP-b-CD concentrations from 0 to
signal of the included organic moiety, which is valuable
from the analytical standpoint. (4).
In the present work, we develop a sensitive, rapid and
simple fluorimetric method for the determination of
11-MeBPHT in aqueous media, based on the formation
of inclusion complexes with b-CD and 2-hydroxypropyl-
b-CD (HP-b-CD). This investigation represents a
follow-up of our previous studies on the fluorescence
properties of b-CD:BPHT and b-CD: 10-Me-BPHT
inclusion complexes (4, 5). In this paper, we report the
effects of b-CD and HP-b-CD on the fluorescence spectra
of 11-Me BPHT. We demonstrate the improvement of the
analytical performances of our fluorimetric method in the
presence of b-CD and HP-b-CD. We provide also an
analytical application to the assay of 11-Me BPHT in
urine.

MATERIAL AND METHODS

Instrumentation
Fluorescence excitation and emission spectra and fluor-
escence intensity (IF) were measured at 20°C on Perkin-
Elmer LS-5 and LS-50 spectrophotofluorometers, using a
quartz cuvette (10 mm optical pathlength).
Figure 1. Fluorescence excitation (EX) and emission (EM)
spectra of 11-Me-BPHT (10 5 mol/L) in various media: (a)
ethanol; (b) water:ethanol (99:1 v/v) (WE) mixture; (c) b-CD
(10 2 mol/L) in WE; (d) HP-b-CD (2.5  10 2 mol/L) in WE.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291


Table 1. Analytical figures of merit for the fluorimetric
determination of 11-Me-BPHT in various media
LDRa LODc
Medium (mg/mL) rb (ng/mL)
Ethanol 0.01–8.00 0.998 0.20
Watere 0.1–13.00 0.990 8.00
HP-b-CDf 0.01–3.00 0.996 1.00
b-CDg 0.001–13.00 0.993 0.06
a
LDR = linear dynamic range; b r = correlation coefficient; c LOD =
IUPAC limit of detection; d RSD = relative standard deviation;
e
water:ethanol (99:1 v/v) mixture; f [HP-b-CD] = 2.5  10 2 mol/L;
g
[b-CD] = 10 2 mol/L.
10 2 mol/L and from 0 to 10 1 mol/L, respectively, the
fluorescence intensities were enhanced regularly, which
indicates the formation of inclusion complexes. By
applying the Benesi–Hildebrand and the Scatchard
methods to our fluorescence data, we determined the
stoichiometry and formation constant (Kf) of the in-
clusion complexes (4, 5). A 1:1 stoichiometry was found
and a Kf value of 141  11 mol/L was obtained for the
HP-b-CD–11-Me-BPHT complex.
Analytical ®gures of merit
Fluorimetric analytical figures of merit were compared
for the determination of 11-Me-BPHT in the various
media under study, using optimal conditions (Table 1).
Linear calibration graphs with three replicates for each
concentration value were established over two to four
orders of magnitude, with larger dynamic ranges in the
presence of cyclodextrins than in WE solutions. Correla-
tion coefficients were satisfactory, ranging between 0.990
and 0.998. The RSD values were rather small (5.2–9.0%).
The IUPAC limits of detection (LOD) were much lower
in the presence of b-CD and HP-b-CD (0.06–1 ng/mL)
than in WE solution (8 ng/mL).
Application to urine analysis
We applied our fluorimetric method to the determination
of 11-Me-BPHT in spiked human urine samples, using
the addition standard procedure. A good linearity and a
good precision were obtained for the standard addition
plots. Satisfactory recoveries ranging from 97% to 108%
were found at various 11-Me-BPHT concentrations for
the urine samples under study.
We can conclude from this work that the presence of b-
CD and HP-b-CD considerably improves the sensitivity
of the fluorescence determination of 11-Me-BPMT, due
to the formation of inclusion complexes, and that it is
possible to apply our method to assay 11-Me-BPHT in
urine.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 251
REFERENCES
1. Foncer GJS, Reej RC, Devonshire R. Photochem. Photobiol. 1990;
RSDd 52: 489.
(%) 2. (a) Motohashi N, Sakagami H, Kamata K, Yamamoto Y. Anticancer
Res. 1991; 11: 1933; (b) Motohashi N, Sasaki Y, Wabayashi YI,
6.2 Molnar J, Kurinara T. Anticancer Res. 1992; 12: 1423.
6.8 3. Aaron JJ, Maafi M, Kersebert C, Parkanyi C, Antonious MS,
9.0 Motohashi N. J. Photochem. Photobiol. A Chem. 1996; 101: 127.
5.2 4. (a) Maafi M, Aaron JJ, Mahedero MC, Salinas F. Appl. Spectrosc.
1998; 52: 91; (b) Maafi M, Mahedero MC, Aaron JJ. Talanta 1997;
44: 2193.
5. Gaye Seye MD, Prot C, Adenier A, Aaron JJ, Motohashi N. New J.
Chem. 2001; 25: 1290.
6. Maafi M, Laasis B, Aaron JJ, Mahedero MC, Muñoz de la Peña A,
Salinas F. J. Incl. Phenom. 1995; 22: 235.
Fluorometric determination of thiobarbituric
acid reactive substances in patients with
coronary artery disease
VladimõÂr BlaÂha, Dagmar SolichovaÂ,
DusÏan CÏernohorskyÂ, Petr ZÏd'aÂnsky and ZdeneÏk ZadaÂk
Department of Metabolic Care and Gerontology, Sokolska 408,
Faculty Hospital Charles University, CZ-50005 Hradec KraÂloveÂ,
Czech Republic. E-mail: blaha@lfhk.cuni.cz
INTRODUCTION
The clinical sequelae of atherosclerosis, ie coronary
artery disease (CAD), stroke and peripheral vascular
disease, are the leading causes of morbidity in the
industrialized countries (1). There is much interest in the
hypothesis that intra-arterial oxidation of lipoproteins
such as low-density cholesterol (LDL-C) contributes to
major risk factors of atherogenesis (2). Since several
dietary components, including antioxidants (3) and
dietary fat saturation (4), influence atherosclerosis, how
these factors relate to the oxidation hypothesis is being
investigated. Thus, the aim of this study was to
investigate the association of lipoperoxidation factors
known to be pro-atherogenic (thiobarbituric acid reactive
substance activity, saturated fatty acids) or anti-athero-
genic (a-tocopherol, polyunsaturated fatty acids) in
coronary artery disease, as assessed by coronary
angiography.
DESIGN AND METHODS
Eighty hyperlipidemic patients (age 33–74 years) who
underwent an elective coronary angiography for CAD
were divided into four groups, based on the severity of
CAD (group 1 = luminal narrowing <50%,n = 21; lumi-
nal narrowing of 50% or more is defined as a significant
lesion, and the patients are referred to as having one-
(group 2, n = 15), two- (group 3, n = 27) or three-vessel
Luminescence 2002;17:191–291
252 ABSTRACTS
Figure 1. Serum thiobarbituric acid reactive substances
(TBARS) were measured in 80 hyperlipidemic patients who
underwent an elective coronary angiography for coronary
artery disease (CAD), and were divided into four groups based
on the severity of CAD (group 1 = luminal narrowing <50%,
n = 21; luminal narrowing of 50% or more is defined as a
significant lesion, and the patients are referred to as having one-
(group 2, n = 15), two- (group 3, n = 27) or three-vessel disease
(group 4, n = 17). Blood samples were taken before coronary
angiography. Results are expressed as mean  SEM; value of a
p < 0.05 was considered to be significant.
disease (group 4, n = 17). Blood samples were taken
before coronary angiography. Thiobarbituric acid reac-
tive substances (TBARS), which are products of lipid
peroxidation, were determined after reaction with thio-
barbituric acid spectrofluorometrically (excitation wave-
length 528 nm, emission wavelength 558 nm) after
extraction with n-butanol treatment with thiobarbituric
acid. Serum lipoprotein fractions were prepared by
density gradient ultracentrifugation (Beckman TL 100,
Palo Alto, CA). The lipoprotein fractions were dis-
tinguished as very low density lipoprotein (VLDL)
<1.006 g/mL; intermediate density lipoprotein (IDL)
<1.019 g/mL; low density lipoprotein (LDL) <1.063
g/mL; high density lipoprotein (HDL) >1.063 g/mL.
Free fatty acids (FFA) in plasma, fatty acid composition
of erythrocyte cell membranes, including saturated fatty
acids (SUFA), mono-unsaturated fatty acids (MUFA) and
polyunsaturated fatty acids (PUFA), and FA of lipo-
protein fractions were measured using capillary gas
chromatography. Vitamin E (a-tocopherol) and vitamin
A (retinol) were analysed by a reversed-phase high-
performance liquid chromatography technique.
RESULTS AND DISCUSSION
TBARS did not differ significantly between groups 1, 2, 3
or 4 (3.36  0.21; 3.31  0.22; 3.03  0.16; 3.56 
0.16 mmol/L), and did not correlate with the degree of
CAD. However, there was significant correlation of
TBARS with total vitamin E ( p = 0.02) and vitamin E in
VLDL ( p = 0.02) and LDL ( p = 0.01) (Figure 1). There
was significant correlation of TBARS with VLDL-
myristic acid ( p = 0.03) and negative correlation with
VLDL-arachidonic acid ( p = 0.03), negative correlation
with IDL-myristic acid ( p = 0.03), LDL-stearic acid
Copyright  2002 John Wiley & Sons, Ltd.
( p = 0.03) and LDL-linoleic acid ( p = 0.01), and HDL-
linoleic acid ( p = 0.02). The differences in vitamins A
and E were not statistically different between groups 1, 2,
3 or 4. However, there was significant correlation of total
vitamin E ( p = 0.01) and VLDL-vitamin E ( p = 0.01)
with the degree of CAD. Pro-atherogenic lipoperoxida-
tion, as revealed by TBARS, was not associated with the
severity of CAD in our group of hyperlipidemic subjects.
Negative impact of CAD was compensated with anti-
atherogenic factors, viz. total a-tocopherol and its VLDL
and LDL fraction.
Acknowledgement
Supported by Grants Nos NB/6822-3, NB/6549-3,
NB/6999-3 and NG/6770-3 from the Ministry of Health,
Czech Republic.
REFERENCES
1. Levy RI. Declining mortality in coronary heart disease. Arterio-
sclerosis 1981; 1: 312–325.
2. Witztum JL, Steinberg DJ. Role of oxidized low-density-lipoprotein
in atherogenesis. J. Clin. Invest. 1991; 88: 1785–1792.
3. Knekt P, Reunanen A, Jarvinen R et al. Antioxidant vitamin intake
and coronary mortality in a longitudinal population study. Am. J.
Epidemiol. 1994; 139: 1180–1189.
4. Esterbauer H, Dieber-Rotheneder M, Striegl G, Waeg G. Role of
vitamin E in preventing the oxidation of low-density-lipoprotein.
Am. J. Clin. Nutrit. 1991; 53: S314–N7321.
Spectroscopic and analytical study of the
photochemically-induced ¯uorescence of
meclofenamic acid and tolfenamic acid.
Application to determination in urine
Leila Bettaieb,1 Jean-Jacques Aaron1 and
Patrice Prognon2
1
ITODYS, Universite Paris 7-Denis Diderot, Associe au CNRS,
UMR 70-86, Rue Guy de la Brosse, F-75005 Paris, France.
E-mail: aaron@paris7.jussieu.fr
2
Laboratoire de Chimie Analytique II, Faculte de Pharmacie,
Universite Paris-Sud, 5 Rue J. B. CleÂment, F-92290 Chatenay
Malabry, France
INTRODUCTION
Meclofenamic acid (MCF) and tolfenamic acid (TLF) are
derivatives of N-phenylanthranilic acid, which belong to
the important class of nonsteroidal anti-inflammatory
drugs (NSAIDs). Several analytical methods based on
high-performance liquid chromatography (HPLC) (1),
gas chromatography–mass spectrometry (GC–MS) (2)
and native fluorescence (3, 4) have been developed for
Luminescence 2002;17:191–291

the determination of these drugs, especially in the case of
mixtures.
In this work, we describe a photochemically-induced
fluorescence (PIF) approach for the quantitative analysis
of MCF and TLF. PIF constitutes a simple, versatile and
very sensitive luminescence method, based on the photo-
chemical transformation of a non-fluorescent analyte into
fluorescent photoproduct(s), which has been already
applied to the determination of a variety of pharmaceu-
ticals, including flufenamic acid, a NSAID, in several
media (5, 6).
MATERIALS AND METHODS
Instrumentation
Fluorescence and PIF excitation and emission spectra
were measured at room temperature on a Kontron
(Zürich, Switzerland) SFM 25 spectrophotofluorimeter,
using a quartz cuvette (10 mm optical pathlength). An
Osram (Germany) 200 W high-pressure mercury arc
lamp powered with a spotlight power supply and located
in a Schoeffel Instrument GmbH (Germany) light-box
was used for the photochemical studies.
Figure 1. Photochemically-induced fluorescence excitation (EX) and emission (EM) spectra of
tolfenamic acid (10 5 mol/L) in dimethyl sulphoxide (tirr = 15 min) (a) and corresponding fluorescence
spectra of TLF (tirr = 0 min) (b). The PIF spectra were recorded with a sensitivity scale divided by 10.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 253
Chemical
Meclofenamic acid and tolfenamic acids were purchased
from Sigma and used as received. Deionized water and
analytical grade solvents (dioxane, ethanol, acetonitrile,
dimethylformamide (DMF) and dimethyl sulphoxide
(DMSO) were utilized for the preparation of solutions.
RESULTS AND DISCUSSION
Fluorescence and PIF spectral properties
MCF (10 5 mol/L) and TLF (10 5 mol/L) displayed
relatively weak, native fluorescence emission spectra at
room temperature in most solvents under study, with
maxima of 374–472 m and 414–490 nm, respectively,
according to the solvent. Upon UV irradiation much
stronger PIF emission bands appeared, with maxima of
376–410 nm for MCF and 396–410 nm for TLF,
depending on the solvent.
In contrast, no significant change of the MCF and TLF
PIF excitation maxima occurred upon changing the
solvent. As an example, Fig. 1 shows the native
fluorescence and PIF excitation and emission spectra of
TLF in DMSO.
Luminescence 2002;17:191–291
254 ABSTRACTS

Optimization of the PIF conditions values were 0.998 and 0.997, respectively, indicating a
good linearity. The relative standard deviation (RSD) was
We investigated the effect of the UV irradiation time and
2.0% for 0.63 mg/mL of MCF and 6.0% for 0.26 mg/mL
solvent on the fluorescence intensity of MCF and TLF.
of TLF. The IUPAC detection and quantification limits
The optimal solvent systems in which the increase of IF
were, respectively, 1.7 and 5.5 ng/mL for MCF, and 9.5
with irradiation time was maximal were DMSO:water,
and 31.5 ng/mL TLF.
70:30 v/v for MCF and DMSO for TLF. In these media,
the PIF enhancement factors reached values of 59 and 35,
respectively. The curves of MCF and TLF fluorescence Analytical applications
intensity vs. UV irradiation time, obtained in the optimal
We applied our PIF method to the determination of MCF
solvent conditions, exhibited an initial rapid increase of
and TLF in fortified human urine samples. A partial
IF, which reached a maximum value after about 6 min for
photolysis of both compounds occurred in urine. In spite
MCF and 12 min for TLF (optimal irradiation times)
of these photostability problems, recovery values of
(Fig. 2). For longer irradiation times, a slower decrease in
98.2 (7)% for MCF and 67.0 (13.4)% for TLF were
IF was observed for both compounds, which probably
found.
indicated photolysis of the fluorescent photoproducts.

Analytical ®gures of merit
The analytical figures of merit were obtained in the
optimal PIF conditions: for MCF, lex/lem = 344/410 nm;
DMSO:water, 70:30 v/v; toptirr = 6 min; for TLF, lex/lem =
opt
334/400 nm; DMSO; tirr = 12 min. Linear calibration
graphs with two replicates for each concentration value
were established in the range 6.4–3200 ng/mL for MCF
and 13–5200 ng/mL for TLF. The correlation coefficient
CONCLUSION
We have demonstrated the analytical usefulness of the
photochemically-induced fluorescence method for the
quantitative analysis of MCF and TLF, which present
only a weak, natural fluorescence in most media. The PIF
method is simple, rapid and particularly sensitive,
allowing detection of concentration of MCF and TLF in
the low ng/mL range. This method is also applicable to
assay both NSAIDs in urine.

Figure 2. Influence of the UV irradiation time on the PIF intensity of (a) meclofenamic acid
(10 5 mol/L) in DMSO:H2O 70:30, v/v (lex = 344 nm; lem = 410 nm); (b) tolfenamic acid
(10 5 mol/L) in DMSO (lex = 334 nm; lem = 400 nm).

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291


REFERENCES
1. Mikali E, Goto T, Ohno T et al. J. Chrom. B Biom. Sci. App. 2000;
744: 81–89.
2. González G, Ventura R, Smith AK, de la Torre R, Segura J. J.
Chrom. A 1996; 719: 251–264.
3. Ruiz TP, Lozano CM, Tomas V, Carpena J. Talanta 1998; 47: 537–
545.
4. Capitan-Vallvey LF, Navas N, Del Olmo M, Consonni V,
Todeschini R. Talanta 2000; 52: 1069–1079.
5. Aaron JJ. Photochemical fluorometry. In Molecular Luminescence
Spectroscopy—Methods and Applications, S. G. Schulman (ed.).
Wiley New York, 1993; 85–131.
6. Bettaieb L, Aaron JJ. Turk. J. Chem. 2001; 25: 165–171.
Fluorometric determination of zearalenone
in corn based on enzymatic reaction
Y. Andreu, S. de Marcos, J. GalbaÂn and J. R. Castillo
Analytical Spectroscopy and Sensors Group (GEAS), Department
of Analytical Chemistry, Faculty of Sciences, University of
Zaragoza, E-50009 Zaragoza, Spain. E-mail:
smarcos@posta.unizar.es
Micotoxins have an extensive impact on humans and
animals due to their ability to contaminate foods and
feeds worldwide. Zearalenone, a toxin produced by
several Fusarium species is of particular interest because
of its widespread contamination of several types of
cereals, especially, corn. In addition, one of its deriva-
tives with toxic properties too, zeranol, is used at large-
scale as a growth promoter in the cattle industry in
several countries.
Current analytical methods for zearalenone determina-
tion, such as thin-layer, gas or liquid chromatography,
require an extended extraction and clean-up step. A direct
competitive ELISA has been recently commercially but
although it does not need previous steps, it is an
expensive alternative for routine application.
We have found that zearalenone is able to react to
3a-hydroxysteroid dehydrogenase enzyme (EC 1.1.1.50)
when b-NADH is present, producing zearalenol. This
reaction can be followed by measuring fluorescence at
NADH wavelengths (excitation and emission wave-
lengths at 334 nm and 453 nm, respectively). On this
way, when the enzymatic reaction occurs, NADH is
consumed producing a gradual decrease of the fluores-
cent intensity which the slope has been found dependent
on the zearalenone concentration.
Different parameters have been optimised. Firstly,
zearalenone has strong absorbance at working wave-
lengths that can originate a strong inner filter effect, so
low zearalenone concentrations are required (below
3  10 5 mol/L  10 mg/ml). Although high co-factor
concentrations increase the velocity of the reaction, it
also reduces sensibility, so low concentrations are also
desired (1.6  10 5 mol/L). About enzyme concentration
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 255
two aspects must be considered. High enzyme con-
centration increases reaction kinetics but it also increases
the cost of the determination, as a consequence 0.125
units/ml are used along the determination. As a result of
working at low concentration of every compound, long
time measurements are required (20 min). Regarding to
the pH, optimal signal variation occurs at pH 6.5. In these
conditions it has been possible to determine up to 1 mg/ml
zearalenone.
Zearalenone determination in corn samples is being
studied. Firstly, corn is grinded and mixed. Then, 20 g
sample is weighed and 2 g NaCl is added. After that,
50 mL methanol is added. The mix is vigorously shaken
for 2 min and filtered. The filtrate is evaporated to
dryness using N2 and then it is dissolved in 500 mL
methanol; however, a problem arises when this solution
is measured because of protein precipitation. The
problem has been resolved by using an ulterior
ultrafiltration step using low adsorption filters.
Fluorometric±enzymatic determination of
choline based on chemically modi®ed
choline-oxidase
A. Aguayo, O. SaÂnchez-Monreal, Y. Andreu,
S. de Marcos, J. GalbaÂn and J. R. Castillo
Analytical Spectroscopy and Sensors Group (GEAS), Department
of Analytical Chemistry, Faculty of Sciences, University of
Zaragoza, E-50009 Zaragoza, Spain. E-mail:
smarcos@posta.unizar.es
Methods based on the combination of enzymatic reaction
and molecular fluorescence are among the most interest-
ing analytical alternatives for substrate determinations.
Although enzymes have fluorescent properties, deter-
minations based on this property have an important
drawback arising from the spectral region where enzymes
absorbs (about 280 nm) and emits (at 330–340 nm). As an
interesting alternative, our research group has previously
determined several analytes by using changes in
fluorescence intensity of a fluorophore, covalently linked
to an enzyme, during the enzymatic reaction (1). In this
work a method for the determination of choline (Ch)
based on enzymatic reaction with chemically modified
choline oxidase (ChOx-FNHS) is purposed. The fluoro-
phore (FNHS) is a fluorescein derivative with a
succinimide group that is able to link covalently to the
primary amines of the enzyme. Apart from its fluorescent
properties (excitation and emission wavelengths at 492
and 516 nm, respectively), the best results, both the
linking procedure and the stability of the bond, were
found with this compound respect to other studied
fluorophores.
Luminescence 2002;17:191–291
256 ABSTRACTS
Figure 1. Variation of fluorescent intensity during the enzymatic reaction for different choline concentrations
(A, blank; B, [choline] = 1  10 4 mol/L; C, [choline] = 2  10 4 mol/L; D, [choline] = 4  10 4 mol/L).
Fig. 1 shows ChOx-FNHS fluorescence variation with
the reaction time for different choline concentrations
at optimal conditions (pH = 9.0, [ChOx-FNHS] =
0.3 IU/ml). The changes observed on the fluorescence
signal are similar to those observed when glucose or
cholesterol react to modified glucose or cholesterol
oxidase, respectively (2, 3).
Since all the mentioned enzymes have FAD as co-
factor, it was thought that the mechanism responsible for
the fluorescence changes of the fluorophore during the
enzymatic reaction should be similar in all of these cases.
From this point we have tried to elucidate the mechanism,
apart from the fact that this methodology could be also
applied to other FAD enzymes. Experiments made with
chemically modified glucose oxidase seem to confirm an
energy transfer process between FAD and the fluoro-
phore.
REFERENCES
1. Galbán J, Andreu Y, Sierra JF, de Marcos S, Castillo JR. Intrinsic
fluorescence of enzymes and fluorescence of chemically modified
enzymes for analytical purposes: a review. Luminescence 2001; 16:
199–210.
2. Sierra JF, Galbán J, de Marcos S, Castillo JR. Fluorimetric–
Copyright  2002 John Wiley & Sons, Ltd.
enzymatic determination of glucose based on labeled glucose
oxidase. Anal. Chim. Acta 1998; 368: 97–104.
3. Galbán J, Sierra JF, López-Sebastián JM, de Marcos S, Castillo JR.
Direct fluorometric determination of total cholesterol in serum using
derivatized cholesterol oxidase. Appl. Spectrosc. 2000; 54: 1157–
1162.
Automated ¯ow-injection and sequential
injection ¯uorimetric determination and
dissolution studies of pharmaceuticals
Petr Solich,1 Hana SklenaÂrovaÂ,1 Zlatka LegnerovaÂ1 and
Christophoros K. Polydorou2
1
Laboratory of Flow Analysis, Faculty of Pharmacy, Charles
University, CZ-500 05 Hradec KraÂloveÂ, Czech Republic. E-mail:
solich@faf.cuni.cz
2
Laboratory of Analytical Chemistry, Department of Chemistry,
University of Athens, Panepistimiopolis, GR-157 71 Athens,
Greece
Dissolution testing, also referred as in vitro availability
tests, is performed under specified conditions of tem-
perature, volume and stirring rate that should mimic
processes in the human gastrointestinal tract. Dissolution
Luminescence 2002;17:191–291

tests of active compounds in pharmaceutical formula-
tions are included among the officially recommended
methods of most pharmacopoeias world-wide, thus their
automation and routine application has become a major
goal for many laboratories. Automated systems required
the following parameters: high sampling rate, low sample
consumption, possibility of on-line analysis, and sensi-
tive detection due to the low concentration of drugs in the
beginning of the dissolution procedure (1).
Both flow injection analysis (FIA) and sequential
injection analysis (SIA) techniques introduced by
Ruzicka et al. in 1975 and 1990, respectively (2), are
computer-aided analytical methods convenient to fulfil
demands for dissolution studies. Characteristic advan-
tages of these methods are their versatility, full computer
compatibility, high sample throughput, and finally the
low sample and reagent consumption.
Prazosin is a pharmaceutically active compound,
which is strongly fluorescent in alkaline medium. That
fact was utilized for the development of a simple,
sensitive and rapid FIA method with fluorimetric
detection, applied to the assay and dissolution studies
in pharmaceuticals. The optimum excitation wavelength
for prazosin was 246 nm and emission wavelength
370 nm. The measurement throughput was 60/h. The
concentration range of the calibration was D0–500
mg . l 1. Measurements were done using automated
flow-injection system, all parts were controlled by the
computer (FIA-MOD 2.2 software). Potential interfer-
ences were tested. Examples of real-time dissolution
profiles for solid pharmaceutical formulation Deprazolin
tablets containing only 1 mg prazosin will be demon-
strated.
Ergotamine tartarate was determined in pharmaceu-
ticals using the SIA technique with fluorescence detec-
tion. The method is based on the formation of a blue
fluorescence of ergotamine tartarate after excitation by
UV radiation in acidic medium. The calibration curve
relating the intensity of fluorescence (F) to concentration
(c) was linear over the range 0.03–0.61 mg/L of
ergotamine tartarate. The linear regression (n = 6) was
calculated: F = 117.7c ‡ 0.80; r = 0.9998. The content
uniformity and dissolution tests of the formulation
Bellaspon (0.3 mg/tablet) were carried out following
the conditions for dissolution tests described in the
British Pharmacopoeia.
The use of the fluorescence detection in FIA and SIA
substantially increases the sensitivity in comparison with
the conventional HPLC–UV methods, that are also time-
consuming.
Acknowledgement
Supported by the Grant Agency, Czech Republic, Grants
Nos 203/00/0515 and 203/02/P013, and by the Grant
Agency of Charles Univ. (No. 250/2002/B-CH).
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 257
REFERENCES
1. Calatayud JM. Flow Injection Analysis of Pharmaceuticals. Auto-
mation in the Laboratory. Taylor & Francis: London, 1996.
2. Ruzicka J, Marshall GD. Anal. Chim. Acta. 1990; 237: 329.
Flow-injection analysis of ¯ufenamic acid, in
the dynamic and stopped-¯ow modes,
using photochemically-induced ¯uorescence
detection
Leila Bettaieb,1 Jean-Jacques Aaron1 and
Patrice Prognon2
1
ITODYS, Universite Paris 7-Denis Diderot, Associe au CNRS,
UMR 70-86, 1 Rue Guy de la Brosse, F-75005 Paris, France.
E-mail: aaron@paris7.jussieu.fr
2
Laboratoire de Chimie Analytique II, Faculte de Pharmacie,
Universite Paris-Sud, 5 Rue J. B. CleÂment, F-92290
Chatenay-Malabry, France
INTRODUCTION
Flufenamic acid (FF) is a non-steroidal anti-inflammatory
drug (NSAID) of major importance. Its determination in
either pharmaceuticals or biological matrices is generally
performed by high performance liquid chromatography
(HPLC) with UV detection (1, 2); fluorimetry has also
been used (3). Recently, we have applied the photo-
chemically-induced fluorescence (PIF) approach to the
quantitative analysis of FF in stationary solutions (4).
This study constitutes the first report on the coupling of
a flow injection analysis (FIA) system with PIF detection
in order to determine FF in a dosage form
(MOVILISIN1). The respective merits of using FIA–
PIF with continuous and stopped-flow modes were also
evaluated (5).
MATERIAL AND METHODS
The FIA device consists of an IPC model, fully pro-
grammed perilstatic pump from Ismatec (Switzerland),
which delivers the chosen flow rates. Samples were
injected through an injection valve equipped, as needed,
with PTFE loops of various volumes. A photo-reactor,
Model 31125 from Knauer (Germany), including a
0.5 mm i.d. coiled tube and a 8 W, 254 nm Osram
(Germany) UV lamp, was used to obtain the photo-
chemically-induced fluorescence of the sample. Detec-
tion was performed by means of a Kontron (Switzerland)
SFM25 spectrophotofluorimeter equipped with a 25 mL,
1.5 mm length optical path cell from Hellma (Germany).
Excitation and emission wavelengths were set at 390 and
424 nm, respectively, and FF solutions were prepared in a
0.1 mol/L HClO4 aqueous medium, as optimized in a
Luminescence 2002;17:191–291
258 ABSTRACTS
Figure 1. Effect of the stopped-flow time on the PIF intensity
of flufenamic acid (2  10 6 mol/L). Flow rate, 1.4 mL/min;
injected volume, 393 mL; reactor length, 200 cm; delay time,
35 s.
previous stationary spectroscopic study (4). Water was
used as mobile phase. In such conditions, the injected
amounts of FF corresponded to concentrations varying
from 14 to 2800 ng/mL.
RESULTS AND DISCUSSION
Optimization of the FIA±PIF parameters
In continuous flow mode, the PIF–FIA signal decreases
upon increasing the flow rate. The best resolution and the
highest PIF signals were achieved for the following set of
optimized parameters: a 0.5 mL/min flow rate, an
injected volume (Vi) of 393 mL and a 400 cm reactor
length (1).
In stopped-flow mode, the optimal injected volume
was similar, whereas the reactor length value was
reduced to 200 cm in order to minimize longitudinal
dispersion. A delay time of 35 s was selected with a flow
rate of 1.4 mL/min. The residence time in the photo-
reactor was also optimized by measuring the change of
the PIF intensity with the stopped-flow time; after a
relatively rapid initial increase of the PIF signal, a plateau
region was reached and a 4 min optimal stopped-flow
Table 1. Analytical figures of merit of the FIA–PIF method of determination of FF, using the
continuous and stopped-flow modes
Mode LDRa (ng/mL) Slopeb
Continuous 28–1125 1.01
Stopped-flow 14–1125 0.96
a
LDR linear dynamic range; b slope of the log-log calibration curve; c r, correlation coefficient; d LOD, limit of
detection calculated for a S/N ratio of 3; e LOQ, limit of quantification calculated for a S/N ratio of 10; f RSD, relative
standard deviation.
Copyright  2002 John Wiley & Sons, Ltd.
time was finally selected (Fig. 1). A significant enhance-
ment of PIF intensity (about 5) was observed relative to
the continuous flow mode.
Analytical ®gures of merit
The analytical characteristics of the FIA–PIF method
with either continuous or stopped-flow mode are reported
in Table 1. In the optimal experimental conditions the
linearity of the calibration curves was demonstrated over
about two decades. The LOD and LOQ values were
relatively low, in the ng/mL range.
Application to real samples
As an illustration, the standard addition method was used
for the determination of FF in MOVILISIN1, a dosage
form containing 30 mg/mL FF. For this purpose,
FIA–PIF in the continuous flow mode was chosen. Our
experiments show that the standard addition regression
line exhibits a perfect parallelism relative to the
calibration standard curve, thus demonstrating the
absence of matrix effect. Satisfactory recovery values,
ranging between 90.5% and 100.2% were found in the
assay of MOVILISIN1 throughout the whole range of FF
concentrations under study.
CONCLUSION
We have shown in this work the analytical interest of the
FIA–PIF method for the determination of FF in a
pharmaceutical. Although the stopped-flow mode leads
to lower detectability, the continuous flow mode is
preferred for application to the assay of drug, because of
its greater rapidity.
REFERENCES
1. Papadoyannis IN, Zotou AC. J. Liq. Chrom., 1992; 15: 1923–1945.
2. Cerretani A, Micheli L, Fiaschi AI, Giorgi G. J. Chrom. B Biomed.
Appl., 1996; 678: 365–368.
3. Albero MI, Sanchez-Pedreño C, Garcia MS. J. Pharm. Biomed.
Anal., 1995; 13: 1113–1117.
4. Bettaieb L, Aaron JJ. Turk. J. Chem., 2001; 25: 165–171.
5. Garcia LF, Eremin S, Aaron JJ. Anal. Lett., 1996; 29: 1447–1461.
rc LODd (ng/mL) LOQe (ng/mL) RSDf (%)
0.998 6.6 21.9 10.0
0.980 2.3 7.9 7.0
Luminescence 2002;17:191–291

Sequential injection analysis applied to the
simultaneous ¯uorometric multiresidue
determination
Graciela de Armas, Manuel MiroÂ, Jose Manuel Estela
and VõÂctor CerdaÁ
1
Department of Analytical Chemistry, Materials and Reagents
Institute (IMRE), University of Havana, Havana, Cuba
2
Department of Chemistry, University of the Balearic Islands, E-
07071-Palma de Mallorca, Spain. E-mail: vcerda@p01.uib.es
A stopped-flow sequential injection (SI) spectrofluori-
metric procedure is presented for the simultaneous
on-line determination of Carbaryl (CBL) (1-naphthyl-
N-methylcarbamate), Fuberidazole (FBZ) (2-(2'-furyl)-
benzimidazole), Thiabendazole (TBZ) (2-(4'-thiazolyl)-
benzimidazole) and Warfarin (W) (3-a-acetonylbenzyl)-
4-hydroxycoumarin) (Fig. 1) in micellar medium through
the chemometric resolution of the recorded variable angle
scanning (VAS) spectra. Regarding to pesticide analysis,
batch or flow injection (FI) methods for the individual
TBZ or FBZ determination have been issued (1–3).
Garcı́a-Sanchez et al. (4) and Panadero et al. (5) reported
the multi-residue batch analysis of W, FBZ and CBL by
VAS-fluorescence, and W and CBL by derivative con-
stant wavelength synchronous fluorescence, respectively.
Nevertheless, to our best knowledge no paper related to
the automated and simultaneous determination of W,
CBL, FBZ and TBZ has been reported to date. Hence, the
potentiality of VAS-spectroscopy coupled to multivariate
least-squares regression (MLR) algorithms, as an alter-
Figure 1. Structures of the selected pesticides.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 259
native to robust chromatographic procedures, for sequen-
tial injection multi-determination of residues with highly
overlapped spectra using a sandwich technique is demon-
strated in this communication.
A suitable number of sequential emission spectra of
the standard solutions of each pesticide and its mixtures
at pH 2 and 7 were registered in order to obtain both
three-dimensional plots and contour maps. These figures
were used to select the proper variable-angle scanning
route, which was optimized to traverse those regions of
three-dimensional spectra with the highest difference
between C, FBZ, TBZ and W and other zones with a high
sensitivity for each pesticide.
The proposed methodology is based upon the segmen-
tation of an aqueous sample slug (400 mL) between two
different buffer zones (viz. pH 7 and 2) (see Fig. 2) in
order to obtain both an improvement of sensitivity and
residual minimization of the target species following a
selected variable-angle route at both pH zones. The
proper zones for analyte determination should be decided
upon the content level in samples. Thus, while FBZ and
CBL may be analysed at either pH 2 or 7, W and TBZ
should be determined at pH 2 and 7, respectively, when
moderately high levels are found. On the other hand,
analysis at pH 7 for W and pH 2 for TBZ and FBZ are
required for samples at low pesticide levels; whereas
CBL sensitivity is independent from pH.
Several chemical, physical and hydrodynamic vari-
ables, such as the pH of the reaction medium, nature of
the buffer, ethanol concentration, length of sample and
reagent slugs, flow-rates, protocol of aspiration, influence
Luminescence 2002;17:191–291
260 ABSTRACTS

Figure 2. (a) Sequential injection manifold for the spectrofluorimetric multiresidue determination. Holding coil (HC) and reaction
coil (RC) lengths are 300 cm and 110 cm, respectively. SV, selection valve; HTAC, hexadecyltrimethylammonium chloride; W,
waste. (b) Schematic representation of stacked plugs in the holding coil for sandwich technique.

of surfactants, geometry of mixing coil and instrumental
parameters of the spectrofluorimeter, have been studied
thoroughly. Dynamic ranges of 13–720 mg/L CBL, 0.10–
14 mg/L FBZ, 0.19–60 mg/L TBZ and 0.05–5 mg/L W
were achieved at the pH of maximum sensitivity.
Relative standard deviations (n = 10) were 0.2% for
100 mg/L CBL and 2.4 mg/L FBZ, 0.7% for 8 mg/L TBZ
and 1.0% for 1 mg/L W. The presented automated
method, which handles 17 samples/h, was validated and
applied to spiked real water samples, with very
satisfactory results.
REFERENCES
1. Garcı́a-Sánchez F, Cruces C. Anal. Chem. 1988; 60: 323–328.
2. Capitán F, Alonso E, Avidad R, Capitán-Vallvey LF, Vilchez JL.
Anal. Chem. 1993; 65: 1336–1339.
3. Capitán-Vallvey LF, Avidad R, Vilchez JL. J. AOAC, 1994; 77:
1651–1654.
4. Garcı́a-Sánchez F, Ramos A, Cerdà V, Oms MT. Anal. Chim. Acta
1990; 228: 293–299.
5. Panadero S, Gómez-Hens A, Pérez-Bendito D. Talanta 1993; 40:
225–230.
Design and characterization of a ¯uorescent
biosensor for glucose determination based
on labelled glucose oxidase
Vanesa Sanz, Yolanda Andreu, Susana de Marcos,
Javier GalbaÂn and Juan R. Castillo
GEAS, Anal. Chem. Dept., Sciences Faculty, University of
Zaragoza, Zaragoza, Spain. E-mail: smarcos@posta.unizar.es
In recent years our research group has studied the use of
enzymes covalently-bonded to different derivatizing
agents whose fluorescence changes during the enzymatic
reaction (1). The best results were obtained with the
fluorescein derivative fluorescein-5(6)-carboxamido-
caproic acid N-hydroxy-succinimide ester (FS), which
exhibits an excitation maximum at 490 nm and an
emission maximum at 520 nm. In this work, the
application of this methodology to the design and
characterization of a fluorescent biosensor for glucose
determination, based on the immobilization of glucose
oxidase derivatized with this compound (GOx-FS), is
presented.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291


Figure 1. Transmission electron microscopy images of colloidal Au nanoparticles synthesized
in SDS system.
The main advantage of this biosensor is that in the
design of the sensor film only the labelled enzyme has to
be immobilized. Apart from that, and due to the use of the
derivatizing agent, the detection is carried out in a
spectral zone where many samples do not have
interference problems.
Different methods of immobilization were tested, such
as adsorption, entrapment and covalent bonding. With
regard to adsorption, different supports, e.g. active glass,
polystyrene and polyacetate, were tested. The fluores-
cence of GOx-FS was only observed in those obtained
with polyacetate, but the fluorescence intensity decreases
after washing the film. The immobilization procedures
based on the formation of covalent-bonding with transi-
tion metals were tested with free enzyme because both
the derivatizing agent and the active groups of the support
are likely to be joined to the same amino groups of the
enzyme. In this case the product obtained exhibits the
intrinsic fluorescence of the enzyme but the reaction with
glucose was not observed due to the fact that the amount
of enzyme immobilized (the maximum allowed) was not
sufficient to this determination technique. Apart from
that, entrapment in polypyrrol, polyaniline and polyacril-
amide were also tested, but only the last exhibits the
GOx-FS fluorescence and reacts reversibly with glucose,
therefore this was the immobilization method chosen for
GOx-FS.
Measurements were perform by means of flow
injection analysis in order to provide a certain auto-
matism to the analytical procedure. The analytical signal
is transient and therefore there are some analytical
parameters which could be related to the glucose
concentration, e.g. regeneration time, peak area and the
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 261
percentage of increase on the fluorescence intensity in the
maximum. The best results were obtained with the latter.
A home-made flow cell (volume 225 mL) was designed
and different parameters were optimized, e.g. a type of
carrier solution (buffer solution of 0.1 mol/L phosphate,
pH 6.5), flow rate (11 ml/s), injection volume (300 mL)
and GOx-FS concentration in the acrylamide mixture
(5000 UI/ml). Under these conditions, the sensor films
are reversible, chemical-resistant and stable for at least 2
months and have a linear range from 0.0027–0.022 mol/L
glucose, as can be seen in Fig. 1.
Furthermore, the effect on the analytical signal from
interfering fluorescent and absorbent species were
investigated and it has been concluded that an absor-
bance value lower than 0.11 does not interfere in the
measure.
Finally, a mathematical model that justifies the
relationship between the percentage of increase on the
fluorescence intensity in the maximum and the inverse of
the glucose concentration is being studied.
Acknowledgement
This work has been supported within the project BQU
2000-1162 of the DGES (Spain).
REFERENCE
1. Sierra JF, Galbán J, de Marcos S, Castillo JR. Direct determination
of glucose in serum by fluorimetry using a labelled enzyme. Anal.
Chim. Acta 2000; 414: 33–41.
Luminescence 2002;17:191–291
262 ABSTRACTS
Control synthesis and luminescence analysis
of gold nanoparticles in regular system of
SDS
Yuan Hong, Liu Xiaoling, Cai Ruxiu and Pang Daiwen
Department of Chemistry, Wuhan University, Hubei 430072,
People's Republic of China. E-mail: Cai_lin@whu.edu.cn
Size provides important control over many of the
physical and chemical properties of nanoscale materials,
including luminescence, conductivity and catalytic
activity. Chemistry has led the discovery of quantum
confinement in colloidal nanocrystals and to the use of
such structures as probes for biological diagnostic appli-
cations, light-emitting diode (LED) materials, lasers, and
Raman spectroscopy-enhancing materials. In Sodium
dodecyl sulphonate (SDS) micelle systems, the new
synthetic technique (1) for colloidal gold (Au) nanopar-
ticles has been improved (Fig. 1). Colloidal Au particles
of mean diameter 5–14 nm were synthesized, which
exhibit improved monodispersivity relative to previously
published methods (2). According to the particular
molecular structure, made up of a water-soluble (hydro-
philic) part and a water-insoluble (hydrophobic) part of
surfactants, and different electron distribution arising
from colloidal small sizes and high surface: volume
ratios, it is found that a complex was probably formed in
the presence of SDS and Au3‡ (or deoxidized Au atoms).
During the synthesis of colloidal gold nanoparticles, SDS
can prevent their further growth as a stabilizer for Au
particles, therefore the colloidal gold nanoparticles are
monodispersed and steadier.
Resonance Rayleigh scattering (RRS) is a special
elastic scattering, which not only has high signal levels
but also can provide new information concerning mol-
Figure 1. Transmission electron microscopy images of
colloidal Au nanoparticles synthesized in SDS system.
Copyright  2002 John Wiley & Sons, Ltd.
Figure 2. Resonance Rayleigh scattering spectra of the three
systems: (1) BSA; (2) Au nanoparticles; (3) BSA–Au nano-
particles.
ecular structure, size, form, charge distribution, state of
combination and so on. This technique (3, 4) has been
increasingly applied to the study and determination of
biological macromolecules, trace amounts of inorganic
ions and inorganic nanoparticles. Based on the high
intensity of RRS, a sensitive and rapid method for the
determination of protein with gold colloid is reported.
Further work concerns the study of resonance Rayleigh
light-scattering caused by the interaction between protein
and gold nanoparticles (Fig. 2). The method is based on
the interaction between proteins and gold nanoparticles in
a certain pH range, which causes different extents of
enhancement of the scattering signal of gold colloid in
the wavelength range 500–650 nm. Into a 10 mL colori-
metric tube, different volume of 5 mg/mL borine serum
albumin (BSA), 2.0 mL Mcllvaine buffer (pH 4.9) and
50 mL gold nanoparticle solution were placed in turn. The
mixture was diluted to 10 mL with water and mixed
thoroughly. The Rayleigh light scattering (RLS) spec-
trum was obtained by synchronously scanning the
excitation and emission monochromators. After optimiz-
ing this method, we can determine different proteins with
satisfactory results. The detection limits is 50 ng/mL for
BSA.
REFERENCES
1. Yuan H, Liu XL, Cai RX, Pang DW, J. Wuhan University (Nat. Sci.
Ed.), 2002; 48: 129–132.
2. Grabar KG, Freeman RG, Hommer MB, Natan MJ. Anal. Chem.
1995; 67: 735–743.
3. Pasternak RF, Collings PJ, Giannetti A et al. J. Am. Chem. Soc.
1993; 115: 5393–5399.
4. Parkash J, Robblee JH, Agnew J et al. Biophys. J. 1998; 74: 2089–
2099.
Luminescence 2002;17:191–291
 ABSTRACTS 263

Merocyanine540 for spectroscopic analysis of RESULTS AND DISCUSSION

the interaction of a peptide sequence of
Absorption spectra of MC540 containing DPPC or DPPG
hepatitis G virus with liposomes
LUVs with or without peptide, were recorded to deter-
M. Alay,1 M. A. Busquets,1 I. Haro,2 N. Rojo,2 M. A. mine the maximum absorption wavelength (not shown).
Alsina1 and J. Prat1 Spectra shape was strongly dependent on the lipid com-
1
Physical Chemistry Department. Faculty of Pharmacy. Avgda position, thus indicating a different location of MC540 in
Joan XXIII, s/n E-08028 Barcelona. Spain. E-mail: the bilayer. However, both DPPC and DPPG spectra
alsina@farmacia.far.ub.es showed two maxima corresponding to the dimer (lower
2
Department of Peptide & Protein Chemistry, IIQAB.CSIC, Jordi wavelength) and monomer form of the probe, respec-
Girona Salgado, 18, E-08034 Barcelona, Spain tively. For MC540/DPPC LUVs they were 530 and
566 nm and for MC540/DPPG LUVs, 500 and 530 nm.
The absorption and fluorescence properties of molecular Fig. 1 shows the fluorescence spectra resulting from the
probes have been extensively used to monitor drug tritation of MC540 containing DPPC liposomes with
interaction with membranes. Although the presence of an increasing concentrations of E2 (125–139) peptide (0,
external probe can disturb the system under study, in 12 mmol/L, 59 mmol/L and 216 mmol/L) at lex of 530 (a)
general, the information derived from its analysis has
shown to be useful in predicting the pharmacological
effect of a wide range of molecules (1). In the present
study, we describe the interaction of a peptide sequence
corresponding to the fragment 125–139 of the E2
structural protein of the hepatitis G virus (HGV) with
lipid bilayers by using merocyanine540 (MC540) as
external probe. MC540 is an amphipathic anionic probe
that has been shown to be useful to examine lipid head
group spacing as well as the surface properties of the
bilayers, where it can be easily incorporated (2, 3). Its
spectral characteristics, both visible and fluorescence, are
highly dependent on the media. In our case, these proper-
ties have been taking into consideration for studying the
perturbing effects of the peptide on liposomes, a bilayer
biomimetic system.

MATERIALS AND METHODS

The peptide with a sequence of (CTIAALGSSDRDTVV)
was manually synthesized, purified and characterized
as described elsewhere. Large unilamelar vesicles
(LUVs) of 1,2-dipalmitoyl-sn-Glycero-3-phosphocholine
(DPPC) or 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-
(1-glycerol)] (DPPG) (both from Avanti Lipids) contain-
ing MC540 were prepared by the extrusion method (4)
(Northern lipids Inc., Canada) by passing the lipid
dispersion (10 mmol/L Tris, pH 7.4) 10 times through
25 mm polycarbonate filters with 0.1 mm pore size
(Nucleopore Corp., Canada). All extrusion procedures
were conducted at 45°C, above the transition temperature
of the lipids. Liposome size was 120  3 with a poly-
disperity index of 0.10  0.02. Final lipid concentration,
assessed by phospholipid analysis, was 4 mmol/L. MC540
(Sigma) and was incorporated to the lipid bilayer by
incubating the probe with the preformed LUVs at 45°C
for 30 min. The final ratio MC540/phospholipid was Figure 1. Effect of increasing (125–139) E2 peptide concen-
tration on fluorescence spectra of MC540 incorporated into
1:300. Fluorescence experiments were performed in an DPPC LUVs. (A) lex = 530 nm; (B) lex = 566 nm. The arrow
AB-2 spectrofluorimeter SLM-Aminco with excitation indicates the direction of increasing peptide concentration (0,
and emission slit-widths of 4 nm. 12, 59 and 216 mmol/L, respectively).

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

264 ABSTRACTS

and 566 (b) nm. In both cases, maximum emission wave- tion spectra in aqueous, micellar and bilayer environments. Eur. J.
Biochem. 1992; 207: 1085–1091.
length was not affected by peptide presence. However, 6. Bernik D, Tymczyszyn E, Daraio ME, Negri RM. Fluorescent
when lex = 530nm, peptide addition resulted in a slight dimers of merocyanine 540 (MC540) in the gel phase of phos-
decrease in fluorescence intensity. This fact was con- phatidylcholine liposomes. Photochem. Photobiol. 1999; 70: 40–48.
comitant with an increase in the monomer form assessed
by the fluorescence intensity increase observed when
exciting the sample at lex = 566 nm. According to these
results, the peptide modifies bilayer organization, loca-
ting the probe in the bilayer interface parallel to the Intrinsic cipro¯oxacin ¯uorescence in acidic
surface (5). As indicated below, the monomer form could micellar media
have a quenching effect on dimer fluorescence. Control
experiments incubating MC540 with the same peptide A. Vercauteren, T. Vankeirsbilck, G. Van der Weken
concentrations did not show any change on fluorescence and W. R. G. Baeyens
spectra. Department of Pharmaceutical Analysis, Faculty of
Pharmaceutical Sciences, Ghent University, Harelbekestraat 72,
On the other hand, spectra for MC540 in DPPG LUVs
B-9000 Ghent, Belgium. E-mail: willy.baeyens@rug.ac.be
showed a decrease in fluorescence intensity upon peptide
addition when exciting at 530 nm (monomer band) while
Ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-
was not significantly affected when lex was 500 nm
oxo-7-piperazin-1-yl-quinoline-3-carboxylic acid), a
(dimer band) (not shown). Considering the results
4-quinolone derivative, is one of the newer compounds
obtained in the absorption spectra, in which we observed
of the fluoroquinolone class of antibacterial agents (Fig.
a decrease in the monomer peak (530 nm) concomitant
1). Ciprofloxacin has a wide spectrum of activity against
with an increase in the intensity of the dimer peak
Enterobacteriaceae, Pseudomonas aeruginosa, Haemo-
(500 nm) after peptide addition, fluorescence decrease
philus and Neisseria spp. but also against Staphylococcus
can be easily explained. Peptide presence increases dimer
and some other Gram-positive bacteria. Several articles
concentration, whose emission properties are character-
have been published on the determination of cipro-
ized by a lower quantum yield than the monomer (6), in
floxacin in different media by HPLC with fluorimetric
fact that appears together with a quenching of the
detection (1–3).
monomer emission. Also, the absorption spectra shape of
MC540 in presence of DPPG LUVs is very similar to that
observed in aqueous media, thus indicating a low degree EXPERIMENTAL
of probe incorporation into the bilayer, probably due to
Chemicals
repulsion forces as both lipid and probe are anionic. The
peptide presence probably favours probe exclusion from Ciprofloxacin hydrochloride was obtained from Alcon-
the bilayer and dimer formation. Couvreur (Puurs, Belgium). Sodium dodecyl sulphate
These results indicate that the peptide has a slight (SDS) was purchased from Panreac (Barcelona, Spain).
effect on the properties of the dye incorporated into Tetramethylammonium bromide (TMAB), tetrabutyl-
DPPC and DPPG LUVs, and thus can probably modify ammonium bromide (TBAB), b-cyclodextrin (BCD)
bilayer surface properties. Complementary studies with and a-cyclodextrin (ACD) were obtained from Janssen
lipid monolayers are in agreement with these data. Chimica (Beerse, Belgium). 1-Octanesulphonic acid
Further experiments with calorimetric techniques are sodium salt (OSH) and 1-heptanesulphonic acid sodium
being performed in order to gain more insight into the salt (HSA) were obtained from Acros (Geel, Belgium).
interaction process.

REFERENCES
1. Lakowicz JR. Principles in Fluorescence Spectroscopy, 2nd edn.
Kluwer Academic/Plenum: New York, 1999.
2. Lelkes PI, Miller IR. Perturbations of membrane structure by optical
probes: I. location and structural sensitivity of merocyanine 540
bound to phospholipid membranes. J. Membrane Biol. 1980; 52: 1–
15.
3. Langner M, Hui SW. Merocyanine 540 as a fluorescence indicator
for molecular packing stress at the onset of lamellar-hexagonal
transition of phosphatidylethanolamine bilayers. Biochim. Biophys.
Acta 1999; 1415: 323–330.
4. New RRC. Preparation of liposomes. In Liposomes: a Practical
Approach, New RRC (ed.). IRL Press: Oxford, 1990; Chapter 2.
5. Kaschny P, Goñi FM. The components of merocyanine 540 absorp- Figure 1. Chemical structure of ciprofloxacin.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291


Cetyltrimethylammonium bromide (CAB) was obtained
from Aldrich (Milwaukee, WI, USA). All other chemi-
cals and solvents used were of analytical grade and used
as such without extra purification.
Apparatus
The pH values of the solutions were controlled with a
Metrohm 691 pH meter (Pleuger, Belgium) with a
combined Ag/AgCl electrode. The fluorescence spectra
and the fluorimetric measurements were carried out
on an RF-5001 PC spectrofluorophotometer (Shimadzu
Benelux, ‘s-Hertogenbosch, The Netherlands). The
spectral data were processed on a PC.
RESULTS AND DISCUSSION
For all experiments ciprofloxacin hydrochloride solutions
with a concentration of 10 mg/mL in distilled water were
used. The excitation wavelength was installed at 279 nm,
emission was measured at 443 nm. To obtain the optimal
pH for maximum fluorescence emission, different buffers
(0.05 mol/L) were used. For pH 2 and 3 a citric acid
buffer was used, for pH 4–8 a phosphoric acid buffer and
for pH 9–13 a borate buffer. Precipitation of the molecule
started from pH 6 onwards, limiting the aqueous
experiments. Maximum fluorescence was obtained at
pH 4. Further experiments were carried out at this
particular pH value. It was attempted to improve native
fluorescence intensity by using some frequently cited
neutral, positively and negatively charged organized
media (ACD, BCD, CAB, HSA, OSH, SDS, TBAB and
Figure 2. Relative fluorescence intensities in function of the used organized medium.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 265
TMAB). They were added in concentrations of 10, 25 and
50 mmol/L at pH 4. BCD 25 and 50 mmol/L were
insoluble in water at pH 4. Fig. 2 shows a plot of relative
fluorescence intensities as a function of the employed
organised medium. The blank stands for a ciprofloxacin
solution without any organized medium added. There are
no significant differences in fluorescence emission
(measured at lEXC = 279 nm, lEM = 443 nm) between
most of the media, except for SDS. The medium
containing SDS provides the highest fluorescence
intensity.
The ciprofloxacin fluorescence intensities for different
concentrations of SDS were measured. A concentration
of 25 mmol/L provides the highest fluorescence emis-
sion, although there is a minimal difference between this
value and those measured for 10 and 30 mmol/L SDS
concentrations.
CONCLUSION
The relative fluorescence intensity of ciprofloxacin is
highest when a pH 4 medium is used in combination with
the SDS surfactant. Thus, so as to enhance fluorescence
emission of ciprofloxacin in HPLC experiments, a mobile
phase of pH 4 containing 10–30 mmol/L SDS is being
suggested as micellar medium for liquid chromato-
graphic work. Apart from the enhanced selectivity of
analysis as offered by the proposed detection system,
further work is in progress so as to find a compromise
between required mobile phase composition (separation)
and measuring conditions (detection).
Luminescence 2002;17:191–291
266 ABSTRACTS

REFERENCES Physicochemical studies

1. Yorke JC, Froc P. Quantitation of nine quinolones in chicken tissues

by high performance liquid chromatography with fluorescence
detection. J. Chromatogr. A. 2000; 882(1–2): 63–77.
2. Garcia MA, Solans C, Aramayona JJ, Rueda S, Bregante MA, de
Jong A. Simultaneous determination of enrofloxacin and its primary
metabolite, ciprofloxacin, in plasma by HPLC with fluorescence
detection. Biomed. Chromatogr. 1999; 13(5): 350–353.
3. Mizuno A, Uematsu T, Nakashima M. Simultaneous determination
of ofloxacin, norfloxacin and ciprofloxacin in human hair by high-
performance liquid chromatography and fluorescence detection. J.
Chromatogr. B Biomed. Appl. 1994; 653(2): 187–193.
4. Zhao Y, Baeyens WRG, Van der Weken G, Garcı́a-Campaña AM.
Fluorimetric study of acyclovir in acidic micellar media. Biomed.
Chromatogr. 1999; 13: 143–144.
5. Baeyens WRG, Vanparys M, Van der Weken G. Optimization of
intrinsic naproxen fluorescence by off-line and flow injection
analysis. Biomed. Chromatogr. 1999; 13: 145–147.
Effect of laminin (GESIKVAVS) active
peptide on the microviscosity of bilayers
A. JuveÂ,1 P. Sospedra,2 L. RodrõÂguez3 and F. Reig1
1 Fusion of vesicles. This experiment was performed
Department of Peptides, Institut for Biological and
Environmental Chemistry, Jordi Girona 18, E-08034 Barcelona,
Spain
2
Physicochemical Department, Faculty of Pharmacy, University
of Barcelona, Avgda. Joan XXIII s/n, E-08028 Barcelona, Spain
3 intensity of both probes recorded. As reference liposomes
Pharmacology Department, Faculty of Medicine, Casanova
143, E-08018 Barcelona, Spain
Surface activity was determined, as surface pressure
increases, in a Langmuir Balance after injection of
different volumes of a 10 3 mol/L solution of peptide so
that concentrations in the bulk of the aqueous phase
ranged between 6.3 and 50.7 mmol/L.
Insertion in monolayers was carried out following the
same procedure described before but, previous to the
peptide injection, a monolayer of DPPC (dipalmitoyl
phosphatidyl choline) or DPPG (dipalmitoyl phosphati-
dyl glycerol) or their mixtures had been spread on the
surface in order to attain 5, 10 or 20 mN/m of initial
surface pressure. Peptide concentration in the sub-phase
was 12 mmol/L and pressure changes were recorded for
60 min.
Fluorescence experiments were performed in an
Aminco Bowman spectrofluorimeter, equipped with a
thermostated 4 cuvette holder. DPPC liposomes were
saturated either with DPH (1,6-diphenyl-1,3,5-hexa-
triene) or ANS (1-anilino-8-naphthalene sulphonic acid).
Details are given in (4). Peptide solutions 10 5 mol/L
were incubated with labelled liposomes and polarization
fluorescence recorded between 25–55°C of temperature
range.
through resonance energy transfer (RET) measurements.
Two types of vesicles composed either of DPPC or DPPG
containing 0.6% of N-NBD-PE or N-Rh-PE were
incubated with peptide solutions and the fluorescence

Adhesion molecules enable cells to contact and specifi-

cally interact with each other. Members of this group of
molecules are critical in several biological processes
among them cancer metastasis. Laminin is a glycoprotein
clearly involved in adhesion processes (1); one of its
sequences (SIKVAV) is specifically recognized by
receptors overexpressed in several types of tumour cells
and shows important effects concerning neovasculariza-
tion of tumor cells (2, 3). In this paper we have
synthesized an extended sequence of this peptide and
studied its physicochemical characteristics as well as its
interaction with model membranes.

MATERIALS AND METHODS

Synthesis
The peptide was synthesized, (as carboxamide C-ter-
minal), on solid phase using a 4-methylbenzhydrylamine
resin, and Fmoc/OBut strategy. Purification was carried
out by HPLC and the peptide was identified by mass
spectrometry–electrospray. Peptide concentration was Figure 1. Time-course for the adsoption of peptide molecules
determined through quantitative amino acid analysis to the air–water interface. Inset: pressure increase vs. log
using Nle as internal standard. concentration measured 60 min after injection.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

 ABSTRACTS 267

Acknowledgement
This work was financed by the Ministerio of Ciencia y
Tecnologial, Spain, Grant No. SAF-97-0174.

REFERENCES
1. Koshikawa N, Moriyama K, Takamura H, Mizushima H. Cancer
Res. 1999; 21:5596–5601.
2. Davel LE, Puricelli LI, Del Carmen C et al. Joffe ED. Oncol. Rep.
1999; 6(4): 907–911.
3. Nomizu M, Weeks BS, Weston CA, Kim W, Kleinman HK,
Yamada Y. FEBS Lett. 1995; 365: 227–231.
4. Sospedra P, Nagy IB, Haro I et al. Langmuir. 1999; 15: 5111–5117.

Figure 2. Polarization values of ANS-labelled liposomes,

incubated with peptide solution, represented vs. temperature.

In¯uence of lipid composition on the

microviscosity of liposomes

containing 0.3% of both fluorophores in the same vesicle N AlminÄana,1 M. A. Alsina2 and F Reig1
1
were prepared. Peptide Department, Institute for Biological and Environmental
Chemistry, Jordi Girona 18, E-08034 Barcelona, Spain
2
Department of Physicochemistry, Faculty of Pharmacy,
RESULTS AND DISCUSSION University of Barcelona, Avgda. Joan XXIII, s/n E-08028
Barcelona, Spain. E-mail: alsina@farmacia.far.ub.es
The peptide is surface active itself, spontaneously
forming an adsorption monolayer after introduction into
Immunoliposomes have been extensively developed in
the sub-phase. The time course of this process is given in
order to improve the delivery of encapsulated drugs to
Fig. 1. Inset represents surface pressure/peptide concen-
their target cells (1). Several alternatives have been
tration dependence. Peptide interaction with DPPC and
described to link the targeting entity either antibodies,
DPPG monolayers was monitored by measurement of the
peptides or proteins to the liposomal surface. In most
surface pressure increases after injection of peptide under
cases, this process involves the presence of a derivatized
the monolayer. Higher initial surface pressures result in
phospholipid on the bilayer (2). Moreover, in vivo studies
smaller pressure increases, indicating a more restricted
require the presence of PEG (polyethylene glycol) on the
penetration. Comparing the results obtained for the
surface to reduce macrophages uptake (3). As a conse-
different monolayers assayed, one can conclude that
quence, liposomes are often composed of four to five
electrical charge and differences in the chemistry of the
components. The presence of different hydrophobic
polar heads have a small influence in the type of inter-
molecules on the bilayer affects the degree of packing
actions determined. Nevertheless, as a common trend,
of the main components (phospholipids), and can also
pressure increases obtained with DPPG monolayers were
determine the overall stability of the liposomal prepara-
lower than those recorded for DPPC. This behaviour can
tions (4). In this paper we describe the physicochemical
be attributed to a neutralizing effect of positively charged
study of three basic lipid compositions, prepared to
peptide molecules that results in a decrease of repulsion
render liposomes able to be coated with peptides, using
forces between negatively charged PG polar heads.
fluorescence polarization techniques to measure the
Fluorescence studies carried out with ANS indicate
fluidity of the vesicles.
that the peptide interacts with the external part of the
bilayer promoting a decrease of microviscosity, but has
no effect on the polarization values recorded for DPH MATERIALS AND METHODS
labelled vesicles (Fig. 2). The influence of this peptide on
Chemicals
the fusion of vesicles determined by RET indicate that
this sequence even at mmol/L concentrations has no Hydrogenated phosphatidyl choline (PC) was from
effect on this process. The lack of fusion confirms that the Asahi. Phosphatidyl glycerol (PG) and Cholesterol
interaction detected with ANS is only restricted to the (Chol) were from Sigma. N-glutaryl phosphatidyl
external zone of the bilayer and do not destabilize the ethanolamine was synthesized following the description
liposomal entity. given in (5). Distearoyl phosphatidyl ethanolamine-poly-

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

268 ABSTRACTS
ethylenglycol monometoxy (DSPE-PEG), MW = 2000,
and bisuccinimidyl PEG [PEG(ss)2] were from Shear-
water Polymers Inc. and distearoyl phosphatidyl ethanol-
amine (DSPE) was from Sigma. PEG(ss)2 and DSPE were
used to synthesize DSPE-PEGCOOH (6).
Fluorescence experiments
The microviscosity of bilayers was determined through
polarization fluorescence measurements. Liposomes
were prepared with the following compositions (molar
ratio): (a) PC:PG:Chol (26:28:46); (b) PC:PG:Chol:
NGPE (26:17:46:11); (c) PC:PG:Chol:NGPE:DSPE-
PEG (26:12:46:10:6); (d) PC:PG:Chol:NGPE:DSPE-
PEGCOOH (26:12:46:10:6), using an ultrasound probe to
reduce size to diameters under 100 nm. Vesicles were
labelled with either 1,6-diphenyl-1,3,5-hexatriene (DPH)
or 1(4-trimethylamino phenyl)-6-diphenyl-1,3,5-hexa-
triene (TMA-DPH). The polarization of fluorescence
was determined in an Perkin-Elmer luminescence
spectrometer equipped with a thermostated four-cuvette
holder.
RESULTS AND DISCUSSION
Polarization ( p) values are directly related to the
anisotropy (r) of the system and reflect the microviscosity
of bilayers:
2p
rˆ …1†
3 p
High values of anisotropy indicate that the motion of the
Figure 1. Fluorescence anisotropy of: (a) PC/PG/Chol;
(b) PC/PG/Chol/NGPE; (c) PC/PG/Chol/NGPE/DSPE-PEG;
(d) PC/PG/Chol/NGPE/DSPE-PEG-COOH liposomes satu-
rated with DPH recorded as a function of temperature.
Copyright  2002 John Wiley & Sons, Ltd.
fluorescent probe is strongly restricted thus indicating a
rigid environment. As a common trend, the microvis-
cosity decreases as temperature increases. This tendency
can be observed in Fig. 1, where anisotropy (measured
for DPH) is represented in the range of 25–60°C. In
general, anisotropy values are higher than those obtained
in equivalent experiments carried out with pure DPPC (4)
and show a softer temperature dependence. This is due to
the presence of cholesterol that promotes a rigidification
of bilayers. Taking composition 1 as reference one can
appreciate that liposomes’ samples containing DSPE-
PEGCOOH are clearly less fluid than those lacking this
compound ( p < 0.001). Other compositions show no
important differences among them. As far as the
anisotropy values determined with TMA-DPH (that
reflects the motion at the external polar zone of the
bilayer) is concerned, the graphics depicted in Fig. 2,
show that in this experience the presence of DSPE-
PEGCOOH chains has also a stabilizing effect in the
ordered state of lipids ( p < 0.001). Besides, with this
probe the presence of DSPE-PEG (as monomethoxy) has
also a rigidifying effect, lower, but still significant
compared to compositions 1 and 2. ( p < 0.05). As a
summary, all these liposomal compositions are stable
enough to be developed as drug carriers.
Acknowledgements
This work was financed by Grant No. 2FD97-0129 from
the Ministerio de Educación y Cultura, Spain, and a
predoctoral grant for N. Almiñana from the Ministerio de
Ciencia y Tecnologı́a, Spain.
Figure 2. Fluorescence anisotropy of: (a) PC/PG/Chol;
(b) PC/PG/Chol/NGPE; (c) PC/PG/Chol/NGPE/DSPE-PEG;
(d) PC/PG/Chol/NGPE/DSPE-PEG-COOH liposomes satu-
rated with TMA-DPH recorded as a function of temperature.
Luminescence 2002;17:191–291

REFERENCES
1. Mastrobattista E, Koning GA, Storm G. Immunoliposomes for the
targeted delivery of antitumor drugs. Adv. Drug Delivery Rev. 1999;
40: 103–127.
2. Allen TM, Moase EH. Therapeutic opportunities for targeted lipo-
somal drug delivery. Adv. Drug Delivery Rev. 1996; 21: 117–133.
3. Papahadjopoulos D, Allen TM, Gabizon A et al. Sterically
stabilized liposomes: Improvements in pharmacokinetics and anti-
tumor therapeutic efficacy. Proc. Natl Acad. Sci. USA 1991; 88:
11460–11464.
4. Polo D, Haro I, Alsina MA, Reig F. Physicochemical characteriza-
tion of poly(ethylene glycol)-coated liposomes loaded with doxo-
rubicin. Langmuir 1997; 13: 3953–3958.
5. Holrnberg E, Maruyama K, Litzinger DC et al. Highly efficient
immuliposomes prepared with a method which is comparable with
various lipid compositions. Biochem. Biophys. Res. Commun. 1989;
165: 1272–1278.
6. Maruyama K, Takizawa T, Yuda T, Kennel SJ, Huang L, Iwatsuru
M. Targetability of novel immunoliposomes modified with amphi-
pathic poly(ethylene glycol)s conjugated at their distal terminals to
monoclonal antibodies. Biochim. Biophys. Acta 1995; 1234: 74–80.
Applications of bioluminescent and
bio¯uorescent microbiological organisms in
the analysis of toxicity
Andrew W. Knight,1 Nicholas Goddard,1
Peter Fielden,1 Nicholas Billinton,2 Paul Cahill,2
Gordon Barker,2 Patrick Keenan2 and
Richard Walmsley2
1 as an enzyme or antibody. Such sensors are usually rapid
Department of Instrumentation and Analytical Science,
University of Manchester Institute of Science and Technology,
PO Box 88, Manchester M60 1QD, UK. E-mail:
a.knight@gentronix. co.uk
2 biological organisms. A more relevant picture is obtained
Department of Biomolecular Sciences, University of
Manchester Institute of Science and Technology, PO Box 88,
Manchester M60 1QD, UK
There is a growing need in a wide range of industries for
rapid, sensitive and inexpensive methods for the
measurement of toxicity and assessment of risk to the
environment and to man. In the environmental field,
water providers need to protect their treatment plants
from the influx of toxic wastes, whilst also ensuring that
the supply of drinking water is uncontaminated. Chemi-
cal industries are increasingly having to monitor the
toxicity of effluents discharged into surface waters as new
regulations governing pollution prevention are enforced.
Chemical manufacturers are bound to assess the toxic
characteristics of any new chemical produced for
industrial or household use. Historically, environmental
toxicity testing involved characterizing the levels of
specific species, such as a heavy metal or pesticide, and
comparing to known toxicity thresholds. However, this
approach misses unexpected toxicants that the analyst is
not specifically looking for, as well as synergistic effects.
Furthermore, this approach often does not take into
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 269
account the bioavailability of the toxicant. Tests are
required to rapidly assess and warn of toxicity in
environmental samples, following which detailed labora-
tory analysis can be made.
In the pharmaceutical industry, most drugs undergo a
lengthy development and approval program lasting up to
10 years. Government agencies set strict guidelines for
toxicological testing, which involve a range of both
cellular (in vitro) and animal studies, to predict how the
drug will behave in man. Such tests are often expensive
and time consuming, and hence there is a need for pre-
screens as predictors for toxicity which would have only
become evident later in the drug development process. In
this way, potentially toxic compounds can be filtered out
early on, saving the costs of bringing them through to
regulated testing. A drug can be toxic by many mech-
anisms; however, drug-induced mutagenesis of DNA is
one of the most significant. Of particular interest are tests
for detecting both general cellular (cyto) toxicity and
specific genotoxicity, a measure of the compound’s
ability to induce DNA damage and hence its potential to
be a carcinogen. Rapid screening tests are required that
consume very low quantities of compound and can be
performed at a high throughput of up to thousands of
compounds/day.
In both the environmental and pharmaceutical fields, to
assess risk to biological species, the use of a biosensor is
the most pharmacologically relevant approach. Many
biosensors for toxicity employ a component of cells, such
and sensitive, but lack robustness and generally have
short shelf-lives. The sensors also reproduce only a small
part of the mechanism of the interaction of toxicants with
when a whole organism is present. Microorganisms such
as bacteria and yeasts provide a neat solution. They can
be cultured easily and inexpensively from frozen or
freeze-dried stocks, and their small size, simple mor-
phology and large surface area increases their sensitivity
compared to larger, more complex organisms. Examples
of microorganisms in toxicity biosensors have been
reported using a variety of measurement techniques,
including amperometric, colourimetric and thermal.
However, optical methods of detection are the most
convenient and are non-invasive and generally the most
free from interference.
Methods of toxicity testing using naturally biolumi-
nescent microorganisms, such as the marine bacterium
Vibrio fischeri, are well characterized and commercially
available as the Microtox2 system, amongst others.
Bioluminescence depends on respiratory metabolism,
hence any substance that disrupts the normal cellular
metabolism and so compromises cell viability leads to a
reduction in light emission. The test is extremely rapid,
giving acute toxicity results in minutes. A significant
disadvantage, however, is that samples must be adjusted
Luminescence 2002;17:191–291
270 ABSTRACTS
to 2% sodium chloride for osmotic protection of the
marine organism.
The advent of genetic engineering has allowed the
creation of genetically modified organisms whose gene
function has been altered or into which genes from
another species have been introduced. The Mutatox2
system uses a genetically modified dark variant of Vibrio
fischeri to detect genotoxins (1). In response to DNA
damage, a protease is expressed that breaks down a
repressor protein of the light-producing pathway, leading
to luminescence, which serves as a measure of genotoxi-
city. Vitotox2, commercialized by Labsystems, is a
method for screening pharmaceuticals for cyto- and
genotoxicity using Salmonella bacteria genetically modi-
fied to contain the Vibrio fischeri luciferase gene, which
is expressed in response to DNA damage (2). The Vitotox
assay also uses a second strain in which the luciferase
gene is under the control of a highly active constitutive
promoter. This ensures that a positive genotoxicity signal
is not a result of cell death or a non-specific enhancement
in luminescence.
Our own work has been to develop a yeast-based
genotoxicity and cytotoxicity test for both automated,
high-throughput, pharmaceutical screening and environ-
mental monitoring (3). Unlike toxicity tests based on
bacteria (prokaryotes), yeast is a eukaryotic organism.
Since humans are eukaryotes, and share many of the
same structural and biochemical characteristics in our
cells, so the results of the test are more relevant for
human risk assessment. The cells are genetically
modified to express a yeast-enhanced green fluorescent
protein (GFP) under the control of the promoter for a
native yeast gene, known to be specifically upregulated
by the cells in response to DNA damage. Thus, on
exposure to a genotoxic agent the cells become increas-
ingly fluorescent as GFP accumulates. GFP has the
advantage of possessing favourable properties, such as
low toxicity, high chemical and photostability, and the
ability to spontaneously form a fluorophore upon
expression without the need for additional reagents and
co-factors. In the assay the sample is combined with a
culture of the yeast in a specialized growth medium and
then incubated overnight. A single measurement is
subsequently made of fluorescence and cell density.
Fluorescence is used to indicate the presence, concentra-
tion or potency of a genotoxin, whilst the cell density
provides a measure of cytotoxicity, which restricts cell
proliferation during incubation.
Initial development of the assay was carried out using
flow-through instrumentation to study the kinetics of
gene expression and fluorescence induction in response
to genotoxic materials. The assay has since been
developed in a variety of formats. For environmental
monitoring, a small portable instrument has been
designed for use with a cuvette-based assay capable of
measuring the cytotoxicity and genotoxicity of aqueous
Copyright  2002 John Wiley & Sons, Ltd.
Figure 1. Schematic of the Gentronix cytotoxicity and
genotoxicity assay. Cytotoxic samples restrict cell proliferation
in a quantitative way. Genotoxic samples also induce the
expression of green fluorescent protein in the yeast cells,
resulting in enhanced green fluorescence upon illumination
with blue light. The figure’s background shows a microscope
image of fluorescent yeast cells.
samples collected in the field. In addition, Gentronix Ltd.,
a spin-off company from UMIST in Manchester, is in the
process of developing this technology to produce an
automated 96- and 384-well microplate-based assay (Fig.
1) for high-throughput pharmaceutical screening, using
commercially available liquid handling robotics. In a
separate project, funded by the European Space Agency,
current work is also focused on the use of cells
immobilized in a gel matrix to form a badge-type sensor.
This is intended for use by pilots and astronauts, to assess
the risk of DNA damage resulting from higher levels of
radiation present in the upper atmosphere and in space.
Our group has also used genetic modification to
incorporate metabolic competency, which is largely
absent in the wild-type strain. Yeast strains have been
modified to include cytochrome P450 genes to allow the
metabolic activation, and hence detection, of pro-
mutagenic species, such as certain polyaromatic hydro-
carbons.
An ever-present challenge is the interference posed by
the analysis of environmental samples with high levels of
particulate or other light absorbing material, or that are
inherently autofluorescent. In the case of luminescence
methods these problems are addressed by the use of
kinetic rather than absolute light measurements, and in
the case of GFP expressing organisms by employing
fluorescence polarization to exploit the high fluorescence
anisotropy of GFP compared to other autofluorescent
species in the sample. Finally, there is the matter of the
need to gain public confidence and acceptance in the use
of genetically modified organisms. This is best addressed
by the use of strains whose proliferative potential is
Luminescence 2002;17:191–291

restricted without specialized growth media, and selec-
tion of markers other than antibiotic resistance genes.
Fluorescent and luminescent microorganisms provide
compact and robust biosensors, with an easily interpreted
response, for the analysis of toxicity. Being whole
organisms, they produce results which more accurately
predict risk to higher animals and to man.
REFERENCES
1. http://www.azurenv.com
2. http://www.thermo.com
3. http://www.gentronix.co.uk
Development and application of
chemiluminescence ¯ow-injection
immunoassays
Gerald GuÈbitz
Institute of Pharmaceutical Chemistry, Karl-Franzens University
Graz, UniversitaÈtsplatz 1, A-8010 Graz, Austria. E-mail:
guebitz@hermes.kfunigraz.ac.at
Immunoassays have found broad application in biochem-
istry, clinical chemistry, pharmaceutical analysis and
environmental analysis due to their selectivity and
sensitivity. Classical immunoassay techniques are rather
time-consuming and not always easy to handle. More and
more research is being done using flow analysis tech-
niques for immunoassays (1). In addition to spectro-
photometric, fluorometric and electrochemical detection,
chemiluminescence detection has attracted increasing
attention in flow-injection immunoassays (FIIA) with
respect of its sensitivity (2). We developed one of the first
chemiluminescence flow-injection immunoassays using
an on-column chemiluminescence detection principle.
The central part in this system is the immunoreactor
column, which consists of a piece of Teflon tubing
Figure 1. Instrumental set-up in the FIA mode: a, assay buffer; b, regeneration buffer;
1, pump; 2, switching valve; 3, injection valve; 4, photomultiplier; 5, immunoreactor; 6, PC.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 271
packed with immobilized antibodies or haptens, respec-
tively. This immunoreactor is mounted instead of the
flow-cell in the front of the PMT. All reactions, including
both the immunoreaction and the chemiluminescence
reaction, take place in this immunoreactor cell. Emitted
light is collected directly from this column. The set-up is
based on a simple one-pump FIA system (Fig. 1). On this
basis we developed different formats of flow-injection
immunoassays. The first type developed was a two-site
assay for mouse immunoglobulin G (IgG) (3). In the first
step, sample mouse IgG is injected, which binds to
immobilized anti-mouse IgG antibodies. In the second
step, anti-mouse IgG antibodies labelled with an
acridinium ester are injected, which bind to second
binding sites of the IgG, followed by hydrogen peroxide
to initiate the chemiluminescence reaction. This type of
assay is applicable to large molecules, such as proteins.
An increase in sensitivity can be obtained by involving
the biotin–streptavidin binding system. In this case
streptavidin is labelled with the acridinum ester and
biotin labelled antibodies are used. Streptavidin can be
labelled to a much higher extent than other proteins. One
molecule, streptavidin, can bind up to eight molecules of
acridinium label. With this modification, the detection
limit can be decreased to the low attomole level. Since
the acridinium-labelled streptavidin can be used for
different analytes, this approach is more flexible. More-
over, several biotin-labelled antibodies are commercially
available. Another variant is a competitive format,
applied to the determination of human IgG, where
sample IgG and acridinium-labelled IgG compete for
the binding sites at the immobilized antibodies (4). This
format is also applicable to small molecules and was
applied, for example, to the determination of triiodothy-
ronine (T3) (5) and digoxin (6). In addition to a FIA set-
up, a modified sequential injection set-up was also used.
Contrary to FIA, where the components are injected into
a flowing stream which transports them to the reactor, in
sequential-injection analysis (SIA) all components are
injected sequentially as plugs directly into the reactor,
Luminescence 2002;17:191–291
272 ABSTRACTS
Figure 2. Scheme of the enantioselective immunoassay.
using a syringe pump system with electronically
controllable valves. In the case of the digoxin assay, the
biotin–streptavidin system was involved. Sample digoxin
and biotin-labelled digoxin compete for the binding sites
at the immobilized antibodies. In the next step,
acridinium-labelled streptavidin is injected, which
strongly binds to the biotin moiety of the labelled
digoxin molecules. The chemiluminescence reaction is
again initiated by injection of hydrogen peroxide.
Recently, we developed enantioselective FIIAs and
SIIAs for amino acids using enantioselective antibodies
(Fig. 2). Antibodies raised against D- or L- p-amino-
phenylalanine as a hapten were found to respond
enantioselectively, not only to the hapten but also to a
broad spectrum of amino acids. The antibodies are
labelled with the acridinium ester and the immunoreactor
contains immobilized D-or L- p-aminophenylalanine. A
mixture of sample amino acid and labelled antibodies,
preincubated for 20 min, is injected. Excess antibodies
bind to the immobilized hapten. The chemiluminescence
signal obtained after injection of hydrogen peroxide is
indirectly proportional to the concentration of amino
acid. If antibodies against D-amino-Phe and as immo-
bilized hapten D-amino-Phe are used, only D-amino acid
enantiomers are recognized by the antibodies, resulting in
a decrease of the signal, while the signal for L-enantio-
mers shows the same height as the blank and vice versa.
One assay cycle in a fully automated system takes 5 min.
These antibodies were also found to respond enantio-
selectively to thyroxin (T4) and triiodothyronine (T3).
Copyright  2002 John Wiley & Sons, Ltd.
While the L-enantiomers are the naturally occurring
thyroid hormones, the D-enantiomers reduce the choles-
terol level in blood. The absence of traces of the
L-enantiomers in pharmaceutical preparations is of great
importance. With this approach it is possible to detect
less than 0.01% of the L-enantiomers in samples of the
D-enantiomers. In another variant, commercially avail-
able anti-L-T3 antibodies were used, which were found to
respond enantioselectively to L-T3 with increased
sensitivity.
REFERENCES
1. Gübitz G, Shellum C. Flow-injection immunoassay: review. Anal.
Chim. Acta 1993; 283: 421–428.
2. Gübitz G, Schmid MG, Silvaieh H, Aboul-Enein HY. Chemilumi-
nescence flow-injection immunoassays. Crit. Anal. Chem. 2001; 31:
167–174.
3. Shellum C, Gübitz G. Flow-injection immunoassay with acridinium
ester-based chemiluminescence detection. Anal. Chim. Acta 1989;
227: 97–107.
4. Hacker A, Hinterleitner M, Shellum C, Gübitz G. Development of
an automated flow injection chemiluminescence immunoassay for
human immunoglobulin G. Fresenius J. Anal. Chem. 1995; 352:
793–796.
5. Dreveny D, Klammer C, Michalowski J, Gübitz G. Flow-injection-
and sequential-injection immunoassay for triiodothyronine using
acridinium ester chemiluminescence detection. Anal. Chim. Acta
1999; 398: 183–190.
6. Dreveny D, Michalowski J, Seidl R, Gübitz G. Development of
solid-phase chemiluminescence immunoassays for digoxin compar-
ing flow injection and sequential injection techniques. Analyst 1998;
123: 2271–2276.
Luminescence 2002;17:191–291

Luminescent bacteria and honeybees:
bioindicators in environmental monitoring
S. Girotti,1 L. Bolelli,1 F. Fini,1 S. Ghini,1 C. Porrini,1
G. Sabatini,1 M. Musiani,2 G. Gentilomi,2
G. Andreani,3 E. CarpeneÂ3 and G. Isani3
1
Istituto Scienze Chimiche, UniversitaÁ di Bologna, Via San
Donato 15, I-40126 Bologna, Italy. E-mail: girotti@biocfarm.
unibo.it
2
Dipartimento di Medicina Clinica Specialistica e Sperimentale,
Via Massarenti 9, Bologna, Italy
3 monitor coastal pollution.
FacoltaÁ di Medicina Veterinaria, Via Tolara di Sopra 50, I-40064
Ozzano Emilia, Bologna, Italy
Toxic pollutants are found everywhere in the environ-
ment and their early detection is of primary importance to
avoid damages. Bioindicators are very useful tools,
serving as early warnings that a community or an
ecosystem is being degraded. Several organisms can
work as bioindicators, and among them are luminescent
bacteria and honeybees.
A bioassay for monitoring of toxic compounds
(mercury, lead and BTEX at several concentrations) has
been developed based on bioluminescent bacteria. In vivo
luminescence is a sensitive indicator of xenobiotic
toxicity: if noxious substances are present, the lumi-
nescence decreases proportionally to their concentration
(1). The assay was performed at room temperature and
proved to be rapid and versatile, even if unspecific.
Bacteria could be easily stored at 20°C and transported
Figure 1. Mussels grown in water polluted with different concentration of cadmium were analysed
using the luminescent bacteria toxicity test. The luminescent emission (RLU) was recorded up to 1 h.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 273
after lyophilization. The use of 96-well microplates
allowed reducing the working volumes and therefore the
cost per assay.
Mussels grown in cadmium-polluted water were also
analysed using the luminescent bacteria toxicity test.
After homogenization, mussels were directly tested and
polluted samples at 4 ppm were distinguished from
controls (mussels grown in non-polluted water) (Fig. 1).
Mussels in association with bioluminescent bacteria
could therefore represent a possible bioindicator to
The luminescent bacteria toxicity test, compared with
other bioassays, proved that its average sensitivity is well
within the same order of magnitude as the other tests (2).
However, it is acknowledged that the ‘battery of tests’
approach, utilizing several different short-term biological
tests, would be preferred in any monitoring scheme.
Honeybees are used in the monitoring of heavy metals
pollution in cities and industrial areas, in the detection of
radionuclides in several other environments, and in
monitoring projects in the control of pesticides in
agroecosystems (3).
Recently our research group demonstrated that honey-
bees can be used for detection of phytopathogenic
microorganisms (4). The method was tested on Erwinia
amylovora, the causal agent of fire blight, the most
destructive bacterial disease of rosaceous plants. A new
molecular diagnostic technique (PCR–ELISA) for the
automated detection of E. amylovora in pollen, based on
the immunoenzymatic determination of PCR products,
Luminescence 2002;17:191–291
274 ABSTRACTS
Figure 2. Determination of Erwinia amylovora in pollen
samples from environmental monitoring analysed with PCR–
ELISA and with the official method.
was developed to improve the specificity and detection
limit of standard analytical methods. At least one pollen
sample from all the beehives placed in infected area
resulted positive to test. A pollen sample collected from a
bordering area that did not appear contaminated was
found positive, and in this area the disease occurred some
months lather (Fig. 2). Border areas hit by ‘fire blight’
can therefore be constantly monitored using beehives,
thus enabling prevention the extension of the disease.
REFERENCES
1. Girotti S, Ferri EN, Bolelli L, Sermasi G, Fini F. Applications of
bioluminescence in analytical chemistry. In Chemiluminescence in
Analytical Chemistry, Garcia-Campaña AM, Baeyens WRG (eds).
Marcel Dekker: New York, 2001; 247–284.
2. Kudryasheva N, Kratasyuk V, Esimbekova E, Vetrova E, Nemtseva
E, Kudinova I. Development of bioluminescent bioindicators for
analysis of environmental pollution. Field Anal. Chem. Technol.
1998; 2: 277–280.
3. Rossi S, Dalpero AP, Ghini S, Colombo R, Sabatini AG, Girotti S.
Multi-residual method for gas chromatography analysis of pesti-
cides in honeybees cleaned by gel permeation chromatography. J.
Chromatogr. A 2001; 905(1–2): 223–232.
4. Merighi M, Sandrini A, Landini S et al. Chemiluminescent and
colorimetric detection of Erwinia amylovora by immunoenzymatic
determination of PCR amplicons (PCR–ELISA) from plasmid
pEA29. Plant Dis. 2000; 84: 49–54.
Chemiluminescence detection for ®eld-¯ow
fractionation
P. Reschiglian,1 A. Zattoni,1 B. Roda,1 M. Guardigli,2
D. Melucci1 and A. Roda2
1
Department of Chemistry `G. Ciamician', Via Selmi 2, I-40126
Bologna, Italy. E-mail: resky@ciam.unibo.it
2
Department of Pharmaceutical Sciences, Via Belmeloro 6,
I-40126 Bologna, Italy
Copyright  2002 John Wiley & Sons, Ltd.
INTRODUCTION
Field-flow fractionation (FFF) is a family of chromato-
graphic-like techniques in which separation is performed,
rather than by the interaction with a stationary phase,
through an external field that is applied perpendicularly
to the mobile phase flow (1). The workhorse for detection
in FFF has been the UV/Vis spectroscopy (2). However,
as in conventional liquid chromatography, an increase in
specificity and sensitivity can be sought through coupling
FFF to other detection devices able to increase the
amount of analytical information obtained for a given
sample. Chemiluminescence (CL) detection has been
already applied successfully to several flow-through
analytical techniques, including flow injection analysis
(FIA) and separative methods such as pre- and post-
column HPLC (3) and capillary electrophoresis (4).
In this work a feasibility study on FFF–CL coupling is
presented for the first time. Model samples consisting of
micron-size polystyrene beads (PS) coated with enzymes
suitable for CL detection are used for the development of
offline and online FFF–CL. Offline FFF–CL potential-
ities are explored with flow FFF (FlFFF), while an online
FFF–CL system with the highly simple subset of
sedimentation FFF, gravitational FFF (GFFF), is pre-
sented.
MATERIALS AND METHODS
Test samples were PS 6.10  0.57 or 3.00  0.14 mm in
diameter (Polysciences, Inc., Warrington, PA), coated
with horseradish peroxidase (HRP; EC 1.11.1.7, Type
VI-A, from Sigma Chemical Co, St. Louis, MO, USA) or
alkaline phosphatase (AP; EC 3.1.3.1, from Roche
Diagnostics, Mannheim, Germany).
The FlFFF fractionator was derived from the commer-
cial model F-1000 Universal Fractionator (FFFractiona-
tion LLC, Salt Lake City, UT) (5), and it was connected
to a conventional UV/Vis detector for HPLC. Samples
were eluted in pure, Milli-Q-grade water. Fractions of
200 mL FlFFF effluent were collected from the outlet of
the UV/Vis detector into a 96-well microtitre plate
(White Cliniplate, Labsystems Oy, Helsinki, Finland).
The proper chemiluminescent substrate (100 mL) was
then added to the fractions in each well. Substrates were
the commercial ECL1 (Amersham Pharmacia Biotech,
Amersham, UK) and LumiPhos1 Plus (Lumigen, South-
field, MI, USA) for the CL detection of HRP and AP,
respectively.
The GFFF channel was home-built (6) and connected
to a conventional UV/Vis detector for HPLC. The outlet
of the UV/Vis detector was directly fed to a flow-cell
luminometer (Lumiflow, Immunotek, Moscow State
University, Moscow, Russia). The mobile phase em-
ployed for GFFF of PS (TRIS buffer 0.01 mol/L, pH 8.6,
‡ SDS) was modified by adding the substrate for HRP
Luminescence 2002;17:191–291

Figure 1. Specificity and detectability in offline FlFFF–CL.
Comparison with UV/Vis detection.
(30 ppm hydrogen peroxide) to induce en route CL from
the oxidation of 1 mmol/L luminol. An enhancer
(10 mmol/L p-iodophenol) was also added to increase
analytical sensibility and CL signal stability.
RESULTS
In Fig. 1 the online FlFFF-UV/Vis and the offline FlFFF-
CL fractograms are compared for a mixture of PS/HRP
3 mm and PS/AP 6 mm. As expected, the UV/Vis
fractogram is not specific in terms of detection of
different coated enzymes. Nevertheless, when the two
specific CL substrates for HRP and AP are independently
used for the CL detection of each enzyme, only the band
relevant to the enzyme in question appeared in the
FlFFF–CL fractogram. This result is a proof of the
interesting applicability of offline FFF–CL to multi-
analyte detection of particles of different size that are
coated with different CL-catalysing enzymes.
Figure 2. Detectability in online GFFF–CL. Comparison
with UV/Vis detection. Two repeated runs (1, 2) are super-
imposed to show the excellent inter-run reproducibility.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 275
Fig. 2 shows the fractograms obtained with the online
GFFF–CL system for the injection of 10 mg PS/HRP
3 mm. The increase in signal detectability achieved by CL
with respect to UV/Vis detection is evident, as well as the
reduced noise level in CL detection. The limit of
detection determined for online GFFF–CL, expressed in
mass of PS, is found to be about 12 ng. This corresponds
to a detectable amount of immobilized HRP as low as
10 pg (ie about 0.2 fmol), if the mass ratio between
immobilized HRP and supporting PS is estimated. If
compared to UV/Vis, a reduction of two orders of
magnitude is observed. These findings indicate interest-
ing perspectives in the framework of future developments
of FFF–CL-based immunoassays.
REFERENCES
1. Giddings JC. Field-flow fractionation: analysis of macromolecular,
colloidal, and particulate materials. Science 1993; 260: 1456–1465.
2. Reschiglian P, Zattoni A, Melucci D, Torsi G. Quantitative analysis
by UV-Vis detection in flow-assisted separation techniques for
dispersed samples. Part I: theory; Part II: experimental tests with
applications to field-flow fractionation. Rev. Anal. Chem. 2001; 20:
239–269.
3. Kuroda N, Kai M, Nakashima K. Chemiluminescence detection in
liquid chromatography. In Chemiluminescence in Analytical
Chemistry, Garcia-Campaña AM, Baeyens WRG (eds). Marcel
Dekker: New York, 2001; Chapter 14, 393–425.
4. Garcia-Campaña AM, Baeyens WRG, Guzman NA. Chemilumi-
nescence detection in capillary electrophoresis. In Chemilumi-
nescence in Analytical Chemistry, Garcia-Campaña AM, Baeyens
WRG (eds). Marcel Dekker: New York, 2001; Chapter 15, 427–472.
5. Reschiglian P, Melucci D, Zattoni A et al. Working without
accumulation membrane in flow field-flow fractionation. Anal.
Chem. 2000; 72: 5945–5954.
6. Reschiglian P, Zattoni A, Casolari S, Chmelı́k J, Krumlova A,
Budinska M. Size characterization of barley starch granules by
gravitational field-flow fractionation: a rapid, low-cost method to
assess the brewing capability of different strains. Ann. Chim. (Rome)
2002; 92 (in press).
Real-time imaging of micronsize particles
separation by ®eld-¯ow fractionation with
chemiluminescence detection: a useful tool
for probing retention mechanisms at
ultra-low detection limits
A. Roda,1 M. Guardigli,1 B. Roda,2 A. Zattoni,2
D. Melucci2 and P. Reschiglian2
1
Department of Pharmaceutical Sciences, Via Belmeloro 6,
I-40126 Bologna, Italy. E-mail: roda@alma.unibo.it
2
Department of Chemistry `G. Ciamician', Via Selmi 2, I-40126
Bologna, Italy
INTRODUCTION
Field-flow fractionation (FFF) is a family of chromato-
graphic-like techniques that are suited for separating and
Luminescence 2002;17:191–291
276 ABSTRACTS
Figure 1. CL images of the GFFF channel obtained during
relaxation and elution of a PS/HRP sample.
characterizing analytes in suspension, such as macro-
molecules, nanoparticles, colloids and micro-sized par-
ticles (1). Sample detection in FFF is usually performed
using spectroscopic tecniques (e.g. UV-Vis (2) and
fluorescence spectrophotometry or light scattering).
However, all these detection systems employ flow-
through detector cells that are positioned downstream
the separation channel. As a consequence, real-time
information on the fractionation mechanisms occurring
while particles are eluting along the FFF separation
channel is lacking.
In this work, we describe for the first time the real-time
imaging of a micron-sized particles separation in FFF.
We employed the gravitational FFF (GFFF) subtech-
nique, which is well suited for separating micron-sized
particles of biological and diagnostic interest (3) and
images the fractionation process using chemilumi-
Figure 2. CL image of the GFFF channel during elution of a mixture of PS/HRP and free HRP (lower left panel), CL signal profile
within the GFFF channel (upper left panel), and reconstructed CL signal at the channel outlet (right panel).
Copyright  2002 John Wiley & Sons, Ltd.
nescence (CL) (4). Thanks to the high detectability of
the CL signal, it was possible to visualize the overall
fractionation process at very low sample loads, thus
providing information on the fractionation mechanisms
in nearly ideal conditions.
MATERIALS AND METHODS
Model samples (PS/HRP) were polystyrene beads
6.10  0.57 mm in diameter (Polysciences Inc., Warring-
ton, PA) covalently coated with a monolayer of horse-
radish peroxidase (HRP; EC 1.11.1.7, Type VI-A, from
Sigma Chemical Co., St. Louis, MO).
The GFFF fractionator was home-built, as recently
described (5), and simply replaced the column in a
conventional HPLC system. The fractionator’s walls
were made of transparent polycarbonate and polyvinyl-
chloride, thus allowing visualization of the CL emission
from within the channel. Mobile phase was Milli-Q-grade
water containing TRIS buffer 10 mmol/L, pH = 8.7, and
0.05% w/v sodium dodecyl sulphate. In order to obtain a
stable HRP-triggered CL signal during elution, the
chemiluminescent HRP substrates (1 mmol/L luminol
and 30 ppm H2O2) and an enhancer (10 mmol/L p-iodo-
phenol) were directly added to the carrier.
The GFFF channel was placed within a low-light
luminograph (Night Owl LB 981, EG&G Berthold, Bad
Wildbad, Germany) equipped with a back-illuminated,
double Peltier-cooled CCD camera. Images of the light
emitted from the channel were collected at fixed time
intervals during elution, using a 5 s acquisition time. The
acquired images were then elaborated using MatLab 6.0
Luminescence 2002;17:191–291

(The MathWorks, Natick, MA) to reconstruct the
fractionation process and to simulate the analytical CL
signal at the fractionator outlet.
RESULTS
Fig. 1 shows two representative CL images obtained
during the relaxation and elution processes of a PS/HRP
sample (7.5 mg). It can be seen that the real-time CL
imaging allows us to obtain information about the
fractionation process that can be hardly achieved from
a standard fractogram, obtained using a post-column
detector. The kinetics profile and the shape of the eluting
bands within the channel can be analysed, providing
more insights into GFFF basics. Direct observation of
sample interaction with the channel walls is also
possible, which can make easier optimization of sample
recovery as well as a more as a more direct analysis of
other factors that can influence sample relaxation before
the elution.
Fig. 2 reports a CL image obtained for the fractionation
process of a mixture of PS/HRP (7.5 mg) and free HRP
(1.25 ng), along with the CL signal profile within the
channel and the corresponding reconstructed CL signal at
the channel outlet. It is shown that bead-immobilized and
free HRP can be easily separated and detected by GFFF-
CL, even if they are present in different relative propor-
tions.
These results suggest the possibility to use real-time
GFFF-CL imaging for the development of multi-analyte
immunoassays or innovative methods for cell sorting,
based on highly specific CL labelling.
REFERENCES
1. Giddings JC. Field-flow fractionation: analysis of macromolecular,
colloidal, and particulate materials. Science 1993; 260: 1456–
1465.
2. Reschiglian P, Zattoni A, Melucci D, Torsi G. Quantitative analysis
of dispersed analytes by flow-through UV-Vis spectroscopy for
field-flow fractionation and other separation techniques. G. Rev.
Anal. Chem. 2001; 20: 239–269.
3. Cardot PJP, Gerota J, Martin M. Separation of living red blood cells
by gravitational field-flow fractionation. J. Chromatogr. 1991; 568:
93–101.
4. Roda A, Pasini P, Musiani M et al. Chemiluminescent low-light
imaging of biospecific reactions on macro- and microsamples using
a videocamera-based luminograph. Anal. Chem. 1996; 68: 1073–
1080.
5. Reschiglian P, Zattoni A, Casolari S, Chmelı́k J, Krumlova A,
Budinska M. Size characterization of barley starch granules by
gravitational field-fractionation: a rapid, low-cost method to assess
the brewing capability of different strains. Ann. Chim. (Rome) 2002;
92 (in press).
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 277
Molecular and atomic laser-induced
¯uorescence with an OPO system:
experimental set-up and applications
P. Giamarchi,1 L. Burel,1 L. Stephan,1 Y. Lijour2 and
A. Le Bihan1
1
UMR - CNRS 6521. E-mail: Philippe.Giamarchi@univ-brest.fr
2
Department of Chemistry, University of Bretagne Occidentale,
6 Av. Le Gorgeu, BP 809, 29285 Brest Cedex, France UMR -
CNRS 6521
A set-up composed of a Nd:YAG laser coupled with an
optical parametric oscillator (LYOPO) (1), connected to
a spectrophotometer and a high-sensitivity camera,
permits one to achieve both molecular and atomic
fluorescence spectra (2, 3). Due to its architecture, it
combines the sensitivity of laser-induced fluorescence
(LIF) with intensively charged coupled device (ICCD)
detection, the selectivity resulting from OPO tunability
and the benefits of pulsed laser-offered time resolution.
The molecular fluorescence of organic pollutants, eg
polyaromatic hydrocarbons (PAHs), at ultra-trace levels
can be measured without any prior preconcentration or
separation procedures. The molecules’ photobleaching
due to the high beam energy is continuously compensated
by the use of a flow cell.
Time resolution enables one to choose the appropriate
time delay between the excitation and fluorescence
measurements. As the matrix fluorescence has a shorter
lifetime than most of the PAHs, this time delay enhances
the method’s sensitivity by eliminating the matrix
fluorescence of natural samples (Fig. 1). By direct
analysis, we measured benzo[a]pyrene detection limits
of 0.7 ng/L in drinking water, where the legislative limit
is 10 ng/L, and 4 ng/L in raw water containing 1 mg/L
humic acids.
Atomic fluorescence of metals ultra-traces was studied
using electrothermal atomization time of 14 ns; the
optimum gate-width and gate delay were selected in
order to maximize the signal-to-noise ratio (Fig. 2).
However, it is possible to avoid the use of nitric acid
modifier to diminate any contamination risk, which
constitutes an important advantage for ultra-trace analy-
sis. Of course, in this case, natural samples have to be
frozen just after sampling.
The detection limit obtained for lead analysis in
seawater was 1 ng/L. In this case, addition of oxalic acid
modifier was needed to separate the atomization of lead
atoms from that of the matrix molecules.
Future developments of the atomic-LIF techniques will
be conducted on iron and other metals in the same matrix.
These results demonstrate the capabilities of the
LYOPO system to quickly detect organic pollutants and
metal contamination at ultra-trace levels through direct
measurements. In the near future our investigations will
focus on increasing sensitivity and improving selectivity
Luminescence 2002;17:191–291
278 ABSTRACTS

Figure 1. Fluorescence spectrum time decrease of a 1.24 mg/L benzo[a]pyrene solution in a matrix of
1 mg/L humic acid at lEX = 295 nm. The gate-width was set to 5 ns and gate time delay was incremented
by 5 ns between successive spectra. The x axes are in nanometers, whereas the y axes are in arbitrary
fluorescence intensity units.

Figure 2. Signal-to-noise ratio variation vs. the gate-width.

by developing multi-component determination in mix-
tures (4), using both the LIF excitation–emission matrix
and temporal resolution with data analysis techniques,
such as principal components regression and partial least
square.
REFERENCES
1. Zhou JX, Hou X, Kang KX, Tsai SJ, Michel RG. Lasers based on
optical parametric devices: wavelength tunability empowers laser-
based techniques in the UV, visible and near IR. Appl. Spectrosc.
1998; 52: 176A–189A.
2. Gooijer C, Mank AJG. Laser spectroscopy in analytical chemistry:
light on the next millennium. Anal. Chim. Acta 1999; 400: 281–
295.
3. Stchur P, Yang KX, Hou X, Sun T, Micher RG. Laser excited
atomic fluorescence spectrometry—a review. Spectrochim. Acta B
2001; 56: 1565–1592.
4. Giamarchi P, Stephan L, Salomon S, Le Bihan A. Multicomponent
determination of polyaromatic hydrocarbons mixture by direct
fluorescence measurements. J. Fluoresc. 2000; 10: 393–402.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

 ABSTRACTS 279

Capability of detection of the ¯uorescence formed at room temperature on a Perkin-Elmer LS50 B

technique with soft calibration for the Luminescence Spectrometer. The excitation range was
analysis of tetracyclines 300–433 nm with an interval of 1 nm and the emission
wavelength was fixed at 510 nm. Neither the emission
Inmaculada GarcõÂa,1 Luis A. Sarabia2 and nor the excitation spectrum was corrected or smoothed.
MarõÂa C. Ortiz1
1
Department of Chemistry, Faculty of Sciences, University of
Burgos, Pza. Misael BanÄuelos s/n, E-09001 Burgos, Spain. RESULTS
E-mail: mcortiz@ubu.es
2 With the zero order signal, 26.37 mg/kg is the minimum
Department of Mathematics and Computation, Faculty of
Sciences, University of Burgos, Pza. Misael BanÄuelos s/n,
detectable net concentration of TC with a probability of
E-09001 Burgos, Spain. E-mail: lsarabia@ubu.es false positives and false negatives of 5%. Using the first-
order signal and PLS calibration, this quantity is reduced
to 15.12 mg/kg in the absence and 10 mg/kg in the
presence of CTC (with five different levels of CTC
INTRODUCTION between 10 mg/kg and 70 mg/kg). In both cases, prob-
abilities of false positives of 5% and false negatives <5%
The antibiotics of the tetracycline group (tetracycline, were established. The probability of false negatives, as a
TC; chlortetracycline, CTC; and oxytetracycline, OTC) function of the minimum detectable net concentration of
are often given to livestock destined for human con- TC, is shown in Fig. 1. The influence of the CTC is clear
sumption. The need to control the daily intake has led the and the best results are obtained with multivariate cali-
European Union (EC No. 281/96) to set the maximum bration.
residue limits of TC, CTC and OTG at 100 mg/kg in milk
and muscle, 200 mg/kg in eggs, 300 mg/kg in liver and
600 mg/kg in kidney. Acknowledgements
This paper shows that the capability of detection of
This work has been partially supported by the Spanish
the fluorescence method is satisfactory for the legal
Ministerio de Ciencia y Tecnologı́a, DGI (BQU2000-
thresholds, both with zero-order analytical signals
0863) and the Junta de Castilla y León (BU15/01). I.
(maximum excitation at fixed emission wavelength, or
Garcı́a thanks the Ministerio de Educación, Cultura y
vice versa) and with higher-order signals. However, when
Deporte for FPU Grant AP2000-1314.
a tetracycline is analysed, the others act as interferents; in
this case, the use of higher-order signals with soft
calibration, e.g. partial least squares (PLS), is the
adequate solution.
The ISO norm (1) and the IUPAC (2) propose
evaluating the capability of detection through the
probability of false negatives and false positives. The
one-way methodology is already well-established (3) and
for higher-order signals it has been developed recently
(4). This, together with the capacity of the spectro-
fluorimetric technique to generate first- or higher-order
signals, allows one to improve the capability of detection
in the presence of interferents.

MATERIALS AND METHODS

Tetracycline standards were obtained from Sigma and
stored below 0°C away from light. Stock solutions (1 g/L
each tetracycline in methanol) were kept at 4°C in brown
glass vials for a maximum period of a month. A dilute
standard solution (10 mg/L each tetracycline) was
arranged in methanol. Working solutions were prepared
immediately before use by dilution of the stock to
appropriate concentrations with EDTA/Britton Robinson
buffer (adjusted to pH 9)–methanol (30 ‡ 70, v/v). All
solutions must be degassed by ultrasonification to avoid
the oxygen quenching. The measurements were per-
Figure 1. Minimum detectable net concentration of TC at
lem = 510 nm. In univariate detection the excitation wavelength
was fixed at 374 nm whereas in multivariate detection the
excitation spectrum was recorded between 300 and 433 nm.
(a) Multivariate detection without interference; (b) multivariate
detection with five levels of CTC; (c) univariate detection
without interference; (d) univariate detection with 10 mg/kg
CTC. The probability of false positive was 5%.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

280 ABSTRACTS
REFERENCES
1. ISO 11843. Capability of Detection. Geneva, Switzerland. Part 1:
1997, and Part 2: 2000.
2. Inczédy J, Lengyel T, Ure AM, Gelencsér A, Hulanicki A. IUPAC,
Compendium of Analytical Nomenclature. Blackwell: Oxford, 1998.
3. Ortiz MC, Sarabia LA. DETARCHI: a program for detection limits
with specified assurance probabilities and characteristic curves of
detection. Trends Anal. Chem. 1994; 13: 1–6.
4. Ortiz MC, Sarabia LA, Herrero A et al. Determination of capability
of detection according to ISO norm with soft calibration method. In
PLS and Related Methods, Esposito V, Lauro C, Morineau A,
Tenenhaus M (eds). CISIA-CERESTA: Montreuil, France, 2001;
235–248.
Regulation of the in¯ammatory response in
mononuclear blood cells in septic patients
T. Zimmermann,1 S. Albrecht,2 S. Kersting,1
I. Alldinger,1 G. von Gagern1 and H. D. Saeger1
1
Department of Surgery, Technical University Dresden,
Fetscherstraûe 74, D-01307 Dresden, Germany. E-mail:
zialblcl@rcs.urz.tu-dresden.de
2
Department of Gynaecology, Technical University Dresden,
Fetscherstraûe 74, D-01307 Dresden, Germany
The redox-sensitive transcription factor NF-kB plays an
important role in amplification and regulation of pro- and
Figure 1. Possible mechanism of activation of transcriptions factors and expression of target genes and role of selenoenzymes.
Copyright  2002 John Wiley & Sons, Ltd.
anti-inflammatory cytokines. The activation of MBCs
(mononuclear blood cells) and the NF-kB-induced
expression of target genes (MIF, TNFa, TF) seem to
trigger the inflammatory response (1–4). (see Fig. 1).
The selenoenzymes phospholipid-hydroperoxid-gluta-
thionperoxidase (Gpx 4) and thioredoxinredulctase are
responsible for the regulation of the redox balance and
the binding of NF-kB to the DNA promotor region.
The activating protein AP-1 is activated by reductive
(c-fos) as well as oxidative (c-jun) processes in the
inflammation. It was the aim of our study to understand
the molecular mechanism of inflammation in MBCs. The
intra- and extracellular path from transcription, mRNA
expression to protein syntheses is shown.
PATIENTS
28 patients suffering from severe sepsis (Apache III score
>69 points) were included. 18 of these died, 10 survived
in the following. The patients were included within 24 h
after the diagnosis of the sepsis. Blood samples were
taken on day 1, 3, 7, 14, 21 and 28.
MONITORING
NF-kB and AP-1 binding activity in MBCs, EMSA;
subfraction of AP-1(c-jun, c-fos) in MBCs, EMSA;
Luminescence 2002;17:191–291
 ABSTRACTS 281

Figure 2. (A) NF-kB/AP-1 binding activity in mononuclear blood cells in septic patients. (B) NF-kB/mRNA TNFa in mononuclear
blood cells and TNFa in plasma in septic patients. (C) NF-kB/mRNA MIF in mononuclear blood cells and MIF in plasma in septic
patients. (D) NF-kB/mRNA tissue factor in mononuclear blood cells and tissue factor in plasma in septic patients.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

282 ABSTRACTS
Figure 2. (Continued).
mRNA expression of TNFa, TF, TNF in MBC, RT–PCR;
protein detection of TNFa, TF, TNF in MBC, Western
blot; protein detection of TNFa, TF, TNF in plasma,
ELISA.
RESULTS
The expression of target genes in MBCs differs mortally
between surviving and not surviving patients. Non-
survivors show a rather weak expression of the mRNA
of TNFa, MIF, TF compared to survivors. However, the
plasma concentration of their proteins is for higher in
non-survivors, which could result from an extracellular
release of the proinflammatory mediators (Fig. 2).
CONCLUSION
MBCs account only in part for the release of inflamma-
tory cytokines. The expression of target genes in MBCs
could be helpful for prediction of prognosis (disturbance
in non-survivors → paralysis of the immune system?
Modulation of the NF-kB binding activity by seleno-
enzymes (phospholipid-hydroperoxid-GPx and thio-
redoxinreduktase)—restoration of the redox balance?
Therapeutic modulation of the activity of MBCs alone is
not sufficient; multimodular therapy is necessary—
antioxidants, IkB-modulation and corticoids
REFERENCES
1. Oeth PA, Parry GCN, Kunsch C, Nantermet P, Rosen CA, Mackman
Copyright  2002 John Wiley & Sons, Ltd.
N. Lipopolysaccharide induction of tissue factor gene expression in
monocytic cells in mediated by binding of c-REL/p65 heterodimers
to a kB-like site. Mol. Cell Biol. 1994; 14: 3772–3781.
2. Zuckerman SH, Evans GF, Guthrie L. Transcriptional and post-
transcriptional mechanisms involved in the differential expression
of LPS-induced IL-1 and TNF mRNA. Immunology 1991; 73: 460–
465.
3. Parry GCN, Mackman N. A set of inducible genes expressed by
activated human monocytic and endothelial cells contain IkB-like
sites that specifically bind c-REL-p65 heterodimers. J. Biol. Chem.
1994; 269: 20823–20825.
4. Nolan GP, Ghosh S, Liou HC, Tempst P, Baltimore D. DNA binding
and IkB inhibition of the cloned p65 subunit of NFkB, a REL-
related polypeptide. Cell 1991; 64: 961–969.
Analysis of urinary 2-hydroxy¯uorene as a
new biological marker of the exposure to
polycyclic aromatic hydrocarbons
Thaneeya Chetiyanukornkul, Akira Toriba, Ryoichi Kizu
and Kazuichi Hayakawa
Graduate School of Natural Science and Technology, Kanazawa
University, 13-1, Takara-machi, Kanazawa, Ishikawa 920-0934,
Japan. E-mail: thaneeya@p.kanazawa-u.ac.jp
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) are contained
in tobacco smoke, diesel engine exhaust particulate and
Luminescence 2002;17:191–291
 ABSTRACTS 283

many kinds of foods. Several PAHs, such as benzo[a]-
pyrene are carcinogenic and/or mutagenic (1). PAHs
readily absorbed into the human body are converted via
intermediate epoxides to phenols, diols and tetrols, and
the last fraction is rapidly excreted into the urine in the
form of glucuronides and sulphates. Measurement of
their metabolites can be used as a feasible way to access
the exposure to PAHs. Recent studies as well as our
experimental data showed that 2-hydroxyfluorene
(2-OHF), a major of the metabolite of fluorene, exhibited
cestrogenic activity by a yeast two-hybrid assay (2, 3).
Hence, a new high-performance liquid chromatographic
(HPLC) method with fluorescence detection for the
quantification of 2-OHF in human urine has been
established for estimating the exposure to PAHs. In the
proposed method, deuterated 2-hydroxyfluorene (2-OHF-
d9), enzymatically synthesized from deuterated fluorene,
has been used as an internal standard (IS).
EXPERIMENTAL
2-OHF-d9 was enzymatically synthesized from deuter-
ated fluorene (Fle-d10) with cytochrome P450. The
human urine sample treatment involved enzymatic
hydrolysis followed by solid phase extraction using a
Sep-Pak C18 cartridge (Waters). The HPLC system
consisted of three pumps, an injector, three columns, a
column oven, two fluorescence detectors and a switching
valve (Fig. 1). The analytes were cleaned up on an ODS
column (C1), and, via a trapping column (C2), separated
on an alkylamide-type reverse-phase column (C3).
Fluorescence detection was performed at 327 nm with
excitation at 270 nm. The mobile phases were aceto-
nitrile: phosphate buffer (pH 7.0) (40:60, v/v), at flow
rates of 1 mL/min. A column oven was set at 40°C. The
switching valve position was automatically changed. 2-
OHF-d9 was eluted closely prior to 2-OHF on C3 column
after the switching (Fig. 2). The detection limit was found
to be 0.2 nmol/L (S/N = 3) and the quantification range
was 5–100 nmol/L with good linearity (r2 = 1). Precision
and accuracy of intra-days and inter-days were deter-
mined by replicate analysis (n = 5) of a single urine
sample added standard at two different concentrations
(10 and 50 nmol/L). The method applied to evaluate

Figure 1. Flow diagram of HPLC system using column-switching. I, injector; D1, 2, fluorescence detectors
(lex 270 nm, lem 327 nm); V, switching valve; P1–3, pumps, flow rates 1.0 mL/min; C1–3, separation columns; GC, guard column;
C1, Cosmosil 5C18 MS (250  4.6 mm i.d., 5 mm, Nacalai Tesque); C2, Discovery RP-Amide C16 (50  4.6 mm i.d., 5 mm, Supelco);
C3, Discovery RP-Amide C16 (250  4.6 mm i.d., 5 mm, Supelco); GC, Cosmosil 5C18 MS (10  4.6 mm i.d., 5 mm, Nacalai Tesque);
CO, column oven, 40°C; MP, mobile phase, AcCN/10 mM phosphate buffer, pH 7.0, 40/60, v/v; WS, washing solvent, AcCN: water,
90:10, v/v.

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284 ABSTRACTS
Figure 2. HPLC chromatoagrams of 2-OHF in a urine sample from a smoker using column-switching.
urinary 2-OHF concentration as a biomarker in smokers
and non-smokers. A significant difference was observed
between on the two groups.
REFERENCES
1. Kizu R, Ishii K, Kobayashi J et al. Materials Sci. Eng. C. 2000; 12:
97.
2. Nishihara T, Nishikawa J, Kanayama T et al. J. Health Sci. 2000; 46.
3. Hirose T, Morito K, Kizu R et al. J. Health Sci. 2001; 47.
Synthesis of a ¯uorescent molecularly
imprinted polymer for selective binding of
histamine
Aijun Tong, He Dong and Longdi Li
Department of Chemistry, Tsinghua University, Beijing 100084,
China. E-mail: tongaj@chem.tsinghua.edu.cn
Copyright  2002 John Wiley & Sons, Ltd.
Molecular recognition using molecularly imprinted
polymers (MIPs) has received increasing attention due
to the highly specific binding sites to the template
molecules. In the most studies, methacrylic acid (MAA)
was used as the functional monomer to synthesize the
non-covalent binding mode of MIPs (MAA–MIP) (1).
There have also been some studies using well-designed
and synthesized fluorescent functional monomers for the
sensing of cAMP (2), 9-ethyladenine (3), fructose (4) as
well as some synthetic compounds (5, 6). In this study,
we chose a market product, zinc(II)-protoporphyrin
(ZnPP), as a new fluorescent functional monomer to
synthesize a fluorescent MIP capable of recognizing
histamine. Using the commercially available ZnPP as the
functional monomer can avoid tedious synthesis. The
reagent has an axial binding site, Zn and two peripheral
carboxylic acid groups capable of hydrogen binding.
Furthermore, It also contains two vinyl groups necessary
for the polymerization.
The polymers were prepared according to the general
Luminescence 2002;17:191–291

Table 1. Binding abilities of ZnPP-MAA-MIP (A), MAA-MIP (D) and ZnPP-MIP (E) determined
from the Scatchard plot
Kass (mol/L 1)
Polymer [His] = 0.1–1mmol/L [His] = 4–10mmol/L
A 4.5  10 3
2.7  102
D 1.2  103 1.3  102
E 2.0  103 2.0  102
imprinting protocol with ethylene glycol dimethacrylate
(EGDMA) as the cross-linking agent in the DMSO
porogen. The functional monomers used were the ZnPP
(MIP-E), MAA(MIP-D) or their mixture (MIP-A).
Aliquots of ZnPP (30 mmol), MAA (450 mmol), EGDMA
(7.95 mmol) and the initiator, AIBN (0.18 mmol) were
mixed with DMSO and purged with nitrogen for
polymerization at 43°C for 24 h. The template in the
MIPs was subsequently removed by water and methanol
containing 10% (v/v) acetic acid until the template was
no longer detected in the elution. Binding ability of the
polymers was evaluated with a batch adsorption method
by measuring the concentration of free histamine in the
supernatant spectrometrically. Fluorescence change of
the polymer after shaking with histamine aqueous
solution was conducted in DMSO and chloroform (1:1)
mixed solvent by using a front-face illumination method.
Association constant (Kass) and the number of
accessible sites (N) between the MIP and the template
were found to be dependent upon the template con-
centration. Using Scatchard’s plot, Kass and N were
determined and summarized in Table 1. The binding
ability was also evaluated more accurately with a multi-
binding model. Higher association constants were
obtained for the polymer with both ZnPP and MAA as
Figure 1. Fluorescence intensity change of the two fluor-
escent MIPs A and E when rebinding with histamine. a, ZnPP-
MAA-MIP(A); b, ZnPP-MIP (E). The data are means  stan-
dard error (n = 3).
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 285
N (mmol/g)
[His] = 0.1–1mmol/L [His] = 4–10mmol/L
1.8  102 1.1  103
2.4  102 1.6  103
4.5  101 1.5  102
the functional monomers (MIP-A), while the Kass values
were lower in the case of using MAA alone (MIP-D) or
using ZnPP (MIP-E) as the functional monomer.
Fluorescence intensity of the MIP decreased with
histamine concentration but saturation behaviour was
observed when the histamine concentration was above
1 mmol/L, as shown in Fig. 1, indicating that histamine
no longer coordinated with ZnPP in the MIP and led to
the low association constant at this condition. Our results
demonstrated that MAA and ZnPP were corporately
arranged by histamine in the ZnPP-MAA-MIP, and using
ZnPP as the fluorescent functional monomer can make
highly specific recognition sites for histamine.
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (Grant No. 29975015).
REFERENCES
1. Kriz D, Ramstrom O, Mosbach K. Anal. Chem. 1997; 69: 345A.
2. Turkewitsch P, Wandelt B, Darling GD, Powell WS. Anal. Chem.
1998; 70: 2025.
3. Takeuchi T, Mukawa T, Matsui J, Higashi M, Shimizu K. Anal.
Chem. 2001; 73: 3869.
4. Wang W, Gao S, Wang B. Organic Lett. 1999; 1: 1209.
5. Rathbone D, Su D, Wang Y, Billington DC. Tetrahedron Lett. 2000;
41: 123.
6. Rathbone D, Ge Y. Anal. Chim. Acta 2001; 435: 129.
Potential of photochemically-induced
¯uorescence detection coupled to ¯ow
injection analysis for the determination of
chlorophenoxyacid herbicides in micellar
medium
Ana M. GarcõÂa-CampanÄa1, Jean-Jacques Aaron2 and
Juan M. Bosque-Sendra1
(1) Department of Analytical Chemistry, Faculty of Sciences,
University of Granada, Campus Fuentenueva, E-18071
Granada, Spain
(2) ITODYS, Universite Paris 7- Denis Diderot, Associe au CNRS,
UMR 7086, 1 Rue Guy de la Brosse, F-75005 Paris, France
Luminescence 2002;17:191–291
286 ABSTRACTS

INTRODUCTION the carrier stream using a rotary injection valve fitted

with an interchangeable PTFE loop. As a photoreactor, a
Chlorophenoxyacid herbicides constitute widely used
commercial Knauer Model 31125 (Berlin, Germany)
compounds in agriculture. They do not show native
equipped with a germicide UV lamp (8 W, 254 nm) was
fluorescence, but, as well as other aromatic pesticides,
used for analyte conversion; it was located between the
they can be photolysed into strongly fluorescent photo-
injection valve and the detector, with a PTFE tubing
products in a mixture of methanol and pH 5 buffer (50/50,
helically coiled around it and connected in series with the
v/v), allowing the establishment of a very rapid and
spectrofluorimeter (Fig. 1).
simple method, called photochemically induced fluores-
cence (PIF) (1). Due to the easy adaptability of this
technique to continuous procedures, a flow-injection Procedure
assembly with PIF detection was proposed for the simple
Standard stock solutions of the chlorophenoxyacid
and faster analysis of five chlorophenoxy herbicides in
herbicides (0.1–0.5 mg/mL) were prepared by dissolving
spiked river waters (2). In this work we have investigated
them in ethanol. Working solutions were prepared by
the effect of micellar media on the PIF intensity of 2,4-
transferring an aliquot of the herbicide solution into a
dichlorophenoxyacetic acid (2,4-D) and Mecoprop
10 mL calibrated flask and by adding 3 mL buffer
(MCPP), considering the enhancement of the signal
solution (pH 3), 0.3 or 0.4 mL cetyltrimethyl ammonium
intensity from native fluorescent compounds in the
chloride (CTAC) aqueous solution (1% m/v) for 2,4-D or
presence of micelle solutions, which provide an increase
MCPP, respectively, and by diluting with deionized
in sensitivity and also a reduction of the need to use
water. For the FIA procedure, an aliquot of sample
organic solvents.
containing the herbicide was injected through the
injection valve into the carrier stream, constituted of
EXPERIMENTAL deionized water. The PIF intensity was monitored at fixed
analytical excitation and emission wavelengths of the
Apparatus
herbicide photoproducts by measuring the FIA peak
Fluorimetric measurements were carried out on a height (Fig. 1).
Kontron SFM-25 spectrofluorimeter, equipped with a
data control and a K-wind 25 acquisition program. The
RESULTS AND DISCUSSION
flow injection analysis (FIA) system included an Ismatec
Model IPC peristaltic pump, poly(tetrafluoroethylene) For both herbicides, the cationic surfactant CTAC was
(PTFE) connecting tubing (0.5 mm i.d.) and end-fittings the only significant enhancer of PIF emission, providing
and connectors (Bioblock). The analyte was injected into micellar enhancement factors (MEF) of 30.9 and 4.1 for

Figure 1. ? caption ?

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

 ABSTRACTS 287

2,4-D and MCPP, respectively, relative to the PIF
intensity in water, therefore improving considerably the
signal measured when a mixture of methanol and pH 5
buffer (50/50, v/v) was utilized (2). The maximum PIF
intensity was obtained at a pH 3, using a 30% (v/v) buffer
concentration, as shown in previous studies (3). The
signal was drastically enhanced as the concentration
increased and reached a maximum, which remained
constant in a wide concentration interval above the
critical micellar concentration (c.m.c). The surfactant
concentration optimal values were 9.4  10 3 mol/L for
2,4-D or 1.3  10 2 mol/L for MCPP, respectively. In the
case of the photodegradation of chlorophenoxyacid
herbicides, a mixture of photoproducts, including lower
chlorinated phenols, polymeric and dihydroxylated
compounds, and phenol, is probably formed (3,4). Exci-
tation and emission wavelengths of 270 and 298 nm were
chosen in cationic micellar media, to develop the screen-
ing method using a flow-injection assembly. Water, an
aqueous micellar solution and a buffer solution (pH 3,
30% v/v) containing the optimal CTAC concentration,
were tested as mobile phase for both herbicides. A
minimum blank signal, a maximum sensitivity and a
better resolution of the FIA peaks were obtained when
deionized water was used as mobile phase, and the buffer
solution and the CTAC optimal concentration were added
to the sample solutions before injection.
In order to optimize the FIA system, the effect of
different parameters on the MEPIF signal, such as the
injected sample volume, carrier flow rate and photo-
reactor length, were investigated. The analytical signal
decreased upon increasing the flow rate because of a too
short UV irradiation time for the formation of fluorescent
photoproducts. To avoid excessive band broadening of
the FIA peaks and to obtain the best analytical signals, a
compromise flow rate value of 1.0 mL/min was selected
for both herbicides. The effect of the photoreactor length
was also examined in the range 100–400 cm, as well as
the effect of the injection volume, which was varied
between 102–393 mL. Due to the slow kinetics of the
photolysis reactions for both herbicides, a good reprodu-
cibility and strong signals were obtained by applying the
highest reactor length (400 cm) in both cases. Also, the
highest injected volume of 393 mL was chosen because of
an increase of the herbicide mean residence time in the
photoreactor.
Analytical performance characteristics
The analytical performance characteristics were evalu-
ated by means of the calibration data sets (5,6), using
linear calibration graphs with three replicates for each
concentration value and three injections for each
concentration level of 2,4-D and MCPP (Table 1). Good
analytical resolution and satisfactory precision expressed
as relative standard deviation (RSD) were obtained for
Table 1. Analytical performance characteristics for the
proposed FIA–MEPIF methods
Feature (units) 2,4-D MCPP
2
r (%) 99.68 99.92
Linear range (ng/mL) 243.8–5000 111.6–5000
Linearity online (%) 98.4 99.4
Lack-of-fit P-value (%) 13.5 21.0
Detection limit (ng/mL) 73.2 33.5
Analytical resolution (SR,c/b) 50.0 28.9
(ng/mL)
Precision (%) (for 2 mg/mL) 3.1 3.7
SR,c, residual standard deviation; b, slope.
both analytes. The IUPAC detection limit low values
assure the applicability of our method to real samples for
future work.
REFERENCES
1. Eremin SA, Laasis B, Aaron JJ. Talanta 1996; 43: 259.
2. Garcı́a LF, Eremin SA, Aaron JJ. Anal. Lett. 1996; 29: 1447.
3. Garcı́a-Campaña AM, Aaron JJ. Luminescence 2000; 15: 110;
Garcia-Campana AM, Aaron JJ, Bosque-Sendra JM. Talanta 2001;
55: 531.
4. Werhoven-Goewie CE, Boon WM, Pratt AJJ, Frei RW, Brikman
UATh, Little CJ. Chromatographia 1982; 16: 53.
5. Cuadros Rodrı́guez L, Garcı́a-Campaña AM, Jiménez Linares C,
Román Ceba M. Anal. Lett. 1993; 26: 1243.
6. Garcı́a-Campaña AM, Cuadros Rodrı́guez L, Alés Barrero F,
Román Ceba M, Sı́erra Fernández JL. Trends Anal. Chem. 1997; 7:
381.
trans-Resveratrol: antioxidant capacity using
phycoerythrin as the ¯uorescent probe
M. LoÂpez-VeÂlez, F. MartõÂnez-Martinez and
C. del Valie-Ribes
Department of Physical Chemistry, Faculty of Pharmacy,
University of Granada, E-18071 Granada, Spain
In recent years, researchers have focused their attention
on the pathologic role of free radicals in a variety of
diseases, among which the most important are athero-
selerosis and cancer. The antioxidant compounds can
neutralize free radicals and may be of great importance in
the prevention of these diseases.
The non-flavonoid phytoalexin trans-resveratrol
(trans-3,5,4'-trihydroxystilbene) has attracted interest as
a wine constituent that may reduce heart disease by
acting as an antioxidant agent (1). In this study, the
described phenolic compound has been subjected to an
antioxidant test by a fluorimetric assay in the aqueous
phase using B-phycoerythrin (B-PE) as the fluorescent
probe and 2,2'-azobis(2-amidinoropane) dihydrochloride
(AAPH) as the peroxyl radical generator (2). Further-

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288 ABSTRACTS
Figure 1. (A) Fluorescence spectra of trans-resveratrol in
methanol at different concentrations (lexcitation, 310 nm). (B)
Change of fluorescence with the concentration of trans-
resveratrol.
more, other fluorescence properties of this compound are
studied using steady-state fluorescence.
CHANGE IN FLUORESCENCE WITH THE
CONCENTRATION OF TRANS-RESVERATROL
The fluorescence of various solutions 0.1  10 5 to 7 
10 5 mol/L was measured at an excitation of 310 nm and
Figure 2. Relative fluorescence of B-PE with time of incubation in the presence of Trolox
at different concentrations of trans-resveratrol.
Copyright  2002 John Wiley & Sons, Ltd.
emission of 380 nm (Fig. 1). A calibration graph for this
compound was constructed. Good linearity (r2 > 0.99)
was obtained for the concentration range 1  10 5 mol/L.
PHYCOERYTHRIN FLUORESCENCE-BASED ASSAY
This assay depends on the detection of chemical damage
to B-PE through the decrease in its fluorescence emission
(excitation 545 nm; emission 575 nm). Under appropriate
conditions, the loss of PE fluorescence in the presence of
reactive species is an index of oxidative damage of
protein. The inhibition of the reactive species action by
trans-resveratrol, which is reflected in the protection
against the loss of PE fluorescence in the fluorimetric
assay, is a measure of its antioxidant capacity against the
reactive species. In addition, the loss of PE fluorescence
in the presence of reactive species, which include peroxyl
radicals generated from AAPH, does not follow zero-
order kinetics, as shown in Fig. 2. As a reference
compound for antioxidant capacity we used Trolox (6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid),
a water-soluble a-tocopherol analogue. This compound
reacts rapidly with peroxyl radicals and, until the Trolox
is consumed, no loss in phycoerythrin fluorescence is
observed. The phenolic compound studied, trans-res-
veratrol, reacts with peroxyl radicals more rapidly than
does phycoerythrin, showing similar effects to Trolox.
Samples of the fluorimetric assay kinetic data obtained
from Trolox at different concentrations and trans-
resveratrol at 0.3 mmol/L are presented in Fig. 2, which
shows that trans-resveratrol had a higher antioxidant
Luminescence 2002;17:191–291

activity than Trolox at the same concentration. The final
antioxidant activity value of trans-resveratrol was
calculated by using a regression equation between the
Trolox concentration and the net area under the B-PE
decay curve and was expressed as Trolox equivalent
[Y(mmol/L) = 0.11695 ‡ 0.15931X (net area)]. A good
linear relationship between net area and concentration of
Trolox was obtained with a correlation coefficient (r2) of
0.99. The antioxidant activity value of trans-resveratrol
at 0.3 mmol/L was 2.48, expressed as Trolox equivalent.
REFERENCES
1. López M, Martı́nez F, Del Valle C, Orte C, Miró M. J. Chromatogr.
A 2001; 922: 359–363.
2. Cao G, Prior RL. Methods Enzymol. 1999; 299: 50–62.
The role of IkBa on regulation of
in¯ammatory response in septic patients
1 2
T. Zimmermann , S. Albrecht , G. v. Gagern , 1 blood cells.
R. GruÈtzmann1, I. Alldinger1, S. Kersting1 and
H.-D. Saeger1
(1) Department of Visceral, Thoracic and Vascular Surgery,
University Hospital `Carl Gustav Carus', Technical University of
Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany
(2) Department of Gynaecology, University Hospital `Carl Gustav
Carus', Technical University of Dresden, Fetscherstrasse 74,
D-01307 Dresden, Germany
The transcription factors NF-kB and AP-1 are sequence-
Figure 1. NF-kB pathway in mononuclear blood cells.
Copyright  2002 John Wiley & Sons, Ltd.
ABSTRACTS 289
specific DNA binding proteins (trans-acting factors).
They lead via binding to the promoter region of the DNA
(cis acting elements) to induction, repression and
expression of target genes. Specific cytoplasmic inhibi-
tory genes (IkBa) prevent the DNA-binding in the
nucleus by association to subunits of NF-kB (Fig. 1).
One of the main representatives of IkB is IkBa, which
inhibits p65 and c-rel, but not p50. Another is IkBb,
which inhibits the p50–p65 complex and IkBb. These
inhibitory proteins are mediated by protein kinases.
Reactive oxygen species lead as second messengers to
phosphorylation and further to separation of IkB to
NF-kB (induction of transcription).
PATIENTS
28 Patients with severe sepsis (18 non-survivors and 10
survivors). The Apache III score was >69 points. The
blood samples were taken on days 1, 3, 7, 14 and 28.
METHODS
• RT–PCR: mRNA expression of IkBa in mononuclear
• Western blot: intracellular protein formation of IkBa.
• EMSA: binding activity of NF-kB in mononuclear
blood cells.
• EMSA: measurement of the protein fraction of p50 and
p65 in the nucleus and cytoplasm of mononuclear
blood cells.
RESULTS
A reduced mRNA–IkBa expression in mononuclear
Luminescence 2002;17:191–291
290 ABSTRACTS

Figure 2. (A) NF-kB binding activity and mRNA-IkBa expression in mononuclear blood cells in septic patients (first day = 100%).
(B) NF-kB binding activity and protein concentration of p50 subunit in nucleus of mononuclear blood cells in septic patients (first
day = 100%). (C) NF-kB binding activity and protein concentration of p65 subunit in nucleus of mononuclear blood cells in septic
patients (first day = 100%). (D) NF-kB binding activity and protein concentration of p50 subunit in cytoplasm of mononuclear blood
cells in septic patients (first day = 100%). (E) NF-kB binding activity and protein concentration of p50 subunit in cytoplasm of
mononuclear blood cells in septic patients (first day = 100%).

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291

 ABSTRACTS 291

Figure 2. Continued.

blood-cells was found on the third day in non-survivors. CONCLUSIONS

A protein formation of IkBa was not detectable in non-
1. NF-kB–IkBa interaction (Fig 2) seems to be intact in
survivors, whereas a low protein formation of IkBa was
surviving patients (course in the same direction).
found in survivors. The p50 protein fraction was clearly
2. Missing protein formation (despite of mRNA expres-
increased in the nuclens of mononuclear blood cells. The
sion of IkBa) could be the reason for uncontrolled
reason could be the missing formation of IkBa. The
activation of NF-kB.
increase of the cytoplasmic p50 formation of the
3. The third day seems to be prognosis-relevant, there-
survivors points to intact IkBa (specific IkBa).
fore IkBa could be a parameter of prognosis.
4. The role of IkBb has to be elucidated in further
experiments.

Copyright  2002 John Wiley & Sons, Ltd. Luminescence 2002;17:191–291