Resonance Light Scattering Study On The Interaction Between ...
Research Article
Received: 11 January 2009,
Revised: 15 March 2009,
Accepted: 25 March 2009,
Published online in Wiley Interscience: 2009
(www.interscience.wiley.com) DOI 10.1002/bio.1139
John Wiley & Sons, Ltd.
Resonance light scattering study on the
interaction between quinidine sulfate and
congo red and its analytical application
Interaction between quinidine sulfate and congo red
Yanbo Zenga, Liling Caia, Haidong Wanga, Lei Lia*, Weijing Youa,
Longhua Guob and Guonan Chenb*
ABSTRACT: The interaction between quinidine sulfate (QDS) and congo red (CR) was studied using resonance light scattering
(RLS) technique, ultraviolet–visual spectrophotometry and fluorimetry. In weak acidic medium, QDS reacts with CR to form a
supermolecular complex which results in the enhanced RLS intensity. Some important interacting parameters, such as the
solution acidity and CR concentration, salt effect and addition order of the reagents, were investigated and optimized. Under
the optimum conditions, it was found that the enhanced RLS intensity was in proportion to the concentration of QDS in the
range 0.2–8.4 mg mL-1. The corresponding detection limit was 12.0 ng mL-1. The results showed that this new method enabled
simple, sensitive and rapid determination of QDS and was used for the determination of QDS in urine and simulated huamn
serum samples. Copyright © 2009 John Wiley & Sons, Ltd.
Keywords: resonance light scattering; quinidine sulfate; congo red
Introduction
much attention has been paid to the study and determination
of nucleic acids,[15–17] proteins[18–20] and inorganic ions[21–23] by use
Quinidine sulfate (QDS) is an antiarrhythmic drug which can be
of this method. In recent years, RLS has been widely applied
used to suppress ventricular fibrillation and prevent antitachy-
to determine some pharmaceuticals.[24–26] Until now, there has
cardia pacing.[1–3] However, when patients were administered
been no report concerning the determination of QDS using RLS.
doses of quinidine sulfate sufficient to produce serum concen-
The aim of this work is to develop a simple, sensitive and rapid
trations of higher than 2.5 μg mL−1, it resulted in side effects such
method for the determination of QDS that gives results com-
as drug allergy, exacerbation of weakness and the development
parable to those obtained by the existing methods.
of respiratory failure.[4] Therefore, to take full advantage of the
Congo red, a kind of bisazo anionic dyes, is a sensitive RLS
antiarrhythmic drug and to decrease its toxicity, it is essential to
probe and has been used to determine many chemicals, such
quantitatively determine QDS in clinical samples.
as proteins,[27] nucleic acids[28] and drugs.[29–31] In the experiments
Several methods have been used for the determination of
of this paper, the interaction between QDS and CR to form a
QDS (the molecular structure of QDS is shown in Fig. 1), includ-
supermolecular complex was studied using RLS. The faint RLS
ing fluorimetry (FLU),[5] high-performance liquid chromato-
intensity of congo red was greatly enhanced by the addition of
graphy (HPLC),[6] capillary electrophoresis (CE)[7] and fluorescence
QDS. Under the optimum experimental conditions, the RLS
microscopic analysis (FMA).[8] FLU exhibits good sensitivity for
intensity change was linear with the concentration of QDS in the
QDS quantification; however, the narrow linear range limits its
range 0.2–8.4 μg mL−1 with a detection limit of 12.0 ng mL−1. The
application range.[5] HPLC and CE have relatively good sensitivity,
proposed method exhibited high precision and was successfully
accuracy and precision, especially low disturbance, but the pro-
applied to the determination of QDS in urine and simulated
cedure is complicated and time-consuming.[6,7] FMA has been
huamn serum samples.
used owing to its high sensitivity and small sample consumption,
but this method is easily affected by large amounts of surfac-
tants and salts.[9]
* Correspondence to: Lei Li, College of Biological and Chemical Engineering,
Analytical procedures applying the RLS method combine
Jiaxing, Zhejiang 314001, People’s Republic of China. E-mail:
the advantages of simplicity, sensitivity and rapidity.[10–14] Since
lileichem@yahoo.com.cn
Huang et al. first used this technique for analytical purposes,[10]
Guonan Chen, The Ministry of Education Key Laboratory of Analysis and
Detection Technology for Food Safety (Fuzhou University), and Department
of Chemistry, Fuzhou University, Fuzhou, Fujian 350002, People’s Republic of
China. E-mail: gnchen@fzu.edu.cn
a
College of Biological and Chemical Engineering, Jiaxing University, Jiaxing,
Zhejiang 314001, People’s Republic of China
b
The Ministry of Education Key Laboratory of Analysis and Detection Tech-
nology for Food Safety (Fuzhou University), and Department of Chemistry,
Figure 1.
The molecular structure of QDS.
Fuzhou University, Fuzhou, Fujian 350002, People’s Republic of China
1
Luminescence 2009
Copyright © 2009 John Wiley & Sons, Ltd.
Y. Zeng et al.
intensity of the system was represented as ΔI
= I − I (I and
Experimental
RLS
RLS
0
RLS
I are the intensities of the systems with and without QDS).
0
Apparatus
Results and discussion
The RLS and fluorescence spectra were measured with a Cary
Eclipse fluorescence spectrophotometer (Varian, USA) with a quartz
RLS spectra
cuvette (1 × 1 cm). A UV2550 spectrophotometer (Shimadzu,
Japan) was used for measuring absorption spectra. A Delta 320
Figure 2 shows the RLS spectra of CR, QDS and QDS-CR system
pH meter (Mettler-toledo instrument corporation, Shanghai,
at pH 4.5. It can be seen from Fig. 2 that RLS intensities of the
China) was used for pH measurement.
CR and QDS are weak in the wavelength range of 230–750 nm.
However, the enhanced RLS intensity can be clearly observed
with three peaks located at 272, 388 and 562 nm when a trace
Reagents
amount of QDS was added to the solution of CR. Moreover, the
A stock solution of 1 g L−1 quinidine sulfate was prepared by dis-
enhanced RLS intensity increased with the increasing QDS
solving 0.105 g quinidine sulfate dihydrate (Sigma, USA) in 100
concentration. The maximum RLS peak was located at 562 nm.
mL water. The concentration of the working solution was made
Therefore, 562 nm was selected as the analytical wavelength.
up to 100 μg mL−1 by diluting the QDS stock solution with water.
The stock and working solutions were stored in dark environment.
Effect of acidity
The stock solution of congo red (analytical-reagent grade,
Shanghai Chemistry Reagent Corporation, China) was made
As shown in Fig. 3, the effect of the solution acidity on the scat-
up to 1.0 × 10−3 mol L−1 by dissolving CR in water. The working
tering intensity of the system was investigated. Variation of
solution was prepared by diluting the stock solution to 1.0 ×
pH from 2.5 to 6.0 at the concentration of 6 μg mL−1 of QDS
10−4 mol L−1 with water.
was studied. At pH 4.5, the enhanced RLS intensity reached its
Britton Robinson buffer solutions with different pH (pH 2.5–
6.0) were prepared by mixing the mixed acid (composed of
0.04 mol L−1 H PO , HAc and H BO ) with 0.2 mol L−1 NaOH in
3
4
3
3
proportion. The buffer was used to control the acidity of the
interacting system.
All other reagents were of analytical-reagent grade and were
used without further purification. Double-distilled water was
used throughout.
Sample preparation
Human serum and urine samples were provided by a local hospital.
To prepare serum samples, a 1.0 mL aliquot of serum sample
and 0.25 mL trichloroacetic acid were mixed thoroughly and
centrifuged at 4000 rpm for 10 min. A 0.5 mL aliquot of the
supernatant fluid was diluted to 1000-fold with water without
further purification. Then QDS in serum samples was deter-
Figure 2.
RLS spectra of the QDS-CR system. Conditions—(1) C
6.0 μg mL−1,
QDS
mined according to the general procedure. Urine samples were
pH 4.5; (2) C 1.0 × 10−5 mol L−1, pH 4.5; (3–8) QDS + CR, C 1.0 × 10−5 mol L−1, C
CR
CR
QDS
directly determined according to the general procedure without
(3–8, μg mL−1): (3) 2.0; (4) 3.0; (5) 4.0; (6) 5.0; (7) 6.0; (8) 7.0, pH 4.5.
simple treatment.
The compositions of simulated serum samples (SMSS) were
as follows: 5.14 μg mL−1 methionine, 5.95 μg mL−1 cysteine,
14.3 μg mL−1 tryptophan, 1.52 μg mL−1 tyrosine, 1.84 μg mL−1
histidine, 12.6 μg mL−1 serine, 11.2 μg mL−1 glycine, 26.4 μg mL−1
phenylalanine, 29.2 μg mL−1 lysine, 38.3 μg mL−1 arginine,
36.5 μg mL−1 alanine, 119.7 μg mL−1 aspartic acid, 0.84 g L−1
NaHCO and 3.85 g L−1 NaCl. The simulated serum samples were
3
diluted 10-fold for the determination and recovery tests.
General procedure
A 1.0 mL aliquot of Britton–Robinson buffer, 1.0 mL CR working
solution and appropriate QDS (or samples) were added to a
10 mL calibrated flask. The resulting solution was diluted to the
mark with water and then mixed thoroughly.
All RLS spectra were obtained by simultaneously scanning the
excitation and emission monochromators (Δλ = 0.0 nm) from
230.0 to 750.0 nm with the excitation and emission slits 5.0 nm.
Figure 3.
Effect of pH on the RLS intensity. Conditions—C
6.0 μg mL−1, C
2
QDS
CR
The RLS intensities were measured at 562 nm. The enhanced RLS
1.0 × 10−5 mol L−1.
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Copyright © 2009 John Wiley & Sons, Ltd.
Luminescence 2009
Interaction between quinidine sulfate and congo red
intensity of the system QDS-CR decreases gradually with the
increasing NaCl concentration. When NaCl concentration reaches
3.0 × 10−2 mol L−1, the RLS signal maintains a constant value. The
variation of RLS signal with ionic strength indicates that electro-
static interaction plays a dominant role in this reaction. When
the concentration of the NaCl increases, the effects of the elec-
trostatic shielding of charges between CR and QDS reduces the
combination between QDS and CR and results in a decreased
RLS signal. Therefore, this system should be under a low ionic
strength condition without adding NaCl.
Effect of addition order
The effect of the addition order of the reagents on the intensity
was investigated. The result shows that mixing buffer solution
and CR solution first and then adding QDS can give a higher
intensity than other sequences of adding the reagents. This may
be explained by the buffer and CR solution mixing first and pro-
Figure 4.
Effect of CR concentration on the RLS intensity. Conditions—CQDS
moting the ionization of CR, benefiting the reaction of QDS with
6.0 μg mL−1, pH 4.5.
CR. Therefore, in this research, the optimal order for addition of
the reagents was: buffer–CR–QDS.
maximum. In addition, we tested a suitable amount of a BR buffer
solution for the system. The results showed that 0.8–1.2 mL of BR
Incubation time and stability
buffer solutions were optimum. Therefore, a BR buffer solution
with pH 4.5 was used in this research and the optimum volume
The stability of the QDS–CR system was investigated by measuring
of buffer was 1.0 mL.
the RLS intensity every 2 min for 2.5 h immediately after mixing.
The results show that the formation time of all reaction products
is 20 min at room temperature and the RLS intensity will remain
Effect of the CR concentration
constant over 120 min (change of RLS intensity within ± 5%).
As shown in Fig. 4, the effect of CR concentration on the RLS
intensity of this system was studied. If the concentration of CR
Selectivity of the method
was too low, the enhanced RLS intensity was small because of
the incomplete reaction between QDS and CR. On the contrary,
Under the optimum conditions, the effects of some foreign
if the concentration of CR was too high, the scattering intensity
coexisting substances on the determination of 6.0 μg mL−1 were
was also faint due to the self-assembling of CR. Therefore,
investigated by pre-mixing QDS with foreign substances and
1.0 × 10−5 mol L−1 CR was used in this research.
the results are listed in Table 1. It can be seen from Table 1 that
sugars, urea, amino acids and some ions in fluids can be allowed
with high concentration. Therefore, the selectivity of the method
Effect of salt effect
is good and the method can be applied to determine QDS in
The effect of salt on the RLS intensity of the system is presented
practical samples.
clearly in Fig. 5. It can be observed from Fig. 5 that the RLS
Calibration and detection limit
According to the above standard procedure, the RLS intensities
were obtained under optimum conditions and a calibration curve
was constructed. The linear range was 0.2–8.4 μg mL−1, and the
linear regression equation was calculated as ΔI
= −84.9 + 105.1C
RLS
(μg mL−1) with regression coefficient r = 0.9987 (n = 6). The detec-
tion limit was 12.0 ng mL−1. The limit of detection is given by 3S /
0
S, where 3 is the factor at the 99% confidence level, S is the
0
standard deviation of the blank measurements (n = 11) and S is
the slope of the calibration curve. Comparisons with some other
assays of QDS are shown in Table 2. The results show that the
proposed assay method exhibits simplicity, high sensitivity and
a wide linear range. This method provides a sufficient sensitivity
for the one-step measurement of trace amounts of QDS in solu-
tion samples.
Mechanism of the reaction
CR, a kind of bisazo anionic dye, is negatively charged in aque-
Figure 5.
Effect of ionic strength on the RLS intensity. Conditions—C
6.0 μg
QDS
mL−1, C 1.0 × 10−5 mol L−1, pH 4.5.
CR
ous solution due to two sulfonic acid groups within its structure,
3
Luminescence 2009
Copyright © 2009 John Wiley & Sons, Ltd.
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Y. Zeng et al.
Table 1.
Effects of foreign substances (conditions: pH 4.5, C 1.0 × 10−5 mol L−1, C
6.0 μg mL−1)
CR
QDS
Foreign
Concentration
Relative
Foreign
Concentration
Relative
substances
of foreign substances
error (%)
substances
of foreign substances
error (%)
(μg mL−1)
(μg mL−1)
Na(I)
900
−2.8
Serine
500
4.8
K(I)
1800
−3.9
Glycin
90
4.6
Mg(II)
60
1.7
Phenylalanine
600
4.1
Ca(II)
900
3.8
Lysine
72
3.6
Cd(II)
150
2.4
Arginine
100
−3.8
Zn(II)
120
3.4
Alanine
600
−4.0
Fe(II)
3
3.0
Aspartic acid
300
−0.3
NO –
230
3.4
Valine
480
3.0
3
HCO –
300
−4.3
Saccharose
1500
4.0
3
Cl–
1200
−2.8
Glucose
3000
4.7
SO 2–
240
1.7
Fructose
1500
3.8
4
Methionine
500
3.3
Lactose
1500
0.5
Cystenine
100
−0.7
Leucine
480
−2.3
Tryptophan
300
−0.9
Starch
30
0.9
Tyrosine
100
−3.6
Vitamin C
150
0.8
Histidine
100
−3.8
Urea
120
3.7
Table 2.
Comparisons with some other assays of QDS
Method
Experimental conditions
Determination
References
limit (ng mL−1)
FLUa
Sulfuric acid, λ /λ
= 353 nm/445 nm,
10.0
[5]
ex
em
extraction by benzene
HPLC Fluorescent
detection
2.4
[6]
CE
Laser-induced fluorescence detection
24.1
[7]
FMA
Poly(vinyl alcohol)-124
20.4
[8]
RLS
Congo red, 562 nm
12.0
This work
aThe linear range of FLU is 0.06–0.8 μg mL−1.
Figure 6.
The formed process of the supermolecular complex between QDS and CR.
shown in Fig. 6. Therefore, it will ionize as an anion in aqueous
owing to the neutralization of charges and the appearance of
solution. On the other hand, QDS will ionize as a cation in aque-
the liquid–solid interface, they reacts with each other to form
ous solution. Before interaction, either CR anion or QDS cation
an ion-association complex, which can be further demonstrated
4
has strong hydrophility. When the hydrophobility is enhanced
by absorption and fluorescece spectra. The formation of the
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Copyright © 2009 John Wiley & Sons, Ltd.
Luminescence 2009
Interaction between quinidine sulfate and congo red
Figure 7.
Comparisons between RLS and absorption spectra. Conditions—(1)
RLS spectra (enhanced I ): C 2.0 × 10−6 mol L−1; C
6.0 μg mL−1; pH 4.5; (2)
RLS
CR
QDS
absorption spectra against the reagent blank (enhanced A): C 2.0 × 10−6 mol L−1;
CR
C
8.0 μg mL−1; pH 4.5; (3) absorption spectra against the reagent blank: C
QDS
CR
Figure 8.
The fluorescence excitation and emission spectra of QDS reacting with
2.0 × 10−6 mol L−1; C
6.0 μg mL−1; pH 4.5.
QDS
variable CR concentrations. Conditions—C
6.0 μg mL−1, C ( × 10−5 mol L−1): (1–2)
QDS
CR
0; (3) 0.2; (4) 0.4; (5) 0.6; (6) 0.8; (7) 1.0, pH 4.5.
hydrophobic interface enhances the RLS intensity. The forma-
tion process of the supermolecular complex between QDS and
CR is shown in Fig. 6. From comparisons between RLS and
cluded that the ion-association complex between QDS and CR is
absorption spectra, shown in Fig. 7, it can be seen that the RLS
formed by function of electrostatic and hydrophobic interaction
spectra of complex for QDS–CR is situated near their absorption
force, which greatly enhances the RLS intensity.
band. RLS is an absorption rescattering process, which is pro-
duced when resonance takes place between the RLS and the
Analytical application
light absorption with equal frequency.[29,32] Therefore, the RLS
peak (562 nm) is close to the corresponding absorption peak
In order to evaluate the applicability and reliability of the pro-
(560 nm). In this case, a resonance scattering effect is produced,
posed method, the present method was applied to determine
which leads to the enhanced RLS intensity.
QDS in human serum and urine samples using the standard
The fluorescence excitation and emission spectra of QDS reacting
addition method. In addition, the proposed method was also
with variable CR concentrations are shown in Fig. 8. It can be
used for the determination of QDS in simulated serum samples.
seen from Fig. 8 that two excitation peaks are located at 254 and
The concentration of each component of simulated serum samples
350 nm and one emission peak is lcated at 451 nm. Figure 8
was chosen to match its normal level in human serum.[33] The
shows the fluorescence emission spectra of QDS reacting with
results are listed in Table 3. The relative standard deviation (RSD)
variable CR concentrations which were operated by 254 nm of
and recovery were examined by using the standard addition
the maximum excitation wavelength. With the various amounts
method. It can be found from Table 3 that the RLS method has
of CR added, a decrease in fluorescence intensity could be
a good repeatability. The RSD of serum is 4.2–2.8%. That of the
observed, which might be attributed to the supermolecular
human urine is 3.2–2.3%. The recovery of serum is 102.2–100.6%.
complex between QDS and CR. This phenomenon indicates
That of human urine is 99.0–100.4%. The results showed that
that interaction between QDS and CR occurs, which is beneficial
this simple, fast and sensitive method can be successfully applied
to the RLS intensity enhancement. Therefore, from the absorp-
to the determination of QDS in urine and simulated huamn
tion, fluorescence and RLS spectra shown above, it can be con-
serum samples.
Table 3.
The results for determination of QDS in serum and urine samples (conditions: pH 4.5, CCR
1.0 × 10−5 mol L−1)
Sample
Found
Added
Total found mean
Recovery
RSD (n = 5)
(μg mL−1)
(μg mL−1)
(n = 5) (μg mL−1)
(%)
(%)
Serum 1
NDa
2.0
2.02
101.0
3.7
Serum 2
ND
5.0
5.03
100.6
2.8
Urine 1
ND
2.0
1.98
99.0
3.2
Urine 2
ND
5.0
5.02
100.4
2.3
SMSSb 1
2.2
2.0
4.26
101.4
4.2
SMSS 2
5.3
1.0
6.44
102.2
3.6
aNot detected; bsimulated serum sample.
5
Luminescence 2009
Copyright © 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/bio
Y. Zeng et al.
Conclusions
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Luminescence 2009
