Fluorescence enhancement effect for the determination of adenosine 5′-monophosphate with 9-anthracene carboxylic acid-cetyl trimethyl ammonium bromide system

Article abstract of DOI:10.1007/s10895-011-0900-9

A fluorimetric method based on fluorescence enhancement effect was developed for the determination of adenosine 5′-monophosphate (AMP) with 9-anthracene carboxylic acid (9-ANCA)-cetyl trimethyl ammonium bromide (CTAB) system. Fluorescence intensity of 9-ANCA was decreased by the addition of CTAB but addition of AMP again rose the intensity of 9-ANCA gradually. The observed fluorescence enhancement is attributed to the competitive binding reaction of 9-ANCA and adenosine to CTAB. The enhancement in the fluorescence intensity was found proportional to the concentration of AMP over the range 2.0∈×∈10^-4 to 1.2∈×∈10^-3 mol dm^-3. The ion pair complex is formed spontaneously between 9-ANCA and CTAB. Since the binding interaction is larger for the adenosine-CTAB pair, the fluorophore 9-ANCA will be released. The quantum yield of free 9-ANCA is higher therefore its fluorescence observed at 417 nm wavelength is enhanced. This mechanism of competitive molecular interaction is further confirmed by conductometric measurements. The method was applied successfully for the determination of AMP from pharmaceutical sample. The method is more selective, sensitive and relatively free from interferences.

Full text of DOI:10.1007/s10895-011-0900-9

J Fluoresc (2011) 21:1997–2003
DOI 10.1007/s10895-011-0900-9
ORIGINAL PAPER
Fluorescence Enhancement Effect for the Determination
of Adenosine 5′-Monophosphate with 9-Anthracene
Carboxylic Acid-Cetyl Trimethyl Ammonium
Bromide System
Mahadev S. Khot & Netaji K. Desai &
Govind B. Kolekar & Shivajirao R. Patil
Received: 19 February 2011 /Accepted: 20 May 2011 /Published online: 28 June 2011
#
Springer Science+Business Media, LLC 2011
.
Keywords Fluorescence enhancement 9-Anthracene
Abstract A fluorimetric method based on fluorescence
enhancement effect was developed for the determination of
adenosine 5′-monophosphate (AMP) with 9-anthracene
carboxylic acid (9-ANCA)–cetyl trimethyl ammonium
bromide (CTAB) system. Fluorescence intensity of 9-
ANCA was decreased by the addition of CTAB but addition
of AMP again rose the intensity of 9-ANCA gradually. The
observed fluorescence enhancement is attributed to the
competitive binding reaction of 9-ANCA and adenosine to
CTAB. The enhancement in the fluorescence intensity was
found proportional to the concentration of AMP over the
range 2.0×10−4 to 1.2×10−3 mol dm−3. The ion pair
complex is formed spontaneously between 9-ANCA and
CTAB. Since the binding interaction is larger for the
adenosine–CTAB pair, the fluorophore 9-ANCA will be
released. The quantum yield of free 9-ANCA is higher
therefore its fluorescence observed at 417 nm wavelength
is enhanced. This mechanism of competitive molecular
interaction is further confirmed by conductometric
measurements. The method was applied successfully for
the determination of AMP from pharmaceutical sample.
The method is more selective, sensitive and relatively
free from interferences.
.
.
carboxylic acid Adenosine 5′- monophosphate
.
Ion-association complex Cetyl trimethyl
ammonium bromide
Introduction
The photochemical interaction of chromophores with
nucleotides, nucleosides and their bases has been the focus
of extensive recent research [1–3]. The studies on inter-
actions between DNA and self-assembled nanostructures,
such as micelles and vesicles, have been major concern,
due to the possibility to use these complexes as transfection
tools [4]. The electrostatic interaction between the nucleo-
tides and fluorophores like coumarine, pyrenes were
extremely studied to develop a method for DNA sequencing
and possible mechanisms of electron transfer are shown to
involve in the interactions [5, 6]. Among the technological
applications, the use of micellar systems for the study of
synthetic nucleolipids is recently coming up [7]. The
electrostatic interactions between the positively charged
cationic surfactant molecules and the negatively charged
mononucleotides are expected to lead to the formation of
complexes. Furthermore by studying these interactions
significant information as to the recognition, sensing of
anionic analytes and supramolecular chemistry are reported
in the literature [8].
M. S. Khot (*)
Ch. Sambhaji Raje Sainik School,
Jamge (Khed),
The quantitative determination of nucleotides is required
in many fields, such as molecular biology, biotechnology
and pharmacodynamics studies. The natural fluorescence
intensity of nucleotides is very weak to use in direct
fluorimetric method [9]. The DNA bases and nucleotides
are known to interact with different fluorescent probe in
Ratnagiri 415709 Maharashtra, India
e-mail: msk_chem77@rediffmail.com
:
:
N. K. Desai G. B. Kolekar S. R. Patil
Fluorescence Spectroscopy Laboratory,
Department of Chemistry, Shivaji University,
Kolhapur 416 004 Maharashtra, India

1998
J Fluoresc (2011) 21:1997–2003
water and other environment also [6, 10]. The enhancement
in fluorescence intensity of ethidium bromide is reported
due to direct interaction with DNA bases and nucleotides
[11–15]. However, quenching of fluorescence probes by
interaction with DNA bases and nucleotides is also seen to
be used in analysis [16–18].
In view of a search for new selective and more
sensitive fluorescent probes for detection of DNA the
electrostatic interactions between the analytes and
oppositely charged probe molecule were made of use.
The 9-anthracene carboxylic acid (9-ANCA) is widely
used as probe molecule because of its high quantum
yield for detection of cationic surfactant and trace metal
ions [19–21]. Adenosine monophosphate consisting of
the phosphate group, the pentose sugar ribose, and the
nucleobase adenine is an intermediary substance formed
during the body’s process of creating energy in the form
of adenosine triphosphate (ATP) from food. The body
creates AMP within cells during normal metabolic
processes. It has medicinal importance and its injection
available commercially is used in postherpetic neuralgia
treatment. The present work aimed to develop fluorimet-
ric method for analysis of adenosine by using 9-ANCA.
The direct molecular interaction between 9-ANCA and
AMP is not possible because both are negatively charged
species. Hence possible electrostatic interactions between
the pairs ANCA-CTAB and CTAB-AMP are used to
develop a new fluorimetric method for analysis AMP
from aqueous solution and applied successfully for
pharmaceutical samples containing AMP. The competi-
tive electrostatic interaction between these two pairs was
examined by conductometric studies. The present method
is selective and more sensitive to other methods such as
spectrophotometry, chemiluminescence, electrochemical
and capillary electrophoresis reported in the literature
[22–26].
Fig. 1 Absorption spectra of 9-ANCA (A), with CTAB (B) and with
CTAB-AMP mixture (C) in aqueous solution
Preparation of Solutions
Stock solution of 9-ANCA of concentration (4.0×
10−4 mol dm−3) was prepared by dissolving 0.0044 g in
50 mL double distilled water and stirring overnight. Stock
solution of AMP (1.0×10−2 mol dm−3) was prepared by
dissolving 0.0977 g in 25 mL double distillated water.
Similarly, stock solution of CTAB (3.0×10−3 mol dm−3)
was prepared by dissolving 0.1093 g in 100 mL double
distillated water. Tris (hydroxymethyl) amino methane
hydrochloride buffer (5.0×10−2 mol dm−3) was prepared
by dissolving 0.7889 in 100 mL deionised water and then
the pH was adjusted to 7.5 using reported procedure [27].
The fluorescence intensity of 9-ANCA is maximum in
solution of tris (hydroxymethyl) amino methane hydrochlo-
ride of pH 7.5 [28]. These solutions were further diluted to
the required concentration in the experiments.
Experimental
Materials
The purity of 9-anthracence carboxylic acid obtained
from Merck-Schuchardt was confirmed by noting its
melting point, electronic spectra and by production of
similar spectra after excitation at different wavelengths.
The adenosine 5′-monophosphate, cetyl trimethyl ammo-
nium bromide and Tris (hydroxymethyl) amino methane
hydrochloride buffer obtained from s.d. fine-chem.
Limited Mumbai were used directly without further
purification. Double-distilled water was used in all
experiments. All measurements were performed at ambient
temperature.
Fig. 2 Steady state fluorescence spectra of 9-ANCA in the absence
(1) and presence of varying concentrations of CTAB in aqueous
solution (2–5), 0.7×10−4 mol dm−3 (2), 3.0×10−4 mol dm−3 (3), 6.0×
10−4 mol dm−3 (4), 1.0×10−3 mol dm−3 (5)

J Fluoresc (2011) 21:1997–2003
1999
O-
O
OH
O
Br-
H3C
N+
CH3
H3
C
N+
CH3
H3C
Br-
H+
H3
C
+
+
ANCA
H3C
H3
C
CTAB
ANCA --- CTAB Complex
Scheme 1 Mechanism of interaction of CTAB with 9-ANCA to form complex
Fluorescence and Absorption Measurements
and to develop a fluorimetric method more sensitive to
spectrophotometry.
Steady-State fluorescence and excitation spectra of the
solutions were recorded on PC based spectrofluorophotometer
(JASCO Model-FP-750, Japan) equipped with 150 W Xenon
lamp source and 1.00 cm quartz cell. The excitation
wavelength 364 nm was obtained from the excitation
spectrum and the emission spectrum was monitored at
this excitation wavelength. The excitation and emission
slit width were kept constant at 10 nm during the
measurements. The absorption spectra of solutions were
recorded on UV-Visible spectrophotometer (UV-3600
SHIMADZU) using 1.00 cm quartz cell. All solutions
were degassed by passing nitrogen gas before subjected
to optical measurements. The absorption and excitation
spectra were found identical. The conductivity of
solution was measured by using conductivity meter
(model No.EQ.660A EQUIP-TRONICS) equipped with
dip type conductivity cell (cell constant 1.00 cm−1).
Fluorescence Spectra of 9-ANCA in Presence of CTAB
The aqueous solution of 9-ANCA of concentration of 4.0×
10−6 mol dm−3, exhibited a strong fluorescence at 413 nm.
The cationic surfactant such as CTAB is known to
solubilize and stabilize the drugs and DNA molecules of
opposite charges and may result in several desirable or
undesirable interactions. Hence effect of CTAB concentra-
tion on fluorescence of 9-ANCA was studied and given in
Fig. 2. From the figure it is observed that the fluorescence
intensity of 9-ANCA was quenched by addition of CTAB
(spectra 1 to 4) below its CMC (6.3×10−4 mol dm−3), due
to the formation of non-fluorescent ion-association complex
between two molecules [29] .The fluorescence spectrum of
9-ANCA is shifted to red from 413 nm to 417 nm, which is
an indication of complex formation as shown in Scheme 1.
However, when the concentration of CTAB was higher than
critical micelle concentration, CTAB micelles were formed
and the fluorescence intensity of the 9-ANCA at 417 nm
seen to increased at higher CTAB concentration in Fig. 2
(spectrum 5). This is because of the fact that the 9-ANCA
Results and Discussion
The interaction between 9-ANCA-CTAB and 9-ANCA-
CTAB-AMP were studied by absorption and fluorescence
spectrometry. Figure 1 shows, absorption spectra of 9-
ANCA alone (A), with CTAB (B) and with CTAB-AMP
mixture (C). The absorption spectrum of 9-ANCA is
banded with maximum at 364 nm. The addition of dilute
solution of CTAB below its critical micelle concentration
(CMC) not only decreases the absorbance but also the
wavelength of maximum absorbance seems to be shifted
slightly from 364 to 368 nm. The observed spectral changes
led to believe the formation of ion-pair complex between 9-
ANCA and CTAB. However, introduction of AMP in the 9-
ANCA-CTAB mixture, again rose the absorbance of 9-
ANCA. It is believed that the presence of AMP act to break
the ion-association complex. The observed absorption
spectral results were used to optimize 9-ANCA-CTAB
system as a fluorescent probe for determination of AMP
Fig. 3 Conductometric titration curve of 9-ANCA with CTAB system
in aqueous solution, [9-ANCA]: 4.0×10−6 mol dm−3

2000
J Fluoresc (2011) 21:1997–2003
Fig. 4 Steady state fluorescence spectra of the interaction between 9-
ANCA-CTAB complexes with varying concentrations of AMP in
aqueous solution (A to G) 0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0×
10−4 mol dm−3 respectively, [9-ANCA]: 4.0×10−6 mol dm−3
,
[CTAB]: 3.0×10−4 mol dm−3. Inset shows the fluorescence spectra
of 9-ANCA as a function of [AMP] in absence of CTAB
Fig. 5 Conductometric titration curves for 9-ANCA-AMP (▲) and
CTAB-AMP (■) system in aqueous solution, [9-ANCA]: 4.0×
10−6 mol dm−3, [CTAB]: 3.0×10−4 mol dm−3
molecules were bound to the core of CTAB micelles and
protected from the ion-association complex formation. The
complex formation between 9-ANCA and CTAB was also
examined and followed by conductometric titration. A
typical conductometry curve for the titration of 9-ANCA
against varying concentration of CTAB shown in Fig. 3,
exhibits the expected interactions between the two mole-
cules below and above CMC. The decrease in specific
O-
O
OH
HO
N
H3C
N+
CH3
H3C
OH
P
N
O
O
OH
N
+
O
N
H2N
AMP
H3C
ANCA---- CTAB Complex
OH
HO
N
OH
P
O-
+
N
H C
3
O
O
CH
3
N
H C
3
N
O
O
OH
N
H2N
+
H C
3
Free ANCA
AMP ---- CTAB Complex
Scheme 2 Mechanism of competitive interaction of CTAB and AMP with 9-ANCA

J Fluoresc (2011) 21:1997–2003
2001
Table 2 Influence of coexisting substances on 9-ANCA-CTAB and
AMP system in aqueous solution [9-ANCA]: 4.0×10−6 (mol dm−3),
[CTAB]: 3.0×10−4 (mol dm−3) and [AMP]: 4.0×10−4 (mol dm−3
)
Sr.No.
Foreign
substance
Concentration
(mg/mL)
Change in
intensity (%)
1
2
Zn2+
Cu2+
Mn2+
La3+
Fe3+
1
1
+2.70
−4.18
+1.93
−4.44
−6.8
−7.82
+0.32
+12.65
−3.60
+11.08
3
1
4
1
5
1
6
Cr3+
1
7
SCN−
EDTA
Glucose
Citrate
1
8
100
100
100
9
Fig. 6 Plot of the enhanced fluorescence intensity as a function of
concentration of AMP in aqueous solution
10
conductivity is because of reduction in charged species due
to complexation before CMC whereas increase in conduc-
tivity is due to separation of 9-ANCA ion from complex
due to CTAB micelle.
The present work required 9-ANCA-CTAB system
having minimum fluorescence intensity. Hence the optimal
CTAB concentration was decided from Fig. 2 indicating
fluorescence intensity changes of 9-ANCA with CTAB
below CMC. The intensity of (4.0×10−6 mol dm−3) 9-
ANCA is minimum present in (3.0×10−4 mol dm−3) CTAB
concentration. The complex forming system of these
concentrations was used further to study the interaction
with AMP solution.
and AMP did not interact directly because both are
negatively charged species. But CTAB-AMP is ion pair of
opposite charges and therefore the possible competitive
binding interaction between them releases 9-ANCA. The
quantum yield of free 9-ANCA is higher therefore its
fluorescence observed at 417 nm wavelength is enhanced.
The conductometric studies between 9-ANCA-AMP and
CTAB-AMP also supports the expected interactions in the
CTAB-AMP. A typical conductometry curves for the
titration of 9-ANCA and CTAB against varying concentra-
tion of AMP are shown in Fig. 5. Figure shows that the
specific conductance of CTAB increases appreciably with
addition of AMP while that of 9- ANCA does not vary with
concentration of AMP. It is concluded that AMP has strong
molecular interaction with CTAB rather than with 9-
ANCA.
Interaction of the 9-ANCA-CTAB Complex with AMP
The fluorescence spectra of 9-ANCA-CTAB system of
optimal concentration as determined above are recorded in
presence of varying concentration of AMP and shown in
Fig. 4. It is observed that the intensity of fluorescence of
emission band at 417 nm increases gradually without any
spectral shift. It demonstrated that AMP interacted with
CTAB and dissociated the non-fluorescent ion-association
complex of the 9-ANCA and CTAB in aqueous solution
shown in Scheme 2. These molecular interactions are
feasible only in ternary 9-ANCA-CTAB-AMP system
because fluorescence spectra of 9-ANCA with AMP
concentration shown in inset of Fig. 4, did not show any
significant spectral changes. It is concluded that 9-ANCA
Calibration Curve for AMP Determination
Under optimal conditions, calibration graph for the
determination of AMP was constructed. The enhanced
fluorescence intensity was represented as ΔF ¼ F ꢀ Fo,
where F and Fo were the fluorescence intensities of the
systems with and without AMP, respectively. The calibra-
tion graph was constructed by plotting the enhanced
fluorescence intensity (ΔF) at 417 nm as a function of
AMP concentration and presented in Fig. 6. The enhanced
fluorescence intensity showed a good linear relationship
Table 1 Analytical characteristics of the AMP determination
Nucleotide
Linear range
(mol dm−3
Linear regression equation
(mol dm−3
Correlation
coefficient (R)
Standard
deviation
Relative standard
deviation (%)
Detection limit
)
)
(mol dm−3
)
ΔF ¼ 3:9333 þ 3:4679 ꢁ 104
AMP
2.0×10−4 to 1.2×10−3
0.9990
0.1209
1.99
1.05×10−5

2002
J Fluoresc (2011) 21:1997–2003
Table 3 Results of determination of AMP from pharmaceutical sample
Sample
Composition
Amount found (mg)
S.D.
R.S.D. (%)
1.97
I
II
III
Adenosine injection Sanofi-synthelabo,
Glaxo wellcome prod. Bondeville, France
(i)Adenosine Ph. Eur. 6 mg per
2 mL (ii) NaCl solution, 0.9% w/v
5.80
6.00
5.80
0.1197
with the concentration of AMP in the range of 2.0×
10−4 mol dm−3 to 1.2×10−3 mol dm−3, and the regression
equation was ΔF ¼ 3:9333 þ 3:4679 ꢁ 104 C, where C is
the concentration, correlation coefficient was 0.9990. The
detection limit given by the equation, Clim: ¼ 3d=k, where
δ is the standard deviation of blank determination (n=6)
and k is the slope of calibration graph. The relative
standard deviation is 1.99% for the determination of 6.0×
10−4 mol dm−3, shown in Table 1.
form ion association pair complex. This system is opti-
mized for fluorescence performance because addition of
CTAB below its CMC has quenched the fluorescence of 9-
ANCA while the fluorescence is increased when CTAB
concentration is above CMC. The optimal 9-ANCA-CTAB
system exhibiting minimum fluorescence was used to study
the interactions with biologically important analytes AMP.
The conductometric studies indicated the competitive
electrostatic interactions between 9-ANCA-CTAB and
CTAB-AMP. Therefore addition of AMP into optimal 9-
ANCA-CTAB enhanced the fluorescence intensity linearly.
The calibration curve obtained between fluorescence
enhancement and concentration of AMP was used for
analysis of adenosine injection. The determination of AMP
by this method is reliable, precise and simple. Therefore,
this method has the potential of practical applications in the
determination of AMP from pharmaceutical sample avail-
able commercially.
Effect of Coexisting Substances and Method of Analysis
Under the optimal conditions, effects of interferences that
usually used as the compatibility of coexisting substances
on the fluorescence intensity of the system were studied
[30]. Initially, coexisting substances to the AMP solution
are taken in large excess and the fluorescence intensities are
measured. When interference was found to be intensive, the
tests were repeated with successive smaller amounts of
coexisting substances. According to the proposed system,
the most of them have little effect on the determination of
AMP. For a relative error of less than 5%, the results are
shown in Table 2.
Acknowledgement The authors gratefully acknowledge, Depart-
ment of Science and Technology (DST) and University Grants
Commission (UGC) New Delhi for providing grants to the Depart-
ment of Chemistry, Shivaji University, Kolhapur under FIST and SAP
program, respectively.
Analysis of AMP from Pharmaceutical Sample
References
The observed fluorescence enhancement of 9-ANCA-CTAB
system was employed for determination of adenosine 5′
monophosphate in pharmaceutical sample namely Adenosine
injection [26]. For the assay of adenosine 5′ monophosphate
the sample must be diluted appropriately within the linear
range of determination of AMP. The sample solution was
analysed by the method developed above. Though adenosine
injection contains NaCl, it does not interfere on fluorescence
intensity measurements because of use of selective Tris–HCl
buffer. The results are in good agreement with certified
values performed in triplicate, which affords good precision
and accuracy. The values are reported in Table 3.
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