Research article

Received: 27 August 2013, Revised: 30 September 2013, Accepted: 3 October 2013 Published online in Wiley Online Library: 21 November 2013

(wileyonlinelibrary.com) DOI 10.1002/bio.2602

Determination of amlodipine using terbium-

sensitized luminescence in the presence of
europium(III) as a co-luminescence reagent
Salma M. Z. Al-Kindy,* Abdalla Al-Snedi, Fakhr Eldin O. Suliman
and Haidar A. J. Al-Lawati
ABSTRACT: A sensitive time-resolved luminescence method for the determination of amlodipine (AM) in methanol and in
aqueous solution is described. The method is based on the luminescence sensitization of terbium (Tb3+) by formation of a ter-
nary complex with AM in the presence of tri-n-octylphosphine oxide (TOPO) as co-ligand, dodecylbenzenesulfate as surfactant
and europium ion as a co-luminescence reagent. The signal for TbAMTOPO is monitored at ex = 242 nm and em = 550 nm.
Optimum conditions for the formation of the complex in aqueous system were 0.015 M Tris (hydroxylmethyl) amino methane
buffer, pH 9.0, TOPO (1.0 104 M), Eu3+ (2.0 107 M), dodecylbenzenesulfate (0.14%) and 6.0 105 M of Tb3+, which allows the
determination of 1050 ppb of AM with a limit of detection of 1.2 ppb. The relative standard deviations of the method range
between 0.1 and 0.2% indicated excellent reproducibility of the method. The proposed method was successfully applied for
the assay of AM in pharmaceutical formulations and in plasma samples. Average recoveries of 98.5 0.2% and 95.2 0.2%
were obtained for AM in tablet and plasma samples respectively. Copyright 2013 John Wiley & Sons, Ltd.

Keywords: sensitized luminescence; amlodipine; plasma; pharmaceutical products; co-luminescence

Introduction
Amlodipine (3-ethyl-5-methyl-2-[2-aminoethoxy) methoxy]-
4-(o-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate
monobenzenesulfonate) (AM) (Fig. 1) is a relatively new and
potent long-acting calcium channel blocking agent. Amlodipine is
a third-generation calcium antagonist, which has a dihydropyridine
ring as part of its structure and it is used alone or in combination
with other medications for treating high blood pressure, certain
types of vasospastic angina, cardiac arrhythmias and coronary heart
failure (1,2). It selectively inhibits the arterial vascular smooth muscle
cell proliferation, which prevents progressive narrowing of arteries
and coronary spasms resulting in increased blood ow with myocar-
dial oxygen supply (13).
Owing to the therapeutic importance of AM, numerous
analytical methods have been developed for its quantitative
determination in pure, pharmaceutical dosage forms and/or bio-
logical uids (4). These methods include high-performance liq-
uid chromatography combined with electrospray ionization
tandem mass spectrometry (57), voltametric (4) and spectro-
photometric methods (810). Spectrouorimetric methods for
the quantication of AM reported recently are based on indirect
methods involving uorescent derivatization techniques with
uorigenic labels such as 7-chloro-4-nitro-2,1,3-benzoxadiazole
and ninhydrin and phenylacetaldehyde in buffered medium
(11). Luminescence methods are simple, sensitive, selective
and well suited for the determination of trace amounts of drugs
in biological materials and in some dosage forms. Among the
luminescence techniques are lanthanide-sensitized lumines-
cence methods that result from complexation of Ln3+ ion with
a suitable ligand. Lanthanide ions exhibit weak molar absorptiv-
ity and low uorescence quantum yields. However, when
chelated with ligands that have broad intense absorption bands,
an appreciable enhancement in the luminescence intensity of
the system has been observed. This arises from the intramolecu-
lar energy transfer through the excited state of the ligand, which
serves as an antenna chromophore, to the emitting level of the
Ln(III) ion. The intramolecular energy transfer from the triplet
state of the organic ligand to the Ln3+ ion depends on the
structure of the ligand and the position of its triplet state (12).
This phenomenon is called the antenna effect and such
complexes are considered to be light conversion molecular
devices because they are able to transform light absorbed by
the ligand into light emitted by the ions via an intramolecular
energy transfer process. As part of our ongoing interest on
method development on drug analysis (1317), we report a novel
method for the assay of AM in pharmaceutical formulations and
in biological uids. The method is based on the luminescence
enhancement and sensitization of terbium brought about by
complexation with AM. The luminescence properties were
investigated in various solvents. Factors affecting complexation
such as the concentration of Tb3+ and the ratio of Tb3+ to AM
as well as the effect of co-ligands and co-luminescence agents
on the luminescence properties of Tb-AM have been carefully
studied. The method was subsequently used to determine the
concentration of AM in pharmaceutical and plasma samples.
* Correspondence to: S. M. Z. Al-Kindy, Sultan Qaboos University, College of
Science, Box 36, Department of Chemistry, Al-Khod 123, Sultanate of Oman.
Email: alkindy@squ.edu.om
Sultan Qaboos University, College of Science, Box 36, Department of
657

Chemistry, Al-Khod 123, Sultanate of Oman

Luminescence 2014; 29: 657662 Copyright 2013 John Wiley & Sons, Ltd.

Figure 1. Chemical structure of amlodipine.
Experimental
Apparatus
Luminescence measurements were recorded with a Perkin Elmer
LS-55 luminescence spectrometer (Beaconseld, UK) in a 1 cm
quartz cuvette. The spectrometer was connected to a computer
with FL WINLAB (version 4.00.02) software. The spectrometer
was equipped with a xenon discharge ash lamp. The instru-
ment was operated in the phosphorescence mode (time
resolved mode) with a delay time of 0.04 ms and a gate time of
1.0 ms. The excitation and emission slits were both set at 10 nm with
a scan speed of 800 nm/min. Absorbance measurements were made
on a Varian CARY 50 Conc UV visible spectrophotometer (Sydney,
Australia) with 1 cm matched quartz cell. The pH values were mea-
sured using Hanna (Romania) HI 8314 membrane pH meter.
Reagents
All reagents used in this work were prepared from analytical grade
reagents and were used without further purication. All solvents
used were of high-performance liquid chromatography grade.
Ultrapure water (Milli-Q Millipore Corporation, Milford, MA, USA)
was used. Tris (hydroxylmethyl) amino methane (TRIS), tri-n-
octylphosphine oxide (TOPO) were obtained from Sigma-Aldrich
(St Louis, MO, USA). The surfactants used in this study include
Tween-20 (TW-20), Tween-80 (TW-80), Triton X-100 and sodium
dodecyl sulfate (SDS) (All from Sigma, Steinheim, Germany) whereas
sodium dodecylbenzenesulfate (SDBS) was from a Chem Heri,
Shanghai, China.
Stock solutions of 1 104 M (AM) provided by the Jordanian
Pharmaceutical Manufacturing Company (Amman, Jordan) was
prepared in methanol and stored in a refrigerator. The solution
was found to be stable for 1 week, after which fresh stock
of AM was prepared. Tb3+ 1 104 M and TOPO 1 103 M
solutions were prepared in methanol. A working standard of
AM (1 106 M) was freshly prepared by appropriate dilution
of the stocks with methanol.
Stocks standards of: 1 103 M Tb3+, 1% SDBS, 0.1 M TRIS were
prepared in ultrapure water. Fresh stock solutions (1 103 M) of
lanthanum(III) chloride, zinc(III) chloride, gadolinium(III) chloride,
samarium(III) chloride and europium(III) chloride were prepared
in ultrapure water. Pharmaceutical formulations of AM were
provided by Sultan Qaboos University Hospital.
Preparation of pharmaceutical formulations
Five tablets of AM were weighed and ground to a ne powder
using a pestle and mortar. An equivalent amount of the powder
containing a known amount of active material was weighed out
and dissolved in methanol, sonicated for 15 min, ltered and
diluted to the mark with methanol to make a stock solution.
Convenient aliquots from the working solutions were taken for
the determination of AM by luminescence.
658
wileyonlinelibrary.com/journal/luminescence Copyright 2013 John Wiley & Sons, Ltd.
S. M. Z. Al-Kindy et al.
Preparation of plasma samples
The plasma samples (donated by the Blood Bank, Muscat)
were stored in the freezer at 10C and thawed at room tem-
perature before processing. Plasma was spiked with conve-
nient amounts of AM stock solutions. Untreated plasma
samples gave turbid solutions, so a deproteinization process
with acetonitrile was employed. One mL of serum was
deproteinized with 2 mL of acetonitrile, by vortexing for 1
min followed by ltration.
Procedure
Aliquots of the working solution were transferred to 10 mL
calibrated asks. To these solutions 0.6 mL of stock Tb3+, 1 mL
of stock TOPO solution and 1.6 mL of stock SDBS solution were
added. Furthermore, 0.2 mL of Eu3+ as co-luminescent agent
was also added to this mixture. The solution was diluted to the
mark with 0.015 M TRIS solution. The pH of TRIS solution was
adjusted to 9. The AM concentration range was 0100 ppb.
The luminescence was measured at 550 nm, using an excitation
wavelength of 242 nm. The concentration of AM in the sample
was determined from a calibration curve prepared under
identical conditions.
Results and discussion
Spectral characteristics
The absorption spectra of AM and its Tb(III) complex were
recorded in methanol. Both the ligand and the complex
exhibited absorption maximum at 240 and 350 nm, with the
maximum absorptivity observed at 240 nm. A slight increase in
absorptivity was noticed for the complex when compared to
AM alone. A weaker absorption was seen for the metal solution
in the ultraviolet (UV) region.
To improve the analytical characteristics for the determination
of AM in pharmaceutical formulations we initially investigated
the complexation of TbAM in methanol. Our aim was to sensi-
tize the luminescence of the terbium ion using AM. The results
shown in Fig. 2 indicate that the luminescence intensity of ter-
bium is very weak. However, upon addition of AM to Tb+3 solu-
tions, the intensity of Tb+3 emission was signicantly enhanced
(Fig. 2). This enhancement is due to the efcient energy transfer
3+
Figure 2. Luminescence excitation and emission spectra of: 1. and 3. Tb in meth-
3+ 3+ 5
anol system, 2. and 4. Tb in presence of AM in methanol. [Tb ] = 8 10 M;
5 3+
[AM] = 1 10 M; ex of Tb AM complex = 242 nm, em = 550 nm. AM, amlodipine.
Luminescence 2014; 29: 657662
Determination of amlodipine using terbium sensitized luminescence
of excitation energy from AM to the encapsulated Tb+3 ions.
Using the Jobs continuous variation method the stoichiometry
of the complex was found to be 1 : 1 Tb3+/AM.
It worth mentioning here that the successful sensitization
process requires the sensitizing moiety to be excited in the near
UV region, as its triplet state energy should be sufcient to
provide efcient energy transfer to the rare earth ion. The
previous result agrees with the successful sensitization process
as AM contains efcient chromophores that absorbs in the UV
region (max = 240 nm).
The absorption spectra of AM, and the excitation spectra of
the TbAM complex exhibited an excellent agreement between
the absorption and excitation spectra (max = 240 nm for
absorption, excitation = 242 nm) for AM. This is an indication of
the efcient sensitization process and that; the antenna chromo-
phore is the only photophysical pathway leading to the
observable luminescence in the system (18,19).
The luminescence emission spectrum of TbAM, when excited
by the -* absorption band at 240 nm of the complex, revealed
the well-known bands of terbium luminescence, based on
5
D47F6 (490 nm), 5D47F5 (550 nm), 5D47F4 (590 nm) and
5
D47F3 (620 nm). Among these bands, that at 5D47F5 at 550
nm was found to be the strongest and hence the peak height
at 550 nm was used to monitor the TbAM complex. Although
this emission band partly overlaps with the second-order scatter
from the excitation wavelength, this was eliminated by the
time-resolved mode, which can be used in these systems as they
have a relatively long luminescence lifetime. Rayleigh scattering
was eliminated using a 0.04 ms td and maximum luminescence
intensity was found with a 1.0 ms tg. Moreover, a large Stokes
shift was observed (> 250 nm), indicating that there is no
overlap of the Tb3+emission band with the antenna
chromophore absorption bands.
Optimization of conditions in aqueous media
The conditions that were developed in methanol media were
further applied in aqueous system but no sensitization of the
Tb3+ luminescence was observed due to the well-known
quenching by water molecules. In aqueous solutions, the
dominant mode of luminescence quenching of the excited ions
occurs via coupling of the Tb(III) excited state to OH oscillators
of water molecules coordinated to the ion. The luminescence
lifetime of an excited state of the Tb(III) ion is linearly
proportional to the number of H2O molecules in the inner
coordination of this ion. An increase in the number of water
molecules in the rst coordination sphere leads to a decrease
in the luminescence (18). Therefore, for this reason we investi-
gated the effect of surfactants, co-ligands and buffer types and
concentration on the luminescence intensity of TbAM system
in aqueous media.
Luminescence emission and excitation spectra of Tb3+, Tb3+
SDBS, Tb3+SDBSAM in aqueous system are shown in Fig. 3.
The excitation and emission spectrum of Tb3+ was increased
by using SDBS, indicating that Tb3+ could interact with SDBS
accompanied by energy transfer from the SDBS to Tb3+. When
AM was added to the Tb3+SDBS solution, the luminescence in-
tensities were markedly enhanced compared to the Tb3+SDBS
system, indicating that the interaction of Tb3+ with AM results
in an efcient energy transfer from the drug to Tb3+. The Tb
(III)AM complex exhibited time-resolved luminescence in
aqueous systems only in the presence of surfactant.
Luminescence 2014; 29: 657662 Copyright 2013 John Wiley & Sons, Ltd.
Figure 3. The effect of SDBS in excitation and emission spectra of TbTOPOTRIS
3+ 3+ 3+
AM complex. 1. Tb TOPOTRIS; 2. Tb TOPOTRISSDBS; 3. Tb TOPOTRIS
+3 5 4
SDBSAM. [Tb ] = 6 10 M; [TOPO] = 1 10 M; [SDBS] = 0.16%; [TRIS] = 0.015 M
(pH 9). AM, amlodipine; SDBS, sodium dodecylbenzenesulfate; TOPO, tri-
n-octylphosphine oxide; TRIS, Tris (hydroxylmethyl) amino methane.
The optimum concentration of SDBS in the present study was
slightly higher than the critical micelle concentration of SDBS
(= 0.63 mmol/L) (20,21). The excitation spectra of Tb3+SDBS in
aqueous system revealed two structural bands and a shoulder,
with the maximum band at 242 nm (Fig. 3). Addition of AM in
the presence of TRIS buffer resulted in a slight blue shift of the
bands. The luminescence enhancement of Tb3+SDBS in the
presence of AM and the shift in the excitation wavelength and
the appearance of new bands indicate complex formation
between the drug and the Tb3+ ion.
It is well known that addition of micelles allows the removal of
the efcient quenching effect of water in aqueous system by
forming an aggregate with the hydrophilic head region in
contact with the surrounding solvent and as a result the possibil-
ity of complexation is more rapid (22). In a previous study (23), it
was reported that micelles provide a discrete volume region for
inclusion of the Ln3+ ion and the ligand; therefore, SDBS used as
a surfactant will act as an energy donor. It is worth noting here
that a number of surfactants (Tween-20, Tween-80, Triton
X-100, SDS in addition to SDBS) were investigated in this study.
The concentration of surfactants was xed at 0.1%, which is
above the critical micelle concentration of all of the used surfac-
tants. Among these surfactants, SDS and SDBS gave the highest
intensity of the luminescence signal. The specic effect of
anionic surfactants on the luminescence intensity can be
associated with the ability of negatively charged micelles to
concentrate Tb3+ ions at their surfaces by electrostatic interac-
tion, while the solubilization of ligands is mainly due to the
hydrophobic interaction of the hydrocarbon skeleton of the mol-
ecule with the hydrocarbon part of the anionic surfactant (20).
Therefore, anionic surfactants SDS and SDBS are more effective
for enhancing the luminescence intensity but SDBS exhibited
the highest intensity due to the interaction of SDBS with Tb3+,
which resulted in the energy transfer from SDBS to Tb3+ and,
additionally, SDBS provides a hydrophobic environment with
relatively low polarity and high viscosity.
The highest signal was obtained when SDBS was used as a
surfactant; hence, it was important to optimize its concentration
to enhance the signal further. An increase in luminescence
intensity was observed when the surfactant concentration of
SDBS increased from 0.12% reaching a maximum at 0.16% and
then decreases at the higher concentration of SDBS as shown
659
wileyonlinelibrary.com/journal/luminescence

in Fig 4. The decrease in the intensity could be due to the
increase in the number of micelles with an increase in the surfac-
tant concentration, which results in a further dilution of the
concentration of the solubilizate per micelle as the surfactant
concentration is increased further. This causes a reduction in
the rate of solubilization (22). Another reason for a decrease in
the luminescence intensity of the complex in the presence of
high concentration of SDBS may be due to competition between
surfactant and AM for Tb+3 to form the complex. Therefore, the
concentration of SDBS in this study was maintained at 0.16%.
A possible strategy to improve luminescence intensity of Tb
AM chelates involves substitution of water molecules in the
coordination sphere with synergic agent. Several co-ligands
were investigated in this study, including TOPO, EDTA,
1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline each
at a concentration of 1 104 M. Maximum luminescence
sensitization was observed in the presence of TOPO.
Furthermore, the optimum concentration of TOPO was stud-
ied by monitoring the luminescence intensity of the TbAM
complex in the presence of various concentrations of TOPO
ranging from 0.05 to 0.17 mM. The luminescence intensity of
the complex was found to increase with an increase in the
concentration of TOPO up to 0.1 mM, after which a decrease in
intensity was observed. Coordinating TOPO with the Tb3+ ion
through its oxygen atom helps to remove water molecules from
the coordinating sphere of the metal ion. The efcacy of the
bulky hydrocarbon tail of TOPO in protecting the luminescence
complex from radiationless deactivations is manifested in the
signicant enhancement of the emission from Tb3+ ions.
However, using higher concentrations of TOPO result in increas-
ing the competition between TOPO and AM for sites at the
coordination sphere of Tb3+ ion and hence a higher enhance-
ment of Tb3+ blank emission relative to the TbAM is the net
result. Hence, in this study 0.1 mmol/L was considered the
optimum concentration of TOPO.
As was previously reported, AM (pKa = 8.6) structural forms are
highly pH dependent in the aqueous medium, where the
deprotonated form AM predominates at a pH above 8.6.
Because a neutral form is expected to be preferred for complex-
ation with Tb3+, it is necessary to investigate the inuence of pH
of the system on the sensitization process. The inuence of pH
on the luminescence intensity of the system was studied by
varying the pH of 0.01 M Tris buffer in the range of 711 while
Figure 4. The inuence of changing the concentration of SDBS on luminescence of
3+ +3 5 7
(Tb AMTOPOTRIS) system. [Tb ] = 6 10 M; [AM] = 8 10 M; [TOPO] =8 10 M;
[TRIS] = 0.01 M (pH 9). AM, amlodipine; SDBS, sodium dodecylbenzenesulfate; TOPO,
660
tri-n-octylphosphine oxide; TRIS, Tris (hydroxylmethyl) amino methane.
wileyonlinelibrary.com/journal/luminescence Copyright 2013 John Wiley & Sons, Ltd.
S. M. Z. Al-Kindy et al.
keeping other experimental factors constant and the results
are shown in Fig. 5. Possibly, the protonation of the amine group
at this pH results in the formation of less favorable species for
complexation with Tb(III) ions. Increasing the pH from 7 to 9 led
to the enhancement of luminescence intensity reaching the max-
imum at pH 9, above which a decrease in the intensity was
observed. The changes in pH would inuence the compositions
and stabilities of the luminescence complexes and resulted in
changes in the luminescence characteristics (24). This decrease
also could be attributed to the competitive hydrolysis of terbium
forming Tb(OH)3. At pH 9, the neutral form of AM is the predom-
inant species, which is favorable for the sensitization process.
The time-resolved spectra of the TbAM complex and of Tb3+
ions were studied in the presence of different types of buffers
such as sodium acetate, sodium phosphate, sodium carbonate
and TRIS buffers at pH 9. A high intensity of the signal of the
terbium complex when compared to the signal of the terbium
blank was observed in the presence of TRIS buffer indicating
favorable complex formation of terbium with the drug. On the
other hand acetate, phosphate and carbonate buffers gave a
very low difference in signal intensity in the presence and
absence of AM suggesting the possibility of competition
between the buffers and AM for terbium. Hence, the assay of
the AM was carried out in the presence of TRIS buffer. The inu-
ence of TRIS buffer concentrations was studied by varying the
concentration of pH 9 buffer in the range 0.0050.03 mol/L.
The concentration of Tb3+, AM and TOPO were maintained at
6 105 mol/L, 8 107 mol/L and 1 104 mol/L, respectively,
while the concentration of SDBS was maintained at 0.16%. On
increasing the concentration from 0.005 to 0.015 mol/L, an in-
crease in the luminescence intensity was observed, after which
the intensity started to decline. This behavior indicated that an
optimum concentration of TRIS buffer is required for maximum
complex formation between Tb3+ and AM. By exceeding the
optimum concentration, the buffer molecules start to compete
with the AM ligand for Tb3+ binding sites. This may result in less
TbAM complex being formed and hence a decrease in lumines-
cence intensity. Hence, the optimum concentration of TRIS
buffer for this study was considered 0.015 mol/L.
The effect of a Tb3+ ion concentration on the luminescence
intensity was investigated in the range 0.020.14 mmol/L. The
luminescence intensity reached a maximum at the Tb3+ ion
concentration of 0.06 mmol/L and the intensity decreased
3+
Figure 5. The inuence of changing pH on luminescence spectra of Tb AM
5 +3 5 7 5
complex in aqueous medium; [Tb ] = 1 10 M; [AM] = 4 10 M; [TOPO] = 8 10 M;
[SDBS] = 0.14%; [TRIS] = 0.01 M. AM, amlodipine; SDBS, sodium dodecylbenzenesulfate;
TOPO, tri-n-octylphosphine oxide; TRIS, Tris (hydroxylmethyl) amino methane.
Luminescence 2014; 29: 657662
Determination of amlodipine using terbium sensitized luminescence

slightly above this concentration. These results suggest that the proposed method compared favorably with most of the
an optimum concentration of Tb3+ is required to facilitate published methods for the determination of AM (Table 1). In
maximum complex formation. A slight decrease in lumines- addition, the method offered an advantage over other lumines-
cence intensity was observed when the Tb3+ concentration cence methods, in that its emission is at a longer wavelength,
was increased above 0.06 mmol/L after which the lumines- free from interference from short-emitting species present in
cence intensity remained constant. This behavior may be these matrices. The relative standard deviation of the method
explained as due to the protective power of TOPO and the was obtained for all standard solutions of AM and was found
surfactant for the AM singlet state against radiationless to be less than 1.0% (n = 8) showing excellent precision.
transitions was nally broken down by collision with free
Tb3+ ions. Therefore, the Tb3+ concentration 0.06 mmol/L
was selected for further study.
Analytical application
The proposed luminescence method was applied for the deter-
Effect of addition of co-luminescence agents
mination of AM in real samples. Sample solutions were analyzed
Effects of the co-luminescence ions such as Ln3+, Sm3+, Zn3+, for AM contents, where solutions were prepared from commer-
Eu3+ and Gd3+ were studied in TbAM system. The results are cial tablets claimed to contain 5 mg and 10 mg of AM as
shown in Fig. 6, from which we can see that Eu3+ is the most described above in the Experimental section and treated using
effective one. Furthermore, all the ions studied resulted in the optimum condition of the calibration curve. The results
enhancement of the luminescence signal except Ln3+ where obtained for these samples of AM are summarized in Table 2.
a decrease of the signal of the complex was observed. To Excellent recoveries ranging from 97.4 to 99.5% with excellent
get further enhancement for the system under study, the precision were observed. Hence, the method can be applied
inuence of the concentration of Eu3+ ions in the range successfully for the determination of AM in commercially
0.051.05 mol/L was assessed. The luminescence intensity available pharmaceutical samples. The method was then applied
increased with an increase in concentration of Eu3+ and for the determination of AM in serum samples. One mL of the
reached a maximum at a Eu3+ concentration of 0.2 mol/L plasma was diluted 10-fold with distilled water and spiked with
then the intensity rapidly decreased above this concentra- a concentration of drug ranging from 0.1 to 0.2 ppm. Appropri-
tion. Hence, 0.2 mol/L was chosen to be the optimum ate aliquots of the plasma were then treated in the same
concentration for this study. manner as for standard solutions. Excellent recoveries ranging
from 95.0 to 96.0 were obtained (Table 2). This indicates that this
method is also suitable for the analysis of biomedical samples
Analytical gures of merit
without interference from the matrix.
To probe whether sensitization is brought about by the
presence of the AM ligand, emission intensities of Tb(III)AM
using concentrations of AM in the range 0100 ppb were
measured while keeping the concentration of Tb3+ and other Table 1. Comparison with other methods that were used
reagents constant at their aforementioned optimum values. An for analysis of amlodipine
increase in luminescence intensity was observed with an
increase in the concentration of AM in the range studied. The Method Linear range LOD Reference
luminescence intensity I, versus AM concentration, C, was found (ppm) (ppm)
to be linear over the range 1050 ppb. The calibration equation
was: I = (10.41 0.04) C 1.10 0.72, with a correlation Spectrouorometric 0.351.8 0.09 (11)
coefcient, R2, of 0.997) indicating good linearity over the tested Spectrouorometric 0.553.0 0.16 (11)
range. The detection limit (signal to noise ratio of 3) was 1.2 ppb Electrochemical 0.0030.6 0.0006 (4)
and the quantication limit was 4.3 ppb. The gures of merit of Electrochemical 0.021 0.007 (25)
Electromembrane 0.010.5 0.003 (26)
extraction
This study 00.05 0.0012

Table 2. Determination of AM in pharmaceutical and in

plasma samples (n = 9)

Amount AM AM found Recovery RSD %

10 mg (tablets) 9. 74 mg 97.4 0.2
5 mg (tablets) 4.98 mg 99.5 0.1
0.10 ppm (plasma) 0.096 ppm 96.0 0.1
Figure 6. The effect of co-luminescence agents on the luminescence spectra of
3+ +3 5
Tb AM system. [Tb ] = 1 10 M; [TRIS] = 0.01 M; [SDBS] = 0.16%; [TOPO] = 8
0.20 (plasma) 0.190 ppm 95.0 0.2
5
10 M. AM, amlodipine; SDBS, sodium dodecylbenzenesulfate; TOPO, tri-n- AM, amlodipine.
661

octylphosphine oxide; TRIS, Tris (hydroxylmethyl) amino methane.

Luminescence 2014; 29: 657662 Copyright 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/luminescence

Conclusion
The development of a sensitive, simple and robust method for the
determination of AM in pharmaceutical preparations and biologi-
cal uids is reported. The proposed method is based on sensitiza-
tion of terbium luminescence in the presence of TOPO, SDBS and
Eu(III). The enhancement of the terbium luminescence upon com-
plexation with AM has enabled the assay of this drug with high
sensitivity and selectivity where short-lived emissions were ef-
ciently eliminated. The procedure was successfully applied for
the determination of AM in tablets and in plasma samples with
excellent reproducibility and no interference was observed from
substances commonly found in pharmaceutical preparations and
from the matrix in plasma samples. The proposed method has
the advantage of being simple, more rapid and suitable for auto-
mation. It is sensitive to the low amount of AM (detection limit is
1.2 ppb). As a result, the proposed method represents a good an-
alytical alternative for the determination of AM in pharmaceutical
formulations and in biological uids.
References
1. De Portu S, Menditto E, Scalone L, Bustacchini S, Cricelli C, Mantovani
LG. The pharmacoeconomic impact of amlodipine use on coronary
artery disease. Pharmacol Res 2006;54:15863.
2. Kario K, Shimada K. Differential effects of amlodipine on ambulatory
blood pressure in elderly hypertensive patients with different noc-
turnal reductions in blood pressure. Am J Hypertens 1997;10:2618.
3. Levine CB, Fahrbach KR, Frame D, Connelly JE, Estok RP, Stone LR,
et al. Effect of amlodipine on systolic blood pressure. Clin Ther
2003;25:3557.
4. Goyal RN, Bishnoi S. Voltametric determination of amlodipine
besylate in human urine and pharmaceuticals. Bioelectrochemistry
2010;79:23440.
5. Ramani AV, Sengupta P, Mullangi R. Development and validation of
a highly sensitive and robust LC-ESI-MS/MS method for simulta-
neous quantitation of simvastatin acid, amlodipine and valsartan in
human plasma: application to a clinical pharmacokinetic study.
Biomed Chromatogr 2009;23:61522.
6. Pilli NR, Inamadugu JK, Mullangi R, Karra VK, Vaidya JR, Rao JV.
Simultaneous determination of atorvastatin, amlodipine, ramipril
and benazepril in in human plasma by LC-MS/MS and its application
to a human pharmacokinetic study. Biomed Chromatogr
2011;25:43949.
7. Zhou Y, Li J, Hea X, Jia M, Liu M, Lia H, et al. Development and
validation of a liquid chromatographytandem mass spectrometry
method for simultaneous determination of amlodipine, atorvastatin
and its metabolites ortho-hydroxy atorvastatin and para-
hydroxyatorvastatin in human plasma and its application in a
bioequivalence study. J Pharm Biomed Anal 2013;83:1017.
8. Darwish HW, Hassan SA, Salem MY, El-Zeany BA. Three different
methods for determination of binary mixture of amlodipine
662
wileyonlinelibrary.com/journal/luminescence Copyright 2013 John Wiley & Sons, Ltd.
S. M. Z. Al-Kindy et al.
and atorvastatin using dual wavelength spectrophotometry.
Spectrochim Acta A 2013;104:706.
9. Mahmoud AM, Abdel-Wadood HM, Mohamed NA. Kinetic
spectrophotometric method for determination of amlodipine
besylate in its pharmaceutical tablets. J Pharm Anal 2012;2:33441.
10. Erk N. Extractive spectrophotometric determination of atorvastatin
in bulk and pharmaceutical formulations. Anal Lett
2003;36:2699711.
11. Abdel-Wadood HM, Mohamed NA, Mahmoud AM. Validated
spectrouorometric methods for determination of amlodipine
besylate in tablets. Spectrochim Acta A 2008;70:56470.
12. Lis S. Luminescence spectroscopy of lanthanide (III) ions in solution.
J Alloys Compounds 2002;341:4550.
13. Al-Kindy SM, Suliman FEO. Determination of ibuprofen in pharma-
ceutical formulations using terbium sensitized luminescence.
Luminescence 2007;22:294301.
14. Al-Kindy SM, Suliman FEO, Al-Wishahi AA, Al-Lawati HAJ, Aoudia M.
Determination of piroxicam in pharmaceutical formulations and
urine samples using europium-sensitized luminescence. J Lumin
2007;127:2916.
15. Al-Kindy SM, Suliman FEO, Al-Wishahi AA. A sequential injection
method for the determination of piroxicam in pharmaceutical
formulations using europium sensitized uorescence. Talanta
2004;64:134350.
16. Al-Kindy SM, Al-Habsy S, Suliman FEO, Al-Lawati HAJ. Determination
of meloxicam using europium sensitized luminescence in the
presence of co-luminescence reagents. J Fluoresc 2012;22:46774.
17. Al-Kindy SM, Al-Harasi Z, Suliman FEO, Al-Hamadi A, Pillay A. Ter-
bium sensitized luminescence for the determination of ketoprofen
in pharmaceutical formulations. J Fluoresc 2009;19:24955.
18. Rieutord A, Prognon P, Brion F, Mahuzier G. Liquid chromatographic
determination using lanthanides as time-resolved luminescence
probes for drugs and xenobiotics: advantages and limitations.
Analyst 1997;122:5966R.
19. Lis S, Kimura T, Yoshida Z. Luminescence lifetime of lanthanide(III)
ions in aqueous solution containing azide ion. J Alloys Comp
2001;323:1257.
20. Yang J, Ma Q, Jie N, Si Z, Ren X, Hu J. Enhanced uorescence of Tb-
Y-BPMPHD-CTMAB system and its analytical application. J Lumin
1996;69:5764.
21. Yang J, Jie N, Ren X, Wang N, Zou H. Study on the co-luminescence
system of Tb-Gd-BPMPHD-CTMAB- and its analytical application.
Spectrochim Acta A 1995;51:18595.
22. Archer RD, Chen H. Synthesis, characterization, and luminescence of
europium(III) Schiff base complexes. Inorg Chem 1998;37:208995.
23. Rosen MJ. Solubilization by solution surfactants: micellar catalysis. In:
Surfactants and Interfacial Phenomena. 3rd edn. New York: Wiley,
2004;178207.
24. Yu F, Li L, Chen F. Determination of adenosine disodium
triphospahte using prulioxacin-terbium (III) as auorescence probe.
Anal Chim Acta 2008;610:25762.
25. Gazy AA. Determination of amlodipine besylate by adsorptive
square-wave anodic stripping voltammetry on glassy carbon
electrode in tablets and biological uids. Talanta 2004;62:57582.
26. Nojavan S, Fakhari ARJ. Electro membrane extraction combined with
capillary electrophoresis for the determination of amlodipine
enantiomers in biological samples. J Sep Sci 2010;33:32318.
Luminescence 2014; 29: 657662