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Received: 27 August 2013, Revised: 30 September 2013, Accepted: 3 October 2013 Published online in Wiley Online Library: 21 November 2013
Introduction chelated with ligands that have broad intense absorption bands,
an appreciable enhancement in the luminescence intensity of
Amlodipine (3-ethyl-5-methyl-2-[2-aminoethoxy) methoxy]- the system has been observed. This arises from the intramolecu-
4-(o-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate lar energy transfer through the excited state of the ligand, which
monobenzenesulfonate) (AM) (Fig. 1) is a relatively new and serves as an antenna chromophore, to the emitting level of the
potent long-acting calcium channel blocking agent. Amlodipine is Ln(III) ion. The intramolecular energy transfer from the triplet
a third-generation calcium antagonist, which has a dihydropyridine state of the organic ligand to the Ln3+ ion depends on the
ring as part of its structure and it is used alone or in combination structure of the ligand and the position of its triplet state (12).
with other medications for treating high blood pressure, certain This phenomenon is called the antenna effect and such
types of vasospastic angina, cardiac arrhythmias and coronary heart complexes are considered to be light conversion molecular
failure (1,2). It selectively inhibits the arterial vascular smooth muscle devices because they are able to transform light absorbed by
cell proliferation, which prevents progressive narrowing of arteries the ligand into light emitted by the ions via an intramolecular
and coronary spasms resulting in increased blood ow with myocar- energy transfer process. As part of our ongoing interest on
dial oxygen supply (13). method development on drug analysis (1317), we report a novel
Owing to the therapeutic importance of AM, numerous method for the assay of AM in pharmaceutical formulations and
analytical methods have been developed for its quantitative in biological uids. The method is based on the luminescence
determination in pure, pharmaceutical dosage forms and/or bio- enhancement and sensitization of terbium brought about by
logical uids (4). These methods include high-performance liq- complexation with AM. The luminescence properties were
uid chromatography combined with electrospray ionization investigated in various solvents. Factors affecting complexation
tandem mass spectrometry (57), voltametric (4) and spectro- such as the concentration of Tb3+ and the ratio of Tb3+ to AM
photometric methods (810). Spectrouorimetric methods for as well as the effect of co-ligands and co-luminescence agents
the quantication of AM reported recently are based on indirect on the luminescence properties of Tb-AM have been carefully
methods involving uorescent derivatization techniques with studied. The method was subsequently used to determine the
uorigenic labels such as 7-chloro-4-nitro-2,1,3-benzoxadiazole concentration of AM in pharmaceutical and plasma samples.
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 * Correspondence to: S. M. Z. Al-Kindy, Sultan Qaboos University, College of
luminescence techniques are lanthanide-sensitized lumines- Science, Box 36, Department of Chemistry, Al-Khod 123, Sultanate of Oman.
Email: alkindy@squ.edu.om
cence methods that result from complexation of Ln3+ ion with
a suitable ligand. Lanthanide ions exhibit weak molar absorptiv- Sultan Qaboos University, College of Science, Box 36, Department of
ity and low uorescence quantum yields. However, when
657
Luminescence 2014; 29: 657662 Copyright 2013 John Wiley & Sons, Ltd.
S. M. Z. Al-Kindy et al.
Reagents
Results and discussion
All reagents used in this work were prepared from analytical grade
reagents and were used without further purication. All solvents Spectral characteristics
used were of high-performance liquid chromatography grade. The absorption spectra of AM and its Tb(III) complex were
Ultrapure water (Milli-Q Millipore Corporation, Milford, MA, USA) recorded in methanol. Both the ligand and the complex
was used. Tris (hydroxylmethyl) amino methane (TRIS), tri-n- exhibited absorption maximum at 240 and 350 nm, with the
octylphosphine oxide (TOPO) were obtained from Sigma-Aldrich maximum absorptivity observed at 240 nm. A slight increase in
(St Louis, MO, USA). The surfactants used in this study include absorptivity was noticed for the complex when compared to
Tween-20 (TW-20), Tween-80 (TW-80), Triton X-100 and sodium AM alone. A weaker absorption was seen for the metal solution
dodecyl sulfate (SDS) (All from Sigma, Steinheim, Germany) whereas in the ultraviolet (UV) region.
sodium dodecylbenzenesulfate (SDBS) was from a Chem Heri, To improve the analytical characteristics for the determination
Shanghai, China. of AM in pharmaceutical formulations we initially investigated
Stock solutions of 1 104 M (AM) provided by the Jordanian the complexation of TbAM in methanol. Our aim was to sensi-
Pharmaceutical Manufacturing Company (Amman, Jordan) was tize the luminescence of the terbium ion using AM. The results
prepared in methanol and stored in a refrigerator. The solution shown in Fig. 2 indicate that the luminescence intensity of ter-
was found to be stable for 1 week, after which fresh stock bium is very weak. However, upon addition of AM to Tb+3 solu-
of AM was prepared. Tb3+ 1 104 M and TOPO 1 103 M tions, the intensity of Tb+3 emission was signicantly enhanced
solutions were prepared in methanol. A working standard of (Fig. 2). This enhancement is due to the efcient energy transfer
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.
wileyonlinelibrary.com/journal/luminescence Copyright 2013 John Wiley & Sons, Ltd. Luminescence 2014; 29: 657662
Determination of amlodipine using terbium sensitized luminescence
Luminescence 2014; 29: 657662 Copyright 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/luminescence
S. M. Z. Al-Kindy et al.
in Fig 4. The decrease in the intensity could be due to the keeping other experimental factors constant and the results
increase in the number of micelles with an increase in the surfac- are shown in Fig. 5. Possibly, the protonation of the amine group
tant concentration, which results in a further dilution of the at this pH results in the formation of less favorable species for
concentration of the solubilizate per micelle as the surfactant complexation with Tb(III) ions. Increasing the pH from 7 to 9 led
concentration is increased further. This causes a reduction in to the enhancement of luminescence intensity reaching the max-
the rate of solubilization (22). Another reason for a decrease in imum at pH 9, above which a decrease in the intensity was
the luminescence intensity of the complex in the presence of observed. The changes in pH would inuence the compositions
high concentration of SDBS may be due to competition between and stabilities of the luminescence complexes and resulted in
surfactant and AM for Tb+3 to form the complex. Therefore, the changes in the luminescence characteristics (24). This decrease
concentration of SDBS in this study was maintained at 0.16%. also could be attributed to the competitive hydrolysis of terbium
A possible strategy to improve luminescence intensity of Tb forming Tb(OH)3. At pH 9, the neutral form of AM is the predom-
AM chelates involves substitution of water molecules in the inant species, which is favorable for the sensitization process.
coordination sphere with synergic agent. Several co-ligands The time-resolved spectra of the TbAM complex and of Tb3+
were investigated in this study, including TOPO, EDTA, ions were studied in the presence of different types of buffers
1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline each such as sodium acetate, sodium phosphate, sodium carbonate
at a concentration of 1 104 M. Maximum luminescence and TRIS buffers at pH 9. A high intensity of the signal of the
sensitization was observed in the presence of TOPO. terbium complex when compared to the signal of the terbium
Furthermore, the optimum concentration of TOPO was stud- blank was observed in the presence of TRIS buffer indicating
ied by monitoring the luminescence intensity of the TbAM favorable complex formation of terbium with the drug. On the
complex in the presence of various concentrations of TOPO other hand acetate, phosphate and carbonate buffers gave a
ranging from 0.05 to 0.17 mM. The luminescence intensity of very low difference in signal intensity in the presence and
the complex was found to increase with an increase in the absence of AM suggesting the possibility of competition
concentration of TOPO up to 0.1 mM, after which a decrease in between the buffers and AM for terbium. Hence, the assay of
intensity was observed. Coordinating TOPO with the Tb3+ ion the AM was carried out in the presence of TRIS buffer. The inu-
through its oxygen atom helps to remove water molecules from ence of TRIS buffer concentrations was studied by varying the
the coordinating sphere of the metal ion. The efcacy of the concentration of pH 9 buffer in the range 0.0050.03 mol/L.
bulky hydrocarbon tail of TOPO in protecting the luminescence The concentration of Tb3+, AM and TOPO were maintained at
complex from radiationless deactivations is manifested in the 6 105 mol/L, 8 107 mol/L and 1 104 mol/L, respectively,
signicant enhancement of the emission from Tb3+ ions. while the concentration of SDBS was maintained at 0.16%. On
However, using higher concentrations of TOPO result in increas- increasing the concentration from 0.005 to 0.015 mol/L, an in-
ing the competition between TOPO and AM for sites at the crease in the luminescence intensity was observed, after which
coordination sphere of Tb3+ ion and hence a higher enhance- the intensity started to decline. This behavior indicated that an
ment of Tb3+ blank emission relative to the TbAM is the net optimum concentration of TRIS buffer is required for maximum
result. Hence, in this study 0.1 mmol/L was considered the complex formation between Tb3+ and AM. By exceeding the
optimum concentration of TOPO. optimum concentration, the buffer molecules start to compete
As was previously reported, AM (pKa = 8.6) structural forms are with the AM ligand for Tb3+ binding sites. This may result in less
highly pH dependent in the aqueous medium, where the TbAM complex being formed and hence a decrease in lumines-
deprotonated form AM predominates at a pH above 8.6. cence intensity. Hence, the optimum concentration of TRIS
Because a neutral form is expected to be preferred for complex- buffer for this study was considered 0.015 mol/L.
ation with Tb3+, it is necessary to investigate the inuence of pH The effect of a Tb3+ ion concentration on the luminescence
of the system on the sensitization process. The inuence of pH intensity was investigated in the range 0.020.14 mmol/L. The
on the luminescence intensity of the system was studied by luminescence intensity reached a maximum at the Tb3+ ion
varying the pH of 0.01 M Tris buffer in the range of 711 while concentration of 0.06 mmol/L and the intensity decreased
3+
Figure 4. The inuence of changing the concentration of SDBS on luminescence of Figure 5. The inuence of changing pH on luminescence spectra of Tb AM
3+ +3 5 7 5 +3 5 7 5
(Tb AMTOPOTRIS) system. [Tb ] = 6 10 M; [AM] = 8 10 M; [TOPO] =8 10 M; complex in aqueous medium; [Tb ] = 1 10 M; [AM] = 4 10 M; [TOPO] = 8 10 M;
[TRIS] = 0.01 M (pH 9). AM, amlodipine; SDBS, sodium dodecylbenzenesulfate; TOPO, [SDBS] = 0.14%; [TRIS] = 0.01 M. AM, amlodipine; SDBS, sodium dodecylbenzenesulfate;
660
tri-n-octylphosphine oxide; TRIS, Tris (hydroxylmethyl) amino methane. TOPO, tri-n-octylphosphine oxide; TRIS, Tris (hydroxylmethyl) amino methane.
wileyonlinelibrary.com/journal/luminescence Copyright 2013 John Wiley & Sons, Ltd. 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
Luminescence 2014; 29: 657662 Copyright 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/luminescence
S. M. Z. Al-Kindy et al.
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