Research paper
PHOTOSTABILITY OF AMLODIPINE BESYLATE IN ALOPRES®
TABLETS
Valentina D. Marinkovic1, Ivana M. Savic2, Ivan M. Savic2, Predrag S. Sibinovic3, Aleksandar
Dosic4, Danica Agbaba1
1
2
3
Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia
Faculty of Technology, Univerzity of Nis, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia
Pharmaceutical and Chemical Industry - Zdravlje Actavis, Vlajkova 199, 16000 Leskovac, Serbia.
4
Faculty of Technology, University of East Sarajevo, 75400 Zvornik, Bosnia and Herzegovina.
*
Corresponding author:
Dr Ivana Savic
Faculty of Technology,
Bulevar oslobodjenja 124, 16000 Leskovac, Serbia.
Tel.: +381 16 242859; fax: +381 16 242859.
E-mail address: [email protected]
Intoduction
Amlodipine belongs to a class of dihydropyridines. This class of dihydropyridines is
generally referred to as calcium channel blockers or calcium antagonists1,2. They act to reduce the
movement of calcium into the cell and are thus able to delay or prevent the cardiac contracture,
which is caused by an accumulation of intracellular calcium under ischemic conditions. Excessive
calcium influx during ischemia can have a number of additional adverse effects that would further
compromise the ischemic myocardium. These include less efficient use of oxygen for ATP
production, activation of mitochondrial fatty acid oxidation and, possibly, promotion of cell
necrosis. Thus calcium antagonists are useful in the treatment or prevention of a variety of cardiac
conditions, such as angina pectoris, cardiac arrhythmias, heart attacks and cardiac hypertrophy.
Calcium antagonists also have vasodilator activity since they can inhibit calcium influx in cells of
vascular tissue and are thus also useful as antihypertensive agents and for the treatment of coronary
vasospasm.
Literature survey revealed HPLC3-5, RP-HPLC6-11, HPTLC12,13, LCMS/MS14, LCMS15,
UPLC–MS/MS16, and simultaneous UV spectrophotometric methods17-20 are reported for the
estimation of amlodipine besylate alone or in combination with other drugs in pharmaceutical
dosage forms or individually in biological fluids.
The investigation of amlodipine besylate stability in different pharmaceutical forms7,8,21 and
in several pharmaceutical inclusion systems22 can be found in the literature. The mechanism of
photochemical degradation of amlodipine23 and its photo-transformation in aqueous solution24 was
studied.
In this case was found that amlodipine besylate is a photo-sensitive and liable in the solution
and in the solid state18.
In order to improve the bioavailability and photostability of amlodipine, was prepared dry
emulsion by spray-drying the oil-in-water emulsion of amlodipine25.
The aim of this work was to study the photostability of Alopres® tablets (Zdravlje-Actavis,
Serbia) to investigate the kinetics of amlodipine besylate degradation. Tablets were exposed to
conditions of accelerated photo decomposition. The analyses of the degraded samples were
performed by developed and validated modified RP-HPLC method. In this paper, the photostability
of this product was compared with commercially available products from the same class.
Material and methods
Samples. Standard of amlodipine besylate was purchased from NOSCH-a Labs Private Limited,
India. Standard of impurity D was purchased from Merck, Darmstadt, Germany. Alopres®
(Zdravlje-Actavis, Serbia), one originator and 3 domestic products were analyzed to compare each
other.
Chemicals and reagents. Chemicals and solvents for analysis: methanol, acetonitrile and water for
HPLC were purchased from Merck, Darmstadt, Germany. Triethylamine was a product of
Qualigens Fine Chemicals, India. Acetonitrile and methanol were HPLC grade. HPLC grade water
was obtained following distillation in glass and passage through a Milli-Q® system (Millipore,
Milford, MA, USA) and was used to prepare all solutions.
Apparaturs. The method was performed by Agilent 1100-Series HPLC system (Agilent
Technologies, USA), consisting of a HP G13141A variable wavelength UV detector and Agilent
1100-Series auto-sampler. The system was controlled and data analyses were performed by Agilent
HPLC Data Analysis software. The reproducibility was performed by another LC system,
consisting of an Agilent 1100-Series binary pump and Agilent 1100-Series DAD detector (Faculty
of Technology, Leskovac). The detector was set at 237 nm and the peak areas were integrated
automatically using the Agilent HPLC Data Analysis software program. The separation of
amlodipine besylate from its impurity D was carried out at 20 °C using C18 column (4.6×150 mm, 5
μm) Agilent Technologies, USA.
Chromatographic conditions. RP-HPLC analysis was performed by isocratic elution with a flow
rate of 1 cm3 min-1. A mobile phase was contained an acetonitrile : methanol : solution of
triethylamine (15:35:50 v/v/v). Procedure for preparation of triethylamine solution is the following:
dissolve a 7.0 cm3 triethylamine in 1000 cm3 of water. Using a concentrated phosphoric acid
(HPLC purity), pH was adjusted to 3.0. All solvents were filtered through a 0.45 μm millipore filter.
The eluent was monitored using UV detection at a wavelength of 237 nm. The column was
maintained at ambient temperature (20 °C), while the injection volume was 10 μl.
Preparation of standard solution. 37.5 mg of amlodipine besylate and 1 mg of impurity D were
dissolved in 50 cm3 volumetric flask in mobile phase by mixing in the ultrasonic bath for 15 min. 1
cm3 of this solution contained a 0.75 mg amlodipine besylate and 002 mg impurity D.
Validation study. The linearity of method was determined by diluting the standard solution of 10
μg cm-3and then plotting a dependency between a response of peak area and the concentration.
Method precision was assessed by measuring a repeatability (or intraday precision) and
intermediate (interday) precision, in accordance with ICH guidelines26. Repeatability was studied
by assay of three different concentrations of the drug (20, 25 and 30 μg cm-3) in one laboratory by
the same analyst on one working day. Intermediate precision was checked by repeating the studies
on three different days. The accuracy of the method was determined as recovery from 20 to 30 μg
cm-3 standard solution of amlodipine besylate. Method robustness was assessed by deliberate
changing mobile phase flow rate and proportion of acetonitrile. Detection limit (DL) and
quantification limit (QL) were determined by the standard deviation (Sy/x) method27. Blank
samples were injected in triplicate and peak area was recorded. DL and QL were determined from
the slope (S) of the linearity plot and the standard deviation of the response to the blank sample,
Sy/x, by use of the formulae DL = 3.3 × Sy/x/S and QL = 10 × Sy/x/S. The flow rate of the mobile
phase was 1.0 cm3 min-1. The flow rate of mobile phase was changed for 0.2 units in the range of
0.8-1.2 cm3 min-1 to investigate its influence on the resolution. In this case, the content of mobile
phase was held a constant. The effect of proportion of acetonitrile on resolution was studied at 10
and 20% instead of 15%.
Preparation of tablets for assay. 15 analyzed tablets were weighed, crushed and mixed in a mortar
and pestle for 20 min. A portion of powder equivalent to the weight of one tablet was accurately
weighed into 200 cm3 volumetric flasks and 50 cm3 of mobile phase was added to flask. The
volumetric flask was sonicated for 30 min to effect complete dissolution of the amlodipine besylate
and then the solutions were made up to volume with mobile phase. 1 mL of this solution contained
a 0.025 mg of amlodipine besylate. After that, the obtained solution was filtered through qualitative
filter paper and then through a membrane filter of 0.45 µm.
Photostability studies. Analyzed tablets were packed in blister (Al/PVC) and exposed to effect of
light to determine the effects of irradiation on the stability of the drugs. All samples for
+
photostability testing were placed in a light cabinet (Suntest CPS/CPS , Atlas Material Testing
Technology, Germany) and exposed to different wavelength (254 and 540 nm) and duration of
radiation (3.5; 7 and 17 days) at 25 °C. Control samples, which were protected from light, were also
placed in the light cabinet and exposed concurrently. Following removal from the light cabinet, all
samples were prepared for analysis as described in the part for preparation of tablets.
Data analyses. The order of the photo-degradation reaction is determined by the least squares
method linear adjustment and by calculation of correlation coefficients, in order to choose between
the zero-order and the first-order kinetics. The degradation rate constant (k) is determined from the
slope of the line of peak area versus time. The degradation rate constant, the half and shelf-life were
calculated in accordance with the determined order of the reaction.
Results and discussion
HPLC method optimization
In this paper, a modified RP-HPLC method for determination of amlodipine besylate in the
presence its impurity D was developed. This method was applied for monitoring photostability of
preparation Alopres® (Zdravlje Actavis, Serbia).
The chromatographic conditions were optimized to provide a good performance of the assay.
During the optimization of the method were used different stationary phases like C18 and C8 and
different content of mobile phase (change proportion of acetonitrile and pH by phosphoric acid).
The chromatographic separation was achieved on a ZORBAX Eclipse XDB-C18 column (4.6×250
mm), with a particle size of 5 μm. Satisfactory separation of used standards (amlodipine besylate
and impurity D) was obtained with a mobile phase consisting of acetonitrile : methanol : solution of
triethylamine (15:35:50 v/v/v). If pH was less than 3.0 the separation of amlodipine besylate and
impurity D was with unsatisfactory resolution. The maximum absorption of amlodipine besylate
was detected at 273 nm and this wavelength was chosen for the analysis. The retention times for the
standard solution of amlodipine besylate and impurity D were 16.529 and 2.575 min, respectively
(Fig. 1). Total time of the analysis was less than 10 min.
FIGURE 1.
The chromatographic parameters, such as efficiency column and peak asymmetry were
reconsidered for the standard of amlodipine besylate. According to the obtained value Number of
Theoretical Plato (N=324), the efficiency of column is satisfactory (HETP=0.786). The asymmetry
peak of 0.44 indicates that the peak is not ideally symmetric, that it is not Gauss’s peak. Having in
mind that Wab<Wbc, this means that there is a certain interaction between the stationary phase and
the investigated component.
Method Validation
Linearity of response was found in the concentration range of 10-75 µg cm-3 for amlodipine
besylate with the correlation coefficient (r) of 0.9993. The regression equation for the calibration
plot was y = (119.525 ± 1.789) x + (37.311 ± 1.621) (n = 3; detection at 237 nm). There was no
significant difference between the slopes of calibration plots prepared on different days (ANOVA, p
< 0.05). The method was precise repeatability and intermediate precision, as RSD, were in the
ranges 0.587–1.352% and 0.791-1.601%, respectively. The accuracy, calculated as percentage
recovery, was 99.76–101.63%. Low values of the RSD (0.472–1.538%) and SEM (0.048–0.221)
and good percentage recovery were indicative of the excellent accuracy of the method. Statistical
validation (Student’s t-test) revealed no significant difference (p < 0.05) between results from study
of accuracy and the theoretical concentration.
DL and QL were 0.2 and 0.66 µg cm-3, respectively, indicating that the method is a
sensitive.
In all the deliberate varied chromatographic conditions (flow rate and proportion of
acetonitrile) no significant change in the assay value was observed. The system suitability
parameters like tailing and the RSD values are well within the limits. Tailing was 1.0 and 0.9 and
RSD was 0.8 and 0.6% for flow rate and proportion of acetonitrile, which confirms the robustness
of the developed method.
Hence, it can be concluded that the validated RP-HPLC method is accurate, precise, and
selective and can be employed successfully for the estimation of amlodipine besylate in the
presence impurity D in irradiated sample of products.
Forced Degradation of Alopres®
The method was capable to separate a degradation product in the presence of amlodipine
besylate. Well separated analyte and degradation peak was obtained after all photo degradation
experiments (Fig. 2).
FIGURE 2.
From the obtained chromatogram, the presence of amlodipine besylate was confirmed at the
retention time of 16.485 min. The occurrence of peak at the retention time of 2.571 min indicates on
the presence of impurity D.
UV radiation led to degradation of amlodipine besylate in Alopres® for 32.38%.
Amlodipine besylate degradation in products, caused by UV radiation, is 21.85%, 12.66%, and
30.04% for domestic products 1, 2, 3, respectively. The decrease of 21.35% was in the case of
originator product. After VIS radiation, the content of amlodipine besylate was decreased for
30.66% for Alopres®, 20.86%, 11.61%, 21.71% for domestic products and 21.35% for the
originator product.
After photo-degradation, the content of amlodipine besylate is the smallest for Alopres®
compared with all other analyzed products (domestic products 1, 2, 3 and originator product). Thus,
this fact indicates that the amlodipine besylate is a more photosensitive in Alopres®. In the aim of
reduction a photosensitivity and increasing of Alopres® stability is necessary to change the existing
primary packaging (transparent Al/PVC blister).
Studies of degradation kinetics
For estimation of drug stability is necessary to know the velocity of drug degradation, as
well as the order of this chemical reaction.
The obtained results for the content of amlodipine besylate were fitted according to the
kinetic model of zero and the first order to assess the stability of the product Alopres®. The models
were described using correlation coefficient (r) and the rate constant of degradation. The calculated
values are summarized in Table 1.
TABLE 1.
Analysis of data (Table 1) shows that the kinetics of photo-degradation for Alopres® tablets
and other analyzed products was the best described by the first-order model. The values of kinetic
parameters for a zero and the first order reactions of degradation were presented in Table 1. The
highest values of k0 and k1 were obtained in the case of photo-degradation Alopres® tablet.
The values of rate constant (Zero-order: 1.725 and 1.740 mg cm-3 day-1, First-order: 0.021
and 0.021 day-1 by UV and VIS radiation, respectively) indicate that the photo-degradation reaction
of Alopres® tablet is faster than other tested commercial products. Higher reaction rate of photodegradation shows on a greater instability of product, packaged in a transparent Al/PVC blister.
The values of half-life (t1/2) and shelf life (t90) for both models were calculated and presented
in Table 1.
For the proposed kinetic models, the shortest half-life and shelf-life were found in the case of
Alopres® product. Low values of half-life and shelf-life for Alopres® indicate on the photoinstability of this product.
The values of t1/2 and t90 have lower values for UV radiation than those obtained after VIS
radiation. Lower values of these parameters in the case of UV radiation indicate that the product is a
more sensitive than after influence of VIS radiation.
Conclusion
A modified RP-HPLC method for assay of amlodipine besylate has been developed, and
validated by use of forced degradation procedures recommended by the ICH. The method is simple,
accurate, precise and specific for separations of the drug from its degradation products. The method
is proposed for use in analysis of samples generated during photo-stability study on amlodipine
besylate and its formulations.
The obtained results indicate on higher photo-sensitivity of amlodipine besylate in
Alopres®. In order to reduce photo sensitivity and increased stability of this product, which are
packed in transparent Al/PVC blister, it is necessary to change the type of primary packaging.
The first order kinetic model was confirmed for reactions of photo-degradation for
Alopres® tablets and other tested products from the calcium antagonists group.
Acknowledgments. The authors are grateful to the colleagues of Pharmaceutical and Chemical
Industry Zdravlje-Actavis, Serbia for the given practical help. This work was supported by the
Ministry of Education and Science of the Republic of Serbia under the project TR-34012. Ivan
Savic is a recipient of a scholarship granted by the Ministry of Education and Science of the
Republic of Serbia.
Conflict of interest. We have no conflict of interest.
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Table 1. Correlation coefficient (r), degradation rate constant (k), half-life (t1/2) and shelf life (t90)
of Alopres®
Zero-order
Model/ preparation
k0
(mg cm-3 day-1)
r
First-order
t1/2
t90
k1
(day-1)
r
t1/2
t90
UV
Alopres®
1.725
0.976
28.98
5.79
0.021
0.978
33.80
5.12
1domestic product
1.177
0.961
42.46
8.49
0.014
0.964
51.33
7.77
2 domestic product
0.693
0.987
72.11
14.42
0.007
0.988
93.65
14.19
3 domestic product
1.518
0.988
32.93
6.58
0.018
0.993
38.50
5.83
Originator
1.719
0.879
29.07
5.82
0.020
0.899
33.00
5.00
VIS
Alopres
1.740
0.947
28.74
5.75
0.021
0.955
33.64
5.10
1 domestic product
1.225
0.926
40.81
8.16
0.014
0.932
50.58
7.66
2 domestic product
0.671
0.927
74.55
14.91
0.007
0.927
97.60
14.78
3 domestic product
1.122
0.938
44.58
8.91
0.012
0.948
55.44
8.40
Originator
1.164
0.948
42.94
8.59
0.013
0.953
53.72
8.14
Figure 1. HPLC chromatogram of standard solution of amlodipine besylate (tr = 16.529 min) and
its pharmacopeia impurity D (tr = 2.575 min)
Figure 2. HPLC chromatogram of the irradiated Alopres® tablets (Zdravlje-Actavis, Serbia)