A selective high performance liquid chromatographic method to follow the hydrolytic degradation of nicardipine hydrochloride

Article abstract of DOI:10.1155/2010/625296

A simple, stability indicating, reverse phase high performance liquid chromatographic method was developed and validated for determination of nicardipine hydrochloride (NC) in the presence of its degradation products. The chromatographic separation was performed on Hypersil, BDS-C18, 30 cm × 3.9 mm id, at ambient temperature with UV-detection at 254 nm. A mixture of 20% (v/v) aqueous 0.01 M sodium acetate/acetic acid buffer (pH 4.5) and 80% acetonitrile was used as the mobile phase at a flow rate of 1.5 mL min^-1, losartan was used as internal standard. The calibration curve is linear over the concentration range 5-40 μg mL^-1, with a regression coefficient of 0.9984 and the % recovery was 99.78±0.17. The method was used to investigate the kinetics of alkaline, acids induced degradation, effect of buffer concentration and temperature. The degradation followed first-order kinetics. The rate constant, half-life time, and activation energy were calculated.

Full text of DOI:10.1155/2010/625296

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
2010, 7(1), 85-92
http://www.e-journals.net
A Selective High Performance Liquid
Chromatographic Method to Follow the Hydrolytic
Degradation of Nicardipine Hydrochloride
K. E. IBRAHIM*, R. M. AL-ASHBAN§ and L. B. BABIKER§
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, University of Khartoum, Sudan,
§Central Laboratory for Food and Drug Analysis, Saudi Arabia.
kamalibrahime@yahoo.com
Received 12 May 2009; Accepted 10 July 2009
Abstract: A simple, stability indicating, reverse phase high performance liquid
chromatographic method was developed and validated for determination of
nicardipine hydrochloride (NC) in the presence of its degradation products. The
chromatographic separation was performed on Hypersil, BDS-C18, 30 cm x 3.9
mm id, at ambient temperature with UV-detection at 254 nm. A mixture of 20%
(v/v) aqueous 0.01 M sodium acetate⁄acetic acid buffer (pH 4.5) and 80%
acetonitrile was used as the mobile phase at a flow rate of 1.5 mL min-1, losartan
was used as internal standard. The calibration curve is linear over the
concentration range 5-40 µg mL-1, with a regression coefficient of 0.9984 and
the % recovery was 99.78 0.17. The method was used to investigate the kinetics
of alkaline, acids induced degradation, effect of buffer concentration and
temperature. The degradation followed first-order kinetics. The rate constant,
half-life time, and activation energy were calculated
Keywords: Nicardipine hydrochloride, HPLC, Hydrolytic Degradation.
Introduction
Nicardipine hydrochloride (Figure 1) is chemically known as 2-(benzyl (methyl) amino)
ethyl methyl 1, 4–dihydro-2, 6-dimethyl-4-(3-nitrophenyl) pyridine-3, 5-dicarboxylate
hydrochloride1. It is a calcium antagonist of the dihydropyridine class and has been widely
used for the treatment of hypertension and angina pectoris2.
Several methods have been reported for determination of nicardipine hydrochloride. These
include spectrophotometry3-5, voltammetry6-8, high performance liquid chromatography9-12, liquid
chromatography mass spectroscopy13-15 and capillary electrophoresis16. Most of these methods
were used for quantitative determination of nicardipine hydrochloride in biological fluids.

86
K. E. IBRAHIM et al.
Drug degradation is a serious problem which renders the job of quality control analyst
very critical and is usually associated with stability of the drug. Investigations on stability
during a laboratory process are necessary to establish the precautions required during
development and control of dosage form as well as for development and validation of
quantitative methods. Generally stability of drugs or pharmaceutical preparations depends
on temperature, concentration, pH, oxygen content, light intensity, wave length and other
factors. Reviewing the literature, revealed that, only the effect of light on nicardipine HCl
has been studied.
The aim of the present work was to develop, validate and apply a stability indicating
HPLC method for determination nicardipine HCl and its alkaline and acid hydrolytics
products. The developed method was used to investigate the kinetics of the drug degradation
in different media at different temperatures.
H
H3C
N
CH3
CH3
N-CH2-C6H5 . HCl
H3COOC
COOCH2CH2
NO2
Figure 1. Structural formula of nicardipine hydrochloride.
Experimental
The pure sample of nicardipine hydrochloride was purchased from Sigma (St. Louis, Mo,
USA). The commercial capsules (Pelcard 50 mg Batch # 65801) were obtained from
commercial sources. Methanol, acetonitrile, (all of HPLC grades), hydrochloric acid
(Analar), monobasic sodium phosphate, ammonium acetate, boric acid, sodium acetate and
glacial acetic acid (Analar) were obtained from BDH (Poole, UK). Phosphoric acid, ethanol,
and potassium hydroxide were obtained from E; Merck (Darmstadt, Germany).Losartan was
purchased from Sigma (St. Louis,Mo,USA).
Apparatus
Seperation was performed on Shimadzu class-vp v6.LC-2010A liquid chromatography,
equipped with UVB50 varian detecter, rheodyne injector and the wavelength used was 254
nm.
Column and mobile phase
Separation was achieved on Hypersil, BDS- C18, (5 µm), 30 cm
X
3.9 mm id. The mobile
phase consists of 20 % (v/v) aqueous 0.01 M sodium acetate/acetic acid (pH 4.5) and 80%
(v/v) acetonitrile.
Standard solution
A stock solution of 0.04% (w/v) of nicardipine HCl reference material in the mobile phase
was prepared and working solutions were prepared by appropriate dilutions.
Internal standard
A stock solution of 0.01% (w/v) of losartan was prepared in the mobile phase and working
solutions were prepared accordingly.

A Selective HPLC Method to Follow the Hydrolytic Degradation
87
preparation of solutions of alkalis and acid degradation
For the effect of alkalis and acids on nicardipine HCl, solutions of the drug with a
concentration of 40 µg/mL were treated with solutions of either sodium hydroxide of
different molar concentrations (0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M and 1.0 M) of HCl. The
experiments were conducted at room temperature. The solutions were then neutralized with
0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.5 M HCl and 1.0 M NaOH and diluted to volume with
mobile phase.
Calibration curve
Aliquots of the standard solution covering the working range (5, 10, 15, 20, 25, 30, 35 and
40 µg/mL-1) of nicardipine were prepared in mobile phase with 20 µgmL-1 of the internal
standard. Ten microliter aliquots were injected (in triplicate) and eluted with the mobile
phase under the reported chromatographic conditions. The calibration curve was constructed
by plotting the peak area ratio of nicardipine HCl to that of losartan (internal standard)
versus concentration. The linear regression equation was derived.
Study of the factors affecting the degradation kinetics of nicardipine HCl
Asolution of nicardipine HCl (40 µgmL-1) was used to study the effect of temperature and
pH. The solutions were treated with different concentrations of NaOH (0.1 M, 0.2 M, 0.3 M,
0
0.4 M and 0.5 M) and kept at different temperatures (70 -100 C).The solutions were then
neuterlized with 0.1 M, 0.2 M, 0.3 M, 0.4 M and 0.5 M HCl and diluted with mobile phase.
For the effect of pH four buffer systems (citrate, phosphate, borate and acetate) of different
pH were used with the same concentration of nicardipine HCl (40 µgmL-1).
Analysis of capsules
A solution of nicardipine HCl was prepared and injected using the prescribed method and
the nominal content of the capsule s were calculated using the calibration graph.
Results and Discussion
Chromatographic separation
Figure 2 is a chromatogram obtained from standard solution of nicardipine and its
degradation products (I and II), resulting from alkaline degradation under the prescribed
chromatographic conditions. The method allows complete base line separation with a good
resolution factor (6.5) between each two adjacent peaks. The proposed method was assessed
for specificity, linearity, precision and recovery (Table 1).
Degradation kinetics study
Effect of acids
The result was obtained by treating a solution of 40 µg/mL with 1.0 M HCl. The kinetics
parameters for degradation of nicardipine HCl in acid medium were found to be first order.
The plotting of log (a/a-x) against time (t) was found to be straight line. The rate constant (k)
and the half-life time (t1/2) were found to be 1.38x10-3 min-1 and 502 min. respectively.
Effect of alkalis
The effect of alkalis on the degradation of nicardipine HCl was studied by using increasing
concentrations of sodium hydroxide: 0.1 M, 0.2 M, 0.3 0.4 M and 0.5 M at room and
elevated temperatures. At room temperature, increasing the concentration of sodium
hydroxide resulted in increasing the rate of degradation and the value of the reaction rate
constant (k) and decreasing in the half-life (t½) (Table 2).

88
K. E. IBRAHIM et al.
Figure 2. Chromatogram of nicardipine HCl and its degradation products.
Table 1. Analytical parameters for the HPLC method of determination of nicardipine
adopting the proposed method.
Parameter
Standard nicardipine
5-40
Concentration range, µg/mL
Correlation coefficient(r)
0.9984
Slope
Intercept
RSD%
%ER
0.121
0.0146
0.17
0.064
% R
99.78+0.17
Table 2. Effect of concentration of sodium hydroxide on the kinetic parameters of
nicardipine HCl (40 µg/mL) at room temperature.
K 103, min−1
1.15
1.84
Conc. of NaOH, M
t , min
½
0.1
0.2
0.3
0.4
0.5
601
376
300
250
188
2.30
2.76
3.68
0
Result of the effect of alkaline degradation at different temperatures (70-100 C) are
shown in Table 3. The values of the reaction rate constant (k, min-1), half-life times (t1/2
min) and activation energy (Ea,kcal.mol-1) are listed. Plotting log Kobs values vs 1⁄T, the
Arrhenius plot was obtained (Figure 3). From these data, the rate of degradation of
nicardipine was found to increase upon increasing both temperature and concentration
of sodium hydroxide.

A Selective HPLC Method to Follow the Hydrolytic Degradation
89
1/T X 10
Figure 3. Arrhenius plots for nicardipine HCl at different concentrations of NaOH.
Table 3. Effect of temperature on the kinetic parameters of nicardipine HCl (40 µg/mL) at
different concentrations of sodium hydroxide.
Conc.of NaOH, M
0.1M
Temperature, 0C
K, min-1
t½ min
Ea, kcal.mol-1
X= 7.13
70
80
3.91
4.83
5.98
8.98
4.83
6.67
8.29
x
x
x
x
x
x
x
10-3
10-3
-10-3
10-3
10-3
10-3
10-3
177
143
115
77
90
100
70
143
103
83
80
0.2M
0.3M
0.4M
0.5M
X =7.20
X =8.16
X=8.0
90
100
70
0.01128
61
5.75
7.36
9.69
x
x
x
10-3
10-3
10-3
120
94
80
90
71
100
70
0.01496
46
7.13
9.44
x
x
10-3
10-3
97
74
80
90
0.01335
0.01893
52
100
70
38
7.83
x
10-3
88
80
0.01036
0.0161
0.0251
67
X=9.97
90
43
100
28

90
K. E. IBRAHIM et al.
Effect of buffer
The result of the effect of type and concentration of buffers on the reaction rate constant at
room temperature are shown in Table 4. Based on the effect of different buffers with
0
different pH values on the rate of degradation of nicardipine HCl at 100 C (Table 5), the
following conclusions are drawn:
•
•
For the same type of buffer: increasing the pH increases the rate of degradation.
Citrate buffers increase the rate of degradation more than phosphate more than
borate and finally acetate.
Table 4. The calculated pseudo-first order rate constant (kobs) and half-lives of degradation
of nicardipine HCl at various pH values at room temperature (at 22 oC).
pH
1.1
Buffer composition
HCl
K
obs day–1
Half life t (day)
½
log ko
– 1.40
– 2.71
– 2.82
– 3.05
– 2.85
– 0.23
– 0.56
– 1
4 × 10–2
1. 92 × 10–3
1.5 × 10–3
8.91 × 10–4
1.41 × 10–3
0.564 × 10–1
27.7 × 10–1
0.1 × 10–1
0.316 × 10–1
1.58
17.30
155.00
462.00
777.00
491.00
8.62
2.45
3.40
4.50
5.20
6.00
7.00
8.30
9.00
10.00
11.20
13.00
Acetate
Citrate
Citrate
Citrate
Citrate
Phosphate
Borate
17.50
6.93
Borate
2.19
– 0.50
0.19
Borate
0.46
NaOH
6.58
1.09 × 10–3
0.21
0.81
NaOH
0.44
–2.96
Table 5. Comparison between the different buffer systems on the degradation of nicardipine
(40 µg/mL) at 100 0C.
Type of Buffer
Citrate buffer pH 6
k , min-1
0.0154
0.0108
0.0069
0.0159
0.013
log k
- 1.81
- 1.96
- 2.11
- 1.79
-1.88
-1.95
-2.04
-1.85
-1.96
-2.04
-2.12
-9.38
t1/2 , min
44.91
64.00
100.00
43.61
52.79
61.41
75.22
49.33
62.69
77.16
91.00
1.67
Citrate buffer pH 5.2
Citrate buffer pH 4.5
Phosphate buffer pH 9
Phosphate buffer pH 8
Phosphate buffer pH 7.5
Phosphate buffer pH 6.5
Borate buffer pH 10
Borate buffer pH 9
0.011
0.0092
0.014
0.011
Borate buffer pH 8
0.0089
0.0076
0.0041
Acetate buffer pH 5
Acetate buffer pH 4.5
Application of the proposed method to analysis of nicardipine in its commercial
capsules
The proposed method was applied to determination of nicardipine in its commercial
capsules. The percentage recoveries were obtained Table 6.

A Selective HPLC Method to Follow the Hydrolytic Degradation
91
Table 6. Application of the proposed HPLC method to the determination of nicardipine in
commercial capsules
Concentration
Concentration
Compound
Pelcard 50 mg
capsule
Recovery, %
taken, µg/mL-1 found, µg/mL-1
20
30
20.04
30.558
40.316
100.20
101.86
100.79
NC HCl (50 mg/capsule) 40
Mean S.D =100.95 0.841
Pathway of degradation
Treatment of nicardipine with sodium hydroxide at elevated temperature resulted in
cleavage of the ester linkage and the production of two compounds, I (acid) and II (alcohol)
as shown blow:
H
H3C
N
CH3
CH3
NaOH
CH2-C6H5
CH3OOC
COOCH2-CH2-N
∆
NO2
CH3
H
H3C
N
1
6
2
3
CH3
5
4
CH3OOC
COONa
NO2
+
HO-CH2-CH2-N
HO-CH2- CH2-
CH2 C6H5
II
I
I
II
Compound I is eluted first, as it is more polar than either compound II or nicardipine. It
is possible that, the ester group in the side chain at position 5 is also hydrolysed. However,
this will not have a pronounced effect the chromatogram developed. Study of kinetic
parameters for the degradation of nicardipine involved measuring the peak heights of
nicardipine and internal standard only.
Conclusion
A rapid, precise, and specific HPLC method using a single isocratic system has been
developed for the determination of nicardipine HCl, either alone or in the presence of its
degradation products. The method was used to determine the degradation kinetics
parameters of nicardipine and the drug was found to be most stable at pH =4.5.
The energy of activation calculated from Arrhenius plot suggests that a typical
hydrolytic reaction is involved. The rate of the decomposition increases considerably in the
alkaline medium. This agrees with the fact that ester linkages are more susceptible to
cleavage in alkaline medium than in acid medium.

92
K. E. IBRAHIM et al.
The catalytic effect of four buffers, namely, phosphate, borate, citrate and acetate buffer
indicated that the citrate exerts a more catalytic activity than the other three buffers of which
acetate apparently has no effect on the rate of degradation of nicadipine hydrochloride.
References
1.
2.
Budavari S, in the Merck index ,13th Ed., Merck & Co Inc., 2001.
Sorkin E M and Clissold S P, Drugs, 1987, 33, 296.
3
.
Escrig-Tena I, Rodriguez L A, Esteve-Romero J and Garcia Alvarez M C, Talanta,
1998, 47, 43-52.
4.
5.
6.
7.
8.
9.
Rango G, Vetuschi C, Risoli A and Ioele G, Talanta, 2003, 59, 375-382.
Marciniec B and Ogrodowczyk M, Acta Pol Pharma., 2006, 63, 477-484.
Obendorf D and Stubauer G, J Pharm Biomed Anal., 1995, 13, 1339-1341.
Ozkan S A, Uslu B and Aboul- Enein H Y, Crit Rev Anal Chem., 2003, 33, 155-181.
Atkosar Z, Altiokka G and Tuncel M, Pharmazie, 1997, 52, 959- 960.
Uno T, Ohkubot T and Sugawara K, J Chromatogr B; Biomed Sci Appl., 1997, 698,
181-186.
10. Lopez J A, Martinez V, Alonso R M and Jimenez R M, J Chromatogr A, 2000,
870, 105-114.
11. Fernandez C M and Veiga F J B, Biomed Chromatogr, 2003, 17, 33-38.
12. Ke Li, Xin Zhang, Yi-sheng Yuan and Fei-lang Zhao, Biomed Chromatogr, 1998, 12,
326-329.
13. Meiling Q, Wang P and Jin X, J Chromatogr B, 2006, 830, 81-85.
14. Bakhtiar R and Tse F L, Rapid Commun.Mass Spectrom., 2000, 14, 1128-1135.
15. Chen Sh-M, Hsieh M-C, Chao Su-Hui, Chang E E, Wang Po-Yu and An-Bang Wu,
Biomed Chromatogr, 2008, 22,1008-1012.
16. Pomponio R, Gotti R, Fiori J, Cavrini V, Mura P, Crri M and Maestrelli F,
Biomed Anal., 2004, 35, 267-275.

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