Simultaneous Determination of Azelnidipine and Olmesartan Medoxomil in Pharmaceutical Dosage Forms by UFLC Method

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Journal of PharmaSciTech
Simultaneous Determination of Azelnidipine and Olmesartan Medoxomil in Pharmaceutical Dosage
Forms by UFLC Method
1 2 3
Alaa El Sayed Ahmed Amin *, Mounir Zaky Saad , Mohamed Aly Amin Ahmed
Azelnidipine (AZL), (±)-3-[1-(diphenylmethyl) azetidin-3-yl] 5-
pro pa n -2 - yl 2- a mi no - 6- me t hy l -4 -( 3 -n itr oph en y l) -1, 4-
dihydropyridine-3,5-dicarboxylate as shown in Figure 1, is a new
dihydropyridine derivative with calcium antagonistic activity [1].
the determination of Azelnidipine includes HPLC [2-3], LC-MS method
[4-5], LC-ESI-MS [6-7], HPLC-MS-MS [8], in which two methods for
formulation and remaining for human plasma. Olmesartan medoxomil
(OLM), is (5-methyl-2-oxo-1,3-dioxol-4-yl) methyl 4-(2-hydroxy-
propan-2-yl )-2-propy l-1-{[2'- (2H-tetra zol-5-yl)biphenyl-4-
yl]methyl}-1H-imidazole-5-carboxylate as shown in Figure 1. A
literature survey revealed that Olmesartan is not yet official in any
pharmacopoeia. Several analytical methods have been reported for
the determination of Olmesartan medoxomil in biological fluids,
which includes LC-MS-MS [9], degradation product HPLC [10],
HPTLC [11] and HPLC with dissolution study [12].
Several clinical trials prove that Olmesartan medoxomil and
Azelnidipine gives better therapeutic effect in essential hypertension
rather than in single dosage form [13]. There was only one first
derivative spectrophotometric method reported for simultaneous
analysis [14] and HPLC simultaneous analysis [15].
Materials and Methods
Materials
All chemicals and reagents used were HPLC grade. Pure standards of
azelnidipine, Zhejiang Gaobang Pharmaceutical Co. Ltd., and
olmesartan medoxomil Qilu Tianhe Pharmaceutical Co. Ltd., were
obtained from Chinese. HPLC grade Methanol was purchased from
Romil. Water for chromatography was purchased from Merck,
Germany.
Chromatographic conditions
The analysis of drugs was carried out on a Shimadzu LC-20 XR,
prominence, equipped with an auto sampler (SIL-20AC XR,
Shimadzu, Japan) and PDA detector (SPD- M20A, Japan) was used
for the analysis. The data was recorded using LC-solution software. A
Phenomenex, Prodigy, ODS3, (250mm x 4.6mm, 5μm) column was
used for the analysis. A NSXX sonics ultrasonic bath (NS-A-12-7H,
Germany) was used for degassing of the mobile phase.
In this UFLC method separation was carried out using a mobile phase
Abstract
A simple, precise, sensitive and rapid reversed phase Ultra-Fast liquid chromatography method was developed for simultaneous determination of
azelnidipine and olmesartan medoxomil in Pharmaceutical Dosage form with greater precision and accuracy has been developed and validated. The
chromatographic separation was achieved by using Phenomenex, Prodigy, ODS3, 5 µm, 100 Å, (250 x 4.6 mm) analytical column with a mobile
phase consisting of methanol and water at the ratio of (85:15% v/v). The chromatographic condition was set at a flow rate of 1.5 ml/min, column
oven temperature 25°C and detector wavelength of 255 nm using a photodiode array detector. An injection volume of 10 µl was used for
2
azelnidipine and olmesartan medoxomil. The calibration curve of azelnidipine was linear with correlation coefficient (r ) = 0.9999; over a
concentration range of 1.0 - 60.0 µg/ml for; with a retention time of 6.80 min. While the calibration curve of olmesartan medoxomil was linear with
2
correlation coefficient (r ) = 0.9998; over a concentration range of 1.0 - 60.0 µg/ml for; with a retention time of 1.72 min. The recovery level of
azelnidipine and olmesartan medoxomil was 99.62% and 100.12%; respectively. The validated UFLC method was successfully applied to the
analysis of azelnidipine and olmesartan medoxomil in pharmaceutical dosage form.
Keywords: UFLC, azelnidipine, olmesartan medoxomil, method validation, pharmaceutical dosage forms.
ISSN: 2231 3788 (Print)
2321 4376 (Online)
Research Article
1Department of Analytical Chemistry, Benha University, Arab Republic of Egypt 13511
2Department of Chemistry, Zagazig University, Arab Republic of Egypt 44519
3Mepaco Pharmaceutical Company, Sheraton Heliopolis Cairo 11361
*Correspondence: asamin2005@hotmail.com (Tel. + 002/01205225223)
Introduction
Figure 1: Chemical structure for azelnidipine and olmesartan
medoxomil
The recommended dosing of Azelnidipine is 16 mg per day. A
literature survey revealed that Azelnidipine is not yet official in any
pharmacopoeia. Very few analytical methods have been reported for
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Amin et al, Simultaneous Determination of Azelnidipine and Olmesartan Medoxomil in Pharmaceutical Dosage Forms by UFLC Method
consisting of HPLC grade methanol and water at the ratio of (85:15%
v/v). The mobile phase was filtered by using a 0.45 µm nylon
membrane filter. The column was maintained at a temperature of 25ºC
with column oven (CTO-20AC) and the flow rate was 1.5 ml/min.
Analysis was performed with injection volume of 10 μl using PDA
detection at 255 nm. The run time was set for 8.0 min. The optimized
chromatographic condition is shown in Table 1.
Preparation of stock and working standard solution
A 10 mg of azelnidipine and A 10 mg of olmesartan medoxomil
working standard were weighed and transferred into a 100 ml
volumetric flask. 85 ml of the methanol was added and shake on
vortex for 2 min; then was sonicated for 10 minutes. Working
standard solutions were prepared and further diluted in methanol to
contain a mixture of azelnidipine and olmesartan medoxomil in over
the linearity range from 1.0 - 60.0 µg/ml and 1.0 - 60.0 µg/ml
respectively.
Method validation
The present method of analysis was validated according to the
recommendations of ICH- 1996 and USP-30 for the parameters like
specificity, system suitability, accuracy, linearity and precision.
Specificity
It provides an indication of the selectivity and specificity of the
procedure. The method is to be selective, if the main peak is well
resoluted from any other peak by resolution of minimum 2. This could
be done injecting placebo and compare it with that of standard and
placebo spiked with standard and sample, then peak purity was
ascertained by use of PDA.
System suitability
System suitability was performed by injecting six replicates of
standard solution at 100% of the test condition at a 100% level to
verify the precision of the chromatographic system. The purposed
UFLC method permits the determination of azelnidipine and
olmesartan medoxomil in sample drug have different retention times.
System suitability data are given in Table 2.
Table 1: Optimized chromatographic conditions
Parameters Conditions
Stationary Phase
Mobile Phase
Flow Rate (ml/min)
Run Time (min)
Column Temperature ( )
Injection Volume (µl)
Detection Wavelength (nm)
Retention Time of Azelnidipine (min)
Retention Time of Olmesartan (min)
ºC
Prodigy, ODS3, 250 x 4.6 mm, 5 µm
Methanol and Water (85:15 v/v)
1.5
8.0
Ambient (25 )
10
255nm
6.80
1.72
ºC
Table 2: System suitability parameters for azelnidipine and olmesartan
medoxomil
S. No. Parameters Azelnidipine Olmesartan
1
2
3
Tailing factor
Retention Time
Theoretical plates
1.08
6.80
7044
0.86
1.72
839
Linearity
Is defined by the correlation coefficient, which should be found NLT
0.99, using peak area responses, Linearity for single point
standardization should extend to at least 20% beyond the
specification range and include the target Conc. This was performed
by preparing 7 different concentrations (2.5%, 5%, 25%, 50%, 100%,
125% and 150%), and then making 3 replicates of each concentration.
The linear working range was determined from the constructed
standard calibration curve.
Intraday Precision
This study was conducted by performing multiple analyses on a
suitable number of portions of a homogeneous sample. This was
performed by assaying multiple aliquots with the same
concentration. The analytical precision of the method was
determined by the relative standard deviation.
Inter-day Reproducibility (Method Ruggedness)
The degree of reproducibility determined by analysis of samples from
homogeneous lot of materials, under different but typical test
conditions The method is to be rugged, at any item if the pooled %RSD
of the total number of replicates that have been made in this item is
within the acceptance criteria, 3 replicates of a single sample of
powder material are used for each determination. First day: 3
replicates, on a second day: 3 replicates, then on third day: 3
replicates of freshly prepared test from the same sample are
analyzed, under the same conditions.
Accuracy
Accuracy was evaluated by spiking standard with sample solution.
The measurements are made at a concentration of standard mix,
which is found to be the target concentration, and at suitable intervals
around this point. The test samples was spiked with known quantities
of standard azelnidipine and olmesartan medoxomil using three
determinations over three concentrations level covering the specified
range. Relative recoveries of standard azelnidipine and olmesartan
medoxomil used in the standards were evaluated by comparing their
peak area with those obtained from the calibration curve equation.
Stability of Analytical solution
The stability of analytical solutions was established by injecting the
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solution and sample solution that were injected at periodic intervals
found to be the specified limit. The values are presented in the Table 3
and Table 4.
standard solution and sample solution at different time intervals up to
24 h (0, 12, and 24 h) by keeping the auto sampler temperature at
room temperature (25ºC). The % differences of peak area of standard
Table 4: Stability of standard and sample solution of olmesartan medoxomil
Time
Interval (h)
Standard Sample
Standard
Peak area
% Difference Sample
Peak area
% Difference
0
12
24
1277723
1277526
1277397
-
0.02
0.03
1277136
1276921
1276858
-
0.02
0.02
Table 3: Stability of standard and sample solution of azelnidipine
Time
Interval (h)
Standard Sample
Standard
Peak area
% Difference Sample
Peak area
% Difference
0
12
24
841726
841683
841662
-
0.01
0.01
841423
841375
841303
-
0.01
0.01
Results and Discussion
The proposed UFLC method required fewer reagents and materials,
and it is simple and less time consuming. This method could be used
in quality control test in pharmaceutical industries. The
chromatogram of azelnidipine and olmesartan medoxomil was shown
in Figure 2. There was clear resolution between azelnidipine and
olmesartan medoxomil with retention time of 6.80 and 1.72 minutes;
respectively.
Figure 2: A typical chromatogram for azelnidipine and olmesartan
medoxomil standard drug
Specificity
Generally, the specificity of a method is its suitability for the analysis
of a compound in the presence of potential impurities. Placebo,
standards, and sample test solutions were all injected at the same
wavelength of 255 nm to demonstrate the specificity of the optimized
method. A comparison of the retention times of azelnidipine and
olmesartan medoxomil in sample solutions and in the standard
solutions were exactly the same. Figures 2, 3 and 4 showed that there
were no interferences at the retention times for azelnidipine and
olmesartan medoxomil due to the placebo.
Figure 3: A typical chromatogram for azelnidipine and
olmesartan medoxomil sample drug
Figure 4: UFLC chromatogram of placebo
Therefore, the proposed method is suitable for the quantification of
the active ingredients in tablet formulation.
Amin et al, Simultaneous Determination of Azelnidipine and Olmesartan Medoxomil in Pharmaceutical Dosage Forms by UFLC Method
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Linearity
The response for the detector was determined to be linear over the
range of 1.0-60.0 µg/ml (1.0, 2.0, 10.0, 20.0, 40.0, 50.0 and 60.0) for
azelnidipine as shown in Figure 5 and data are shown in Table 5. The
response for the detector was determined to be linear over the range
Figure 5: Calibration curve of azelnidipine
Table 5: Statistical data of calibration curves of azelnidipine
S. No. % test
Concentration
Concentration
(µg/ml)
Average
Peak area
1
2
3
4
5
6
7
2.5
5
25
50
100
125
150
1
2
10
20
40
50
60
23094
44592
210639
421299
841858
1049211
1253045
Regression co-efficient = 0.9999
of 1.0-60.0 µg/ml (1.0, 2.0, 10.0, 20.0, 40.0, 50.0 and 60.0) for
olmesartan medoxomil as shown in Figure 6 and data are shown in
Table 6.
Figure 6: Calibration curve of olmesartan medoxomil.
Each reading was average of three determinations. They were
represented by the linear regression equation.
2
Y = 20894.15891x + 2872.55984, r = 0.9999
Azelnidipine
2
Y = 31481.83273x + 3849.89660, r = 0.9998
Olmesartan medoxomil
Slopes and intercepts were obtained by using regression equation
(Y = mx + c) and least square treatment of the results used to
confirm linearity of the method developed.
Table 6: Statistical data of calibration curves of olmesartan medoxomil
S. No. % test
Concentration
Concentration
(µg/ml)
Average
Peak area
1
2
3
4
5
6
7
2.5
5
25
50
100
125
150
1
2
10
20
40
50
60
37007
71212
317174
618424
1278919
1578673
1886715
Regression co-efficient = 0.9998
Each of the concentrations was injected in triplicate to get
reproducible response. Calibration curves were constructed by
plotting peak area versus concentration.
Table 7: Results of accuracy for azelnidipine and olmesartan medoxomil
Level (%) Amount of
drug
spiked (mg)
Azelnidipine Olmesartan
Found (mg) Recovery (%)
(n=3)
Amount of
drug
spiked (mg)
Found (mg) Recovery (%)
(n=3)
50
100
150
2.96
5.92
8.88
2.94
5.90
8.87
Average Recovery
SD
% RSD
99.29
99.66
99.91
99.62
0.312
0.313
2.88
5.76
8.64
2.89
5.77
8.63
Average Recovery
SD
% RSD
100.35
100.17
99.85
100.12
0.253
0.253
Accuracy
Accuracy was calculated by addition of standard drugs to
preanalyzed sample at 3 different concentration levels (50%, 100%
and 150%) and computing percentage recoveries. Standard limit of %
recovery study is 98 - 102 % as per ICH guideline. From the studies it
was concluded that % recovery study of azelnidipine and olmesartan
medoxomil complies with standard limit of ICH guideline. Results of
accuracy were proven by the Table 7 and % RSD is 0.313 and 0.253 of
Amin et al, Simultaneous Determination of Azelnidipine and Olmesartan Medoxomil in Pharmaceutical Dosage Forms by UFLC Method
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azelnidipine and olmesartan medoxomil respectively which is within
the acceptable limit (less than 2.0).
Inter-day Precision
Solution containing 40 μg/ml and 40 μg/ml of azelnidipine and
olmesartan medoxomil was prepared from their respective standard
stock solution. Analysis was replicated for 3 different days. The result
of inter-day precision studies was shown in Table 8.
Table 8: Inter -day precision data of azelnidipine and olmesartan medoxomil
Assay ( % labeled amount)
Azelnidipine Olmesartan
Sample
ID
(Day 1) (Day 2) (Day 3) (Day 1) (Day 2) (Day 3)
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6
Average
SD
% RSD
99.23
99.22
98.62
100.02
98.99
99.22
99.22
0.459
0.463
99.44
99.36
100.11
100.15
99.97
99.45
99.75
0.368
0.369
98.88
99.14
99.61
99.55
98.77
99.31
99.21
0.344
0.347
99.12
99.18
99.88
98.45
98.96
99.33
99.15
0.468
0.472
99.62
99.23
100.06
99.65
98.98
99.45
99.50
0.373
0.375
98.98
99.13
99.53
99.41
98.74
99.42
99.20
0.305
0.308
Inter-day Reproducibility (Method Ruggedness)
Three replicates of a single sample of powder material are used for
each determination. First day: 3 replicates, on a second day: 3
replicates, then on third day: 3 replicates of freshly prepared test from
the same sample are analyzed, under the same conditions. The result
of inter-day reproducibility studies was shown in Table 9.
Table 9: Inter-day reproducibility data of azelnidipine and olmesartan medoxomil
Assay ( % labeled amount)
Azelnidipine Olmesartan
Sample
ID
(Day 1) (Day 2) (Day 3) (Day 1) (Day 2) (Day 3)
Sample-1
Sample-2
Sample-3
Average
SD
% RSD
99.44
99.26
99.58
99.43
0.160
0.161
99.56
98.36
99.26
99.06
0.624
0.630
100.12
99.56
99.72
99.80
0.288
0.289
98.86
99.46
98.60
98.97
0.441
0.446
98.89
98.78
98.58
98.75
0.157
0.159
99.56
99.49
98.66
99.24
0.501
0.504
Quantification limit
The limit of detection (LOD) and limit of quantification (LOQ) of the
developed method was determined by injecting progressively low
concentrations of the standard solutions using the developed
methods. The LOD is the lowest concentration of the analyte that can
be detected with signal to noise ratio (3:1) and LOQ is the lowest
concentration that can be quantified with acceptable precision and
accuracy with signal to noise ratio (10:1). The LOD of azelnidipine and
olmesartan medoxomil found to be 0.167 µg/ml and 0.170 µg/ml
respectively. The LOQ of azelnidipine and olmesartan medoxomil
found to be 0.50 µg/ml and 0.51 µg/ml respectively.
Stability of analytical solution
In this study, the mobile phases, the standard solutions, and the
sample solution were subjected to long term (24 h) stability studies.
The stability of these solutions was studied by performing the
experiment and looking for changes in separation, retention, and
asymmetry of the peaks which were then compared with the pattern
of the chromatogram of freshly prepared solutions
System suitability
The system suitability was determined by injecting six replicates of
the standard solutions and analyzing each active ingredient for its
peak area, peak tailing factor, resolution, number of theoretical plates,
and capacity factor. The values obtained demonstrated the suitability
of the system for the analysis of the above drug combinations System
suitability parameters might be fall within ±2% standard deviation
range during routine performance of the methods.
Conclusion
The validated UFLC method developed for the quantitative quality
control determination of azelnidipine and olmesartan medoxomil in
combination was evaluated for system suitability, specificity,
Amin et al, Simultaneous Determination of Azelnidipine and Olmesartan Medoxomil in Pharmaceutical Dosage Forms by UFLC Method
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linearity, range, accuracy (recovery), precision (repeatability and
intermediate precision). This method enables simultaneous
determination of azelnidipine and olmesartan medoxomil because of
good separation and resolution of the chromatographic peaks. As a
result, the proposed UFLC method could be adopted for the
quantitative quality control and routine analysis of tablet dosage form.
Acknowledgement
The authors are grateful to Mepaco-Medifood Pharmaceutical
Company (El Sharkia, Egypt) for her ultimate support for Research and
Development team.
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