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STABILITY INDICATING LIQUID
CHROMATOGRAPHIC METHOD FOR
THE SIMULTANEOUS
DETERMINATION OF OLMESARTAN
MEDOXOMIL AND AZELNIDIPINE IN
COMBINED TABLET DOSAGE FORM
Raveendra Babu Ganduri1*, Jayachandra Reddy Peddapapireddigari2, Himabindu Vurimindi3 and
Rampraksah1
1. Cadila Healthcare Ltd, Ahmedabad, Gujarat.
2. Krishnateja Pharmacy College, Renigunta Road, Tirupati, Andhra Pradesh.
3. Jawaharlal Nehru Technological University, Kukatpally, Hyderabad.
E-mail: raveendrababu@zyduscadila.com
Phone: 02717-666385.
Abstract:
A stability indicating RP-HPLC method for the simultaneous determination of olmesartan medoxomil (OLM)
and azelnidipine from combined tablet dosage form was developed. The separation was accomplished on Inertsil
3V (4.6 mm X 100 mm; particle size 3 µm) column using a mobile phase consisting of potassium dihydrogen
phosphate buffer (pH adjusted to 3.0 with orthophosphoric acid) and acetonitrile in gradient elution mode. The
analytes were monitored by a photo diode array (PDA) detector set at 255 nm and the flow rate was kept at 2.0
mL min-1. The retention time for olmesartan medoxomil and azelnidipine were 3.148 and 3.704 respectively.
Linearity was observed in the concentration range of 10-60 µg/mL for olmesartan medoxomil and 4-24 µg/mL
azelnidipine. Both the drugs were subjected to acid, alkali and neutral hydrolysis, oxidation, dry heat and
photolytic degradation. The degradants were well resolved from the pure drugs. The method could be used for
simultaneous determination of olmesartan medoxomil and azelnidipine in bulk and combined dosage form.
Key words – olmesartan medoxomil, azelnidipine, RP-HPLC, stability-indicating, validation.
1.0 INTRODUCTION
Olmesartan medoxomil (OLM), 2,3-dihydroxy-2-butenyl 4-(1 hydroxy-1-methylethyl)-2-propyl-1-[p-(o-1H-
tetrazol-5-ylphenyl)benzyl]imidazole-5 carboxylate, cyclic 2,3-carbonate, a prodrug, is hydrolyzed to
olmesartan during absorption from the gastrointestinal tract. It is practically insoluble in water and sparingly
soluble in methanol. It is available as film coated tablets containing 5 mg, 20 mg, or 40 mg of olmesartan
medoxomil in US and Europe. It is indicated for the treatment of hypertension. Olmesartan medoxomil is a
white to light yellowish-white powder or crystalline powder with a molecular weight of 558.59.
Azelnidipine (AZL), 3-[1-(Diphenylmethyl)-3-azetidinyl] 5-isopropyl 2-amino-6-methyl-4-(3-nitrophenyl)-1,4-
dihydro-3,5-pyridinedicarboxylate, is a dihydropyridine calcium channel antagonist with selectivity for L-type
calcium channels. Azelnidipine is offered under the registered trademark CALBLOCK® by Sankyo Co. Ltd. of
Japan. CALBLOCK® is offered as an oral tablet administered once daily for the treatment of hypertension and
related diseases. Azelnidipine is only slightly soluble in methanol and water and soluble in ethanol, dimethyl
sulfoxide, acetic acid and dimethyl fluoride. Olmesartan medoxomil and azelnidipine combination is offered
under the registered trademark REZALTAS® by Sankyo Co. Ltd. of Japan. REZALTAS ® is offered as oral
tablets containing 10mg/8mg and 20mg/16mg of olmesartan medoxomil and azelnidipine and administered once
daily for the treatment of hypertension.
A thorough literature survey has revealed that either olmesartan medoxomil or azelnidipine or combination is
not yet official in any pharmacopeia. Few spectrophotmetric1, potentiometric2, LC3-9 methods were reported in
the literature for the determination of azelnidipine. Reported analytical methods for the determination of
olmesartan medoxomil include spectroscopic10, HPLC11-17, UPLC18, LC-MS19. One spectroscopic method was
reported for the simultaneous determination of OLM and AZL in combined pharmaceutical dosage forms. To
the best of our knowledge, a complete validated stability indicating RP-HPLC method for the simultaneous
estimation of OLM and AZL in combined pharmaceutical dosage form was not reported. Therefore, it was
Raveendra Babu Ganduri et al. / International Journal of Pharma Sciences and Research (IJPSR)
ISSN : 0975-9492
Vol 5 No 06 Jun 2014
275
thought worthwhile to develop a simple, precise, accurate reverse phase high performance liquid
chromatographic method for the simultaneous determination of OLM and AZL in combined tablet dosage form.
2.0 EXPERIMENTAL
2.1 Chemicals & Reagents
All the reagents were of analytical-reagent grade unless stated otherwise. Milli-Q-water was used throughout the
experiment. HPLC-grade acetonitrile, potassium dihydrogen phosphate, orthophosphoric acid were procured
from Merck Ltd, Mumbai. All the solvents and solutions were filtered through a membrane filter and degassed
before use. Reference standards of OLM, AZL and combined tablets were received from the research
development department of Cadila Health care Ltd, Ahmedabad, India.
2.2 Instrumentation
The HPLC system used was of model Waters 2695 equipped with quaternary pump, auto sampler, thermostated
column compartment and variable wavelength detector controlled by the empower software. The column used
was Inertsil ODS 3 (100 mm X 4.6 mm, 3µm). Column temperature was maintained at 250C.
2.3 Chromatographic conditions
Stationary phase Inertsil ODS 3 (250 mm X 4.6 mm, 3µm) column
Detection wavelength 255 nm
Injection volume 25µL
Flow rate 2.0 mL/min
Column temperature 25°C
Sample temperature 5°C
Buffer 2.0 gm of potassium dihydrogen phosphate is dissolved in water and pH adjusted
to 3.0 with orthophosphoric acid
Diluent Acetonitrile: water (50:50)
Mobile phase Solvent A: Buffer pH 3.0: Acetonitrile(80:20)
Solvent B: Buffer pH 3.0: Acetonitrile(20:80)
Gradient program Time (Min.) Solvent A Solvent B
0.0 90 10
4.0 2 98
4.5 90 10
6.0 90 10
2.4 Preparation of standard stock solutions
The stock solutions OLM and AZL were prepared separately by dissolving accurately weighed quantity of 60
mg of OLM and 15mg of AZL in 100 mL of diluent. Working solution containing 40 µg/mL of OLM and 16
µg/mL of AZL was prepared from above stock solution.
2.5 Preparation of sample solution
Twenty tablets, each containing 40mg of OLM and 16mg of AZL were accurately weighed and grounded to fine
powder. An amount equivalent to 400 mg of OLM was transferred into a 100 mL volumetric flask and about 50
mL of diluent was added, sonicated for not less than 30 min with occasional shaking and made up the volume
with diluent. The above solution was filtered through 0.45 μm millipore PVDF filter. The above solution was
further diluted to 100 mL with diluent to obtain a concentration of 40 µg/mL of OLM and 16 µg/mL of AZL.
2.6 Stress degradation studies
In order to establish the analytical assay method as stability indicating, the following stress conditions were
studied on the combination tablet dosage form containing 40 mg of OLM and 16 mg of AZL as per ICH
stability guidelines.
a) Acid hydrolysis: Drug solution in 1N HCl at 70°C for 4 hours.
b) Alkaline hydrolysis: Drug solution in 1N NaOH at 70°C for 4 hours.
c) As such: Sample solution as such for 6 hours
d) Oxidative degradation: Drug solution in 3% hydrogen peroxide at room temperature for 4 hours.
e) Thermal degradation: Tablets were subjected to dry heat at 105°C for 10 hours.
f) Photolytic degradation: The photo degradation was carried out by exposing the tablets samples in solid state
to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet
energy of not less than 200 W h/m2 for 24 hours.
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3.0 Results and Discussion
3.1 Method development and optimization
Stressed samples prepared by a systematic forced degradation study were used for method development trials to
optimize the method as a stability-indicating method for simultaneous determination of OLM and AZL. In our
preliminary experiments, OLM and AZL were subjected to separation by RP-HPLC, on different commercial
columns. Water with acetonitrile and methanol as organic modifiers was used as mobile phase. Broad peaks and
tailing were observed for OLM and few degradants. So to improve the peak shapes, phosphate buffer was used
in place of water. As the compounds were having varying polarities, a gradient method was tried. The HPLC
conditions were optimized by studying the effects of different columns, organic modifiers, and pH of buffer on
the separation of OLM, AZL and their degradation products. The separation of OLM, AZL and their degradants
was achieved on Inertsil ODS 3 (250 mm X 4.6 mm, 3µm) column and potassium dihydrogen phosphate and
acetonitrile combination as mobile phase at a detection wavelength of 255 nm. Fig 1 and Fig 2 shows the
separation of OLM and AZL in standard and sample solutions respectively.
Fig 1: Chromatogram of Standard solution of OLM and AZL
Fig 2: Chromatogram of sample solution of OLM and AZL
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Vol 5 No 06 Jun 2014
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3.2 Method Validation
The developed method was validated as per ICH guidelines.
3.2.1 System suitability and precision
System suitability and precision was demonstrated by making five replicate injections of standard solution. The
peak area of OLM and AZL for replicate injection was recorded. The tailing factor and number of theoretical
plates was evaluated for the analyte peak. The precision was evaluated by computing the relative standard
deviation for the analyte peak area of these replicate injections. The results were within the acceptable limits and
were shown in Table 1.
Table 1: System suitability Data
Injection No Peak area
Olmesartan medoxomil Azelnidipine
1 639206 307026
2 639409 307153
3 639480 306748
4 641346 307172
5 639326 307135
Average 639753.4 307046.8
%RSD 0.140071 0.057466
USP tailing factor 1.01 1.02
No.of Theoretical plates 12842 17394
3.2.2 Specificity
The specificity was evaluated by studying the interference from placebo and degradants with the OLM and AZL
peak. Interference from placebo was demonstrated by comparing the chromatograms of placebo, standard
solution and sample solution and no interference was observed from placebo. Interference from degradants was
demonstrated by performing stress study on formulation under the different stress conditions. The stressed
samples of above conditions were prepared as per the test method and chromatographed into HPLC system
equipped with a diode array detector. The peak purity of OLM and AZL peak and the assay of formulation were
evaluated under each stressed condition. In all the stress conditions, purity angle is less than purity threshold and
no purity flag was observed. The study indicates that there is no interference of any degradants with the OLM
and AZL peak, which proves that the method is specific and stability-indicating for estimation of OLM and
AZL in the formulation.
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ISSN : 0975-9492
Vol 5 No 06 Jun 2014
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Fig 3: Stress degradation behaviour of OLM+AZL in various stress conditions.
3.2.3 Precision
Method precision was demonstrated by preparing six samples as per the test method. The assay of these samples
was determined and the precision of the method was evaluated by computing the percentage-relative standard
deviation of the assay results. The % RSD was found to be less than 0.13. The results were shown in Table 2.
The intermediate precision (ruggedness) of the method was demonstrated by carrying out precision study in six
replicates of assay on a single batch sample by two different analysts, on two different days and on two different
instruments. The % RSD was found to be 0.26 and 0.3 for OLM and AZL respectively.
Table 2: Method precision data
Sample % Assay
OLM AZL
1 100.2073 100.043
2 99.97606 99.95636
3 100.0066 99.72445
4 100.1164 99.97427
5 99.85623 99.84399
6 100.1006 99.89545
Average 100.0439 99.90625
%RSD 0.123543 0.112247
3.2.4 Linearity
The linearity of detector response for OLM and AZL was demonstrated by preparing solutions of OLM and
AZL working standard over the range of 25 % to 150 % of target concentration. These solutions were injected
into the system and the peak area of OLM and AZL was recorded. A graph of concentration vs peak area of
OLM and AZL was plotted separately. The correlation co-efficient between concentration & peak area and y-
intercept of the correlation plot were evaluated. The results were shown in Table 3.
Table 3: Linearity Data
Linearity
Level Olmesartan medoxomil Azelnidipine
Concentration (µg/mL) Peak area Concentration (µg/mL) Peak area
25% 9.6 156618 4.2 81937
50% 20.4 334098 7.8 151338
75% 30.0 478691 12.0 233780
100% 39.6 639462 16.2 306473
125% 49.2 787410 20.4 392996
150% 60.0 958641 24.0 457239
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3.2.5 Accuracy
The accuracy of the test method was demonstrated by preparing recovery samples (i.e. spiking placebo with
known quantities of API) at the level of 50 %, 100 % and 150 % of target concentration. The recovery samples
were prepared in triplicate in each level. The above samples were chromatographed and the percentage recovery
for the amount added was estimated. The precision of the recovery at each level was determined by computing
the relative standard deviation of triplicate recovery results. The recovery results are between the range of 98.0
% to 102.0 %, with RSD at each level of less than 0.4 %, proves the method is accurate for the estimation of
OLM and AZL in OLM and AZL tablets over the range of 50 % to 150 % of target concentration. The results
were shown in Table 4.
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Table 4: Accuracy Data
Olmesartan medoxomil Azelnidipine
Amount
Added
(mg)
Amount
Recovered
(mg)
%
Recovery % RSD Amount
Added
(mg)
Amount
Recovered
(mg)
%
Recovery % RSD
20.41 20.54 100.64 0.137 7.87 8.01 101.7789 0.126
19.98 20.24 101.3 7.78 7.86 101.0283
20.51 20.62 100.54 7.92 8.02 101.2626
39.65 40.06 101.03 0.212 16.25 16.38 100.8 0.133
38.74 39.04 100.77 16.05 16.12 100.4361
40.05 40.85 102 16.94 17.15 101.2397
60.01 59.78 99.617 0.318 24.15 24.21 100.2484 0.26
59.88 60.15 100.45 23.85 24.14 101.2159
60.85 61.78 101.53 24.84 25.29 101.8116
3.2.6 Robustness
To determine the robustness of the developed method, experimental conditions were purposely altered. The flow
rate was altered by 0.2 units, column temperature was altered by 5 units, organic phase ratio of mobile phase
was altered by 2 units and pH was altered by 0.2 units. Theoretical plates, tailing factor and % RSD of five
replicate standard injections were evaluated. The results prove that the method is robust. The results were shown
in the Table 5.
Table 5: Robustness Data
Condition % RSD Tailing
factor Theoretical plates % RSD Tailing
factor Theoretical
plates
Normal Condition 0.14 1.01 12842 0.057 1.02 17394
Column Temperature changed by – 5°C (i.e.,
20°C) 0.07 1.0 11945 0.056 1.0 18020
Column Temperature changed by + 5°C (i.e.,
30°C) 0.04 1.02 13229 0.055 1.04 18622
Organic phase ratio of mobile phase changed
by – 2 % absolute [i.e., Buffer solution:
Acetonitrile (81: 19) for mobile phase A and
Buffer solution : Acetonitrile (21: 79) for
mobile phase B].
0.13 1.0 13044
0.056 1.0 18444
Organic phase ratio of mobile phase
changed by + 2 % absolute [i.e., Buffer
solution: Acetonitrile (79: 21) for mobile
phase A and Buffer solution : Acetonitrile
(19: 81) for mobile phase B].
0.12 1.0 11910
0.03 1.01 16845
Flow rate changed by – 10 % (i.e. 1.8
mL/min) 0.06 1.0 13202 0.01 1.0 18922
Flow rate changed by + 10 % (i.e. 2.2
mL/min) 0.05 1.0 12564 0.03 1.01 17643
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3.2.7 Stability of analytical solution
Stability of standard and sample solution at vial thermostat temperature (i.e., 5°C) was established as mentioned
below. Standard and sample solution was prepared as per the test method. The solution thus prepared was
chromatographed at regular intervals up to 48 hours. The response of OLM and AZL peak was monitored for
both standard and sample solutions. The % deviation of OLM and AZL response from initial for both standard
and sample solutions was found to be less than 0.4%. Hence both standard and sample solutions were stable at
vial thermostat temperature (i.e.5°C) upto 36 hours.
4.0 CONCLUSION
A new stability-indicating RP‐HPLC method was proposed for the simultaneous determination of OLM and
AZL in combined dosage form and validated as per the ICH guidelines. The method was found to be simple,
selective, precise, accurate and robust. Therefore, this method can be used as routine testing as well as stability
analysis of OLM and AZL in synthetic mixtures and combined dosage form.
ACKNOWLEDGEMENT
The authors thank Cadila Healthcare Ltd for providing necessary facilities for the completion of this research
work and kind permission to communicate the results for publication.
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