Impurity profiling of Azelnidipine and Telmisartan in Fixed Dose Combination using Gradient RP-HPLC Method
Manish Kumar1*, Umesh Chandra1, Arun Garg1 , Pankaj Gupta1
1,* Department of Pharmacy, School of Medical and Allied Sciences, K.R. Mangalam University, Sohna Road, Gurugram, Haryana-122003.
E mail: [email protected] ABSTRACT
The objective is to develop and validate, stability indicating RP-HPLC method used for the determination and quantification of possible degradants & impurities present in Azelnidipine and Telmisartan in 8mg/40mg fixed-dose combination (FDC) tablets. The method was developed using RP-HPLC, Inertsil C-18 Column with 150×4.6 mm×5 µm, flow rate 1.5 mL/min, Injection volume10 µL, column oven temperature 40°C and run time 40.0 minutes at 254 nm using Acetonitrile and buffer as mobile phase in gradient mode. The developed method was accurate, repeatable, and detectable towards determination of Impurties present in Azelnidipine and Telmisartan combination without any unwanted interference. When evaluated on various parameters like system suitability, precision, accuracy, linearity, force degradation study, solution stability, it can be concluded that the method is efficient in separating drugs from their Impurties and can be utilized for analyzing the samples of Azelnidipine and Telmisartan.
Keywords: Azelnidipine, Telmisartan, RP-HPLC, Method Validation, Stability, Impurties.
1. INTRODUCTION:
Various regulatory guidelines like International Conference on Harmonization (ICH), European Medicines Agency (EMA) and United States Food and Drug Administration (USFDA) have specification limits for impurities present in API and Finished formulations.
Impurties means presence of any foreign matter or substance which differs from the drug substance in terms of its structure, pharmacological, and toxicological effects and any component in drug product i.e. not Active Pharmaceutical Ingredients (API) or excipient. Impurties may be organic, inorganic, process related, formulation related and residual solvents.
Description of known and unknown impurities in drug substance and drug product is called impurity profiling [1-4].
From the extensive literature search, it can be inferred that UV-Spectroscopy, HPLC, LC-MS/MS [5- 12] are the only techniques used for estimating Azelnidipine and Telmisartan either alone or in combination with other drugs. To the best of our knowledge that, no report has been documented so far for determination of impurities of Azelnidipine and Telmisartan in FDC.
2. MATERIAL AND METHODS:
2.1. Chemicals and reagents:
The working standard of Azelnidipine, Telmisartan were received as gift samples from M/s. Synokem Pharmaceutical Limited, Haridwar, Uttrakhand and FDC tablets of Azelnidipine and Telmisartan were prepared with label claim of 8 mg and 40 mg respectively. Azelnidipine impurties viz AZE-4, AZE IMP A, AZE INB Acetoacetate and Telmisartan impurties viz TEL IMP A, TEL IMP B and Other reagents including Acetonitrile, Methanol (HPLC grade) and Ammonium dihydrogen orthophosphate, Orthophosphoric acid, Hydrochloric acid, Sodium hydroxide, Hydrogen peroxide (Analytical grade) and Milli-Q water were obtained from M/s. Kimia Biosciences Limited, Gurugram, Haryana.
2.2. HPLC method development:
2.2.1. Chromatographic Conditions and instrument:
For chromatographic analysis, HPLC instrument equipped with Photodiode-Array Detection (PDA) detector was used (Agilent make, model-LC-1210 with empower software). The stationary phase is Inertsil C-18 column 150×4.6 mm×5 µm, HPLC column oven temperature was 40°C, autosampler temperature 10°C, with the flow of 1.5 mL/min. The injection volume was kept at 10 µL with a run time of 40.0 minutes and a wavelength of 254 nm was optimized. The mobile phase in gradient mode as shown in (Table 1). Other instruments used in the validation like analytical balance, ultra sonicator, and pH meter, Hot air oven etc. were calibrated.
2.2.2. Preparation of Buffer solution:
Accurately weighed and transfer about 2.0 gm of Ammonium dihydrogen orthophosphate in suitable flask, add 1000 mL of HPLC grade water, mix well and dissolve. Adjust the pH with dilute Ortho- phosphoric acid to 3.0±0.05. Filter a solution through 0.45 µm millipore membrane filter and sonicate the solution to degas.
2.2.3. Preparation of Diluent:
The buffer and Acetonitrile solution was filtered and degassed in the ratio of (25:75 % v/v) used as diluent and blank solution.
2.2.4. Preparation of 0.1N sodium hydroxide solution:
Accurately weighed and transfer about 4.0 gm pellets of sodium hydroxide, poured in a 1000 mL volumetric flask containing HPLC grade water, dissolved properly and diluted with HPLC grade water to made up the final volume.
2.2.5 Gradient program:Various combinations of Acetonitrile and Buffer (Ammonium dihydrogen orthophosphate pH 3.0 ± 0.05) for mobile phase as described in (Table 1)
Table 1:Gradient Program.
Gradient Time Pump A % (Buffer) Pump B % (Acetonitrile)
0.01 80 20
5.00 80 20
20.00 30 70
30.00 30 70
35.00 80 20
40.00 80 20
2.2.5. Preparation of stock and standard solutions:
Accurately weighed and transfer about 14.12 mg Azelnidipine and 149.70 mg Telmisartan in to a 50 mL calibrated volumetric flask. Add 20 mL of diluent sonicate to dissolve and made up volume with diluent. Labelled this solution as stock solution.
From this stock solution, accurately pipette and transferred 5 mL of aliquot into a 25 mL volumetric flask, diluted to final volume with the diluent. All the contents were mixed well to get the final concentration of 56.24 µg/mL Azelnidipine and 596.76 µg/mL of Telmisartan. The resultant solution was filtered using a 0.45 µm PVDF membrane filter and labelled this solution as the standard solution.
2.2.6. Preparation of placebo solution:
Weighed accurately about 1030 mg of the placebo (weight equivalent to 5 tablets) powder in a 100 mL volumetric flask, add 30 mL of diluent, sonicate for 15 minutes with intermittent shaking, cool to room temperature and made up the volume with diluent, mix and filter through 0.45 µm PVDF membrane filter.
2.2.7. Preparation of sample solution:
Weighed and powder 10 tablets of Azelnidipine and Telmisartan and transferred powder weight equivalent to 5 tablets in to a 100 mL volumetric flask. To this mixture around 30 mL of diluent was added and sonicated for 20 minutes and made up with diluent. Filter through a 0.45 µm PVDF filter.
2.2.8 Preparation of Impurity stock solution:
Impurity stock solution was prepared by individually weighed 1mg of each Impurties{AZE 4, Azelnidipine (AZE) IMP A, AZE INB Acetoacetate, Telmisartan (TEL) IMP A, TEL IMP B} and transferred in to 10 mL each seprate volumetric flasks. Add 5 mL diluents and sonicate for 15 minutes with intermittent shaking, cool to room temperature and made up the volume with diluent. Mix and filter through 0.45 µm PVDF membrane filter (each impurity concentration:100 µg/mL).
2.2.9 Preparation of System Suitability Solution:
2 mL standard solution was taken into 20 mL volumetric flask and add 2 mL of each Impurity stock solution and made up the volume with diluent. (Final concentration: Azelnidipine: 5.62 µg/mL, Telmisartan: 59.68 µg/mL, AZE 4: 10 µg/mL, AZE IMP A: 10 µg/mL, AZE INB Acetoacetate: 10 µg/mL, TEL IMP A : 10 µg/mL and TEL IMP B: 10 µg/mL.)
2.2.10 Preparation of Identification Solution:
Prepared individual identification solution of Azelnidipine, Telmisartan and their impurities equal to the concentration as mentioned in system suitability solution.
3. Result and Discussion:
The developed method proposed in this study was validated for various parameters like specificity, force degradation study, precision (system, method), accuracy, linearity, stability in analytical solution (SIAS).
3.1.System Suitability:
System Suitability acceptance criteria are theoretical plates not less than 3000 and tailing factor should not be more than 2.0. Resolution between any two peaks in system suitability solution not less than 2.0 (Figure 2). % RSD obtained from 6 replicate injections of system suitability solution for peak area should be NMT 5.0%. Details of system suitability parameters of Azelnidipine and Telmisartan and their impurities are mentioned in (Table 2 and 3).
Table 2: System Suitability Parameters:
Compound name RT Area USP Resolution
USP Plate Count
USP Tailing
Purity Angle
Purity Thershold
AZE 4 4.092 10339 - 7256 1.2 0.436 0.701
AZE IMP A 9.750 148393 34.16 85460 1.2 0.297 0.396 TELMISARTAN 14.078 389558 30.54 150791 1.1 0.100 0.272 TEL IMP B 15.764 290650 11.46 189798 1.0 0.152 0.328
AZE INB
Acetoacetate
16.748 73375 6.50 190911 1.0 0.408 0.611
AZELNIDIPINE 18.054 112094 7.83 175153 0.8 0.587 0.829 TEL IMP A 25.813 222794 30.41 99260 1.0 0.153 0.339
Table 3: System Suitability with Peak Area Counts:
Com poun d name
AZE 4 AZE IMP A
AZE INB ACETOAC
ETATE
AZELNI DIPINE
TEL IMP A
TEL IMP B
TELMISA RTAN
10339 148393 73375 112094 222794 290650 389558
Peak Area
10370 145360 73447 111970 221924 298148 389243
10056 143645 73023 112077 222476 298884 390248
9998 142435 67482 110819 221394 299128 390151
10433 140908 73949 109504 222765 299391 390175
9841 139073 73752 109500 222327 298021 390123
Mean 10172.83 143302.3 3
72504.67 110994.00 222280.0 0
297370.3 3
389916.33 SD 240.209 3305.712 2481.260 1250.458 538.847 3336.306 413.877
% RSD
2.36 2.31 3.42 1.13 0.24 1.12 0.11
SampleName: Blank Solution
AU
-0.0008 -0.0007 -0.0006 -0.0005 -0.0004 -0.0003 -0.0002 -0.0001 0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: Common Placebo
AU
-0.0008 -0.0007 -0.0006 -0.0005 -0.0004 -0.0003 -0.0002 -0.0001 0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0.0008
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
Fig. 1: The chromatogram of (A) Blank, (B) common placebo, (C) standard solution under optimized chromatographic conditions.
Fig. 2: The chromatogram of system suitabilty under optimized chromatographic conditions.
3.2. Specificity:
The specificity is determined by injecting single run of Blank, common placebo, Standard solution and spiked sample solution. No interference was observed at the retention time (Rt) of Azelnidipine, Telmisartan and their impurities (Figure 1). The purity angle of Azelnidipine and Telmisartan peak is less than the purity threshold in the chromatogram of the sample solution (Table 2).
3.3. Identification of Drugs and their Impurties:
Single injection of Blank, common placebo and individual identification solution of Azelnidipine, Telmisartan and their impurities was injected. The recorded well resolved chromatograms were proved that developed method is suitable for impurity profiling of Azelnidipine and Telmisartan in FDC dosage form (Figure 3).
SampleName: Standard Solution TELMISARTAN - 14.062 AZELNIDIPINE - 17.646
AU
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: System suitability solution
AZE 4 - 4.092 AZE IMP A - 9.750 TELMISARTAN - 14.078 TEL IMP B - 15.764 AZE INB Acetoacetate - 16.748 AZELNIDIPINE - 18.054 TEL IMP A - 25.813
AU
0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055 0.060 0.065 0.070
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
The retention time (Rt) of Telmisartan and Azelnidipine was 14.07 and 18.05 respectively.
Azelnidipine impurities i.e. AZE 4 , Azelnidipine IMP A, Azelnidipine INB Acetoacetate were well seprated at Rt 4.09, 9.75 and 16.74 respectively, whereas Telmisartan impurities i.e. Telmisartan Imp B and Telmisartan Imp A were well seprated at 15.76 and 25.81(Table 2).
Fig 3 (A)Chromatogram of Impurity AZE 4 (Rt 4.088)
3 (B)Chromatogram of Telmisartan (Rt 14.072)
3 (C)Chromatogram of Telmisartan Impurity B (Rt 15.758)
SampleName: ID-AZE4
AZE-4 - 4.088
AU
-0.0010 -0.0005 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.0040 0.0045 0.0050 0.0055 0.0060 0.0065 0.0070 0.0075 0.0080 0.0085 0.0090
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: ID-TEMISARTAN
TELMISARTAN - 14.072
AU
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: ID-TELMISARTAN IMP B
TELMISARTAN IMP-B - 15.758
AU
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
3 (D)Chromatogram of Impurity AZE INB Acetoacetate (Rt 16.740)
3 (E)Chromatogram of Azelnidipine (Rt 18.050)
3 (F)chromatogram of Telmisartan Impurity A (Rt 25.700)
SampleName: ID-AZE INB
AZE INB ACETOACETATE - 16.740
AU
-0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: ID-AZELINIDIPINE
AZELNIDIPINE - 18.050
AU
-0.0005 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.0040 0.0045 0.0050 0.0055 0.0060 0.0065 0.0070 0.0075 0.0080 0.0085 0.0090
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: ID-TELMISARTAN IMP A
TELMISARTAN IMP-A - 25.798
AU
0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
Fig. 4: The chromatogram of method precision under optimized chromatographic conditions.
3.4. Precision:
System precision was calculated by injecting six replicate injection of standard solution and % RSD was observed as 0.1% and 0.1% for Azelnidipine and Telmisartan respectively.
Method precision was performed by injecting 6 independently prepared sample solution and % RSD of peak response of six replicate injections was NMT 5%(Table 4).
In method precision sample peak of Telmisartan, Telmisartan Imp B, Azelnidipine INB Acetoacetate (Impurity) and Azelnidipine was found at the Rt of 14.08, 15.47, 16.74 and 17.64 respectively.
Table 4: Peak Area counts of Method Precission Sample:
Name of Drug and their Impurties
AZE INB ACETOACETATE
AELNIDIPINE TEL IMP B TELMISARTA N
Peak Area 50816 4669918 7407 10176936
50727 4691509 7142 10106446
50727 4633170 7421 10215136
51456 4621257 7691 10127028
53984 4597788 7972 9975701
55287 4562495 7932 10187472
Mean 52166.17 4629356.17 7594.17 10131453.17
SD 1975.541 47022.002 327.334 86154.852
% RSD 3.79 1.02 4.31 0.85
SampleName: Method Precision-1 TELMISARTAN - 14.068 TEL IMP B - 15.472 AZE INB Acetoacetate - 16.742 AZELNIDIPINE - 17.645
AU
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
3.5. Accuracy (Recovery):
To demonstrate the accuracy, prepared accuracy samples by taking known quantity of working standard at level 80%, 100%, and 120% in triplicate. Mean % Recovery should not be less than 98%
and not more than 102%. Result of Mean % recovery mentioned in (Table 5).
3.6. Linearity:
Prepare solutions corresponding to 50%, 80%,100%,120% and 150% in diluent by using stock solution.
Linearity graph shall be plotted between area response and concentration. Regression coefficient, Correlation coefficient, Slope and Y-intercept shall be calculated (Table 5). The calibration curve as shown in Figure 5 (A, B, C and D).
Table 5: Summary of Linearity and Recovery parameters.
Name AZELNIDIPINE TELMISARTAN AZE INB
ACETOACETATE
TEL IMP B
Linearity range (μg/mL)
22.50-67.49 238.71-716.12 4.05-12.15 23.40-70.19
Slope 15537.1 6369.7 6832.6 197.7
Y-intercept -65599.7 11003 240.3 -2355
Regression Cofficent (R²)
0.99674 0.99911 0.99880 0.99835
Limit of Detection
(μg/mL)
16.41 15.34 0.79 0.06
Limit of Quantitation
(μg/mL)
49.73 46.49 2.38 0.19
% Recovery 99.8-101.2 99.1-99.7 99.1-100.9 97.9-101.4
Figure 5 (A): Linearity graph of Azelnidipine
Figure 5 (B): Linearity graph of Telmisartan y = 15537.1x -65599.7
R² = 0.99674
0 200000 400000 600000 800000 1000000 1200000
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00
Area
Concentration (µg/mL) Linearity curve of Azelnidipine
y = 6369.7x + 11003 R² = 0.99911
0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 5000000
0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00
Area
Concentration (µg/mL)
Linearity Curve of Telmisartan
Figure 5 (C): Linearity graph of AZE INB acetoacetate
Figure 5 (D): Linearity graph of TEL IMP B 3.7. Forced Degradation Study
3.7.1. Acid hydrolysis:
For this analysis samples of drug products as well as placebo were treated with 5 mL of 5N HCl , Heat at 80°C for 1 hr. and then neutralized with 5mL of 5N NaOH (Figure 6 A).
3.7.2. Base Hydrolysis:
Here the drug product and placebo were treated with 5 mL of 5N NaOH, Heat at 80°C for 1 hr. and then neutralized with 5mL of 5N HCl (Figure 6 B).
y = 6832.6x +240.3 R² = 0.99880
0 10000 20000 30000 40000 50000 60000 70000 80000 90000
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Area
Concentration (µg/mL)
Linearity curve of AZE INB ACETOACETATE
y = 197.7x -2355 R² = 0.99835
0 2000 4000 6000 8000 10000 12000 14000
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00
Area
Concentration (µg/ml) Linearity Curve of TEL IMP B
3.7.3. Oxidative degradation:
The drug product and placebo were treated 5 mL of Hydrogen peroxide ( 30%), Heat at 80°C for 1 hr (Figure 6 C).
3.7.4. Thermal degradation:
The test, as well as placebo, were kept inside the oven for 48 hr. at 80ºC (Figure 6 D).
3.7.5. Photolytic degradation:
The samples of drug product and placebo were placed under ultraviolet at 254 nm (short wavelength) in an ultraviolet region chamber for 48 hr (Figure 6 E).
.
3.7.6. Humidity exposure:
The sample of the drug product and placebo was exposed for 78 hr above 75% RH (Figure 6 F).
Details of Force degradation parameters are mentioned in (Table 6, 7)
Fig. 6 (A): Acid degradation of the sample as well as placebo in acidic conditions.
SampleName: Control_Acid
AU
-0.0004 -0.0003 -0.0002 -0.0001 0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0.0008 0.0009 0.0010 0.0011 0.0012 0.0013 0.0014 0.0015
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: Sample_Acid Telmisartan - 14.437 Tel IMP B - 15.768 AZE INB Acetoacetate - 16.872 Unknown - 17.270 AZELNIDIPINE - 18.070
AU
-0.012 -0.010 -0.008 -0.006 -0.004 -0.002 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020 0.022 0.024 0.026 0.028 0.030 0.032 0.034
Minutes
9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00
Fig. 6 (B): Force degradation of Azelnidipine and Telmisartan in basic environmental conditions.
Fig. 6 (C): Chromatograms of Placebo and sample solution showing peroxide degradation pattern.
SampleName: Control_Base
AU
-0.0010 -0.0009 -0.0008 -0.0007 -0.0006 -0.0005 -0.0004 -0.0003 -0.0002 -0.0001 0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0.0008
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: Sample_Base
Telmisartan - 14.450 15.725Tel IMP B - 15.772 AZE INB Acetoacetate - 16.869 Unknown - 17.271 AZELNIDIPINE - 18.063
AU
-0.005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055
Minutes
10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00
SampleName: Control_Peroxide
AU
0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090 0.100 0.110 0.120 0.130 0.140 0.150 0.160
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: Sample_Peroxide Unknown-1 - 13.284 Unknown-2 - 13.872 Telmisartan - 14.427 Tel IMP B - 15.772 AZE INB Acetoacetate - 16.874 Unknown - 17.277 AZELNIDIPINE - 18.059
AU
-0.005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035
Minutes
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00
Fig. 6 (D): Chromatogram showing thermal degradation of Placebo and sample solution
Fig. 6 (E): The chromatographic representation of photolytic degradation of Placebo and sample solution
SampleName: Control_Thermal
AU
-0.0008 -0.0007 -0.0006 -0.0005 -0.0004 -0.0003 -0.0002 -0.0001 0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0.0008 0.0009 0.0010
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: Sample_Thermal Unknown-1 - 13.281 TELMISARTAN - 14.429 TEL IMP B - 15.720 AZE INB Acetoacetate - 16.869 AZELNIDIPINE - 18.061
AU
-0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00
SampleName: Control_Photolytic
AU
-0.0010 -0.0008 -0.0006 -0.0004 -0.0002 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 0.0012
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: Sample_Photolytic
Unknown-1 - 11.464 Unknown-2 - 13.275 Telmisartan - 14.427 Tel IMP B - 15.712 AZE INB Acetoacetate - 16.867 AZELNIDIPINE - 18.058
AU
-0.020 -0.010 0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070
Minutes
9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00
Fig. 6 (F): The chromatographic representation of humidity at 75%RH of Placebo and sample solution
Table 6: Focre degradation conditions:
Sample Name
Telmisartan (Rt 14.437)
Tel IMP B (Rt 15.768)
AZE INB Acetoacetate (Rt
16.872)
Azelnidipine (Rt 18.070)
Purity Angle
Purity Threshold
Purity Angle
Purity Threshold
Purity Angle
Purity Threshold
Purity Angle
Purity Threshold Acid
Degradation
3.761 3.917 6.466 90.000 1.469 90.000 0.493 1.176 Base
Degradation
3.000 4.234 4.162 39.485 0.801 8.077 0.557 0.705 Peroxide
Degradation
0.401 2.379 - - 1.203 45.727 1.071 1.154
Thermal degradation
0.865 3.05 3.041 6.051 0.439 1.192 0.185 0.376 UV
Degradation
2.253 3.026 5.128 6.872 0.647 1.423 0.245 0.376 Humidity
degradation
2.156 3.19 4.135 5.207 0.54 1.287 0.307 0.364
SampleName: Control_Humidity_75%RH
AU
-0.0010 -0.0009 -0.0008 -0.0007 -0.0006 -0.0005 -0.0004 -0.0003 -0.0002 -0.0001 0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0.0008 0.0009 0.0010 0.0011
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00
SampleName: Sample_Humidity_75%RH Unknown-1 - 11.462 Unknown-2 - 13.272 Telmisartan - 14.423 Tel IMP B - 15.709 AZE INB Acetoacetate - 16.851 AZELNIDIPINE - 18.045
AU
-0.010 0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090
Minutes
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00
Table 7: Summary of degradation of Azelnidipine and Telmisartan Degradation
Condition
% Assay % Degradation
Telmisartan Azelnidipine Telmisartan Azelnidipine
Acidic hydrolysis 95.9 96.2 4.7 3.7
Basic hydrolysis 96.3 98.5 4.3 1.4
Oxidation 94.8 96.7 5.8 3.2
Thermal 96.1 99.5 4.5 0.4
Photolytic 97.2 99.5 3.4 0.4
Humidity 97.3 99.8 3.3 0.1
3.8. Solution stability (SIAS):
The stability of the analytical solution was performed by injecting sample solution (Method precision sample) initially and at specific time intervals and no significant change observed in concerning initial Rt, area, and resolution showing stability in aqueous solution over 25 hrs. and no significant change was observed in system suitability parameters.
CONFLICT OF INTEREST:
The author declares no conflict of interest for the present manuscript.
ACKNOWLEDGMENTS
The authors are thankful to M/s. Synokem Pharmaceutical Limited, Haridwar, Uttrakhand, M/s. Kimia Biosciences Limited, Gurugram, Haryana, and School of Medical and Allied Sciences, K.R. Mangalam University, Gurugram, for their support towards the completion of the present analytical investigation.
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