A VALIDATED REPORT ON FORMULATION STABILITY STUDIES OF BENDAMUSTINE HYDROCHLORIDE
HTML Full TextA validated report on formulation stability studies oF Bendamustine Hydrochloride
Niharika Sridasam*, Ravinder Reddy Y. and V. Kavya
Teegala Ram Reddy College Of Pharmacy, #4-202, Meerpet, Saroor Nagar (M), Ranga Reddy (Dist), Hyderabad-500079, Telangana, India
ABSTRACT: The objective of this study was to adopt the analytical method. This adoption procedure was carried out by validating the method parameters and conducting the forced degradation studies to check the interference of the chromatographic response of the degradation products with the drug. In the present study an attempt was made to studies related to system suitability, linearity, accuracy, precision, and assay content, forced degradation, acid and base degradation, hydrolysis degradation and photo stability studies were conducted as per the standard procedures. System suitability conditions meet the recommended criterion. Linearity of the solution was demonstrated between 0.0002577 mg/ml and 0.51540 mg/ml concentration range. Recovery and % RSD are within the recommended limits. Injection reproducibility was demonstrated and the % relative standard deviation for retention time and area were within the limits. Limit of detection value is at 0.00004165mg/ml and limit of quantisation is at 0.00012621mg/ml. Solution was stable up to 38hrs at 5°C temperature. Precision was demonstrated and the percentage relative standard deviation for assay was within the limits. Specificity was also demonstrated and there was no interference with the diluent & mobile phase. Under forced degradation studies, the peak purity angle is always lower than purity threshold for Bendamustine hydrochloride and all degradant peaks were well resolved. Hence the method can be adopted for the formulation screening studies as a stability indicating method.
Keywords: |
Bendamustine Hydrochloride, Linearity, Accuracy, Precision, Photo Stability Studies.
INTRODUCTION: Pharmaceutical analysis comprises those procedures necessary to determine the “identity, strength, quality and purity” of the drug. Again it may be defined as the application of analytical procedures used to determine the purity, safety and quality of drugs and chemicals. It also deals the analysis of raw materials and intermediates in the manufacture of drugs1, 2. The pharmaceutical analyst must therefore, have a firm background in basic organic analysis and in addition he should have special skills in the quality evaluation of drug products.
Bendamustine is a white, water soluble microcrystalline powder with amphoteric properties. It acts as an alkylating agent causing intra-strand and inter-strand cross-links between DNA bases. After intravenous infusion it is extensively metabolised in the liver by cytochrome p450. More than 95% of the drug is bound to protein - primarily albumin. Only free Bendamustine is active.
Elimination is biphasic with a half-life of 6–10 minutes and a terminal half-life of approximately 30 minutes. It is eliminated primarily through the kidneys3. The objective of this study was to adopt the analytical method. This adoption procedure was carried out by validating the method parameters and conducting the forced degradation studies to check the interference of the chromatographic response of the degradation products with the drug.
MATERIALS AND METHODS:
Standards and solvents
Bendamustine Hydrochloride drug substance was obtained as gift sample from Euticals Pharma Limited. All the other reagents were of analytical grade.
Chromatographic conditions:
Chromatographic system :Waters Alliance 2695 with PDA/UV 2995 detector with Empower software.
Column : Zorbax SB C18 250mm x 4.6mm x 5µm
Mobile Phase-A : Water: Acetonitrile: TFA:: 90:10:0.1
Mobile Phase: B : Water: Acetonitrile: TFA:: 50:50:0.1
Flow rate : 1.0 ml/min
Detection wavelength : 230nm
Column Temperature : 30°C
Auto sampler temperature : 5°C
Injection volume : 10ml
Run time : 45 min.
Diluent : Methanol
Gradient programme :
Pump B. Conc. Time (minutes)
0.01 0.00
3.00 0.00
16.0 50.0
33.0 70.0
35.0 90.0
40.0 90.0
41.0 0.00
45.0 0.00
System Suitability4:
Preparation of System suitability solution: Bendamustine hydrochloride drug substance (12.52mg) was accurately weighed into a 50ml volumetric flask, dissolved and made up to volume with diluents (concentration 0.2504mg/ml).
Procedure: Bendamustine hydrochloride system suitability solution (10ml) was injected into the chromatographic system connected to a Zorbax SB C18 250mm x 4.6mm x 5µm for 6 times and the relative standard deviation for area, theoretical plates and tailing factor for the replicate injections were calculated.
TABLE 1:
Acceptance Criteria: Tailing factor for Bendamustine hydrochloride peak was < 2.5, Theoretical plates for Bendamustine hydrochloride was ≥ 2500 and %RSD for peak area was < 2.0%.
Linearity4:
Preparation of Bendamustine hydrochloride Stock and Level Solutions for Linearity:
Bendamustine hydrochloride Stock Solution for Linearity: Bendamustine hydrochloride drug substance (128.85mg) was accurately weighed into a 50ml volumetric flask, dissolved and made up to volume with diluent (concentration 2.5770mg/ml).
Level Solutions for Linearity: A series of solutions with concentrations of 0.1%, 5%, 10%, 20%, 50%, 80%, 100%, 120%, 150% and 200% were prepared. Level 200% was prepared with 4.0ml of Bendamustine hydrochloride stock solution was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.51540mg/ml). This solution was prepared in duplicate. Level 150% was prepared with 3.0ml of Bendamustine hydrochloride stock solution was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.38655mg/ml).
This solution was prepared in duplicate. Level 120% was prepared with 2.4ml of Bendamustine hydrochloride stock solution was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.30924mg/ml). This solution was prepared in duplicate. Level 100% was prepared with 2.0ml of Bendamustine hydrochloride stock solution was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.25770mg/ml).
Injection Number | Peak Area | Theoretical plates | Tailing factor |
Injection – 1 | 10688971 | 25834 | 1.74 |
Injection – 2 | 10613684 | 25241 | 1.71 |
Injection – 3 | 10622986 | 24865 | 1.71 |
Injection – 4 | 10592403 | 25024 | 1.71 |
Injection – 5 | 10618844 | 24740 | 1.68 |
Injection – 6 | 10593310 | 24853 | 1.69 |
Mean | 10621700 | 25093 | 1.71 |
Acceptance | < 2.0% | ≥ 2500 | < 2.5 |
Result | Pass (RSD – 0.33) | Pass | Pass |
This solution was prepared in duplicate. Level 80% was prepared with 1.6ml of Bendamustine hydrochloride stock solution was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.20616mg/ml). This solution was prepared in duplicate.
Level 50% was prepared with 1.0ml of Bendamustine hydrochloride stock solution was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.12885mg/ml). This solution was prepared in duplicate. Level 20% was prepared with 0.4ml of Bendamustine hydrochloride stock solution was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.05154mg/ml). This solution was prepared in duplicate. Level 10% was prepared with 0.2ml of Bendamustine hydrochloride stock solution was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.02577mg/ml).
This solution was prepared in duplicate. Level 5% was prepared with 0.1ml of Bendamustine hydrochloride stock solution was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.012885mg/ml). This solution was prepared in duplicate. Level 0.1% was prepared with 0.2ml of level 10% solution for linearity was transferred into a 20ml volumetric flask and made up the volume with the diluent (concentration 0.0002577mg/ml). This solution was prepared in duplicate.
1.1.
Demonstration of Linearity 5:
3.1
The linearity of the method was demonstrated by chromatographic analysis of the above prepared solutions containing 0.1%, 5%, 10%, 20%, 50%, 80%, 100%, 120%, 150% and 200% of the target concentration of 0.2577mg/ml. 10µl of each of the above prepared solutions were injected into the chromatographic system connected to a Zorbax SB C18 250mm x 4.6mm, 5 µm and the average peak area in each of the cases was calculated. Statistical evaluation was carried out by a graph between the concentration and the average area was plotted. Points for linearity were observed. Using the method of least squares, a line of best fit was taken and the correlation coefficient, slope, and y-intercept were calculated. Acceptance criteria were taken as R2 value should not be less than 0.98.
Accuracy 5:
Preparation of Bendamustine hydrochloride Stock and Level Solutions for Accuracy:
Bendamustine Hydrochloride Stock Solution for Accuracy:
Bendamustine hydrochloride drug substance (25.20mg) was accurately weighed into a 10ml volumetric flask, dissolved and made up to volume with diluent (concentration 2.520 mg/ml). Level Solutions for Accuracy was prepared with a series of solutions with concentrations of 80%, 100% and 120% were prepared as described below. Level 80% was prepared with 0.8 ml of Bendamustine hydrochloride stock solution was transferred into a 10ml volumetric flask and made up to volume with the diluent (concentration 0.2016mg/ml).
This solution was prepared in triplicate. Level 100% was prepared with 1.0ml of Bendamustine hydrochloride stock solution was transferred into a 10ml volumetric flask and made up to volume with the diluent (concentration 0.2520mg/ml). This solution was prepared in triplicate. Level 120% was 1.2ml of Bendamustine hydrochloride stock solution was transferred into a 10ml volumetric flask and made up to volume with the diluent (concentration 0.3024mg/ml). This solution was prepared in triplicate.
Demonstration of Accuracy 5:
Accuracy was calculated with respect to above prepared solutions at the levels of 80%, 100% and 120% of the normal or target concentration. The accuracy of the method was demonstrated by chromatographic analysis through recovery experiment on 3 samples of the above prepared solutions containing 80%, 100% and 120% of the target concentration of 0.25mg/ml of Bendamustine hydrochloride employed in the usual procedure. 10ml of each of the above prepared solutions were injectedinto the chromatographic system connected to a Zorbax SB C18, 250 x 4.6mm, 5µm and the average peak area in each of the cases was recorded.
The amount of Bendamustine hydrochloride peak area found in each of these test solutions were calculated using the calibration curve. The table below summarizes the amount added vs. amount found and the percentage recoveries. The average percentage recovery should be between 98-102% and relative standard deviation of these recovery concentrations should be less than 2%.
Demonstration of Precision5:
The precision of the method was demonstrated through two parameters which are injection reproducibility and the method precision. Bendamustine hydrochloride drug substance, 12.59mg was accurately weighed into a 50ml volumetric flask, dissolved and made up to volume with diluent (concentration 0.2518 mg/ml). The relative standard deviations of retention time and peak areas of the above system suitability solutions were calculated for injection reproducibility. Acceptance Criteria: The injection reproducibility requirements are met if the relative standard deviation for retention time is not more than 1% and area is not more than 2.0%.
Injection Reproducibility:
For injection reproducibility, considered six injections from the same system suitability solution and the relative standard deviation for the replicate injections was calculated. The relative standard deviations of retention time and peak areas of the above system suitability solutions were calculated for injection reproducibility. Acceptance Criteria: The injection reproducibility requirements are met if the relative standard deviation for retention time is not more than 1% and area is not more than 2.0%.
Method Precision6:
For method precision, six individual preparations were made from the same batch of Bendamustine hydrochloride drug substance with a target concentration of about 0.25mg/ml and individual peak areas were measured. The relative standard deviation was calculated for these six preparations.
Preparation–1 was made with Bendamustine hydrochloride drug substance (12.48mg) was accurately weighed into a 50ml volumetric flask, dissolved, made up to volume with the diluent and labelled as Preparation–1 (concentration – 0.2496mg/ml). Preparation–2 was made with Bendamustine hydrochloride drug substance (12.50mg) was accurately weighed into a 50ml volumetric flask, dissolved, made up to volume with the diluent and labelled as Preparation–2 (concentration – 0.2500mg/ml).
Preparation–3 was made with Bendamustine hydrochloride drug substance (12.52mg) was accurately weighed into a 50ml volumetric flask, dissolved, made up to volume with the diluent and labelled as Preparation–3 (concentration – 0.2504mg/ml). Preparation–4 was made with Bendamustine hydrochloride drug substance (12.55mg) was accurately weighed into a 50ml volumetric flask, dissolved, made up to volume with the diluent and labelled as Preparation–4 (concentration – 0.2510mg/ml). Preparation–5 was made with Bendamustine hydrochloride drug substance (12.45mg) was accurately weighed into a 50ml volumetric flask, dissolved, made up to volume with the diluent and labelled as Preparation–5 (concentration – 0.2490mg/ml).
Preparation–6 was made with Bendamustine hydrochloride drug substance (12.47mg) was accurately weighed into a 50ml volumetric flask, dissolved, made up to volume with the diluent and labelled as Preparation–6 (concentration – 0.2494mg/ml) was made with 0ml of each of the above solutions were injected in duplicate into the chromatographic system connected to a Zorbax SB C18 250mm X 4.6mm X 5µm column and recorded the chromatographic response. The peak areas due to Bendamustine hydrochloride peaks were measured.
Acceptance Criteria was relative standard deviation for the assay preparations should not be more than 2.0%. The amount of Bendamustine hydrochloride in each preparation was calculated using the formula given.
Assay Content6:
Test Solution: Sample average area was taken from 6 sample solutions in the method precision study. The % of the Bendamustine hydrochloride individually in each sample solution was calculated using the following formula:
Where,
AT = Average area of the Bendamustine hydrochloride in the test preparation
AS = Average area of the Bendamustine hydrochloride in standard preparation
WT = Concentration (mg/ml) of the test solution
WS = Concentration (mg/ml) of the standard/system suitability solution
P = Potency of the Bendamustine hydrochloride working standard/drug substance
Specificity6:
The specificity of the method was demonstrated through establishing interference check by injecting the diluent blank and mobile phase to determine whether any peaks are co-eluting with the Bendamustine hydrochloride peak. 10ml each of diluent blank and mobile phase solutions were injected separately onto the chromatographic system connected to a Zorbax SB C18 250 x 4.6mm, 5μm and observed the chromatographic responses.
There should not be any interference of peaks, eluted in the diluent blank and mobile phase with the Bendamustine hydrochloride peak retention time. It was observed that peaks eluted in the chromatographic response of diluent blank and mobile phase does not show any interference with those eluted in the Bendamustine hydrochloride.
Limit of Detection and Limit of Quantification (LOD and LOQ) 7:
Preparation of Bendamustine hydrochloride Stock and Level Solutions for LOD and LOQ:
Preparation of Stock Solution for LOD and LOQ: Bendamustine hydrochloride drug substance, 12.78mg was accurately weighed into a 50ml volumetric flask, dissolved and made up to volume with diluent (concentration 0.2556 mg/ml). Transfer 1ml of this solution into a 100ml volumetric flask, dissolved and made up to volume with diluent (concentration 0.002556mg/ml).
Level solutions for LOD and LOQ: A series of the test solutions (level solutions) were prepared as described below at different concentrations using the above stock solution close to LOD and LOQ level.
Level 0.05% was made by transferring 2.5ml of the stock solution into a 50ml volumetric flask and made up to volume with the diluent (concentration 0.0001278mg/ml). Level 0.10% was made by transferring 5.0ml of the stock solution into a 50ml volumetric flask and made up to volume with the diluent (concentration 0.0002556mg/ml). Level 0.15% was made by transferring 7.5ml of the stock solution into a 50ml volumetric flask and made up to volume with the diluent (concentration 0.0003834mg/ml). Injected 10ml of each of the above prepared solutions on to the chromatographic system connected to a Zorbax SB C18 250 x 4.6 mm, 5µm for 6 times and calculated the average area and standard deviation in each of the cases.
Statistical evaluation:
Plot two different graphs, concentration vs. average area and concentration vs. drug substance deviation and calculate the slope, y-intercept and correlation coefficient.
Solution Stability8:
System Suitability Solution for Solution Stability was taken Initial, 4, 24 and 38hours. The stability of assay solutions was demonstrated for a period of 38 hours at 5°C temperature of sample cooler by chromatograph the same solution at periodic time intervals.
The system suitability solution injected initially was considered as solution stability initial and the same solution was injected at specified time intervals of 24 and 38hours.
Procedure: 10ml of the above prepared solution was injected into the chromatographic system connected to a Zorbax SB C18 250 x 4.6mm, 5µm. Vial was kept in auto sampler which is maintained at 5°C temperature for further study and was injected into the chromatographic system at periodic time intervals (initial, 24 and 38hrs).
Forced Degradation8:
Analytical method was tested by the forced degradation studies to determine adequate separation of degradant from Bendamustine hydrochloride peak.
Acid degradation8:
Preparation of the stock solution for acid degradation:
Weighed accurately 10.25mg of Bendamustine hydrochloride drug substance into a 20ml volumetric flask and added 0.01N HCl, dissolved and made up to volume with 0.01N HCl. This solution was analyzed initially and stock solution was stored at 60°C for further study. Test solutions were prepared as per the following procedure prior to analysis.
Preparation of the test solutions for analysis:
At appropriate time interval, 2.5ml of acid degradation stock solution was transferred into a 5ml volumetric flask and made up to volume with diluent and analyzed by HPLC.
Preparation of Acid Blank solution for acid degradation:
10ml of 0.01 N HCl was marked as blank solution. This blank solution was analyzed initially and stored along with the test solution. At appropriate time interval, blank solution was also prepared similarly as sample solution and injected into chromatograph.
Base Degradation8:
Preparation of the stock solution for base degradation:
Weighed accurately 10.14mg of Bendamustine hydrochloride drug substance into a 20ml volumetric flask and added 0.1N NaOH, dissolved and made up to volume with 0.1N NaOH. This solution was analyzed initially and stock solution was stored at 60°C for further study. Test solutions were prepared as per the following procedure prior to analysis.
Preparation of the test solutions for analysis:
At appropriate time interval, 2.5ml base degradation stock solution was transferred into a 5ml volumetric flask and made up to volume with diluent and analyzed by HPLC.
Preparation of Base Blank solution for base degradation:
10ml of 0.1 N NaOH was marked as blank solution. This blank solution was analyzed initially and stored along with the test solution. At appropriate time interval, blank solution was also prepared similarly as sample solution and injected into chromatograph.
Oxidative degradation9:
Preparation of the stock solution for oxidative degradation:
Weighed accurately 10.22mg of Bendamustine hydrochloride drug substance into a 20ml volumetric flask and added 5% hydrogen peroxide, dissolved and made up to volume with 5% hydrogen peroxide. This solution was analyzed initially and stock solution was stored at 60°C for
Preparation of the test solutions for analysis:
At appropriate time interval, 2.5ml oxidative degradation stock solution was transferred into a 5ml volumetric flask and made up to volume with diluent and analyzed by HPLC.
Preparation of oxidative Blank solution for oxidative degradation:
10ml of 5% hydrogen peroxide was marked as blank solution. This blank solution was analyzed initially and stored along with the test solution. At appropriate time interval, blank solution was also prepared similarly as sample solution and injected into chromatograph.
Hydrolysis Degradation9:
Preparation of the stock solution for Hydrolysis:
Weighed accurately 10.22mg of Bendamustine hydrochloride drug substance into a 20ml volumetric flask and added water, dissolved and made up to volume with water. This solution was analyzed initially and stock solution was stored at 60°C for further study. Test solutions were prepared as per the following procedure prior to analysis.
Preparation of the test solutions for analysis:
At appropriate time interval, 2.5ml hydrolysis degradation stock solution was transferred into a 5ml volumetric flask and made up to volume with diluent and analyzed by HPLC.
Preparation of Hydrolysis Blank solution for hydrolysis:
10ml of water was marked as blank solution. This blank solution was analyzed initially and stored along with the test solution. At appropriate time interval, blank solution was also prepared similarly as sample solution and injected into chromatograph.
Photo Stability Studies10:
Preparation of Bendamustine hydrochloride for Photo Stability Studies:
Bendamustine hydrochloride drug substance (50mg) was weighed into 4vials of 12.5mg each. The vials were, labelled as Uncovered (1 vial), Covered with Aluminium foil (1 vial) & Covered with Aluminium foil and placed in Carton (1 vial), were exposed to UV (200 uw/cm2) & fluorescence light (1.2 million Lux hours) in the Photo stability chamber. The remaining 1 vial was kept in dark as control.
Preparation of Test Solutions for Analysis:
The content from the vial was transferred into a 50ml volumetric flask. The vial was rinsed with the diluent, adding the rinsing to the volumetric flask and made up to volume with the diluent. Directly transferred an aliquot of the solution into an HPLC vial and injected into chromatographic system. All the vials were prepared and injected individually.
RESULTS AND DISCUSSIONS:
Linearity of Bendamustine hydrochloride
TABLE 2. LINEARITY OF BENDAMUSTINE HYDROCHLORIDE
Level Solutions for Linearity | Concentration (mg/ml) | Area Response | Average Area Response |
0.1% | 0.0002577 | 7446 | 7481 |
7516 | |||
5% | 0.0128850 | 564415 | 566738 |
569060 | |||
10% | 0.0257700 | 1129591 | 1133521 |
1137451 | |||
20% | 0.0515400 | 2297303 | 2292939 |
2288574 | |||
50% | 0.1288500 | 5463240 | 5454803 |
5446366 | |||
80% | 0.2061600 | 8860187 | 8852616 |
8845044 | |||
100% | 0.2577000 | 11197387 | 11171780 |
11146172 | |||
120% | 0.3092400 | 13722462 | 13687557 |
13652652 | |||
150% | 0.3865500 | 16882903 | 16883836 |
16884768 | |||
200% | 0.5154000 | 22150335 | 22107914 |
FIGURE 1. LINEARITY OF BENDAMUSTINE HYDROCHLORIDE
Accuracy of Bendamustine hydrochloride
TABLE 3. ACCURACY OF BENDAMUSTINE HYDROCHLORIDE
Level solution for Accuracy | Amount added (conc. mg/ml) | Mean Area Response | Amount found (conc. mg/ml) | % of Recovery |
80% | 0.2016 | 8704999 | 0.20158 | 100.0 |
0.2016 | 8707994 | 0.20165 | 100.0 | |
0.2016 | 8673314 | 0.20080 | 99.6 | |
100% | 0.2520 | 10808272 | 0.25276 | 100.3 |
0.2520 | 10787913 | 0.25227 | 100.1 | |
0.2520 | 10798086 | 0.25251 | 100.2 | |
120% | 0.3024 | 12854829 | 0.30257 | 100.1 |
0.3024 | 12891915 | 0.30347 | 100.4 | |
0.3024 | 12765480 | 0.30039 | 99.3 | |
Mean | 100.0 | |||
SD | 0.35 | |||
% of RSD | 0.35 |
Demonstration of Precision:
TABLE 4. DEMONSTRATION OF PRECISION
Injection Number | Peak Area | Theoretical plates | Tailing factor |
Injection – 1 | 10626548 | 26353 | 1.69 |
Injection – 2 | 10563880 | 26759 | 1.70 |
Injection – 3 | 10693966 | 25999 | 1.67 |
Injection – 4 | 10729380 | 28071 | 1.77 |
Injection – 5 | 10803995 | 27795 | 1.79 |
Injection – 6 | 10689694 | 27771 | 1.74 |
Mean | 10684577 | 27125 | 1.73 |
Acceptance | < 2.0% | ≥ 2500 | < 2.5 |
Result | Pass (RSD – 0.78) | Pass | Pass |
Injection Reproducibility for Bendamustine hydrochloride
TABLE 5. INJECTION REPRODUCIBILITY FOR BENDAMUSTINE HYDROCHLORIDE
S. No. | Injection Number | Retention Time | Peak Area |
1 | Injection – 1 | 29.64 | 10626548 |
2 | Injection – 2 | 29.63 | 10563880 |
3 | Injection – 3 | 29.64 | 10693966 |
4 | Injection – 4 | 29.63 | 10729380 |
5 | Injection – 5 | 29.63 | 10803995 |
6 | Injection – 6 | 29.63 | 10689694 |
Mean | 29.63 | 10684577 | |
Acceptance | < 1.0% | < 2.5% | |
Result | Pass (RSD – 0.02) | Pass (RSD – 0.78) |
Method Precision for Bendamustine hydrochloride
TABLE 6. METHOD PRECISION FOR BENDAMUSTINE HYDROCHLORIDE
S. No. | Solution ID | Conc. (mg/ml) | Bendamustine hydrochloride Peak Area | Bendamustine hydrochloride Mean Area | % of Assay |
Standard | 0.2518 | 10626548 | 10684577 | *96.1 | |
10563880 | |||||
10693966 | |||||
10729380 | |||||
10803995 | |||||
10689694 | |||||
Preparation 1 | 0.2496 | 10557376 | 10550888 | 95.7 | |
10544400 | |||||
Preparation 2 | 0.2500 | 10613664 | 10583567 | 95.9 | |
10553470 | |||||
Preparation 3 | 0.2504 | 10565237 | 10589609 | 95.8 | |
10613981 | |||||
Preparation 4 | 0.2510 | 10572735 | 10636968 | 96.0 | |
10701201 | |||||
Preparation 5 | 0.2490 | 10574537 | 10567355 | 96.1 | |
10560173 | |||||
Preparation 6 | 0.2494 | 10580373 | 10577275 | 96.1 | |
10574176 | |||||
Mean | 95.9 | ||||
SD | 0.16 | ||||
% of RSD | 0.17 |
Note: *Potency of Bendamustine hydrochloride drug substance/ working standard.
Statistical evaluation:
TABLE 7. STATISTICAL EVALUATION
S. No | % of Level Solution | Concentration (mg/ml) | Mean Area | Standard Deviation |
1 | 0.05 | 0.0001278 | 5423 | 56.6 |
2 | 0.10 | 0.0002556 | 10600 | 322.7 |
3 | 0.15 | 0.0003834 | 13505 | 119.9 |
FIGURE: 2 STATISTICAL EVALUATION
FIGURE 3. STATISTICAL EVALUATION
Limit of detection value is at 0.00004165mg/ml and limit of quantisation is at 0.00012621mg/ml.
Solution Stability of Bendamustine hydrochloride
TABLE 8. SOLUTION STABILITY OF BENDAMUSTINE HYDROCHLORIDE
Time Period at 5°C | Average Peak Area | Impurity Area% | RRT | Impurity ID | % of Initial |
Initial | 10626548 | 0.10 | 0.58 | HP1 | 100.0 |
24 hrs | 10586758 | 0.11 | 0.57 | HP1 | 99.6 |
38 hrs | 10678544 | 0.12 | 0.57 | HP1 | 100.5 |
Acid Degradation of Bendamustine hydrochloride at 60°C
TABLE 9. SUMMARY OF ACID DEGRADATION OF BENDAMUSTINE HYDROCHLORIDE AT 60°C
Time period | BDM Area % | Degradant RRT | Degradant area % | Degradant ID | BDM Peak Purity Angle | BDM Peak Purity Threshold |
Initial | 98.3 | 0.57 0.79 1.26 1.30 | 1.29 0.21 0.16 0.05 | HP1 Unknown Unknown Unknown | 0.06 | 0.29 |
60⁰C 1hr | 71.7 | 0.22 0.57 0.68 0.79 1.26 1.30 | 2.15 25.8 0.05 0.12 0.13 0.06 | HP2 HP1 Unknown Unknown Unknown Unknown | 0.07 | 0.29 |
Base Degradation of Bendamustine hydrochloride at 60°C
TABLE 10. SUMMARY OF BASE DEGRADATION OF BENDAMUSTINE HYDROCHLORIDE AT 60°C
Time period | BDM Area % | Degradant RRT | Degradant area % | Degradant ID | BDM Peak Purity Angle | BDM Peak Purity Threshold |
Initial | 24.4 | 0.22 0.38 0.42 0.55 0.57 0.58 0.78 1.06 | 13.3 18.1 2.16 7.43 16.1 0.05 18.3 0.08 | HP2 Unknown Unknown Unknown HP1 Unknown Unknown Unknown | 0.21 | 0.41 |
Oxidative Degradation of Bendamustine hydrochloride at 60°C
TABLE 11. SUMMARY OF OXIDATIVE DEGRADATION OF BENDAMUSTINE HYDROCHLORIDE AT 60°C
Time period | BDM Area % | Degradant RRT | Degradant area % | Degradant ID | BDM Peak Purity Angle | BDM Peak Purity Threshold |
Initial | 96.1 | 0.57 0.66 0.79 1.30 | 3.24 0.07 0.51 0.05 | HP1 Unknown Unknown Unknown | 0.07 | 0.29 |
60⁰C 1hrs | 58.2 | 0.21 0.22 0.33 0.39 0.56 0.57 0.65 0.79 1.30 | 0.06 5.79 0.31 0.08 0.19 34.3 0.74 0.18 0.14 | Unknown HP2 Unknown Unknown Unknown HP1 Unknown Unknown Unknown | 0.09 | 0.30 |
Hydrolysis Degradation of Bendamustine hydrochloride at 60°C
TABLE 12. SUMMARY OF HYDROLYSIS DEGRADATION OF BENDAMUSTINE HYDROCHLORIDE AT 60°C
Time period | BDM Area % | Degradant RRT | Degradant area % | Degradant ID | BDM Peak Purity Angle | BDM Peak Purity Threshold |
Initial | 94.5 | 0.22 0.57 0.79 1.30 | 0.05 4.76 0.67 0.06 | HP2 HP1 Unknown Unknown | 0.06 | 0.28 |
60⁰C 1hrs | 56.4 | 0.22 0.39 0.57 0.79 0.86 0.95 1.30 | 6.95 0.12 36.1 0.16 0.05 0.06 0.18 | HP2 Unknown HP1 Unknown Unknown Unknown Unknown | 0.10 | 0.31 |
Photo Stability Studies of Bendamustine hydrochloride
TABLE 13. SUMMARY OF PHOTO STABILITY STUDIES OF BENDAMUSTINE HYDROCHLORIDE
Sample ID | BDM Area % | ImpurityArea % | RRT | Name of the Impurity | BDM Peak Purity Angle | BDM Peak Purity Threshold |
Control in dark | 99.8 | 0.11 0.06 | 0.57 1.30 | HP1 Unknown | 0.06 | 0.28 |
Uncovered | 97.6 | 0.170.47
0.27 1.13 0.09 0.17 0.07 |
0.270.29
0.30 0.56 0.57 0.67 1.26 |
UnknownUnknown
Unknown Unknown HP1 Unknown Unknown |
0.07 | 0.29 |
Covered with Aluminium Foil | 99.8 | 0.110.06 | 0.571.29 | HP1 Unknown | 0.07 | 0.29 |
Covered with Aluminium Foil and placed in Carton | 99.8 | 0.11 0.05 | 0.57 1.30 | HP1 Unknown | 0.06 | 0.28 |
Forced Degration Chromatograms:
FIGURE 4: CHROMATOGRAM OF BENDAMUSTINE HYDROCHLORIDE STANDARD
FIGURE 5: ACID DEGRADATION CHROMATOGRAM OF INITIAL SAMPLE
FIGURE 6: ACID DEGRADATION CHROMATOGRAM OF 60°C – 1HR SAMPLE
FIGURE 7: BASE DEGRADATION CHROMATOGRAM OF INITIAL SAMPLE
FIGURE 8. OXIDATIVE DEGRADATION CHROMATOGRAM OF INITIAL SAMPLE
FIGURE 9. OXIDATIVE DEGRADATION CHROMATOGRAM OF 60°C – 1HR SAMPLE
FIGURE 10. HYDROLYSIS CHROMATOGRAM OF INITIAL
FIGURE 11: HYDROLYSIS CHROMATOGRAM OF 60°C 1hr
FIGURE12: PHOTO STABILITY STUDIES –CONTROL IN DARK
FIGURE13. PHOTO STABILITY STUDIES –COVERED WITH ALUMINUM FOIL
FIGURE14: PHOTO STABILITY STUDIES –COVERED WITH ALUMINUM FOIL AND PLACED IN CARTON
FIGURE 15. PHOTO STABILITY STUDIES –UNCOVERED
CONCLUSIONS: System suitability conditions meet the recommended criterion. Linearity of the solution was demonstrated between 0.0002577 mg/ml and 0.51540 mg/ml concentration range. Accuracy was demonstrated as reported. Recovery and % RSD are within the recommended limits. Injection reproducibility was demonstrated and the % relative standard deviation for retention time and area were within the limits. Limit of detection value is at 0.00004165mg/ml and limit of quantisation is at 0.00012621mg/ml. Solution was stable up to 38hrs at 5°C temperature. Precision was demonstrated and the % relative standard deviation for assay was within the limits. Specificity was also demonstrated and there was no interference with the diluent & mobile phase. Under forced degradation studies, the peak purity angle is always lower than purity threshold for Bendamustine hydrochloride and all degradant peaks were well resolved. Hence the method can be adopted for the formulation screening studies as a stability indicating method.
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How to cite this article:
Sridasam N, Reddy YR and Kavya V: A Validated Report on Formulation Stability Studies of Bendamustine Hydrochloride. Int J Pharm Sci Res2015; 6(1): 410-20.doi: 10.13040/IJPSR.0975-8232.6 (1).410-20.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Article Information
52
410-420
1071KB
0
English
IJPSR
Niharika Sridasam*, Ravinder Reddy Y. and V. Kavya
Teegala Ram Reddy College Of Pharmacy, #4-202, Meerpet, Saroor Nagar (M), Ranga Reddy (Dist), Hyderabad-500079, Telangana, India
niharikasridasyam@gmail.com
02 June 2014
12 August, 2014
22 September, 2014
http://dx.doi.org/10.13040/IJPSR.0975-8232.6(1).410-20
01 January, 2015