INTRODUCTION:
APR and PSR combination is available as capsule dose form with brand names Prax A 75 (Torrent Pharmaceuticals Ltd) and Prasita A 10 (Sun Pharmaceutical Industries Ltd)5,6. Both capsule dose forms are labeled with 75 mg APR and 10 mg PSR.
APR and PSR combination treatment was used in patients with cardiovascular disorders receiving a percutaneous coronary operation to avoid blood clot problems and plaque destruction. Few methods employing spectrophotometry7,8 and HPLC9-11 was found for the simultaneous estimation of APR and PSR. Among the methods reported only one HPLC method11 is stability indicating. This method suffers many shortcomings including higher retention time, low linearity range, costly and time-consuming.
A crucial analytical aspect of the drug production plan is the forced degradation analysis also called stress testing. Stress testing is conducted to prove the analytical method's specificity and stability-indicating features. Stability tests must be carried out to recommend the shelf-life of new drug substances and/or drug products12,13. So it is required to develop a cost effective, rapid and reliable stability indicating RP-HPLC method for APR and PSR combination in capsule dose for routine analysis.
MATERIALS AND METHODS:
Instrumentation:
APR and PSR combination analysis was done using Waters model 2695 model HPLC system consisted of a Waters model 2998 photodiode array detector. For separation of APR and PSR reverse phase Symmetry C18 (250mm × 4.6mm × 5μm Make: Waters) column was used. Using Empower 2 program software, APR and PSR chromatography data was analysed.
Chemicals and Reagents:
APR and PSR standard API’s were procured from Megafine Pharma (P) Ltd (Mumbai, India) and Dr. Reddy’s Laboratories (Hyderabad, India), respectively. Capsule dose form claimed as 75mg of APR and 10 mg of PSR was acquired from resident market. Methanol was procured from Merck Chemicals (India). Potassium dihydrogen phosphate and phosphoric acid were purchased from Ranbaxy Fine Chemicals (New Delhi, India). Water used throughout the study was purified using Millipore system (Millipore Corp., Bangalore, India).
Chromatographic Condition:
0.1M Potassium dihydrogen phosphate buffer (pH 3.2 units) and methanol (65:35 v/v) at the flow rate of 1.2 mL/min was used as mobile phase. Waters Symmetry C18 was maintained at 30°C. The detection and analysis of APR and PSR was carried out at 220nm with sample injection quantity of 20µl. Mobile phase, standard solutions, sample solutions and degraded sample solutions were filtered by way of 0.22μm filter paper and degassed before infusion into column. The mix of 0.1M Potassium dihydrogen phosphate buffer (pH 3.2 units) and methanol (65:35 v/v) is also used diluent.
Preparation of Stock and Standard Solutions:
Accurately weighted PSR 20mg and APR 150mg are placed into 100mL volumetric flask, poured 25 mL of diluent and sonicated over 30 minutes, and filled with diluent to the volume. Moved 5mL of stock solution to 25mL volumetric flask and filled with diluent to the volume.
Preparation of Capsule Sample Solution:
Twenty PRAX A 75mg capsules were emptied. Transferred quantity of the powder equivalent to 150mg of APR and 20mg of PSR are placed into 100mL volumetric flask, poured 25 mL of diluent and sonicated over 30 minutes, and filled with diluent to the volume. Transferred 5 mL of stock capsule solution into 25mL flask and filled with diluent to obtain 300µg/mL and 40 µg/mL final concentrations of APR and PSR.
Forced degradation studies:
This test was aimed to make sure that APR and PSR peaks are effectively segregated from their degradation product peaks, as well as testing the system features such as stability indicating and specificity14.
Acidic degradation:
625.01mg of capsule powder equivalent to 150mg APR and 20mg PSR was taken into 100mL flask followed by adding 10mL of 0.1N HCl. At room temperature in dark, the flask contents were sonicated over 2 hr. and followed by neutralization of contents with 10mL of 0.1N NaOH.
Alkaline degradation:
625.01mg of capsule powder equivalent to 150mg APR and 20mg PSR was taken into 100mL flask followed by adding 10mL of 0.1N NaOH. At room temperature in dark, the flask contents were sonicated over 2 hr. and followed by neutralization of contents with 10mL of 0.1N HCl.
Oxidative degradation:
625.01mg of capsule powder equivalent to 150mg APR and 20mg PSR was taken into 100mL flask followed by adding 10mL of 3% peroxide. At room temperature in dark, the flask contents were sonicated over 2 hr.
Thermal degradation:
625.01mg of capsule powder equivalent to 150mg APR and 20mg PSR was taken petri dish and kept in oven for 2 hr at 105°C.
Photolytic degradation:
625.01mg of capsule powder equivalent to 150mg APR and 20mg PSR was taken petri dish and kept for 24 hr in direct sunlight.
The samples obtained after degradation with acid, alkali, peroxide, thermal and photo were diluted appropriately with diluent to obtain 300µg/mL and 40µg/mL final concentrations of APR and PSR for analysis by the suggested method.
RESULTS AND DISCUSSION:
RP-HPLC Method Development:
In this investigative process, many columns have been checked. Finally, the Waters C18 Column at 30°C temperature was deemed more appropriate over other columns. Waters C18 Column supported excellent peak geometry, increased reproducibility with good resolution for APR and PSR peaks. 0.1M Potassium dihydrogen phosphate buffer (pH 3.2 units) and methanol (65:35 v/v) at the flow rate of 1.2ml/min was chosen as mobile phase as these conditions ideally resolve the peaks of APR and PSR with complete separation. The retention values of APR and PSR were 2.464 min and 3.772 min, respectively. The wavelengths for detection and analysing APR and PSR were set as 220nm. At this nanometers, APR and PSR gave good response. Typical chromatograms of APR and PSR standard solution and capsule dose solution are shown in Figure 1.
Figure 1: HPLC chromatogram of APR and PSR [A] Standard solution [B] Capsule dose solution
Method Validation:
The method has been validated for its linearity, specificity, accuracy, precision, limit of detection, limit of quantitation and robustness. The testing will be carried out in compliance with the international harmonization guidelines meeting15.
Selectivity:
For determining method selectivity, capsule sample solution (300µg/mL APR and 40µg/mL PSR) was injected into the column. The chromatogram of capsule solution (Figure 1B) was compared with standard APR and PSR chromatogram (Figure 1A). In the chromatogram (Figure 1B), there were no interruptions caused by the capsule excipients and the chromatographic features was not affected. Thus, the selected method was selective for the determination of APR and PSR in marketed capsule dose formulation.
Linearity:
Calibration curves were generated by plotting peak area versus APR and PSR concentration. The calibration curves for APR and PSR, respectively, were observed as linear over 5 varying concentrations within the range of 150-450μg/mL, and 20-60μg/mL The regression equations for APR and PSR were calculated. The findings suggest that there was an excellent relationship between the APR and PSR peak areas with APR and PSR concentrations inside the concentration range of analysis.
· Regression equation for APR = y =46476 x - 3600
· Regression coefficient for APR = 0.999
· Regression equation for PSR = y =33721 x + 16645
· Regression coefficient for APR = 0.999
Limit of detection (LOD) and limit of quantification (LOQ):
The LOQ and LOD for APR and PSR were finalized by computing signal to noise relation of 10:1 and 3:1, respectively. The LOD was calculated as 1.198µg/ml for APR and 0.9449µg/ml for PSR. The LOQ of APR and PSR were computed as 3.995µg/ml and 3.1496µg/ml, respectively. The values of LOQ and LOD supported the sensitivity of method.
Precision:
The precision was checked by six replicate infusions of standard solution (300µg/mL APR and 40µg/mL PSR). The precision was evaluated by computation of standard deviation and percent relative standard deviation for the APR and PSR response. The details are pointed up in Table 1. The preciseness of method is illustrated by the low percent relative standard deviation values of APR and PSR response.
Table 1: Precision Studies for Aspirin and Prasugrel
|
Area (Aspirin) |
Precision evaluation |
Area (Prasugrel) |
Precision evaluation |
|
4641536 |
Mean: 4645819 |
3414412 |
Mean: 3414666 |
|
4649770 |
3417410 |
||
|
4643971 |
SD: 3245.209 |
3418496 |
SD: 2948.448 |
|
4645505 |
3411716 |
||
|
4644610 |
RSD: 0.070 |
3414828 |
RSD: 0.086 |
|
4649524 |
3411134 |
Accuracy:
The accuracy was determined by recovery experiments. A known amount of APR and PSR was added to the fixed amount of pre-analysed capsule solution. Percent recoveries of APR and PSR were calculated after the addition of known amount of APR and PSR. The recovery tests were conducted at a level of 50%, 100% and 150%. The percent recovery of APR and PSR are given in Table 2. The accuracy of the method has been verified through high recovery values.