A VALIDATED STABILITY INDICATING RP-HPLC METHOD OF ESTIMATION OF PIOGLITAZONE HCL IN DOSAGE FORMHTML Full Text
A VALIDATED STABILITY INDICATING RP-HPLC METHOD OF ESTIMATION OF PIOGLITAZONE HCL IN DOSAGE FORM
T. Pramila * 1, Alka Agarwal 2 and Tamizh Mani 3
Pacific University 1, Pacific Hills, Udaipur -313003, Rajasthan, India.
US Ostwal Institute of Pharmacy 2, Mangalwad, Chittorgarh - 313603, Rajasthan, India.
Bharathi College of Pharmacy 3, Bharathi Nagar, Mandya - 571412, Karnataka, India.
ABSTRACT: This present study aims to develop an accurate, precise, and linear reverse-phase High-Performance Liquid Chromatographic (RP-HPLC) method for the estimation of Pioglitazone HCL in Pharmaceutical dosage form. Method: The chromatographic system employs a reverse-phase Hypersil BDS, C8 250 × 4.6 mm, 5 columns using 0.01M Potassium phosphate buffer and acetonitrile (40:60) as mobile phase, methanol as a diluent in isocratic mode. A flow rate of 1.0 ml/min was optimized with a detection wavelength at 225 nm. The retention time (Rt) was around 4.72 ± 0.2 min. Results: The method was validated with respect to specificity, selectivity, linearity, accuracy, precision, and robustness as per ICH guidelines. The assay method was observed linear in the concentration range of 0.079-0.318 mg/ml with a Correlation coefficient (r2) of 0.9999. The percentage recovery of active pharmaceutical ingredients from tablet dosage form ranged from 99.40-100.40%. Stress conditions of degradation in acidic, alkaline, peroxide, thermal, and UV radiation were studied.
Pioglitazone, HPLC, Validation, Forced Degradation studies
INTRODUCTION: Pioglitazone HCl is an anti hyperglycaemic agent that, in the presence of insulin resistance, increases hepatic and peripheral insulin sensitivity, thereby inhibiting hepatic gluconeogenesis and increasing peripheral and splanchnic glucose uptake. It is a potent and highly selective agonist for the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR- γ). PPARs are found in tissues like adipose tissues, skeletal muscle, and liver, which are critical to insulin action. Activation of (PPAR- γ) modulates the transcription of a number of insulin-responsive genes involved in the control of glucose and lipid metabolism 1, 2.
It is administered orally; insoluble in water and ether; slightly soluble in acetone, acetonitrile, and alcohol; and soluble in dimethylformamide and dimethyl sulfoxide. It selectively stimulates the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) and, to a lesser extent PPAR-α.
It modulates the transcription of the insulin-sensitive genes involved in glucose and lipid metabolism in the muscle, adipose tissue, and the liver. As a result, Pioglitazone HCl reduces insulin resistance in the liver and peripheral tissues, increases the expense of insulin-dependent glucose, decreases withdrawal of glucose from the liver, and reduces the quantity of glucose, insulin, and glycosylated hemoglobin in the bloodstream. It is not chemically or functionally related to the alpha-glucosidase inhibitors, the biguanides, or the sulfonylureas. It addresses the main patho-physiological defect, i.e., insulin resistance, so it is used alone or in combination with insulin, metformin, or a sulfonylurea (glimepiride and glibenclamide) as an agent to treat diabetes. It reduces peripheral and hepatic resistance to insulin, resulting in increased insulin‐dependent glucose disposal and decreased hepatic glucose output.
Pioglitazone HCl is generally well tolerated, weight gain and oedema are the most common emergent adverse events, and there are no known drug interactions between Pioglitazone HCl and other drugs. Pioglitazone HCl was also effective in reducing some measures of cardiovascular risk and arteriosclerosis. Pioglitazone HCl thus offers an effective treatment option for the management of patients with type 2 diabetes. It is chemically (±) 5-[[4-[2-(5-Ethyl -2-pyridinyl) ethoxy] phenyl] methyl] -2, 4] thiazolidinedione monohydro-chloride.
Its molecular weight is C19H20N2O3S•HCl The molecular weight is 392.90 Da 3. The influence of pioglitazone on DNA oxidative damage and metabolism of SOD (superoxide dismutase) was evaluated 4. Effect of drug on lipid metabolism and glucose metabolism in type 2 diabetic patient showed a significant decrease in the plasma glucose level and improved tissue being sensitivity to insulin 5. The solid pioglitazone dispersion study was developed, which proposed to improve the rate of dissolution and to develop tablets of pioglitazone with effective and fast dissolution characters 6. Pioglitazone is an oral anti-hyperglycemic agent. It is used for the treatment of diabetes mellitus type 2. It selectively stimulates nuclear receptor pero-xisome proliferator-activated receptor gamma (PPAR-gamma). It was the tenth-best-selling drug in the U.S. in 20087. Attempts were made to study whether the usage of pioglitazone as an antidiabetic increases the risk of cancer.
This study revealed that the use of the drug did not show any significant risk in causing bladder cancer but is associated with the increase in the risk of prostate cancer and pancreatic cancer 8. FDC (fixed-dose combination) of 15 mg of the drug pioglitazone and 850 mg of metformin was used for 24weeks in type 2 diabetic patients to study the anti-diabetic property and patients were not prescribed with or medicated with any diabetic drug 9. Simultaneous estimation of drugs glimeperidine, pioglitazone HCl, and metformin HCI was done by using derivative spectro-photometry method 10. Chromatographic separation was achieved on a C8 column. The mobile phase was methanol–water 45:55% (v/v) containing 0.2% (w/v) n-heptane sulfonic acid and 0.2% (v/v) triethylamine; the pH was adjusted to 3.0 with orthophosphoric acid. The flow rate was 1 mL min-1 and the photodiode-array detection wavelength was 267 nm 11.
Piogliptazone hydrochloride in the tablet form was analysed and validated by HPLC with C18 column, wave length 245 nm, mobile phase 50% of 10 mM phosphate buffer and 50% of acetonitrile. Recovery was more than 90% with LOD and LOQ value being 10 ng/ml and 2.5 ng/ml respectively. The linear regression coefficient being 0.992112.
FIG. 1: STRUCTURE OF PIOGLITAZONE
The present study was aimed for establishing a simple, accurate, and rapid RP-HPLC method for determination of Pioglitazone in presence of its degradation products or other pharmaceutical excipients. The method was validated following analytical performance parameters suggested by ICH guidelines 13.
MATERIAL AND METHODS:
Chemicals and Reagents: The working standard of Pioglitazone was procured from PADM124 and the sample of Pioglitazone PIO/27030004. HPLC grade acetonitrile and methanol were purchased from Rankem. Milli Q water from Merck India Pvt. Ltd. Potassium dihydrogen phosphate obtained from S.D. Fine Chemicals Ltd.
Preparation of Solutions:
Standard Solution: Weigh accurately 100 mg of Pioglitazone HCl standard in a 50 ml volumetric flask. Add about 30 ml of Methanol and sonicate for 3 min. Dilute to volume with Methanol and mix. Dilute 5.0 ml of this solution to 50 ml Methanol.
Sample Solution: Weigh accurately 100 mg of Pioglitazone HCl sample in a 50 ml volumetric flask. Add about 30 ml of Methanol and sonicate for 3 min. Dilute to volume with Methanol and mix. Dilute 5.0 ml of this solution to 50 ml Methanol.
Procedure: Separately inject 10 µL of Standard and Sample solution into the chromatograph, record the chromatograms, and measure the area for the major peaks.
Apparatus and Chromatographic Conditions: The analytical technique was developed using Waters HPLC equipment, Model- HPLC-269, fitted with a Hypersil BDS C8 (250 × 4.6 mm, 5 μ). The mobile phase consisted of a mixture of 0.01M potassium dihydrogen phosphate buffer and acetonitrile in the ratio 40:60. The mobile phase was filtered through a 0.22-mm nylon filter and degassed using an ultrasonic bath sonicator for 30 min before running the experiment.
All experiments conducted on the HPLC were carried out in isocratic mode. Injection volume was 10 μL with a flow rate of 1.0 mL/min. The column temperature was maintained at 30 ºC and elution was monitored at 225 nm using a UV detector. All chromato-graphic data were acquired and processed with the Empower 3 software.
Validation of the Analytical Method: The developed method was validated as per the ICH guidelines for linearity, accuracy and precision, and specificity. Limit of detection (LOD) and limit of quantification (LOQ) were determined using the serial dilution method.
Linearity: Different aliquots of a standard solution of Pioglitazone was transferred into set of 50 ml volumetric flasks and diluted up to the mark with a diluent such that the final concentrations of Pioglitazone were in the linearity range of 0.08-40.32 mg/ml. Evaluation of the drug was performed with a PDA detector at 225 nm, and peak area was recorded. The response for the drug was linear and the regression equation was found to be y = 22750541, 96495x + 39279.17, and the correlation coefficient value of Pioglitazone was found to be 0.9999. The results showed that an excellent correlation exists between peak area and concentration of drug within the specified range.
Accuracy: Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of Pioglitazone in the drug product. The study was carried out in triplicate at 50, 100, and 150%.
The percentage recovery in each case was calculated. The percentage recovery ranges from 99.40-100.40%, and the mean recovery of Pioglitazone was 99.73% that shows there is no interference from excipients, and the lower values of %RSD of assay indicates the method is more accurate.
Precision: The precision was determined for Pioglitazone in terms of system precision, method precision, and intermediate precision. For system precision evaluation, a standard solution of fixed concentration was injected six times at different time intervals. Method precision was studied on six test solutions of the single batch were analyzed, the inter-day precision was studied by injecting the same concentration of the standard solution on consecutive days, and the %RSD for Pioglitazone was 0.0%, 0.3, and 1.1% (limit %RSD < 2.0%).
System Suitability: System suitability parameters like retention time, theoretical plates, and tailing factor were calculated and compared with standard values.
Robustness: The robustness of the method was determined by making slight changes in the chromatographic conditions like flow rate, detection wavelength, and organic phase in the mobile phase composition. It was observed that there were no marked changes in the chromato-grams, which demonstrated that the developed HPLC method is more robust.
Solution Stability: The stability of the solution under study was established by keeping the solution at room temperature for 24 h. The result showed no significant change in concentration and thus confirmed the stability of the drug in the solvent used for the analysis.
Forced Degradation Studies: Forced degradation study was carried out on Pioglitazone HCl. Conditions employed and the results obtained from forced degradation studies are summarized below.
Acid Degradation: The sample was separately treated with 10 mL of 4 N Hydrochloric acid at room temperature for 30 minutes. Cooled and neutralized with 10 mL of 4 N sodium hydroxide solution and further analyzed by the proposed method.
Base Degradation: The sample was separately treated with 10 mL of 4 N sodium hydroxide solutions at room temperature for 30 min. Cooled and neutralised with 10 mL of 4 N Hydrochloric acid. Further analysed by the proposed method.
Peroxide Degradation: Sample was separately treated with 5 mL of 30% v/v H2O2 at Room temperature for 30 min. Further analysed by the proposed method.
Thermal Degradation: Thermal degradation study was carried out by exposing the sample was subjected to thermal degradation by keeping at 105 °C for 48 h followed by analysis by the proposed method.
Humidity Degradation: Humidity degradation study was carried out by exposing the sample at 25 °C / 90% RH for 7 days.
Photolytic Degradation: Photolytic degradation study was carried out by exposing the sample 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-watt hours/square meter. The results obtained are given in Table 3. The chromatograms obtained are presented in a list of Annexures (D to I)
RESULTS AND DISCUSSION: In the present work a simple reverse-phase high-performance liquid chromatographic method has been developed, optimized and validated for the estimation of Pioglitazone in pharmaceutical formulations. Chromatographic separation was achieved on Hypersil BDS, C8 250 × 4.6 mm, 5 column using 0.01M Potassium dihydrogen phosphate buffer and acetonitrile (40:60) as mobile phase, methanol as a diluent in isocratic mode. Flow rate of 1.0 ml/min was optimized with detection wavelength at 225 nm. The retention time (Rt) was around 4.72 ± 0.2 min. The method was validated with respect to specificity, selectivity, linearity, accuracy, precision, and robustness as per ICH guidelines.
The assay method was observed linear in the concentration range of 0.079-0.318 mg/ml with a Correlation coefficient (r2) 0.9999. The linearity and Chromatogram of Standard Chromatogram were shown in Fig. 2 and 3. The percentage recovery of active pharmaceutical ingredients from tablet dosage form ranged from 99.40-100.40%. The results were shown in Tables 1 and 2.
The %RSD for method precision and inter-day precision for Pioglitazone was found to be NMT 2, which indicates the method is precise. The results of precision studies were shown in Tables 3 and 4. A system suitability test was performed to evaluate the chromatographic parameters. The typical chromatograms of the degradation behavior of Pioglitazone in different stress conditions were shown from Fig. 4 to 8.
During the acidic and alkaline degradation, 24.5% and 18.1% were decomposed, respectively. Pioglitazone has undergone oxidative 2%, thermal 1.9%, and photostability was 4.5%. The results of the degradation studies were shown in Table 5. The summary of Validation parameters was shown in Table 6.
FIG. 2: LINEARITY GRAPH OF PIOGLITAZONE
FIG. 3: CHROMATOGRAM OF STANDARD
|S. no||Name||RT||Area||% Area||USP plate count||USP tailing|
FIG. 4: CHROMATOGRAM OF ACID STRESSED SAMPLE (PURITY PLOT)
FIG. 5: CHROMATOGRAM OF BASE STRESSED SAMPLE (PURITY PLOT)
FIG. 6: CHROMATOGRAM OF OXIDATION STRESSED SAMPLE (PURITY PLOT)
FIG. 7: CHROMATOGRAM OF PHOTOLYTIC STRESSED SAMPLE (PURITY PLOT)
FIG. 8: CHROMATOGRAM OF THERMAL STRESSED SAMPLE (PURITY PLOT)
TABLE 1: LINEARITY OF STANDARD PIOGLITAZONE
|S. no.||Level||Pioglitazone HCl Concentration (in mg)||Area||Mean Area|
|Correlation Coefficient (r)||0.999951977|
|% deviation of y-intercept||0.7|
TABLE 2: RESULTS OBTAINED FROM ACCURACY FOR PIOGLITAZONE HCL (% RECOVERY)
|Accuracy solution (50%)-1||49.719||49.623||99.8||100.4||0.5|
|Accuracy solution (50%)-2||49.749||50.145||100.8|
|Accuracy solution (50%)-3||49.828||50.098||100.5|
|Accuracy solution (100%)-1||99.339||98.757||99.4||99.4||0.3|
|Accuracy solution (100%)-2||99.418||99.146||99.7|
|Accuracy solution (100%)-3||99.250||98.488||99.2|
|Accuracy solution (150%)-1||149.028||147.627||99.1||99.4||0.4|
|Accuracy solution (150%)-2||148.909||148.564||99.8|
|Accuracy solution (150%)-3||148.998||147.878||99.2|
TABLE 3: RESULTS OF SYSTEM PRECISION
|Injection||Area of Pioglitazone HCl|
TABLE 4: RESULTS OBTAINED FROM SIX SAMPLE PREPARATIONS FROM METHOD PRECISION
|S. no.||% Assay|
TABLE 5: RESULTS OBTAINED FROM FORCED DEGRADATION STUDY
|Mode of degradation||Condition||Purity
|% Assay||% Degradation|
|Acid stress||10 mL, 4 N HCl for 30 min. at RT||0.392||1.00||74.1||24.5|
|Humidity stress||25 °C/90% RH for NLT
|Photolytic stress||1.2 million lux hrs / 200-watt hrs / square meter||0.137||0.273||93.7||4.5|
|Base stress test||10 mL, 4N NaOH for
30 mins. at RT
|Oxidation stress||5 mL, 30% v/v H2O2 for
30 min at RT
|Thermal stress||105 °C for 48 hours||0.151||0.276||96.2||1.9|
TABLE 6: SUMMARY OF VALIDATION PARAMETERS
|S. no.||Parameter||Experiment||Acceptance criteria||Results|
|1||Specificity & Forced degradation||Blank, standard solution and test solution||There should not be any interfering peaks due to diluent at the retention time of Pioglitazone HCl peak.||There is no interference due to diluent at the retention time of Pioglitazone HCl peak in standard and Sample Solution.|
|Name||Retention time (min)|
|Peak purity angle should be less than Peak purity threshold of Pioglitazone HCl peak in standard, and test solution.
There should be no tick mark in the purity flag column.
|Sample||Purity Angle||Sample||Purity Angle|
|Standard solution||0.158||Standard solution||0.158|
|Blank, standard solution, test solution and Stressed solutions||There should not be any interfering peaks due to diluent at the retention time of Pioglitazone HCl peak.
Peak purity angle should be less than Peak purity threshold for Pioglitazone HCl peak in standard, test solution and stressed solution.
There should be no tick mark in the purity flag column.
|There is no interference due to diluent at the retention time of Pioglitazone HCl peak in standard and stressed solution|
|Sample||Purity Angle||Sample||Purity Angle|
|Control Sample||0.149||Control Sample||0.149|
|Acid Stress||0.392||Acid Stress||0.392|
|Base Stress||0.335||Base Stress||0.335|
|Oxidation stress||0.155||Oxidation stress||0.155|
|Thermal stress||0.151||Thermal stress||0.151|
|Photolytic stress||0.137||Photolytic stress||0.137|
|Humidity stress||0.165||Humidity stress||0.165|
CONCLUSION: The proposed stability-indicating HPLC method was validated as per ICH guidelines. The method was found to meet all the predetermined acceptance criteria. The validated HPLC method for Pioglitazone HCl is specific, stable, linear, accurate, precise, rugged, and robust. Based on the validation study results, it has been concluded that the HPLC method for Pioglitazone HCl is suitable for the intended purpose. At the same time, the chromatographic elution step is undertaken in a short time (< 5 min). There is no interference from any components of the pharma-ceutical dosage form. The chromatograms of diluent (blank) indicate that there is no interference at the retention time Pioglitazone HCl peak.
The peak purity angle was less than the peak purity threshold, and there was no tick mark in the purity flag column for Pioglitazone HCl peak in all final stressed test solutions. The peak purity plot of stressed sample solutions indicates that Pioglitazone HCl peaks are homogeneous and have no co-eluting peaks ensuring the specificity of the method. Hence it can be successfully applied to perform the routine analysis of the drug in pharmaceutical formulations.
ACKNOWLEDGEMENT: The authors are thankful to Padm Labs as they provided lab facilities to experiment.
CONFLICTS OF INTEREST: The authors declare no conflicts of interest.
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How to cite this article:
Pramila T, Agarwal A and Mani T: A validated stability indicating RP-HPLC method of estimation of pioglitazone HCI in dosage form. Int J Pharm Sci & Res 2021; 12(2): 859-67. doi: 10.13040/IJPSR.0975-8232.12(2).859-67.
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