Analytical Method Development and Validation of Pioglitazone hydrochloride by RP-HPLC in Pharmaceutical Dosage Form

Diabetes mellitus is a chronic metabolic disorder characterized by persistently elevated blood glucose levels resulting from inadequate insulin secretion, impaired insulin action, or a combination of both. Among its various forms, type 2 diabetes mellitus (T2DM) is the most common and represents a major global health challenge due to its rapidly increasing prevalence and the development of serious long-term complications such as retinopathy, nephropathy, neuropathy, and cardiovascular disease^(1,2,3). Effective control of blood glucose levels is essential to reduce the risk of these complications and improve the quality of life of affected individuals. Consequently, the availability of accurate and reliable analytical methods for the quality assessment of antidiabetic medications is crucial during pharmaceutical development, manufacturing, and routine quality control⁽⁴⁾.

Pioglitazone hydrochloride is a widely prescribed oral antidiabetic drug belonging to the thiazolidinedione class. It exerts its therapeutic effect by acting as a selective agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ), thereby improving insulin sensitivity in peripheral tissues and reducing hepatic glucose production⁽⁵⁾. Because of its proven efficacy in achieving glycemic control and its extensive clinical use, pioglitazone hydrochloride has become an important therapeutic option for the management of T2DM, both as a single agent and in fixed-dose combination formulations with other antidiabetic drugs.

The quality, safety, and therapeutic efficacy of pharmaceutical products depend on accurate and reliable analytical techniques for quantitative estimate of active pharmaceutical constituents. The methodical process of developing analytical methods involves optimizing chromatographic settings for effective separation, precise quantification, and repeatable outcomes. The choice of chromatographic column, mobile phase composition, pH, flow rate, detection wavelength, injection volume, and column temperature are important factors affecting technique performance. Adequate resolution, symmetrical peak form, suitable retention time, and excellent analytical sensitivity are all ensured by properly optimizing these parameters⁽⁶⁾.

Reverse phase high performance liquid chromatography (RP-HPLC) has become one of the most popular analytical techniques for quantitative determination of pharmaceutical substances due to ease of use, accuracy, precision and reproducibility. RP-HPLC benefits include excellent separation efficiency, very short analytical times, minimal sample preparation and compatibility with a wide range of pharmaceutical substances. These features make RP-HPLC the most popular analytical technique for routine quality control, assay determination, dissolution testing, impurity profiling, and stability investigations in pharmaceutical companies and research labs⁽⁷⁾.

Prior to the routine use of an analytical method for pharmaceutical analysis, it must be validated to establish that it routinely produces accurate and repeatable results. Validation of an analytical method is a particular set of procedures to prove that a method is suitable for its intended purpose. According to the International Council for Harmonization (ICH) recommendation Q2(R2), the analytical techniques should be tested for specificity, linearity, range, accuracy, precision, detection limit, quantitation limit, robustness, and system suitability. Validation guarantees that the method developed will yield analytical data for pharmaceutical quality assurance that is regulatory compliant and scientifically acceptable⁽⁸⁾.

Pioglitazone hydrochloride has been quantified by various analytical methods including UV-visible spectrophotometry, HPLC, HPTLC, UPLC, capillary electrophoresis and LC-MS/MS either alone or in combination with other antidiabetic drugs. While many of these techniques show good analytical performance, some call for complicated mobile phase compositions, costly solvents, lengthier chromatographic run durations, sophisticated instruments, or time-consuming sample preparation techniques. The development of simple, rapid, accurate, precise, affordable and reliable RP-HPLC methods for routine quality control analysis of pioglitazone hydrochloride in pharmaceutical dosage forms is still needed^(9-12).

The present study was therefore undertaken to develop and validate a simple, rapid, accurate, precise, economical, and robust RP-HPLC method for the quantitative estimation of pioglitazone hydrochloride in pharmaceutical dosage forms. The developed method was optimized to achieve efficient chromatographic separation and validated according to the current ICH analytical method validation guidelines. The validated method is expected to provide a reliable, cost-effective, and reproducible analytical procedure suitable for routine quality control, assay determination, stability studies, and pharmaceutical research involving pioglitazone hydrochloride.

1. MATERIALS AND METHODS
   1. Materials

Reference standards for Pioglitazone hydrochloride was obtained from Synokem Pharmaceuticals Ltd, Delhi. Pioglitazone formulation (Pioglit 15mg) was purchased from local drug store. HPLC grade Acetonitrile, Methanol and water Merck specialities (Pvt) Ltd. Mumbai.

1. 2. Instrumentation

The method employed for this study utilized the Agilent 1220 Infinity LC (G4288C) which consist of a pump, injector and variable wavelength detector. The analytical column used was the Agilent Eclipse Plus C18 with dimensions of 4.6×100mm and a particle size of 3.5µm. the Shimadzu analytical balance was utilized for weighing both the standards and samples. Merck Millipore membrane filter with pore size of 0.45 µm was used for mobile phase filtration. Additionally, Agilent Technologies PVDF syringe filters with 0.45 µm was used for sample filtration.

1. 3. Methodology
      1. Preparation of solutions

Preparation of stock solution: Accurately weighed 10mg of pioglitazone and added to a 10ml standard flask, dissolved in methanol and was made upto volume to obtain a concentration of 1000 µg/ml.

Preparation of working standard solution: Diluted the stock solution with methanol to obtain working standard solutions of concentrations 10-50 µg/ml.

Study of spectral characteristics of Pioglitazone

The UV absorption spectrum for Pioglitazone was recorded using UV-Visible    spectrophotometer by scanning 50 µg/ml working standard solution in the range of 200-400nm. 269nm was found to be the λmax.

Method Development

Based on the various physicochemical properties of Pioglitazone hydrochloride such as the solubility, pKa, molecular weight, log P values etc…a number of chromatographic trials were carried out to initiate the method development for the determination of Pioglitazone Hydrochloride.

1. 1. 2.  Method Optimization

Various chromatographic parameters like mobile phase composition, column type, column length, pH, flow rate etc… were assessed for obtaining well resolved, sharp, symmetrical peaks with satisfactory system suitability parameters for pioglitazone hydrochloride.

1. 1. 3. Method validation

The current developed method was validated as per ICH guidelines. The various parameters validated are as follows:

Linearity: It is the ability of an analytical procedure to achieve test results which are directly proportional to the amount of analyte in the sample. This was carried out by injecting and recording the responses of standard solutions of pioglitazone hydrochloride in the range of 10-50µg/ml. Then calibration curve was constructed by plotting concentration against corresponding peak area values and determined the correlation coefficient and regression line equation.

Accuracy: It’s the degree of agreement between the value found and the value that is recognized as either an approved reference value or a conventional actual value. The accuracy of current method was evaluated by recovery studies. Spiked standard pioglitazone solution to sample solution at three concentration levels (80%, 100%, 120%). Prepared three replicates of each level which were injected into HPLC and recorded the responses. % recovery was determined.

Precision: Describes the degree of agreement (or scatter) between a set of measurements made from several samplings of the same homogenous sample under specific circumstances. It was evaluated by injecting six replicates of Pioglitazone Hydrochloride solution of concentration 10µg/ml on the same day (intra day) and subsequent days (inter day) under the same experimental conditions. Recorded the peak area responses for each six replicates from the chromatogram and % RSD was found.

Limit of detection is the lowest concentration of analyte in a sample that is detectable but may not be precisely quantified whereas the Limit of quantification is the lowest concentration of analyte in a sample that can be accurately and precisely measured quantitatively. Both LOD and LOQ were determined according to the ICH guidelines.

            LOD (Detection limit)        = 3.3 σ/Slope

            LOQ (quantification limit) = 10 σ/Slope , where σ is the standard deviation.

Robustness: It is the measure of capacity of an analytical procedure to remain                unaffected by small but deliberate changes in method parameters and provides an indication of its reliability during normal usage. It was assessed by injecting three replicates of 10µg/ml Pioglitazone Hydrochloride standard solution by changing the flow rate at 0.6±0.1 ml/min. Peak area responses were recorded and % RSD was calculated.

System suitability test: Five replicates of Pioglitazone Hydrochloride working standard solutions were prepared and injected into the instrument and evaluated the system suitability parameters by determining the %RSD of Rt, peak areas, theoretical plates and tailing factors. All of these were found to be within the acceptable limit.

Assay of pioglitazone tablet dosage form

20 tablets of Pioglitazone hydrochloride(15mg) were weighed to calculate the average weight per tablet. An amount of powder equivalent to 10mg of Pioglitazone was weighed and transferred to a 10ml volumetric flask and added sufficient amount of mobile phase and sonicated. Then volume was made up to the mark using mobile phase. Filtered the solution through 0.45µm membrane filter and from this solution 0.1ml was pipetted out into a 10ml volumetric flask, diluted using mobile phase to produce a sample solution of concentration 10µg/ml, which was injected into the HPLC and obtained the chromatogram.  Assay was determined using the peak area.

2. RESULTS AND DISCUSSIONS
   1. Selection of wavelength and study of spectral characteristics

 

 

Fig.no.1 UV spectrum for Pioglitazone Hydrochloride

 

 

Fig. no.2 Chromatogram for 10µg/ml

 

 

Fig.no.3 Chromatogram for 20µg/ml

 

 

Fig.no.4 Chromatogram for 30µg/ml

 

 

Fig. no.5 Chromatogram for 40 µg/ml

 

 

Fig.no.6 Chromatogram for 50µg/ml

 

 

Fig.no.7 Calibration plot of Pioglitazone Hydrochloride (Conc. v/s peak area)

Percentage label claim of Pioglitazone in the marketed formulation (Pioglit 15mg) was found to be 99.57%.

CONCLUSION

A simple, rapid, accurate and precise RP-HPLC method was developed and validated as per ICH guidelines for the quantitative estimation of Pioglitazone Hydrochloride. The developed chromatographic conditions provided satisfactory separation of Pioglitazone Hydrochloride with well defined symmetrical peak and suitable retention time. The proposed method demonstrated acceptable linearity, accuracy, precision, robustness within the validated range, complying with the requirements of ICH guidelines. The developed method also offers the advantages of simplicity, reproducibility and relatively short analysis time, making it suitable for routine quality control analysis. Therefore, the current RP-HPLC method can be effectively applied for the quantitative estimation of Pioglitazone Hydrochloride in bulk drug and pharmaceutical formulations. 

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