Comparison of the Robustness of Spectrophotometric Method and High-Performance Liquid Chromatographic Method for the Determination of Pioglitazone in Solid Dosage Form

Pioglitazone is a drug that reduces the amount of glucose (sugar) in the blood. It is included in the class of anti-diabetic drugs called “thiazolidinedione” that are used in the treatment of type II diabetes. It attaches to the peroxisomes proliferatedactivated receptor gamma (PPARΥ) on tissues throughout the body and causes the cells to become more sensitive to insulin. As a result, more glucose is removed from the blood.The aim of the study is more precisely to find out the better analytical method for the quantitative measurement of the content of pioglitazone in commercially available drugs using two analytical methods i-e Spectrophotometric method and HPLC method.The analytical method for the pioglitazone hydrochloride was developed by HPLC, Original Research Article Zahid et al.; JPRI, 33(14): 37-45, 2021; Article no.JPRI.66035 38 and then validated the method according to compendial requirements. Pioglitazone in various Dowglit and Gliden tablets was determined by this developed methodPrecision, Accuracy and stability of Pioglitazone was checked which came out to be100.40% and 100.19% respectively. The analysis of pioglitazone hydrochloride in solid dosage form using the HPLC method shows the results are more sensitive, accurate, validated and economical and can be easily applied to raw materials and finished goods compared to the Spectrophotometer method.

Antidiabetic mechanism of the drug depends upon the presence of insulin. Pioglitazone is responsible to decline insulin resistance in the periphery and in the liver resulting in glucose disposal which is insulin dependent and reduces hepatic glucose output. In contrast to sulfonylureas, Pioglitazone is not an insulin secretagogue. It is a potent agonist for peroxisome proliferator-activated receptorgamma (PPARγ) [2,3]. Peroxisome proliferatoractivated receptor-gamma are located in liver, skeletal muscles and adipose tissues where transport of glucose is insulin dependent. Stimulation of PPARγ nuclear receptors modulates the transcription factors of many insulin responsive genes involved in glucose control and lipid metabolism. In diabetic animal model [4].It has been found that pioglitazone decreases the clinical manifestation of Type II diabetes included hyperglycemia, hyperinsulinemia, and hypertriglyceridemia.
Responsiveness of insulin-dependent tissues increased due to metabolic changes produced by pioglitazone. As pioglitazone increases the response of circulating insulin by reducing insulin resistance, it does not decrease blood sugar level in animal models that are deficient in endogenous insulin [5,6].
Pioglitazone is extensively metabolized by oxidation and hydroxylation; the metabolites also partly convert to glucuronide or sulfate conjugates. Many metabolites including M-II and M-IV (hydroxyl derivatives of pioglitazone) and M-II (Keto derivative of pioglitazone) are found to be active pharmacologically in animal model of Diabetes type-II. In Vitro data describe that multiple isoforms of CYP are involved in biotransformation of pioglitazone. CYP2C8 is involved majorly in the metabolism of Pioglitazone.
Approximately 15% to 30% of pioglitazone is recovered in urine after oral administration. Elimination of drug through renal route is negligible, and drug primarily excreted in the form of conjugates produced during Phase II reactions. It is assumed that major proportion of oral dose is excreted through bile salts and faeces [7,8]. The mean serum half-life of pioglitazone and total pioglitazone ranges from 3 to 7 hours and 16 to 24 hours, respectively. Pioglitazone has an apparent clearance ranges between 5 to 7 L/hour. Pioglitazone is available in various strengths including 15 mg, 30 mg and 45 mg tablets. The recommended dosing schedule of pioglitazone is once a day; it may also be initiated in combination of insulin at 15 mg or 30 mg once daily. The current dose of insulin can be continued with pioglitazone treatment [9,10). In such patients who are receiving combination of insulin and pioglitazone, insulin dose can be reduced upto 25%. [11,12]. Further adjustment based upon glucose level. Various analytical techniques characterize assay method of drugs. Sample distillation is helpful in determination of alcohol contents of glanicals or other substances being volatile in steam including methanol, Thymol and even certain alkaloids such as ephedrine. Dessicator or heat oven has used for determination of moisture contents though most specific and convenient being Karl-Fisher [13,14].Chromatography can be employed for separation of pharmaceuticals.Spectrophotometer can be considered an essential analytical tool. Which incorporate features such as microprocessor control and diode array detector. Fluorometer measures the fluorescence that may be present in the sample such as riboflavin or may be developed into sample as thiamine hydrochloride [15,16].

Instrumentation
The HPLC system consisted of a 515 pump, 710 plus Autosampler, and a variable 480 UV Detector. The data processing system was a multi-channel Chrome and Spec software for chromatography [17].Set up the instrument according to standard conditions. The blank was run. The following suitability of system criteria met prior to the injection of the sample preparation. Six repeat injection of standard preparation were made. %age relative standard deviation of five injections should not be greater than 2.0%. Performed three samples preparation injections for generic product and assay percentage was calculated.

MATERIALS AND METHODS
Dowglit and Gliden tablets (15 mg) containing hydrochloride was purchased from Martin Dow Pharmaceuticals (PVT)Ltd and Reko Pharmacal (PVT) Ltd respectively. Ammonium acetate, Acetonitrile, Tetrahydrofolate (THF) of HPC grade and glacial acetic acid were purchased from Merck (Damstadt, Germany).
The HPLC system consisted of a 515 pump, 710 plus Autosampler, and a variable 480 UV Detector. The data processing system was a channel Chrome and Spec software for chromatography [17].Set up the instrument ditions. The blank was run. The following suitability of system criteria met prior to the injection of the sample preparation. Six repeat injection of standard preparation were made. %age relative standard deviation of five injections should not be greater than 2.0%. Performed three samples preparation injections for generic product and assay

Chromatographic Conditions
1.54 g of sodium acetate was measured accurately and then dissolved in1000 ml of water. pH of buffer was adjusted with glacial acetic acid. 550 ml of this buffer was taken and made a volume of 1000 ml by using 450 ml of Acetonitrile (ACN). This mixture was filtered through Whatman filter paper No. 42 with pore size 45 micrometre and used as mobile phase.The column used was 5µm (250 x 4.6 mm). The optical density was set at 269 nm.

Preparation of Standard Solutions
Stock solution of Pioglitazone HCl by dissolving 11.5 mg of accurately weighed Pioglitazone HCl (100%) into 10 ml Tetrahydrofolate (THF) to dissolve and made a volume of 50 ml by using mobile phase. Solution was filtered through Whatman filter paper No. 42 [18].Calibration curve samples were prepared from the stock solution to get six standard concentrations i-e 0.1, 0.2, 0.3, 0.5 1.0 and 2.0 mg/ml.

Chromatographic Conditions
1.54 g of sodium acetate was measured accurately and then dissolved in1000 ml of of buffer was adjusted with glacial acetic acid. 550 ml of this buffer was taken and made a volume of 1000 ml by using 450 ml of Acetonitrile (ACN). This mixture was filtered through Whatman filter paper No. 42 with rometre and used as mobile phase.The column used was 5µm (250 x 4.6 mm). The optical density was set at

Sample Preparation
20tablets were taken and their average weight was calculated. The tablets were crushed to fine powder. Dose equivalent to 59 mg (active ingredient) was transferred to a 250 ml volumetric flask, and dissolved in 10 ml of tetrahydrofolate (THF) and then the volume of solution was made up to 100 ml by using mobile phase. 10 ml of this solution was taken and made up the volume 25 ml by using mobile phase [19].

Precision
The precision of an analytical procedure is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of homogeneous sample.Six injections of sample solution (concentration 0.2 mg/ml) gave the results in the range of (98.52% -101.05%) which are shown in the Table 1. (Limit = SD NMT 2.0).

Accuracy
The accuracy of an analytical procedure is the closeness of test results obtained by that procedure to the true value. The accuracy of an analytical procedure should be established across its range.Weight of the Pioglitazone HCL (100% or 0.2 mg/ml) was added to the amount of placebo mixture equivalent to the amount of placebo present in the sample solution preparation. Three solutions were prepared and spiked and results were calculated and given in Table 2.

Stability
Stability of the solution is also tested to ensure the validity of analytical procedure.Injections of the sample solutions at room temperature over the period of 5hrs showed no significant difference in peak area of Pioglitazone hydrochloride over this time period, results are given in Table 3.

Linearity
The linearity of an analytical procedure is its ability to elicit results directly or by a well-defined mathematical transformation, proportional to the concentration of analyse in samples within the given range.Solutions of Pioglitazone hydrochloride were produced in mobile phase having concentrations in the range of active/inactive 0.0008 -0.24 mg/ml (0.40 -120%) of the theoretical concentration in the sample preparation 90.2 mg/ml). This method shows the linear response with these concentrations shown in Table 4.

Specificity
Specificity is the ability to assess the analyte in the presence of components that may be expected to the present, such as impurities, degradation products, and matrix components.A weight of placebo mixture equivalent to the amount present in the sample solution preparation was taken through HPLC procedure. No interference was observed in the chromatogram. Hence specificity is confirmed.

Quantitation Limit
The quantitation limit is a characteristic of quantitative essays for low levels of compounds in sample matrices, such as impurities in bulk substances and degradation products in finished pharmaceuticals. The quantitation limit is expressed as the concentration of analyte.Detection limit is the slowest amount of analyte in a sample that can be determined  with acceptable precision and accuracy under the stated experimental conditions. The detection limit is a characteristic of limit tests. It is the lowest amount of analyte a sample that can be detected, but not necessarily quantities under the stated experimental conditions. Thus, limit test shows the amount of analyte is above or below a certain level. The detection limit is usually expressed as the concentration of analyte in the sample.Linearity curve shows that upto concentration of 0.02 mg/ml, curve is linear. So,pioglitazone HCl was quantified at this concentrationi-e 0.02 mg/ml. Beyond this concentration we can detect the pioglitazone HCl at 0.0008 mg/ml ( Table 6).

DISCUSSION
The aim of the study was to determine a simple, cost effective HPLC method that could be conveniently used for the routine analysis of large number of samples usually obtained in pharmacokinetic studies. Only few HPLC methods are available for the quantification of Pioglitazone, therefore, the present work illustrates the robustness and ruggedness ofHPLCanalytical method as compared to spectrophotometric method for the analysis of Pioglitazone hydrochloride in solid dosage form.Before starting the work, a thorough search of literature was carried out using various search engines to study methods available for the quantification of pioglitazone in biological samples. To the best of our knowledge, the HPLC methods reported to-date for pioglitazone are either difficult or requires larger samples after extraction. The liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) methods, although sensitive enough but are costly and not suitable for routine analysis.This analytical method for the pioglitazone hydrochloride was developed by HPLC, and then validated the method according to compendia requirements. Pioglitazone in various pharmaceutical products was determined by this developed method. Different concentrations of standard solution were prepared and run on HPLC and also prepared the sample solution and compare its chromatograms with standard solutions. Assay of these products was in the official limits.And finally, when the results obtained by this method were crosschecked with U.V. method.The precision of the developed method was checked by intra-day repeatability of responses after replicate injections of sample solution with percentage of average as 98.6%, 98.52%, 99.0%, 100.83%, 101.05% and 101.04% showing high precision rate ( Table 1). The specificity of the developed method was observed by analysing placebo (including all the ingredients of the formulation except the analyte), standard solution and market formulation containing pioglitazone. No peaks were observed close to the wavelength of detection and hence proved high specificity of the method. (Table 4). The accuracy was evaluated at three different successive concentrations using the proposed developed method and the value as expressed as percentage of recovery (R%) between the average concentrations. The average percentage of recovery was found to be 100.90%, 100.0% and 99.19% for 0.15, 0.200 and 0.240 mg/ml respectively ( Table 2).The quantitation of the active drugs was determined for two commercial formulations (Dowglit and Gliden tablets) and the potency of pioglitazone by developed HPLC method was found to be 100.40% and 100.9% respectively as compared to the UV/visible spectrophotometric method (98.44% and 100.988% respectively). Therefore, it was found that HPLC method is a precise and accurate,highly sensitive, rapid and effective, mixture of substance can be analysed without any interference and gave results with less errors.

CONCLUSION
The objective of better quantitative determination of pioglitazone in solid dosage form was obtained by more sensitive high performance liquid chromatographic method which was approved and validated by the guidelines of FDA, ICH and USP. In spite of the good separation and resolution of the chromatographic peaks, it is concluded that HPLC is more sensitive, rapid and effective method for the determination of Pioglitazone hydrochloride in raw material and finished pharmaceutical products. Although it requires high operating techniques but it is more reliable for both qualitative and quantitative analysis of pioglitazone hydrochloride in raw material and solid dosage forms. Also, its accuracy has proved with comparative study with U.V. Visible spectroscopy.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.