Development and Validation for the Simultaneous Estimation of Pioglitazone Hydrochloride and Glimipride in Mixed Dosage Form by RP-HPLC Method

Rajini.Talla*, M. Akiful Haque, K.Veena, D. Sireesha, S. Harshini, B. Vasudha

Department of Pharmaceutical Analysis and Quality Assurance, School of Pharmacy, Anurag Group of Institutions, Hyderabad Telangana, India.

*Corresponding Author E-mail: rajini.talla796@gmail.com

ABSTRACT:

A simple, accurate, precise and sensitive reverse phase high performance liquid chromatography (RP-HPLC) method has been developed, which can separate and quantitatively estimate Pioglitazone Hydrochloride and Glimipride in mixed dosage form. The chromatographic separation for Pioglitazone Hydrochloride and Glimipride was achieved with mobile phase containing acetonitrile and phosphate buffer P^H 4 (70:30 % v/v), reverse phase Thermo scientific^TMHypersil^TMBDS 5µ C18 120A (250 × 4.60 mm i.d ) column in isocratic mode at room temperature and UV detection 232nm. The compounds were eluted at a flow rate of 1.2 ml/min. The retention times of Pioglitazone Hydrochloride and Glimipride were found to be 3.301min and 3.854min respectively. The above method was validated in terms of System suitability, linearity, accuracy, precision, Limit of Detection (LOD), Limit of Quantification (LOQ) in accordance with ICH guidelines. The method was rapid, simple, economical and suitable for routine quality control analysis.

KEYWORDS: Pioglitazone Hydrochloride and Glimipride, Analytical method development, validation, RP-HPLC

INTRODUCTION:

Pioglitazone Hydrochloride (PIO) is a thiazolidinedione derivative. Chemically, it is 5-({4-[2-(5-ethylpyridin-2-yl) ethoxy] phenyl} methyl)-1, 3-thiazolidine-2, 4-Dione. It is used for the treatment of diabetes mellitus type 2 (previously known as non-insulin-dependent diabetes mellitus, NIDDM) in monotherapy and in combination with a sulfonylurea, metformin, or insulin and Glimipride.^[1]

Fig 1. Structure of Pioglitazone Hydrochloride

Glimepiride is a sulfonylurea derivative chemically 3-ethyl-2, 5-dihydro-4-methyl-N [2-[4-s (trans-4-methyl cyclohexyl) amino] carbonyl] amino] sulfonyl] phenyl] ethyl]-2-oxo-1Hpyrrole-1-carboxamide1, widely used in patient with type 2 diabetic mellitus.^[2]

Fig 2. Structure of Glimipride

Both the drugs are marketed as combined dose tablet formulation in the ratio of 15:2 mg PIO: GLM. Literature survey revealed that there is no method reported for the simultaneous estimation of these drugs, some methods for estimation of individual drugs or with other drugs UV-Spectrophotometry^[3-8], RP-HPLC^[9] are available. HPLC method is more sensitive compared to UV, Therefore, there is a challenge to develop RP-HPLC method for the simultaneous estimation of Pioglitazone Hydrochloride and Glimipride. The present study was involved in a research effort aimed at developing and validating a simple, accurate, precise and economical RP-HPLC method for the simultaneous estimation of Pioglitazone Hydrochloride and Glimipride in mixed dosage form.

MATERIALS AND METHODS:

Methanol (HPLC grade) was obtained from Merck specialties private limited, Mumbai, India. Water (HPLC grade) was obtained from Merck specialties private limited, Mumbai, India. Ortho-phosphoric acid (GR grade) was obtained from S.D. Fine Chemicals Ltd, Mumbai, India. Potassium Dihydrozen orthophosphate (KH2PO4) was obtained from Merck specialties private limited Mumbai, Dipotassium hydrogen phosphate (K2HPO4) S.D Fine Chemical ltd. Mumbai. Pure drugs of Pioglitazone Hydrochloride and Glimipride were obtained as gift samples from comprime labs (Hyderabad, India). PIOGLAR-G tablets manufactured by Ranbaxy Pharmaceutical Limited purchased from pharmacy is used for the analysis. The label claim states that this formulation contains 15mg of Pioglitazone Hydrochloride and 2mg of Glimipride of each tablet.

Instrumentation and Chromatographic Condition:

HPLC method development and validation were done on SHIMADZU (Japan) liquid chromatograph equipped with LC-20AD pump, LC 20A UV/Vis detector and Rheodyne 7725i injection with a 20 µL loop. For instrument control, data acquisition and processing, the chromatographic system was interfaced to LC solutions software. Other instruments included are Shimadzu electronic balance BL-220H (SHIMADZU corporation, Japan), Value 1 stage vacuum pump Model: VE115, Fast clean ultrasonic cleaner. The column used for chromatographic separations was reverse phase Thermo Scientific^TMHypersil^TMBDS 5µ C18 120A (250 × 4.60 mm i.d). The analytical wave length was set as 232nm and samples of 20 µL were injected. The chromatographic separations were accomplished using mobile phase consisting of Acetonitrile:phosphate buffer PH-4 (70:30 %v/v), filtered through 0.45 µm filter using Value 1 stage vacuum pump and desecrated in fast clean ultrasonic cleaner. Mobile phase was pumped in isocratic mode at a flow rate of 1.2 ml/min at room temperature.

Preparation of standard solutions:

Stock solutions were prepared by dissolving 15mg of Pioglitazone Hydrochloride, 2mg of Glimipride in 10 ml of methanol separately. Aliquots of the standard stock solutions of Pioglitazone Hydrochloride and Glimipride were transferred into 10 ml volumetric flasks and solution was made up to the volume to yield required concentrations of Pioglitazone Hydrochloride and Glimipride. A typical chromatogram obtained from the analysis of drugs using the developed method is shown in Fig.3.

Preparation of sample solution for assay:

Twenty Pioglar-G tablets each containing 15mg of Pioglitazone Hydrochloride and 2mg of Glimipride were weighed, average weight was calculated and powdered. A quantity equivalent to 15mg of Pioglitazone Hydrochloride and 2mg of Glimipride was weighed and transferred into 10 ml volumetric flask. It was extracted with methanol. The volumetric flask was sonicated for 5 minutes to affect the complete dissolution of the drugs and the solution was made up to the volume with methanol and filtered. Suitable aliquots of formulation solution were prepared and injected to HPLC to obtain concentration in the linearity range.

RESULTS AND DISCUSSION:

Method Development:

For the RP-HPLC, chromatographic conditions were optimized to get best resolution and peak shape. The selection of mobile phase was based on peak parameters like symmetry, theoretical plates and capacity factor. Symmetrical peaks with good separation (retention time for Pioglitazone Hydrochloride was 3.301 min and Glimipride was 3.854min) were obtained with reverse phase Thermo Scientific^TMHypersil^TMBDS 5µ C18 120A (250 × 4.60 mm i.d ) column. The mobile phase containing Acetonitrile:phosphate buffer PH-4(70:30 %

v/v) was used at a flow rate of 1.2 ml/min. The optimum wavelength for detection and quantification was at 232nm, at which good detector response was obtained for both the the results are given in Table 1.

Method validation:

As per ICH guidelines, the method validation parameters checked were linearity, precision, accuracy, limit of detection and limit of quantification.^[10]

Linearity and Range:

Linearity was established by least squares linear regression analysis of the calibration curve. The calibration curves were linear over the concentration range of 1-35μg/ml for Pioglitazone Hydrochloride, 1-10μg/ml for Glimipride. Peak areas were plotted versus respective concentrations and linear regression analysis was performed on the resultant curves. Correlation coefficients were found to be 0.999 and 0.999 for Pioglitazone Hydrochloride and Glimipride respectively (Fig.5 and Fig.6).The results are given in Table 1.

Precision:

The precision of the analytical method was studied by multiple sampling of the homogenous sample. The precision was done at two levels (intraday and inter day). Intraday precision was done by analyzing the intermediate concentration of each drugs (Pioglitazone Hydrochloride 15µg/ml and Glimipride 2µg/ml) for six times. Interday precision was measured over three consecutive days for the same drug concentrations for six times. The %RSD values were found to calculated for each of them and the low RSD values indicate that the method is precise. The results are given in Table 1.

Table 1: Summary of validation parameters

Parameters

Pioglitazone Hydrochloride

Glimipride

Linearity range(μg/ml)

Correlation coefficient

Slope

Intercept

LOD(μg/ml)

LOQ(μg/ml)

Recovery (%)

100

120

Precision (RSD %)

Intraday(n=6)

Interday(n=6)

Theoretical plates

Tailing factor

Resolution

1-35

0.9985

34199

27740

0.317

0.961

99.2%

99%

100%

1.05

1.67

4818.213

0.900

2.610

1-10

0.999

30551

4169

1.46

4.29

102.5%

102%

99.3%

1.43

0.47

4415.030

1.010

Accuracy:

Recovery studies were carried out by applying the method to drug sample to which known amount of standard Pioglitazone Hydrochloride and Glimipride corresponding to 80, 100 and 120 % of label claim had been added. At each level of the amount three determinations were performed. The results are given in Table 1

Sensitivity:

LOD and LOQ decide about the sensitivity of the method. LOD is the lowest detectable concentration of the analyte by the method while LOQ is the minimum quantifiable concentration. LOD and LOQ were calculated by standard calibration curves. The results are given in Table 1.

System Suitability Studies

System suitability parameters like number of theoretical plates (N), peak asymmetry factor (As), tailing factor etc., were studied. The results are given in Table 1.

Analysis of Marketed Formulation:

The proposed procedures were successfully applied for the simultaneous estimation of Pioglitazone Hydrochloride and Glimipride in the formulation and the drug contents in each sample were calculated by comparison with the appropriate standard solution of the drug. The results obtained were in agreement with label claim. The results of analysis are given in (Table 2) (Fig. 4).

CONCLUSION:

RP-HPLC methods enable the quantization of Pioglitazone Hydrochloride and Glimipride in oral dosage form with good accuracy and precision, either in laboratory prepared samples or in pharmaceutical dosage forms. The good recoveries were obtained in all cases as well as the reliable agreement with the reported procedure proved that the proposed methods could be applied efficiently for determination of Pioglitazone Hydrochloride and Glimipride in oral dosage form with satisfactory precision. This method is considered simple, reliable, economical providing satisfactory accuracy, precision with lower limits of detection and quantification more sensitive. More over the shorter duration of analysis for Pioglitazone Hydrochloride and Glimipride makes the reported method suitable for routine analysis in mixed dosage form

ACKNOWLEDGEMENT:

I am very thankful to Anurag group of institution (Formerly Lalitha college of pharmacy), Hyderabad. For giving permission to carry out my work

REFERENCES:

1. http://www.drugbank.ca/drugs/DB01132.

2. http://www.drugbank.ca/drugs/DB00222.

3. Sakala Bhargavi, Gopisetty Suryasagar, Dantu Krishna Sowmya, Kota Ashok, Sreekanth Nama.UV Spectrophotometric Method for Determination of Glimepiride in Pharmaceutical Dosage Forms. International Journal of Pharmaceutical Sciences Review and Research, 21(2), July – August 2013; nᵒ 23, 131-133.

4. Seema M.Dhole, Pramod B. Khedekar, Nikhil D. Amnerkar.UV Spectrophotometric Absorption Correction Method for the Simultaneous Estimation of Pioglitazone HCl,Metformin HCl and Glibenclamide in Multicomponent Formulation. International Journal of Analytical and Bioanalytical Chemistry 2013; 3(1): 18-22.

5. L Adhikari, S Jagadev, S Sahoo P.N Murthy U.S Mishra. Development and Validation of UV-Visible Spectrophotometric Method for Simultaneous Determination of pioglitazone Hydrochloride, Metformin Hydrochloride and Glipizide in its Bulk and Pharmaceutical Dosage Form (Tablet). International Journal of ChemTech Research. April-June (2012) Volume 4, No.2, pp 625-630.

6. Kottu P. K., Gadad A.P. and Dandagi P. M. Method development and validation for the simultaneous estimation of pioglitazone and glimepiride – a uv spectrophotometric approach. Indian drugs 49(11) november 2012,pp-30-35.

7. Mamdouh R. Rezk, Safa'a M. Riad, Ghada Y. Mahmoud and Abdel-Aziz El Bayoumi Abdel Aleem. Simultaneous determination of pioglitazone and glimepiride in their pharmaceutical formulations. Scholars Research Library,Der Pharma Chemica, 2011, 3 (5):176-184.

8. Indrajeet Singhvi, Khushboo Mehta and Nidhi Kapadiya, Analytical method development and validation for the simultaneous estimation of pioglitazone and glimepiride in tablet dosage form by multiwavelength spectroscopy. Journal of Applied Pharmaceutical Science 01 (06); 2011: 159-161.

9. K.Sujana, G.Swathi Rani, M.Bhanu Prasad, M.Saheethi Reddy. Simultaneous Estimation of Pioglitazone Hydrochloride and Metformin Hydrochloride using UV Spectroscopic Method. Journal Biomed Science and Research(JBSR)., Vol 2 (2), 2010, 110-115.

10. Freddy H. Havaldar and Dharmendra L.Vairal. Simultaneous Estimation of Glimepiride, Rosiglitazone and Pioglitazone Hydrochloride in the Pharmaceutical Dosage Form. E-Journal of Chemistry 2010, 7(4), 1326-1333.

Received on 16.10.2015 Modified on 06.11.2015

Asian J. Research Chem. 8(12): December 2015; Page 711-715

DOI: 10.5958/0974-4150.2015.00114.5

Drug	Amount labeled(mg)	Amount found(mg)	%Assay	%RSD*
Pioglitazone Hydrochloride	15	15.2	101	0.65
Glimipride	2	2.05	102.5	1.31