Pioglitazone belongs to the class Thiazolidinediones. Pioglitazone is an antihyperglycemic used as an adjunct to diet, exercise, and other antidiabetic medications to manage type 2 diabetes mellitus.  Pioglitazone is a selective agonist at peroxisome proliferator-activated receptor-gamma (PPAR?) in target tissues for insulin action such as adipose tissue, skeletal muscle, and liver. Activation of PPAR? increases the transcription of insulin-responsive genes involved in the control of glucose and lipid production, transport, and utilization. Through this mechanism, pioglitazone both enhances tissue sensitivity to insulin and reduces the hepatic production of glucose (i.e. gluconeogenesis) - insulin resistance associated with type 2 diabetes mellitus is therefore improved without an increase in insulin secretion by pancreatic beta cells.

Glimepiride is a member of the second-generation sulfonylurea (SU) drug class used for the management of type 2 diabetes mellitus (T2DM) to improve glycemic control. Glimepiride blocks the ATP-sensitive potassium channel by binding non-specifically to the B sites of both sulfonylurea receptor-1 (SUR1) and sulfonylurea receptor-2A (SUR2A) subunits as well as the A site of SUR1 subunit of the channel to promote insulin secretion from the beta cell.

MATERIALS AND METHODS

• Materials: Pioglitazone, Glimepiride, combination of Pioglitazone and Glimepiride, HPLC grade Methanol, acetonitrile, water.
•   Instrument: HPLC instrument used was of WATERS HPLC SYSTEM 2690/5 with Auto Injector and PDA Detector. Software used is Empower 2. UVVIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz was be used for measuring absorbance for Pioglitazone and Glimepiride solutions.
• Preparation of solutions:

Preparation of a stock solution: 100 mg of Pioglitazone and Glimepiride each were weighed accurately and dissolved separately in 100 ml volumetric flasks,  sonicated for 20 min to obtain 1000g/ml of Pioglitazone and Glimepiride respectively.

Preparation of sample stock solution: Twenty tablets were weighed and the average weight of each tablet was calculated, and a quantity of tablet powder equivalent to 100 mg of Pioglitazone and 100mg Glimepiride were weighed and dissolved in 100 ml of diluent with the aid of ultra sonication for 20 min to furnish a sample stock solution.

Preparation of working standard solution: From the above standard stock solution 4 ml from each solution was taken into a 100 ml volumetric flask then made up the volume with diluents and sonicated for 10 min and filtered through 0.45?m membrane filter.

Preparation of sample working solution: The sample stock solution was filtered through a 0.45 ?m nylon syringe filter and 4 ml of the filtrate was diluted into 100 ml volumetric flask to give a sample solution containing 40?g/ml Pioglitazone and 40?g/ml Glimepiride.

Method Validation:

System suitability parameters: The system suitability parameters were determined by preparing standard solutions of Pioglitazone (40ppm) and Glimepiride (40ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined. The % RSD for the area of six standard injections results should not be more than 2%.

Specificity: Specificity is checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Linearity: Linearity for the drugs Pioglitazone and Glimepiride was determined by preparing the standard solutions at seven concentrations levels in six replicates in the range of 20-80?g/ml for Pioglitazone and 20-80?g/ml for Glimepiride from stock solution.

Accuracy: Accuracy was performed by spiking known amounts of standard solution to sample solution at three different concentrations levels (50%, 100%, 150%). % recovery should be between 98-102%.

Precision: The precision of the analytical method was studied by injecting six replicates of standard containing 40?g/ml of Pioglitazone and 40?g/ml of Glimepiride which were injected into HPLC system. The % RSD should not be more than 2.0%

 Limit of Detection (LOD) and Limit of Quantification (LOQ):

The limit of detection was defined as the concentration which yields a signal - to – noise ratio 3:1 where as the limit of quantification was calculated to be the lowest concentration that could be measured with signal - to – noise ratio10:1. LOD and LOQ were calculated from slope and standard deviation.  

Robustness: The smallest deliberate changes in method like change in flow rate are made but there were no predictable changes in the results and are in the range as per ICH guidelines. Conditions like flow rate minus (0.8 ml/min), flow rate plus (1.2 ml/min) was maintained and sample were injected in duplicate manner. System suitability parameters should not be much affected.  

Assay: Assay was conducted on marketed formulation and mean % assay was found.

RESULTS AND DISCUSSION

Optimized wavelength selected was 289nm.

METHOD DEVELOPMENT:

Method development was done by changing various, mobile phase ratios, buffers etc.

Trial - 1 Chromatographic conditions:

Flow rate                           : 1.0ml / min

Column                              : Inertsil-C18, ODS

Mobile Phase                     : Degassed Acetonitrile : Water (90:10) .

Detector Wave length        : 289nm

Injection Volume                : 20?l

Run time                            : 6 min

Retention time                   : 3.156 min for Pioglitazone and 4.417 min for Glimepiride

Result                                : Tailing and fronting was observed for both the drugs.So next

                                        trail was performed.          

       
           
       
Fig. 1: Chromatogram of trial 1

Mobile phase         :  Water: Methanol (45:55)

Flow rate               : 1.0ml / min

Column                 :  Inertsil-C18, ODS

Wave length         :  289nm

Injection volume   : 20?l

Run Time              : 6 min

Retention time      : 2.838 min for Pioglitazone and 3.602 min for Glimepiride.

Result      : The drug peaks were merged and tailing was observed.  So next trail was performed.                                                        

       
           
       
Fig. 2: Chromatogram of trial 2

Flow rate                         : 1.0ml / min

Column                            : Inertsil-C18, ODS

Detector wave length      : 289nm

Mobile phase                   :  Acetonitrile: Methanol (10:90)

Injection volume             :  20?l

Run Time                         : 6 min

Retention time                 : 2.902 min for Pioglitazone and 3.618 min for Glimepiride.

Result                              : The  two peaks were merged and tailing was observed. So, next

                                           trial was performed

       
           
       
Fig. 4: Optimized Chromatogram

METHOD VALIDATION

1. System Suitability

       
           
       
Fig. 5: System Suitability Chromatogram

Table 1: System Suitability data of Pioglitazone and Glimepiride

Discussion: The % RSD for peak areas of standard solutions of Pioglitazone and Glimepiride was 0.053 and 0.073 respectively. The number of theoretical plates for standard solutions of Pioglitazone and Glimepiride was 7537 and 8359 respectively. The Tailing factor for the standard solutions of Pioglitazone and Glimepiride was 1.063 and 1.060 respectively.

2. Specificity

           
           
       
Fig. 6: Chromatogram of Blank

       
           
       
Fig. 7: Chromatogram of Standard

3. Precision:  

1. System Precision     

       
           
       
Fig. 8: System Precision Chromatogram

Table 2: System precision Data of Pioglitazone and Glimepiride

Inference: The % RSD for System Precision of Pioglitazone and Glimepiride was 0.042 and 0.057 respectively.

(b)Method precision:

       
           
       
Fig. 9: Method precision chromatogram

Table 3: Method Precision data of Pioglitazone and Glimepiride

Discussion: The % RSD for Method Precision of Pioglitazone and Glimepiride was 0.054 and 0.052 respectively.

4. Accuracy (Recovery):

       
           
       
Fig. 10:  Chromatogram of Accuracy-50%

       
           
       
Fig. 11: Chromatogram of Accuracy-100%

       
           
       
Fig. 12:  Chromatogram of Accuracy-150%

Table 4: Accuracy Data of Pioglitazone

Table 5: Accuracy Data for Glimepiride

Discussion: Mean % Recovery of Pioglitazone and Glimepiride was 100.16% and 100.29 % respectively.

5. Linearity:

Table 6: Linearity data of Pioglitazone

       
           
       
Fig. 13: Calibration curve of Pioglitazone

Table 7: Linearity data of Glimepiride

       
           
       
Fig. 14: Calibration curve of Glimepiride

6. Robustness:

       
           
       
Fig. 16: Chromatogram of flow rate of 1.0ml/min

       
           
       
Fig. 17: Chromatogram of flow rate of 1.2ml/min

Table 8: Robustness data of Pioglitazone with change in flow rate

Table 9:  Robustness data of Glimepiride with change in flow rate

Discussion: The tailing factor for change in flow rate for Pioglitazone and Glimepiride was 0.023 and 0.027 respectively.

7. Limit of Detection and Limit of Quantitation: 

Table 10: Sensitivity data of Pioglitazone and Glimepiride

        
           
       
Fig. 18: Assay chromatogram of Pioglitazone and Glimepiride

Table 11: Assay data of Pioglitazone and Glimepiride

Discussion:  Mean % assay of Pioglitazone and Glimepiride 100.15 and 100.45 respectively.

SUMMARY AND CONCLUSION

Table 12: Summary table