1. INTRODUCTION:^1-6

Diabetes mellitus type 2 – generally –known as non-insulin dependent diabetes mellitus (NIDDM) or also as an adult-onset diabetes.

It is a metabolic issue involving high blood glucose (hyperglycemia) in the setting of insulin resistance and relative insulin lack. Metformin hydrochloride (initially sold as Glucophage) is an oral antidiabetic medication in the biguanide class. Pioglitazone hydrochloride act as an anti-diabetic medication of thiazolidinedione-type, likewise called "glitazones".

Pioglitazone hydrochloride and Metformin hydrochloride tablets are used to upgrade glycemic control in adults with Type 2 diabetes mellitus. It is the first line medication for the treatment of type 2 diabetes.

HPTLC is enhanced form of thin layer chromatography. It is a powerful separation tool for quantitative analysis.

Different changes can be made in the methods of thin layer chromatography to robotize the diverse steps, to expand the determination achieved and to permit more exact quantitative estimations.

HPTLC is an effective instrument for the chromatographic data of complex blend of organic, inorganic and natural compound.

Widely used instrument software for the HPTLC is from CAMAG, Switzerland. It provides automated sample application, plate development, detection and documentation.

2. MATERIALS AND METHODS⁶:

Camag HPTLC system was used during the study. The chromatographic separation were carried out using silica gel precoated aluminium plate 60 F₂₅₄, (20×10 cm² and 10×10 cm²) as stationary phase and mobile phase consisting of butanol:1,4-dioxane:glacial acetic acid in the ratio of 5:3:2 v/v/v. The wavelength selected for detection was 226 nm. This method was used for determination of Metformin hydrochloride and Pioglitazone hydrochloride from pure powder mixture and tablet dosage form. Stability indicating HPTLC method was performed by keeping sample solution in different stress condition. The stability study was performed for acid degradation, photolytic degradation and thermal degradation.

2.1 Preparation of Standard Stock Solution^7-21:

2.1.1 Metformin hydrochloride:

Accurately weighted 250 mg of Metformin hydrochloride standard drug was transferred into a 25 mL volumetric flask. Drug was dissolved in few mL of methanol and volume was made up to the mark with methanol. 2 mL of this prepared solution was withdrawn and transferred into 10 mL volumetric flask. The volume was made up to mark with methanol to give a final standard stock solution of 2000 ng/μL of Metformin hydrochloride.

2.1.2 Pioglitazone Hydrochloride:

Accurately weighted 7.5 mg of Pioglitazone hydrochloride standard drug was transferred into a 25 mL volumetric flask. Drug was dissolved in few mL of methanol and volume was made up to the mark with methanol. 2 mL of this prepared solution was withdrawn and transferred into 10 mL volumetric flask. The volume was made up to mark with methanol to give a final standard stock solution of 60 ng/μL of Pioglitazone hydrochloride.

2.1.3 Preparation of Standard Stock Solution for Mixtures of Metformin Hydrochloride and Pioglita-zone Hydrochloride:

A 2 mL stock solution each of Metformin hydrochloride (2000 ng/μL) and Pioglitazone hydrochloride (60 ng/μL) were transferred in to 10 mL volumetric flask and volume was made up to mark with methanol, so the final concentration of mixture becomes 400 ng/μL of Metformin hydrochloride and 12 ng/μL of Pioglitazone hydrochloride.

2.2 Analysis of Powder Mixture:

A 10 μL sample of mixed standard of Metformin hydrochloride and Pioglitazone hydrochloride containing 400 ng/μL of MET and 12 ng/μL of PIO (4000 ng/band MET and 120 ng/band PIO) was applied on pre-coated silica gel 60 F₂₅₄TLC plates (E. Merck) using Camag Linomat V (Switzerland) automatic sample spotter. The plate was developed in the solvent system consisting of butanol, 1, 4-dioxane, glacial acetic acid in a ratio of (5:3:2 v/v/v) and two drops of formic acid. After complete separation of two drug, the plate was dried at room temperature and scanned using CAMAG TLC scanner 3 at UV 226 nm and Rf values, absorption spectra of bands and their area were recorded and concentrations of each drug from standard drug mixture were calculated using standard calibration curve and data was tabulated.

2.3 Linearity^7-21:

2.3.1 Preparation of Calibration Curve of Metformin Hydrochloride:

1 µL (2000 ng), 3.0 µL (i.e., 6000 ng), 5.0 µL (i.e., 10000 ng), 7.0 µL (i.e., 14000 ng) and 9.0 µL (i.e., 18000 ng) samples of Metformin hydrochloride solution of 2000 ng/µL was spotted on pre-coated silica gel 60 F₂₅₄TLC plates (E. Merck) using Camag Linomat V (Switzerland) automatic sample spotter. The plate was developed in the solvent system consisting of butanol, 1, 4-dioxane, glacial acetic acid in a ratio of 5:3:2 v/v/v and two drops of formic acid. The plate was dried at room temperature and scanned using CAMAG TLC scanner 3 at UV 226 nm and Rf values, absorption spectra of bands and their area were recorded. Calibration curve was plotted using area obtained at 226 nm (Figure 1).

2.3.2 Preparation of Calibration Curve of Pioglitazone Hydrochloride:

1 µL (i.e., 60 ng), 3.0 µL (i.e., 180 ng), 5.0 µL (i.e., 300 ng), 7.0 µL (i.e., 420 ng) and 9.0 µL (i.e., 540 ng) samples of Pioglitazone hydrochloride solution of 60 ng/µL was spotted on pre-coated silica gel 60 F₂₅₄TLC plates using Camag Linomat V (Switzerland) automatic sample spotter. The plate was developed in the solvent system consisting of butanol, 1, 4-dioxane, glacial acetic acid in a ratio of 5:3:2 v/v/v and two drops of formic acid. The plate was dried at room temperature and scanned using CAMAG TLC scanner 3 at UV 226 nm and Rf values, absorption spectra of bands and their area were recorded. Calibration curve was plotted using area obtained at 226 nm (Figure 2).

2.4 Precision^7-21:

In intraday precision, standard solution containing Metformin hydrochloride (2000 ng/µL) and Pioglitazone hydrochloride (60 ng/µL) were analysed six times on the same day and area of band were measured as per analysis of standard drug mixture and % RSD was calculated. Interday precision was obtained by the assay of six sample sets on different days. (Table 6, 7, 8, 9)

2.5 Analysis of marketed Formulation:^7-21

Accurately 20 tablets containing Metformin hydrochloride and Pioglitazone hydrochloride were weighed. Their average weight was determined. A tablet powder equivalent to 500 mg MET and 15 mg of PIO was weighed and transferred to a 50 mL volumetric flask. Few mL (around 20 mL) of methanol was added to the flask, shaked well and sonicated for 30 min. The volume was made up to the mark with methanol. The solution was filtered with Whatman filter paper. The filtrate was collected in a beaker. Then 2 mL of filtrate was transferred into 10 mL volumetric flask. The volume was made up to the mark with methanol to obtain a concentration of 2000 ng/μL of Metformin hydrochloride and 60 ng/μL of Pioglitazone hydrochloride. A 2 μL sample of tablet containing 2000 ng/μL of Metformin hydrochloride and 60 ng/μL of Pioglitazone hydrochloride (4000 ng/band MET and 120 ng/band PIO) was applied on pre-coated TLC plate and their area were recorded. Amount of MET and PIO were determined from standard calibration curve and data was tabulated (Table 12, 13, 14).

2.6 Accuracy/Recovery:^7-21

Recovery studies were carried out by applying the method to drug sample present in tablet dosage form to which known amount of Metformin hydrochloride and Pioglitazone hydrochloride corresponding to 80%, 100% and 120% of label claim was added (standard addition method). In 80% recovery study, amount of standard added is 400 mg of Metformin hydrochloride and 12 mg Pioglitazone hydrochloride per tablet. In 100% recovery study the amount of standard added is 500 mg of Metformin hydrochloride and 15 mg Pioglitazone hydrochloride per tablet. In 120% recovery study the amount of standard added is 600 mg of Metformin hydrochloride and 18 mg Pioglitazone hydrochloride per tablet. In each case, after the addition of pure standards into tablet triturate, the required amount of mixed powder (powder equivalent to 500 mg MET, 15 mg of PIO) was weighed and transferred to a 50 mL volumetric flask. Few mL (around 20 mL) of methanol was added to the flask. It was shaked well and sonicated for 30 min. The volume was made up to the mark with methanol. The solution was filtered with whatman filter paper. The filtrate was collected in a beaker. Then 2 mL of filtrate was transferred into 10 mL volumetric flask. The volume was made up to the mark with methanol to obtain a concentration of 2000 ng/μL of Metformin hydrochloride and 60 ng/μL of Pioglitazone hydrochloride.

A 2 μL sample of recovery studies (containing 4000 ng/band MET, 120 ng/band PIO) was applied on pre-coated TLC plate and their area were recorded. Amount of MET and PIO were determined from standard calibration curve and data was tabulated (Table 15, 16, 17, 18). At each level three different determinations were performed for recoveries study.

2.7 Stability Indicating Study of Metformin Hydrochloride and Pioglitazone Hydrochloride:^13,14,22-28

Stability study of Metformin hydrochloride and Pioglitazone hydrochloride were done in various conditions. Both drugs were kept in acidic, photolytic and thermal condition for particular period of time.

2.7.1 Acid Degradation:

Accurately 250 mg Metformin hydrochloride and 7.5 mg of Pioglitazone hydrochloride pure powder were weighed and 40 mL methanol and 40 mL of 0.5 N HCl solution were added. Then 10 mL of solution was withdrawn in a series of 4 different glass beakers. The solution was refluxed for 15 min., 30 min. and 60 min. and kept at normal temperature for 60 min. At the end of this degradation process, 10 mL of sample was taken and neutralized with 0.5 N NaOH from each beaker separately. The volume was made up to the mark with methanol in 50 mL volumetric flask in order to obtain a concentration of 2000 ng/μL of Metformin hydrochloride and 60 ng/μL of Pioglitazone hydrochloride. A 2µL solution was applied on TLC plate, so concentration becomes 4000 ng/band of Metformin hydrochloride and 120 ng/band of Pioglitazone hydrochloride (Table 19).

2.7.2 Photolytic Degradation:

Accurately 250 mg Metformin hydrochloride and 7.5 mg of Pioglitazone hydrochloride pure powder were weighed. The powder was kept in UV chamber for 3 hrs, 6 hrs and 24 hrs. The powder was transferred in a 25 mL volumetric flask. Volume was made upto the mark with methanol. From these solutions 2 mL was transferred in a series of 3 different 10 mL volumetric flask. Serial dilutions were performed in order to obtain a concentration of 2000 ng/μL of Metformin hydrochloride and 60 ng/μL of Pioglitazone hydrochloride. A 2µL solution was applied on TLC plate, so concentration becomes 4000 ng/band of Metformin hydrochloride and 120 ng/band of Pioglitazone hydrochloride (Table 20).

2.7.3 Thermal Degradation:

Accurately 250 mg Metformin hydrochloride and 7.5 mg of Pioglitazone hydrochloride pure powder were weighed. The powder was kept in an oven at 80⁰C for 3 hrs, 6 hrs and 24 hrs. The powder was transferred in a 25 mL volumetric flask. Volume was made upto the mark with methanol. From these solutions, 2 mL was transferred in a series of 3 different 10 mL volumetric flask. Serial dilutions were performed in order to obtain a concentration of 2000 ng/μL of Metformin hydrochloride and 60 ng/μL of Pioglitazone hydrochloride. A 2µL solution was applied on TLC plate, so concentration becomes 4000 ng/band of Metformin hydrochloride and 120 ng/band of Pioglitazone hydrochloride (Table 21).

3. RESULTS:

3.1 Linearity:

3.1.1 Linearity for Metformin Hydrochloride:

Table 1. Calibration Table of Metformin Hydrochloride at 226 nm

Sr. No.	Concentration (ng/band)	Area under Curve(AUC)	Rf Value	Rf Avg.
1	2000	14390.66	0.15	0.15± 0.02
2	6000	30087.36	0.15
3	10000	39586.50	0.15
4	14000	51337.80	0.15
5	18000	62642.90	0.15

Figure 1. Standard Calibration Curve of Metformin Hydrochloride

3.1.2 Linearity for Pioglitazone Hydrochloride

Table 2. Calibration Table of Pioglitazone Hydrochloride at 226 nm

Sr. No.	Concentration (ng/band)	Area under Curve(AUC)	Rf Value	Rf Avg.
1	60	3129.9	0.72	0.72± 0.02
2	180	6502.1	0.72
3	300	10273.0	0.72
4	420	12973.4	0.72
5	540	17847.8	0.72

3.1.3 Regression data for linearity of Metformin Hydrochloride and Pioglitazone Hydrochloride

Table 3. Regression data for linearity of Metformin Hydrochloride and Pioglitazone Hydrochloride

Drug	Linearity Range (ng/band)	Slope*	Intercept*	Regression coefficient (r²)*
MET	2000 to 20000	2.87	10171.78	0.9943
PIO	60 to 540	29.87	1167.31	0.9932

*n=6

Figure 2. Standard Calibration Curve of Pioglitazone Hydrochloride

3.2 Powder mixture analysis

Table 4. Table of powder Mixture Analysis

Sr. No.	Amount of drug present (ng/band)		Amount of drug found (ng/band)		% Drug found
Sr. No.	MET	PIO	MET	PIO	MET	PIO
1	4000	120	4010.3	119.8	100.25	99.83
2	4000	120	3942.9	120.7	98.50	100.58
3	4000	120	3969.8	119.1	99.24	99.25
4	4000	120	4001.2	120.4	100.03	100.34
5	4000	120	3992.7	119.7	99.81	99.75
6	4000	120	3989.7	119.9	99.74	99.92

3.2.1 Statistical Validation of powder mixture analysis

Table 5. Data for Statistical Validation of powder mixture analysis

Drug	Mean % Drug found*	Standard Deviation*	% Relative Standard Deviation*	Standard Error*
MET	99.98	0.2154	0.21	0.0879
PIO	100.015	0.6067	0.61	0.2477

* n = 6

3.3 Precision

3.3.1 Intra-day Precision

Table 6. Intra-day Precision for Metformin Hydrochloride and Pioglitazone Hydrochloride

Sr. No.	Amount of drug present (ng/band)		Amount of drug found (ng/band)		% Drug found
Sr. No.	MET	PIO	MET	PIO	MET	PIO
1	4000	120	3956.1	119.6	99.96	99.67
2	4000	120	4013.3	119.2	100.33	99.34
3	4000	120	3992.6	120.6	99.81	100.5
4	4000	120	4007.1	119.3	100.17	99.41
5	4000	120	3989.6	120.8	99.74	100.67
6	4000	120	3990.9	120.6	99.77	100.5

3.3.1.1 Statistical Validation of Intra-day precision

Table 7. Statistical Validation of Intra-day Precision

Drug	Mean % Drug found*	Standard Deviation*	% Relative Standard Deviation*	Standard Error*
MET	99.53	0.7588	0.7623	0.3098
PIO	99.94	0.4678	0.4680	0.1910

* n = 6

3.3.2 Inter-day Precision

Table 8. Inter-day Precision for Metformin Hydrochloride and Pioglitazone Hydrochloride

Sr No	Amount of drug present (ng/band)		Amount of drug found (ng/band)		% Drug found
Sr No	MET	PIO	MET	PIO	MET	PIO
1	4000	120	4013.3	120.7	100.33	100.58
2	4000	120	3992.4	120.4	99.81	100.34
3	4000	120	4006.1	119.6	100.15	99.67
4	4000	120	3989.7	119.6	99.74	99.50
5	4000	120	3994.2	119.4	99.85	99.53
6	4000	120	3987.1	119.9	99.68	99.92

3.3.2.1 Statistical Validation of Inter-day precision

Table 9. Statistical Validation of Inter-day Precision

Drug	Mean % Drug found*	Standard Deviation*	% Relative Standard Deviation*	Standard Error*
MET	99.92	0.2560	0.2562	0.1045
PIO	99.93	0.4479	0.4482	0.1828

*n=6

3.4 Analysis of Marketed Formulation

3.4.1 Chromatogram for Metformin Hydrochloride and Pioglitazone Hydrochloride

Figure.3. Chromatogram for Marketed Formulation in butanol: 1, 4-dioxane: glacial acetic acid (5:3:2) + 2 drops of formic acid at 226 nm

3.4.2 Analysis of Marketed Formulation data for Metformin Hydrochloride

Table 10. Analysis of Marketed Formulation data for Metformin Hydrochloride

Sr. No.	Amount Present (mg/tab)	Amount of drug found (mg/tab)	% Drug found in formulation
1	500	499.20	99.84
2	500	497.90	99.50
3	500	500.10	100.02
4	500	498.90	99.78
5	500	500.51	100.10
6	500	499.40	99.88

3.4.3 Analysis of Marketed Formulation data for Pioglitazone Hydrochloride

Table 11. Analysis of Marketed Formulation Data for Pioglitazone Hydrochloride

Sr. No.	Amount Present (mg/tab)	Amount of drug found (mg/tab)	% Drug found in formulation
1	15	14.92	99.46
2	15	15.12	100.8
3	15	15.09	100.6
4	15	14.97	99.8
5	15	14.90	99.33
6	15	14.98	99.87

3.4.4 Statistical Validation of Marketed Formulation

Table 12. Statistical Validation of Marketed Formulation

Drug	Mean % Drug found*	Standard Deviation*	% Relative Standard Deviation*	Standard Error*
MET	99.85	0.2096	0.21	0.0855
PIO	99.97	0.5990	0.59	0.2445

*n=6

3.5 Accuracy/Recovery

3.5.1 Recovery study for Metformin Hydrochloride

Table 13. Recovery Data for Metformin Hydrochloride from Formulation

Sr. No.	Concentration level (%) Recovery	Amt. present in formulation (mg/tab)	Amount of standard added/Spiked (mg/tab)	Concentration of prepared sol. (ng/band)	Conc. of sol. Found (ng/band)	Total Amount Recovered (mg)	% Recovery
1	80	500	400	4000	3996.8	889.28	99.92
2	80	500	400	4000	3995.5	899.01	99.89
3	80	500	400	4000	4020.8	904.68	100.52
4	100	500	500	4000	3998.8	999.70	99.97
5	100	500	500	4000	3987.2	996.11	99.68
6	100	500	500	4000	4003.2	1000.80	100.08
7	120	500	600	4000	4008.0	1102.31	100.2
8	120	500	600	4000	3996.1	1098.80	99.90
9	120	500	600	4000	3944.8	1084.80	98.62

3.5.1.1 Statistical Validation of Recovery Studies for Metformin Hydrochloride

Table 14. Statistical Validation of Recovery Studies for Metformin Hydrochloride

Sr. No.	Concentration level (%) Recovery	Amt. present in formulation (mg/tab)	Amount of standard added/Spiked (mg/tab)	concentration of prepared sol. (ng/band)	Conc. of sol. Found (ng/band)	Total Amount Recovered (mg)	% Recovery
1	80	15	12	120	118.70	26.71	98.92
2	80	15	12	120	119.56	26.90	99.64
3	80	15	15	120	120.04	27.01	100.04
4	100	15	15	120	120.74	30.18	100.62
5	100	15	15	120	119.86	29.96	99.89
6	100	15	15	120	119.91	29.97	99.93
7	120	15	18	120	120.02	33.00	100.02
8	120	15	18	120	119.61	32.89	99.68
9	120	15	18	120	119.72	32.92	99.77

3.6 Stability Study

3.6.1 Acid degradation Table 17. Acid degradation Data for Metformin Hydrochloride and Pioglitazone Hydrochloride

Sr. No.	Condition of degradation	Conc. of drug (ng/band)		Area		Amount Retained (%)		Degradation (%)
Sr. No.	Condition of degradation	MET	PIO	MET	PIO	MET	PIO	MET	PIO
1	Normal temp.(For 60 min.)	4000	120	3637.2	107.85	90.93	89.88	9.07	10.12
2	Reflux for 15 min.	4000	120	3530.8	106.07	88.27	88.39	11.73	11.61
3	Reflux for 30 min.	4000	120	3498.4	104.84	87.46	87.37	12.54	12.63
4	Reflux for 60 min.	4000	120	3408.1	101.53	85.20	84.61	14.80	15.39

3.6.2. Photolytic degradation

Table 18. Photo degradation Data for Metformin Hydrochloride and Pioglitazone Hydrochloride

Sr. No.	Condition of degradation	Conc. of drug (ng/band)		Area		Amount Retained (%)		Degradation (%)
Sr. No.	Condition of degradation	MET	PIO	MET	PIO	MET	PIO	MET	PIO
1	Exposure for 3 hrs.	4000	120	3597.2	106.76	89.93	88.97	10.07	11.03
2	Exposure for 6 hrs.	4000	120	3506.4	103.77	87.66	86.48	12.34	13.52
3	Exposure for 24 hrs.	4000	120	3339.2	98.26	83.48	81.89	16.52	18.11

3.6.3 Thermal degradation

Table 19. Thermal degradation Data for Metformin Hydrochloride and Pioglitazone Hydrochloride

Sr. No.	Condition of degradation	Conc. of drug (ng/band)		Area		Amount Retained (%)		Degradation (%)
Sr. No.	Condition of degradation	MET	PIO	MET	PIO	MET	PIO	MET	PIO
1	Exposure for 3 hrs.	4000	120	3531.2	105.6	88.28	88.08	11.72	11.92
2	Exposure for 6 hrs.	4000	120	3486.4	104.38	87.16	86.99	12.84	13.01
3	Exposure for 24 hrs.	4000	120	3413.2	99.04	85.33	82.54	14.67	17.46

4. DISCUSSION:

Proposed method for Metformin hydrochloride and Pioglitazone hydrochloride determination in HPTLC method was found to be simple, precise and sensitive. Validation data for linearity are given in table 1, 2, and 3. Validation data for precision are given in table 6, 7, 8, 9.Validation data for powder mixture analysis are given in table 4 and 5.

Validation data for marketed formulation are given in table 10, 11, 12. Validation data for recovery are given in table 13, 14, 15, 16. Stability study data for Metformin Hydrochloride and Pioglitazone Hydrochloride are given in table 17, 18, 19. The standard deviation (S.D.), relative standard deviation (%R.S.D.) and standard error (S.E.) calculated are low, indicating high degree of precision of the method. %RSD was found to be less than 2 which comply with ICH. Metformin hydrochloride and Pioglitazone hydrochloride degrades significantly in acidic, photolytic and thermal condition.

5. CONCLUSION:

From the present study it can be concluded that the proposed method is simple, sensitive, precise, specific, accurate and reproducible and offers several advantages such as rapidity, usage of simple mobile phase and easy sample preparation steps. Result of validation parameters demonstrated that the analytical procedure is suitable for its intended purpose. Further, improved sensitivity makes it specific and reliable for its intended use. Hence this method can be successfully applied for determination of amount of Pioglitazone hydrochloride and Metformin hydrochloride from its pure powder mixture and tablet dosage form.

6. REFERENCES:

1. Tripathi K.D., Essential of medical pharmacology, New Delhi, Jaypee brother, 6th ed, 2003: 235-253.

2. Rang HP, Dale MM, Ritter JM, Flower RJ. Rang and Dale's Pharmacology. 6thed. New York: Churchill Livingstone; 2007: 693.

3. https://www.idf.org/WHO_IDF_definition_diagnosis_of_diabetes.pdf (access on 3^rd March. 2016).

4. Craig CR, Stitzel RE. Modern Pharmacology with Clinical Application, New Delhi, Wolter Kluwer India Pvt. Ltd. 6thed, 2003: 598-600.

5. Sethi PD. HPTLC- Qualitative Analysis of Pharmaceutical Formulation. 1^st Ed. New Delhi: CBS Publisher and Distributors; 1996: 3-30.

6. Braun RD. Introduction to Instrumental analysis. 2nded. Hyderabad: Pharma Med Press; 2012: 949-952.

7. Darshana K. Modi et al. A rapid, simple, and sensitive HPTLC method for simultaneous estimation of metformin hydrochloride and pioglitazone hydrochloride, in tablet formulation by HPTLC method; Journal of Liquid Chromatography and Related Technologies. 36(5); 2013: 618-627.

8. Jajow Swapna, et al. Analytical Method Development and Method Validation for the Simultaneous estimation of Metformin hydrochloride and Pioglitazone hydrochloride in Tablet Dosage Form by RP-HPLC; Asian Journal Pharmceutical Analysis. 2(3); 2012: 85-89.

9. Shweta Havele, et al. Estimation of Metformin in Bulk Drug and in Formulation by HPTLC; Journal of Nanomedicine and Nanotechnology. 1(1); 2010: 102-104.

10. Kalpana G. Patel, et al. Validated HPTLC Method for the Simultaneous Determination of Metformin Hydrochloride and Sitagliptin Phosphate in Marketed Formulation; International Journal of Analytical and Bioanalyticla Chemistry. 3(1); 2013: 47-51.

11. Sunil R. Dhaneshwar, et al. Simultaneous determination of Metformin hydrochloride and Glipizide by Application of High performance thin layer chromatography-densitometry method in the bulk drug and tablet formulation; Der Pharmacia Sinica. 2 (1); 2011: 40-48.

12. A. Ghassempour, et al. Simultaneous Determination of Metformin and Glyburide in Tablets by HPTLC; Chromatographia. 64(1); 2006: 101-104.

13. http://www.ich.org/home.html. (Access on 2^nd March, 2016).

14. http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html (cited on 2^nd March. 2016).

15. Potdar MA. Pharmaceutical Quality Assurance. 2nd ed. Nirali Prakashan. 2007: 8.28-8.40.

16. Sharma PP. Validation in Pharmaceutical Industry. 1^stEd. New Delhi: Vandana Publications Pvt ltd. 2007: 361-65.

17. Bhusari KP, Shivhare UD, Goupale DC. Pharmaceutical Quality Assurance and Management. 1^st Ed. Hyderabad: Pharmamed press. 2011: 292-98.

18. International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use. Validation of Analytical Procedures: Text and Methodology ICH Q2 (R1). 2005.

19. Sawant R, Hapse S. Fundamentals of Quality Assurance techniques. 1st ed. Nashik: Career Publications. 2011: 32-5.

20. Iyer S. Guidelines on cGMP and Quality of Pharmaceutical Products. 1st ed. Mumbai: D.K. Publications. 2003: 145-58.

21. Riley CM, Rosanske TW. Development and Validation of Analytical Methods. 1st ed. Noida: Elsevier. 2009: 20-68.

22. Krishna karthik Peruru, et al. Stability indicating RP-HPLC method for Simultaneous determination of metformin hydrochloride and pioglitazone hydrochloride in dosage form, Malaysian Journal of Pharmaceutical Sciences. 12(1); 2014: 33-46.

23. Omnia Ismaiel, et al. Development and Validation of stability-indicating HPLC- UV method for determination of Pioglitazone hydrochloride and Metformin hydrochloride in bulk drug and combined Tablet Dosage Form; Asian j. of Pha. And clinical research. 6(4); 2013: 116-120.

24. Mukesh Chandra Sharma, et al. Study of stressed degradation behavior of pioglitazone hydrochloride in bulk and pharmaceutical formulation by HPLC assay method; Journal of Optoelectronics and Biomedical Materials. 1(1); 2010: 17-24.

25. Jain Pritam, et al. Stability- Indicating HPTLC Densitometric method for determination of Metformin hydrochloride in tablet formulation; J. Pharm. BioSci. 1; 2013: 51-58.

26. Asha Byju Thomas, et al. Stability-indicating HPTLC method for simultaneous determination of nateglinide and metformin hydrochloride in pharmaceutical dosage form; Saudi pharamceutical Journal. 2011: 221-231.

27. Lakshmi Sivasubramanian, et al. Stability indicating HPTLC method for simultaneous determination of telmisartan and ramipril in tablets; International Journal of Pharmacy and Pharmaceutical Science. 2(4); 2010: 127-129.

28. Sachin L. Patil, et al. Development and validation of a stability indicating RP-HPLC method for simultaneous determination of sitagliptin and metformin in tablet dosage form; International Journal of Ressearch in Pharmaceutical and Biomedical Science. 2(5); 2013: 590-596.

Received on 29.03.2016 Modified on 04.04.2016

Research J. Pharm. and Tech 2016; 9(10):1555-1561.

DOI: 10.5958/0974-360X.2016.00305.X

Concentration level (%) Recovery	Mean % Drug found*	Standard Deviation*	% Relative Standard Deviation*	Standard Error*
80	100.11	0.3554	0.3550	02052
100	99.74	0.5768	0.5692	0.2355
120	99.58	0.8391	0.8426	0.4845

Concentration level (%) Recovery	Mean % Drug found*	Standard Deviation*	% Relative Standard Deviation*	Standard Error*
80	99.53	0.5676	0.570	0.3277
100	100.14	0.4104	0.409	0.2369
120	99.82	0.1762	0.176	0.1017