Simultaneous estimation of pioglitazone, glimepiride & metformin hydrochloride in bulk & tablet dosage form by UV, RP-HPLC method

Introduction

Metformin Hydrochloride is a anti-diabetic drug which belongs to Biguanides drug class. The chemical formula for Metformin hydrochloride is C₄H₁₂ClN₅. Metformin is prescribed with other medication to control high blood sugar with instruction to follow proper diet and exercise program. It is used to treat patients with type 2 diabetes. Metformin exerts its anti-diabetic action through suppression of generation of glucose in the liver.1, 2, 3 Till date, two important molecular targets of metformin have been identified, both of which are present in mitochondria.2, 3, 4 Metformin acts on mitochondrial respiratory complex I by inhibiting its function, which results in increasing in the cellular ratio of adenosine monophosphate (AMP) to adenosine triphosphate (ATP) as a result of a reduction in the efficiency of ATP production. This increase in the ratio of AMP: ATP triggers the activation of AMP‐activated protein kinase (AMPK), which has alot of effects on energy metabolism, also down regulation of the expression of gluconeogenic genes is controlled by it.2 The activity of adenylate cyclase is also thought to be inhibited by increase in AMP concentration5, it is an important mediator of glucagon action, therefore results in the inhibition of gluconeogenesis. The second target of metformin in mitochondria is mitochondrial glycerol‐3‐phosphate dehydrogenase, 4 which plays a key role in the glycerophosphate shuttle. This oxidoreduction shuttle between the cytosol and mitochondria is necessary for production of the oxidized form of coenzymes required for biochemical reactions, such as nicotinamide adenine dinucleotide in gluconeogenic reactions. The activity of mitochondrial glycerol‐3‐phosphate dehydrogenase is also inhibited by metformin, which suppresses gluconeogenic reactions including the conversion of lactate to pyruvate.4

Figure 1

Structure of metformin hydrochloride

Figure 2

Structure of pioglitazone

Pioglitazone hydrochloride is another anti-diabetic drug with chemical formula CHNOS. Pioglitazone hydrochloride belongs to drug class thiazolidinedione that depends on the presence of insulin for its mechanism of action. Pioglitazone hydrochloride decreases insulin resistance in the periphery and in the liver which results in increasing the insulin-dependent glucose disposal and decreasing hepatic glucose output. Pioglitazone isecretagogue. 6Pioglitazone has agonist action for peroxisome proliferator-activated receptor-gamma (PPARgamma). PPAR receptors are found in tissues such as adipose tissue, skeletal muscle, and liver which are important for insulin action.7, 8, 9, 10, 11, 12, 13, 14, 15, 16PPARgammametabolism.

Glimepiride is a potent anti-diabetic drug which belongs to Sulfonylurea drug class. Its chemical formula is C₂₄H₃₄N₄O₅S.It is used to treat patients with type 2 diabetes. It is prescribed with other medication with proper diet and regular exercise routine. Route of administration is oral it is available in tablet form and the elimination half life of glimepiride is 5-8 hours. Glimepiride exerts its action by increasing insulin release from the pancreas and by improving glucose tolerance. It acts on the "sulfonylurea receptors" on pancreatic β-cell membrane and reduces conductance of ATP sensitive K+ channels and thus causes depolarization. Due to this the Ca2+ influx and degranulation is enhance which in turn increases insulin secretion rate at any glucose concentration.17 It primarily increases 2nd phase insulin secretion and has little effect on 1st phase. The hepatic degradation of insulin and minor action of reducing glucagon release and increasing somatostatin release is also slowed down. It sensitizes the target tissues such as liver to insulin action and exerts strong extrapancreatic action. Number of insulin receptors are increased and by a post receptor action translation of receptor activation is enchanced.

Figure 3

Structure of glimepiride

Materials and Methods

All the three drugs i.e Pioglitazone, Glimepiride & Metformin Hydrochloride were obtained from RAP Analytical research and training centre and all the chemicals used were of HPLC grade were purchased from Thermofisher pvt ltd. The balance used for weighing is a high precision balance from Wensar with model no. PGB 100. The mobile phase was degassed using Wensar Ultra sonicater and was filtered using Ultra filtration with a membrane filter size of 0.45 microns. For UV spectroscopy, double beam instrument maufactured by Analytical Technologies Ltd. with Model no- UV-2012 was used. The software used for analysis was UV-VIS Analyst.18, 19, 20, 21, 22, 23, 24, 25 The instrument used for the High Performance Liquid Chromatography consisted of binary gradient system. The software used of the HPLC instruments for the method development and the validation purpose was HPLC workstation. The maker of the HPLC system is analytical technologies Pvt. Ltd. with model number HPLC 3000 series. Reciprocating type of pump was used for the analysis of model no.P-3000M. The detector used was UV-VIS detector of single wavelength with the model no. of UV3000M. The C18 type of column was used for the separation which was form cosmosil make having the dimension of 250mm X 4.6mm (ID), particle size of 5micron.

Method development

In order to obtain resolution of the three drugs that is to develop sharp and well resolved peaks various trials were carried out. By changing the ratio of mobile phase methanol and water various trial were taken and it was observed that all the three drugs were well resolved at the 70% methanol and 30% water at 229 nm. The flow rate of the mobile phase was taken at 0.8 ml/min. Optimised results was shown below of all three drugs

Figure 4

Chromatogram of optimised trail

Table 0

Rank	Time	Area	Resolut.	T.Plate	Num asymmetry
1	4.033	1618046	6.25	6955	1.03
2	5.523	327268	4.64	7446	1.00
3	6.951	148683	0.00	7768	1.16

Result

All the System Suitability parameters i.e resolution, number of theoretical plates, asymmetry of peaks were found to be within the range which is  indicating the performance of the system.

Linearity

Statistically the response of the analyte  and the concentration were calculated in order to check the linearity of all the three drugs.

Concentration range from 100-500 ug/ml were taken for metformin, 3-15 ug/ml for pioglitazone, 0.4-1.2 ug/ml for glimepiride and the calibration curve was plotted as shown below

Figure 5

Calibrationcurve for metformin

Figure 6

Calibration curve for pioglitazone

Figure 7

Calibration curve for glimepiride Regression value for all the three drugs were found within the limit.

Ruggedness

Wide range of concentration were injected for all the three drugs and their calibration curve was plotted as the response of analyte with the range if concentration.

Table 22

Statistical data of ruggedness of metformin

Conc.	Area
100	2043536
200	2896093
300	3889571
400	4907803
500	5822486

Figure 8

Calibration curve of ruggedness for metformin

Table 23

Statistical data of ruggedness of pioglitazone

Conc.	Area
3	150608
6	221964
9	295029
12	370883
15	441183

Figure 9

Calibration curve of ruggedness for pioglitazone

Table 24

Statistical data of ruggedness of glimepiride

Conc.	Area
0.4	16812
0.6	28766
0.8	39607
1.0	49589
1.2	61511

Figure 10

Calibration curve of ruggedness for glimepiride

Results are showing that the developed method is stable as regression coefficient of all are within the limit hence change doesn't affects the results.

Table 25

Statistical data of degradation studies of metformin

Sr. no.	Degradation	Area of Standard	Area of degraded Sample	Degraded up to %	Actual % degradation
1	Acid Degradation	5822073	5566418	95.60886646	4.391133536
2	Basic Degradation	5822073	5445290	93.52837039	6.471629607
3	H2O2 Degradation	5822073	5818895	99.94541463	0.054585368
4	Thermal	5822073	5819926	99.9631231	0.036876899
5	Photolytic	5822073	5821547	99.99096542	0.009034583

Table 26

Statistical data of degradation studies of pioglitazone

Sr. NO.	Degradation	Area of Standard	Area of degraded Sample	Degraded up to %	Actual % degradation
1	Acid Degradation	440904	426845	96.81132401	3.188675993
2	Basic Degradation	440904	413594	93.80590786	6.194092138
3	H2O2 Degradation	440904	440145	99.82785368	0.172146318
4	Thermal	440904	440233	99.84781268	0.152187324
5	Photolytic	440904	440374	99.8797924	0.120207574

Table 27

Statistical data of degradation studies of glimepiride

Sr. NO.	Degradation	Area of Standard	Area of degraded Sample	Degraded up to %	Actual % degradation
1	Acid Degradation	61560	59446	96.56595192	3.434048083
2	Basic Degradation	61560	58265	94.64749838	5.352501624
3	H2O2 Degradation	61560	59546	96.72839506	3.271604938
4	Thermal	61560	61541	99.9691358	0.030864198
5	Photolytic	61560A	60563	98.38044185	1.619558155

Limit of detection (LOD) and limit of quantification (LOQ)

It is calculated by slope and the standard deviation. Following are the formulas used to estimate the LOD and LOQ:

LOD= 3.3σ/S

1. Where, σ = the standard deviation of the y-intercept

2. S = slope of calibration curve of analyte

LOQ= 10σ/S

1. Where, σ = the standard deviation of the y-intercept

2. S = slope of calibration curve of analyte

3. The result of LOD and LOQ of respective drugs are as follows:

Metformin

1. LOD =1.3608,

2. LOQ = 4.1239

Pioglitazone

1. LOD = 0.04710,

2. LOQ = 0.14274

Glimepiride

1. LOD = 0.00468,

2. LOQ = 0.01421

Discussion

For simultaneous estimation of metformin, pioglitazone and glimepiride, an analytical method was developed and validated by UV spectroscopy and reverse phase chromatography. The proposed method was found to be specific, accurate and precise. The method was found to be linear which was performed by linearity study for all the three drugs which was studied as per their respective concentration. Around more than 0.99 regression coefficient value was observed which was within the limit. The retention time for metformin was observed at around 4.0 min, for pioglitazone it was observed 5.5 min and for glimepiride it was observed around 6.8 min. The recovery for the method was also accurate within the limit as per the guidelines only which was observed within 98% to 102%. The percent relative standard was also observed below 2 value which falls under the guidelines. No degradation interference was observed while developing and validating the analytical method. It was also observed that the results are unaffected for the small variations in the method showing the repeatability of the developed analytical method. The method was found to be simple, rapid and robust for the analysis of metformin, pioglitazone and glimepiride.

Conclusion

For the simultaneous estimation of metformin, pioglitazone and glimepiride of stability indicating analytical method development and validation in the formulation by RP-HPLC technique, the method was found to be accurate, sensitive, reproducible, linear and precise. The results which was observed was found to be relevant that are carried out as per International Council on Harmonisation guidelines. Hence, for the quality control analysis the proposed method was carried out for the routine analysis in the tablet dosage form.

Source of Funding

None.

Conflict of Interest

None.