DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR METFORMIN HYDROCHLORIDE AND NATEGLINIDE IN BULK AND COMBINED DOSAGE FORM

PRASANTHI CHENGALVA*1, ANGALA PARAMESWARI S.1, ARUNA G.2

1Department of Pharmaceutical Analysis, Krishna Teja Pharmacy College, Tirupathi, Andhra Pradesh, India, 2Department of Quality Assurance, Krishna Teja Pharmacy College, Tirupathi, Andhra Pradesh, India
Email: prashanthi.chengalva87@gmail.com

Received: 11 Jan 2016 Revised and Accepted: 27 Feb 2016

ABSTRACT

Objective: To develop an accurate, precise and linear RP-HPLC method for simultaneous quantitative estimation of Metformin hydrochloride and Nateglinide in tablets and validate as per ICH guidelines.

Methods: The method used a reverse phase column, Inertsil C18-ODS 3V (2504.6 mm, 5 m), a mobile phase comprising of phosphate buffer (pH 4.0): Acetonitrile: methanol (30:60:10) flow rate of 1.0 ml/min and a detection wavelength of 221 nm using a UV detector.

Results: The developed method resulted in elution of Metformin hydrochloride at 2.45 min and Nateglinide at 4.21 min. The calibration curves were linear (r=0.999) in the concentration range of 60-140 g/ml and 14.4-33.2 g/ml for Metformin hydrochloride and Nateglinide respectively. The percentage recoveries were found to be 99.59-101.36 for Metformin hydrochloride and 98.43-101.38 for Nateglinide. The LOD was found to be 2.18 g/ml and 1.55 g/ml for Metformin hydrochloride and Nateglinide respectively. LOQ was found to be 8.52g/ml and 4.69g/ml for Metformin hydrochloride and Nateglinide respectively.

Conclusion: A simple, accurate, precise, linear and rapid RP-HPLC method was developed for simultaneous quantitative estimation of Metformin hydrochloride and Nateglinide in bulk and pharmaceutical formulation and validated as per ICH guidelines. Hence, the method holds good for the routine analysis of Metformin hydrochloride and Nateglinide in various pharmaceutical industries as well as in academics.

Keywords: Metformin hydrochloride, Nateglinide, RP-HPLC, Method development, Validation

2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Metformin hydrochloride (fig. 1) is 1, 1-Dimethyl biguanide monohydrochloride. It is the first-line medication for the treatment of type 2 diabetes. It is a biguanide antihyperglycemic agent used for treating noninsulin dependent diabetes mellitus (NIDDM). It improves glycemic control by decreasing hepatic glucose production, decreasing glucose absorption and increasing insulin-mediated glucose uptake. Metformin is the only oral anti-hyperglycemic agent that is not associated with weight gain.



Fig. 1: Structure of metformin hydrochloride

Nateglinide (fig. 2) is 2-(4-isoopropylcyclohexanecarboxamido)-3-phenylpropanoic acid. It is an oral antihyperglycemic agent used for the treatment of NIDDM. It belongs to the Meglitinide class of short-acting insulin secretagogues. It lowers blood glucose by stimulating the release of insulin from the pancreas, by closing ATP-dependent potassium channels in the membrane of the  cells. This depolarizes the  cells and causes voltage-gated calcium channels to open. The resulting calcium influx induces fusion of insulin-containing vesicles with the cell membrane and insulin secretion occurs. Nateglinide is extensively metabolized in the liver and excreted in urine (83%) and feces (10%).

A detailed literature survey revealed that there was a spectrophotometric method for simultaneous estimation of Metformin with other combination [1] and Nateglinide in tablet dosage form [2], and HPTLC method [3] have been established for simultaneous analysis of Metformin hydrochloride and Repaglinide. There were various RP-HPLC methods have been developed for the determination Metformin hydrochloride in combination with other drugs [4-11]. However, there was no RP-HPLC Method has been reported for simultaneous estimation of Metformin hydrochloride and Nateglinide, we presented easy, rapid, accurate and specific HPLC method for simultaneous estimation of RP-HPLC assay procedure for the analysis of Metformin hydrochloride and Nateglinide in bulk and tablet dosage form. The developed method was validated as per ICH guidelines [12].



Fig. 2: Structure of nateglinide

MATERIALS AND METHODS

Pharmaceutical grade Metformin hydrochloride and Nateglinide were supplied as a gift sample by Chandra labs, Hyderabad and marketed formulation (Netop plus, Metformin-500 mg, and Nateglinide-120 mg) was purchased from the local market. Methanol, Orthophosphoric acid (OPA), Acetonitrile and HPLC grade water were obtained from Merck. All solvents used in this work are HPLC grade. RP-HPLC Shimadzu (LC 20ATVP) model with Spin chrome (LC SOLUTIONS) software was employed in this method. Analytical column used for the separation of analytes is Inertsil ODS C18 (250x4.6 mm, 5).

Methods

Selection of wavelength

Standard solutions of Metformin hydrochloride and Nateglinide were prepared at the concentration of 10 g/ml scanned by UV/Vis spectrophotometer at the range of 200-400 nm. UV spectrums of Metformin hydrochloride (fig. 3) and Nateglinide (fig. 4) were shown below. The isosbestic point selected for simultaneous estimation was 221 nm (fig. 5).



Fig. 3: UV spectrum of Nateglinide



Fig. 4: UV spectrum of metformin hydrochloride



Fig. 5: UV overlaps spectrum of Nateglinide and Metformin hydrochloride

Chromatographic conditions

The developed method used a reverse phase C18 column, Waters Inertsil ODS 3V (250x4.6 mm, 5), a mobile phase of phosphate buffer (pH 4.0): Acetonitrile: methanol (30:60:10), flow rate of 1.0 ml/min and a detection wavelength of 221 nm using a UV detector.

Preparation of phosphate buffer

Accurately 2.7 g of potassium dihydrogen phosphate (KH2PO4) was weighed and dissolved in water and volume was made up to 1000 ml with water. The PH was adjusted to 4.0 by using Orthophosphoric acid. The buffer was filtered to remove all fine undissolved particles.

Preparation of mobile phase

A mixture of 30 volumes of phosphate Buffer, 60 volumes of Acetonitrile and 10 volumes of methanol was prepared. The mobile phase was sonicated for 10 min to remove gasses.

Diluent

The mobile phase was used as diluent.

Preparation of standard solutions

Standard stock solutions of Metformin hydrochloride and Nateglinide were prepared by dissolving 100 mg of Metformin and 24 mg of Nateglinide insufficient mobile phase. After that, the solution was filtered and sonicated for 5 min and diluted to 100 ml with mobile phase. Further dilutions were prepared in 5 replicates of 100 g/ml of Metformin and 24 g/ml of Nateglinide, which was made by adding 1 ml of stock solution to 10 ml of mobile phase. This has been treated as 100 % target concentration.

Preparation of sample solution

20 tablets each containing 500 mg of Metformin hydrochloride and 120 mg of Nateglinide were weighed and taken into a mortar and crushed to fine powder and uniformly mixed. Primary stock solutions of Metformin hydrochloride and Nateglinide (g/ml) were prepared by dissolving a weight equivalent to 500 mg of Metformin hydrochloride and 120 mg of Nateglinide and dissolved in sufficient mobile phase. After that, the solution was filtered by syringe filter and sonicated for 5 min and dilute to 100 ml with mobile phase. Further dilutions were prepared in 5 replicates of 100 g/ml of Metformin hydrochloride, and 24 g/ml of Nateglinide was made by adding 1 ml of stock solution to 10 ml of mobile phase. That has been treated as 100 % target concentration.

RESULTS AND DISCUSSION

Method development

Different chromatographic conditions were tried for better separation and resolution. Inertsil C18-ODS 3V (2504.6 mm, 5 m) column was found satisfactory. Peak purity of Metformin hydrochloride and Nateglinide was checked using UV detector and 221 nm was considered satisfactory for detecting both the drugs with adequate sensitivity. A number of solvents in the different ratio over a wide range of pH were tried, but either peak shape was broad or resolution was not good. Repeated trials to obtain good, sharp peak with an efficient resolution between two peaks of Metformin hydrochloride and Nateglinide done on a C18 column in isocratic HPLC gave satisfactory results. The run time was good in isocratic trial with mobile phase consisting of phosphate buffer (pH 4.0): Acetonitrile: methanol (30:60:10) and C18-ODS 3V (2504.6 mm, 5 m) column, flow rate 1.0 ml/min and detection wavelength 221 nm gave the satisfactory results in terms of retention time, resolution, symmetry and sensitivity. A typical RP-HPLC chromatogram for simultaneous determination of Metformin hydrochloride and Nateglinide from standard preparation and from pharmaceutical formulation was shown in (fig. 6 and 7).



Fig. 6: Typical chromatogram of standard solution



Fig. 7: Typical chromatogram of sample solution

Method validation

The developed RP-HPLC method was validated for parameters like system suitability, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) and robustness according to ICH guidelines.

System suitability

Standard solutions were prepared as per the test method and injected into the chromatographic system. The system suitability parameters like theoretical plates, resolution and asymmetric factor were evaluated. The system suitability parameters were tabulated in table 1. All the parameters were found to be within the limits.

Precision

Method precision

The precision of the method was verified by precision method studies. The sample solution was prepared at working concentration and analysis was carried out at replicating. The sample solutions of Metformin hydrochloride and Nateglinide were prepared as per the test method and injected 6 times into the column. The results of precision were tabulated in table 2. The average was taken, and % RSD was calculated and reported. % RSD values were within the limits, and the method was found to be precise.

Linearity

The linearity of the test solutions for the assay method was prepared from Metformin hydrochloride and Nateglinide standard stock solution at five concentration levels from 50% to 150% of assay concentration. The peak area versus concentration data was treated by least-squares linear regression analysis (fig. 8 and 9). The results have shown an excellent correlation between peak areas and concentration within the concentration range of 60140 g/ml for Metformin hydrochloride, 14.433.2 g/ml for Nateglinide (tables 3-4). The correlation coefficients were found to be 0.999 for both the drugs, which meet the method validation acceptance criteria and hence the method was said to be linear for both the drugs.


Table 1: Results of system suitability studies

Parameters

Acceptance limits

Metformin hydrochloride

Nateglinide

Retention time (min)

-

2.453

4.217

Resolution

NLT 2

-

14.232

Theoretical plates

NLT 2000

8489

7583

Tailing factor

NMT 2

1.706

1.556

# min: minutes


Table 2: Method precision data

n

Metformin hydrochloride

Nateglinide
 

Rt

Peak area

Rt

Peak area

Injection 1

2.459

3984.852

4.253

399.316

Injection 2

2.456

3983.028

4.243

396.285

Injection 3

2.453

3986.756

4.227

380.420

Injection 4

2.457

3983.864

4.210

390.391

Injection 5

2.459

3955.954

4.300

385.999

Injection 6

2.460

3915.924

4.367

385.427

Average

2.456

3968.396

4.267

389.640

SD

0.004

28.176

0.058

7.132

% RSD

0.164

0.710

1.35

1.83

# Rt: Retention times, # n: number of injections=6



Fig. 8: Linearity chart for Metformin hydrochloride

Accuracy

The accuracy of the method was determined by recovery studies by the determination of % mean recovery of both the drugs at three different levels (80 %, 100 % and 120%). At each level, three determinations were performed. The percentage recovery and mean percentage recovery were calculated for the drug was shown in table 4. The observed data were within the required range, which indicates good recovery values and hence the accuracy of the method developed.



Fig. 9: Linearity chart for Nateglinide


Table 3: Linearity data for Metformin hydrochloride and Nateglinide

% Level

Metformin hydrochloride concentration (g/ml.)

Metformin peaks hydrochloride area

Nateglinide concentration (g/ml)

Nateglinide peak area

50

60

2088.713

14.4

201.895

75

80

3002.121

19.2

292.713

100

100

3941.042

24

385.139

125

120

4890.891

28.8

476.353

150

140

5854.513

33.2

560.788

Correlation Coefficient

0.9998

  

0.9996

  

Slope

37.68

  

3.60

  

Table 4: Results of accuracy

Level (%)

Metformin hydrochloride

  

Nateglinide

  
 

% Recovery

% Mean

% Recovery

% Mean

80

98.59

99.59

100.74

100.42

80

99.63

99.90

80

100.57

100.63

100

99.48

100.46

99.53

99.30

100

100.54

98.43

100

101.36

99.95

150

101.20

100.09

101.38

101.14

150

99.08

100.78

150

100.01

101.27

Table 5: Results of robustness

Parameter

Metformin hydrochloride

Nateglinide
 

Plate count

Tailing

Plate count

Tailing

Less flow rate (0.8 ml/min)

8828

1.765

7336

1.500

More flow rate (1.2 ml/min)

8789

1.706

7114

1.556

Less wavelength 216 nm

8828

1.765

7180

1.519

More wavelength 226 nm

8451

1.667

7123

1.538

Table 6: LOD and LOQ

Sample name

LOD(g/ml)

LOQ(g/ml)

Metformin hydrochloride

2.18

8.52

Nateglinide

1.55

4.69

Robustness

To determine the robustness of the developed method, experimental conditions were deliberately altered, and the system suitability parameters were evaluated. The solutions prepared as per the test method and injected at different variable conditions like flow rate (0.8, 1.2 ml/min.) and wavelength (219 nm, 223 nm), system suitability parameters were compared with that of method precision. The results were tabulated in table 5. At the flow rate of 1.0 ml/min shows, a sharp peak with good resolution and rest of the flow rates were found to be not satisfactory. The method passed all system suitability parameters indicating that the method was robust.

Detection limit and quantification limit

Limit of detection (LOD) which represents the concentration of the analyte at S/N ratio of 3 and limit of Quantification (LOQ) at which S/N was 10 were determined experimentally for the proposed methods and results were given in table 6.Hence, the detection limits and quantitation limits of the drugs were given S/N ratios of 3 and 10 respectively.

CONCLUSION

The proposed RP-HPLC method was found to be simple, specific, accurate, precise, robust, rapid and economical. This method gives good resolution between all the two compounds with a short analysis time. The proposed RP-HPLC method can be useful for routine analysis of Metformin hydrochloride and Nateglinide in the tablet dosage form.

ACKNOWLEDGEMENT

The author expresses sincere thanks to the Principal and Head of Pharmaceutical Analysis department, Krishna Teja Pharmacy College for excellent guidance and encouragement.

CONFLICT OF INTERESTS

Declare none

REFERENCES

  1. Jain S, Bhandari A, Purohit S. Spectrophotometric determination of Nateglinide in bulk and tablet dosage forms. Asian J Pharm 2009;3:218-9.
  2. Patel JR, Suhagia BN, Patel BH. Simultaneous spectrophotometric estimation of Metformin and Repaglinide in a synthetic mixture. Indian J Pharm Sci 2007;69:844-6.
  3. Keyur B, Emanual M, Patelia, Arpit S.Simultaneous estimation of Metformin hydrochloride and repaglinide in pharmaceutical formulation by HPTLC-densitometry method. J Chromatogr Sep Tech 2013;4:166-71. 
  4. Dalia Rashad W. Simultaneous determination of Metformin, Nateglinide and Gliclazide in pharmaceutical preparations using micellar liquid chromatography. Int J Biomed Sci 2012;8:14451.
  5. Mallikarjuna Rao N, Gowri Sankar D. RP-HPLCmethod for simultaneous estimation and stability indicating the study ofMetformin and Linagliptinin pure and pharmaceutical dosage forms. Int J Pharm Pharm Sci 2015;7:191-7.
  6. Deepali AN, Vidhya K, Sunil R. Dhaneshwar validated HPLC method for simultaneous quantitation of Benfotiamine and Metformin hydrochloride in bulk drug and formulation. Int J Pharm Pharm Sci 2013;5:138-42.
  7. Ramanji Reddy T, Dhachinamoorthi D, Chandra Sekhar KB. Development of RPHPLC method for Metformin and Repaglinide in rabbit plasma. Int J Pharm Pharm Sci 2012;4:311-3.
  8. Ediga Sasi K, Krishna Reddy V, Chandra K. A new simple RP-HPLC method for simultaneous estimation of Metformin hydrochloride and Gliclazide tablet dosage form. Int J Pharm Biol Sci 2012;2:277-83.
  9. VaniR, Vijaya Kumer B, Krisshna Mohan G. Analytical method development and validation for the determination of Sitagliptin and Metformin using reverse phase HPLC method in bulk and tablet dosage form. World J Pharm Pharm Sci 2014;3:1803-11.
  10. Sasi kiran E, Krishna reddy V, Chandra K. A new simple RPHPLC method for simultaneous estimation of Metformin hydrochloride and Gliclazide tablet dosage form. Int J Pharm Biol Sci 2012;2:277-83.
  11. Deepa R, Laxmanbhai J, Madhabhai M. Stability indicating HPLC method for simultaneous determination of Repaglinide and Metformin hydrochloride in pharmaceutical dosage form. Int J ChemTech Res 2011;4:500-5.
  12. 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.