Method Development and Validation of RP-HPLC Method for Estimation of Nateglinide in Bulk Drug and Pharmaceutical Formulation.

 

Kalpana Vasanthan*, R. Vijayageetha and A. Shantha Arcot

Department of Pharmaceutical Analysis, C. L. Baid Metha College of Pharmacy, Jyothinagar, Thoraipakkam .Chennai-600 097, Tamilnadu, India.

*Corresponding Author E-mail: kalpana.vasanthan@gmail.com

 

ABSTRACT:

A rapid, simple, precise and sensitive RP-HPLC method was developed for estimation of nateglinide in pharmaceutical bulk drug and pharmaceutical formulation. A phenomenex Gemini C18, 5m particle size column having 250 x 4.6 mm .i.d in isocratic mode, with mobile phase containing acetonitrile: 0.1%w/v sodium dihydrogen ortho phosphate (70:30v/v) was used. The flow rate was 2ml/min and the effluent was monitored at 220nm. The retention time of nateglinide was 6.70.2 minutes. The method was validated for linearity, accuracy, precision, specificity, LOD, LOQ and robustness. Nateglinide showed linear response in the concentration range 160-240g/ml. the correlation coefficient for nateglinide was 0.9999 and the percentage recovery was found to be 99.26to 100.81.

 

KEYWORDS: Nateglinide, bulk drug, pharmaceutical formulation, RP-HPLC.

 


INTRODUCTION

Nateglinide is an ant diabetic agent which lowers blood glucose by stimulating the release of insulin from the pancreas. It achieves this by closing ATP-dependent potassium channels in the membrane of the β cells. This depolarizes the β 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 occurs1. It is chemically (R)-2-(4-isopropylcyclohexanecarboxamido)-3-henylpropanoic acid 2 with molecular formula C19H27NO3 and molecular weight 317.423g /mol. The structure of Nateglinide is shown in figure: 1. Literature survey revealed that various methods have been reported for estimation of nateglinide in biological samples such as plasma by LC method with UV detection 3, 4. Two methods have been reported for estimation of enantiomer of nateglinide in bulk drug5 substance and in rat intestine6 by HPLC method, a spectrophotmetric method have been reported for determination of nateglinide in bulk drug substance and in tablet dosage form7. Also, literature revealed only one method for determination of nateglinide in tablet dosage form and stability studies in bulk drugs using 0.025M potassium hydrogen phosphate and 1%triethylamine (pH3) adjusted with dilute Orthophosphoric acid and methanol.

 

In this present study an attempt was made to develop an alternative rapid RP-HPLC for estimation of nateglinide in bulk drug and pharmaceutical formulation using acetonitrile: sodium dihydrogen ortho phosphate (70:30v/v) and UV detector. The method was validated as per ICH guidelines9, 10.

 

EXPERIMENTAL:

Instrument:

A high performance liquid chromatograph (Shimazdu prominence) composed of LC-20AT prominence solvent delivery module, a manual rheodyne injector with 20l fixed loop and SPD 20A prominence UV detector. Separation was performed on a C18 column (5m particle size column having 250 x 4.6 mm .i.d; phenomenex Gemini) at an ambient temperature. Chromatographic data was recorded and processed using Synchrom software.

 

Chemical and reagents:

Working standard of nateglinide was obtained as a gift sample from madras pharmaceutical ltd., Chennai (purity-99.69%). Sodium dihydrogen phosphate, orthophosphoric acid (S.d fines .chem. Ltd), Acetonitrile HPLC grade was used. Milli-Q water was used for the mobile phase preparation. Market sample Glinate 60mg (Glenmark pharmaceuticals LTD, Mumbai. India.

 

Preparation of buffer solution:

1g of sodium dihydrogen ortho phosphate was weighed and dissolved in water and volume was made up to 1000ml. pH is adjusted to 2.5 with ortho phosphoric acid. Filtered through 0.45m membrane filter and degassed it.

Figure: 1: Structure Of Nateglinide.

 

Preparation of mobile phase:

Mix 300ml of above buffer with 700ml of acetonitrile and degas by filtering through 0.45m membrane filter.

 

Preparation of standard stock solution:

50 mg of nateglinide drug substance was weighed and transferred into a 50 ml volumetric flask. Dissolved and diluted to volume with mobile phase.

 

Chromatographic conditions:

Chromatographic conditions of developed method was shown table: 1.

 

Table: 1. Optimized chromatographic conditions.

Parameters

Conditions

Column

C18 phenomenex

Mobilephase

Acetonitrile : 0.1% sodium dihydrogen phosphate (70:30), pH adjusted to 2.1.

Temperature

ambient

Flow rate

2ml/min

Injection volume

20l/min

Wavelength

220nm

Operation mode

isocratic

Retention time

6.7 minutes.

 

From the standard stock solution 5 ml was pipetted in to 25ml standard flask and dilute to volume with mobile phase, to obtain a final concentration of 200mcg/ml. Filtered through 0.45 m finer porosity membrane filters. This solution was injected in to chromatograph and retention time was found to be at 6.7 0.2 minutes. The chromatogram shown in figure: 2.

 

Figure: 2: Chromatogram showing the peak of Nateglinide (6.5min)

 

Linearity:

Appropriate aliquots of standard stock solution were taken in different 25ml volumetric flask and diluted up to mark with mobile phase to obtain final concentration 160,180,200,220,240g/ml. These solutions were injected in to chromatographic system and chromatogram was obtained and peak area was determined for each concentration of drug solution. Calibration curve of nateglinide was constructed by plotting peak area Vs applied concentration of nateglinide and regression equation was computed. The graph was shown in figure: 3 .The optical parameters are shown table: 3.

 

Figure: 3: Linearity Plot of Nateglinide.

 

Analysis of marketed formulations:

Twenty tablets were weighed and crushed to fine powder. The tablet powder equivalent to 50mg of nateglinide was transferred to a 50ml volumetric flask. From this solution 5ml was pipetted and transferred to 25 ml volumetric flask. Volume was made up with mobile phase. The solution was filtered through 0.45m membrane filter. A 20l of sample solution was injected in to sample injector for six times under optimized chromatographic condition. The effluents are measured at 220nm. The amount of drug present in the sample was determined from peak area of nateglinide drug substance and the percentage label claim and the standard deviation was calculated. The results are given in table: 2.

 

Table: 2. Result of analysis of marketed sample and recovery studies

Drug

Label claim

Amount present

% label claim*

% Recovery

Nateglinide

60mg

59.70

99.24 0.64

99.26-100.81%w/w

*average of six determinants.

 

VALIDATION:

The proposed RP-HPLC method was validated as per ICH guidelines.

 

Specificity:

The specificity of the method was evaluated by injecting the blank and standard solution prepared as per proposed method to check for interference, if any, at the retention time of nateglinide from the blank. There was no peak eluting at the retention time of nateglinide from the blank.

Precision:

The system precision was evaluated by measuring the peak response of nateglinide from six replicate injections of the standard solution, prepared and analyzed as per the proposed method and the method precision was determined by preparing sample solutions of a single batch of nateglinide tablet six times and analysing for the content of nateglinide as per the proposed method. The percentage relative standard deviation for method precision and system precision was calculated and shown in table: 3.

 

Table: 3: Validation Parameters

Parameters

Nateglinide

Linearity range

160-240g/ml

Correlation coefficient

0.9999

System precision (%RSD)

1.57

Method precision (%RSD)

0.36

Robustness

Robust

LOD andLOQ

0.67 and2.05g/ml

Theoretical factor

7267

Asymmetry

1.23

Resolution

16.018

 

Accuracy (recovery studies):

Recovery studies were performed by standard addiction method at three levels i.e. 80%, 100%, 120%, known amount of standard nateglinide were added to pre-analyzed sample and they are subjected to proposed HPLC method. Results are shown in table: 2.

 

Limit of Detection and Limit of Quantitation:

The LOD and LOQ of the developed method were determined by injecting progressively low concentration of the standard solution using the developed RP-HPLC method. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio 3). The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (S/N ratio of 10). The LOD and LOQ of nateglinide are shown in table: 3.

 

Robustness:

The robustness study was done by making small changes in the optimized method parameter like 10% change in the flow rate, 2nm change in the wavelength. There was no significant impact on tailing factor and retention time.

 

RESULTS AND DISCUSSION:

The aim of the study was to develop simple, accurate and precise HPLC method for the analysis of nateglinide in the bulk and tablet dosage form using mobile phase (ph2.1 buffer and acetonitrile (30:70 v/v) and commonly employed in RP-HPLC with UV detector at 22nm.

 

The regression data showed a good linear relationship over a concentration range of 160 to240 g/ml. The LOD and LOQ was found to be 0.67 g/ml and 2.07 g/ml. as per USP XXIII11, system suitability test for HPLC were carried out on freshly prepared standard stock solution of nateglinide and parameter obtained with 20 l injection volume are summarized in table :3. The other validation parameters are studied and their results are summarized in table: 3.

 

The developed method is simple, precise and accurate. The statistical data proved that method are reproducible and selective for the analysis of nateglinide in bulk drug and its pharmaceutical formulation.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Madras pharmaceutical Ltd., Chennai, for providing gift sample of nateglinide. The authors are also thankful to Dr. Ceeal laboratories, Chennai, for providing facilities for the research work.

 

REFERENCE:

1)       American Diabetes Association: clinical Practice Recommendation 2000. Diabetes care 2000; 23: 1-116.

2)       Budhavari S. The Merck Index. 13th Edn 2003:1151.

3)       Jolly M.S, Mayur G.S, Vijay B.S and Rajashree C.M. Nateglinide quantification in rabbit plasma by HPLC: Optimization and application to pharmacokinetic study, Journals of pharmaceutical and biomedical 2007; 44: 197-204.

4)       Danai M, Evagelos G, Andreas V, Michael K , Konstantinos D , Dimitra G and Irene P. Determination of nateglinide in human plasma by high-performance liquid chromatography with pre-column derivatization using a coumarin-type fluorescent reagent. Analytical Chimica acta 2007; 599:143-50.

5)       Meiling Q, Peng W,  Yujing S, Ying L.Determination of the L-Enantiomer of Nateglinide in a Bulk Drug Substance by Chiral Reversed-Phase Liquid Chromatography. Journal of Liquid Chromatography and Related Technologies 2003; 26: 1839 1845.

6)       Srinivas M, Srisailam K, Chandra ME, Madhusudan RY, Gerhard KE . Development and validation of a stereo selective HPLC method for the determination of the in vitro transport of nateglinide enantiomers in rat intestine. Journal of Separation Science 2007; 30: 1875-1880.

7)       Sagar GV, Rao NVS, Sastry BS. Spectrophotometeric method for the determination of nateglinide in tablets. Indian Journals of Pharmaceutical Science 2004; 66: 219-221.

8)       Pathare DB, Jadhav AS, Shingare MS. A Validated Stability Indicating LC Method for Nateglinide Drug Development and Industrial Pharmacy 2007; 33: 551 557.

9)       ICH, Q2A Text on Validation of Analytical procedure, International Conference on Harmonization. October; 1994.

10)    ICH, Q3B Validation of Analytical procedure: methodology, International Conference on Harmonization. November; 1996.

11)    The United State Pharmacopoeia XXIII National Formulary XVIII US Pharmacopoeial Convention Inc., Rockville M.D 1995: 1776.

 

 

 

Received on 25.01.2010 Modified on 20.02.2010

Accepted on 22.03.2010 RJPT All right reserved

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 804-806