RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF AZELNIDIPINE

RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF AZELNIDIPINE

Megha G. Gore * and Pratap S. Dabhade

SND College of Pharmacy, Babhulgaon, Yeola, Maharashtra, India.

ABSTRACT: A simple and precise method was developed for the assay of azelnidipine from tablet formulation. The solvent system and wavelength were optimized in order to maximize the sensitivity of the proposed method, azelnidipine shows the maximum absorbance at 257 nm. The separation was achieved on HPLC binary gradient system equipped with HPLC 3000 series. The mobile phase was prepared with Methanol: Water (80: 20%v/v) o-phosphoric acid used for the pH adjustment (pH-3). The method was validated for accuracy, precision, linearity, LOD & LOQ of sample solution. Linearity was observed in the concentration range of 20-100 µg/ml & gave mean correlation coefficient 0.998. The developed RP-HPLC method was found to be accurate, precise and was successful applied to a pharmaceutical tablet formulation for qualitative estimation of Azelnidipine.

RP-HPLC, Methanol, O-Phosphoric acid

INTRODUCTION: RP-HPLC involves the separation of molecules on the basis of hydrophobicity. Excellent resolution that can be achieved under a wide range of chromatographic conditions. Chromatographic selectivity can be manipulated through changes in mobile phase characteristics. High recoveries and high productivity. Excellent reproducibility of repetitive separations carried out over a long period of time. Azelnidipine, (±)-3-[1-(diphenylmethyl)azetidin-3-yl] 5 - propan -2  - yl 2 – amino – 6 – methyl – 4 -(3-nitrophenyl) - 1, 4 dihydropyridine - 3, 5 - di carboxylate,  is a new  dihydropyridine  derivative  with  calcium  antagonistic activity. Only limited analytical methods have been reported  for  the  determination  of  Azelnidipine,  which includes  HPLC,  LC-MS  and  LC-ESI-MS.

Chemical Structure of Azelidipine:

FIG.1: AZELIDIPINE

MATERIALS AND METHODS: The pure drug Azelnidipine and marketed formulation were obtained from reputed firm with the certificate of analysis. Methanol was taken from Hexon Lab, Pune India. Standard and sample solution were prepared in diluent of methanol:water (80:20)v/v.

Instrumentation: The HPLC system used was HPLC binary gradient system equipped with HPLC3000 series and detector UV-3000-M. A PGB100 analytical balance was used and WUC-41L sonicator used.

Preparation of standard stock solution: 10mg of azelnidipine was weigh accurately and transferred to volumetric flask of 10 ml capacity. Small amount of methanol was added to dissolve the sample and further the volume made using methanol. This solution was filtered using Whatmann filter paper to remove the undissolved matter. From this solution, 0.75 ml solution was pipette out and placed into 10 ml of volumetric flask. The volume was made upto mark with methanolto give a solution containing 75 µg/ml. further the absorbance of resulting solution was determined at 257 nm.

0.1 gm of pure drug dissolve in 10 ml methanol gives 1000 ppm. The concentration of 20,40,60,80,100 ppm was prepare using above stock solution. The calibration curve was taken. (Fig. 4)

Chromatographic Conditions: Chromatographic separation was performed on a reverse phase Hexon C8 shield. The mobile phase was a mixture of methanol and water (80:20 % (v/v)). The flow rate of the mobile phase was adjusted to 1.0 ml /min. The detection was carried out at wavelength 257 nm. (Fig. 2) The injection volume of the standard and sample solution was set at 20 μl.         

Analysis of Marketed Formulation: Assay was performed by using Tablets of Azelnidipine for the preparation of the sample solution the 10 tablets were used. The powder equivalent to 10 mg of Azelnidipine was weighed accurately and transferred to volumetric flask of 10 ml capacity. Small amount of methanol was added to dissolve the sample and further the volume made using methanol. From this solution, 0.75 ml solution was pipette out and placed into 10 ml of volumetric flask. The volume was made upto mark with methanol to give a solution containing 75µg/ml.

FIG. 2: UV SPECTRA OF AZELNIDIPINE

Method Validation: The method was validated for linearity, accuracy, precision and limit of detection, and limit and quantitation.

FIG. 3: TYPICAL CHROMATOGRAM OF AZELNIDIPINE (STANDARD)

FIG. 4: TYPICAL CHROMATOGRAM OF AZELNIDIPINE (SAMPLE)

Accuracy: The accuracy of the method was assessed by determination of the recovery of the method at 3 different concentrations (50%, 100% and 150% concentration) by addition of known amount of standard to the placebo. For each concentration three sets were prepared.

Precision: The instrument precision was evaluated by determining the absorbance of the standard solution six times repeatedly. The results are reported in terms of relative standard deviation. The intra-and inter-day variation for the determination was carried out in triplicate for the standard solution.

Limit of Detection &Quantification: LOD and LOQ were calculated using following equation as per ICH guidelines. LOD = 3.3 × σ/ S and LOQ = 10×σ /S, where σ is the standard deviation of response and S is the slope of the calibration curve.

RESULT & DISCUSSION: In this work HPLC method for assay in tablet formulation was developed and validated. Methanol as diluent was selected which give sharp spectra.

FIG. 5: CALIBRATION CURVE FOR LINEARITY

TABLE 1: LINEAR REGRESSION DATA FROM CALIBRATION CURVE

TABLE 2: SUMMARY OF VALIDATION PARAMETERS

*Based on S.D. of response and slope of Regression curve

REFERENCES:

1. Sethi P.D. HPLC, ‘High Performance Liquid Chromatography’, Quantitative Analysis of Pharmaceutical Formulations, 1^st edition, CBS publishers and distributors, New Delhi, 2001: 3-39, 116-120.
2. Christen G.D., Analytical Chemistry, 6^th edition, John Wiley & Sons, 2003: 1-15.
3. Skoog D.A., Principles of Instrumental Analysis, 5^th edition, Eastern press, Banglore, 2004: 1-2, 674-688, 695-698.
4. Sharma B.K., Instrumental Methods of Chemical Analysis, Introduction to Analytical Chemistry, Goel publishing House, Meerut, 19^th edition, 2000: 1-4, 200-2003.
5. Sharma B.K., Instrumental Methods of Chemical Analysis, Introduction to Analytical Chemistry, Goel publishing House, Meerut, 21^th edition, 2002: 3-5, 10.
6. Willard H.H., Instrumental methods of Analysis, CBS publishers & distributors, New Delhi, 7^th edition, 1986: 1-53, 513-522, 530-535.
7. Staut T, High Performance Liquid Chromatography. In: Ohnnesian L, Streeter AJ. Hnadbook of Practical Analysis, 1^st New York: Marcel Dekker, Inc; 2005: 117:87-90.
8. Hamilton R. J. and Swell  A.,  Introduction  to  HPLC.  Chapman and Hall; London, 1982, 2, 189-190.
9. Sethi P.D., et al., HPLC-Quantitative analysis of Pharmaceutical formulations, CBS publisher, & distributors, New Delhi, 2011, 11.
10. Yuri Kazakevich, Rosario Lobrutto, HPLC for Pharmacetical Scientists, A John Wiley & Sons, Inc., Publication, Canada, 2007, 10-22, 26-34.
11. Settle, et al., Handbook of Instrumental Techniques for Analytical Chemistry, NJ: Prentice-Hall, 1997, 58.
12. Validation of Analytical procedure; Text and methodology Q2 (R1), International Conference on Harmonisation of technical requirements for human use, ICH Harmonized tripartite guidelines, 2005.
13. Ravisankar et al, A Review on Analytical Method Development, Indian Journal of Research in Pharmacy and Biotechnology. 2014, 1183-1195.
14. Kiyoshi Kawabata, et al. Simultaneous determination of Azelnidipine and two metabolites in human plasma using liquid chromatography-tandem mass spectrometry, Drug metabolism and pharmacokinetics, Resaerch Laboratories Sankyo, Co. Ltd. 1-2-58, Hiromachi, Shingawa-ku, Tokyo, Japan, 2006, 46-52.

15. Zou Jian-Jun, et al, Determination of Azelnidipine by LC-ES-MS and its application to a pharmacokinetic study in healthy Chinese volunteers, Pharmazie 63, 2008, 568-570.
16. Nilam Patel, et al, Spectrophotometric method  for  the  simultaneous  determination  of Azelnidipine  and  Olmesartanmedoxomil by First  Derivative  Spectrophotometric  method, Scholar Research Library, 2012, 1080-1084.
17. Hiroyuki Daikuhara, et al, The combination of Olmesartan and a Calcium Channel Blocker (Azelnidipine) or candesartan and a Calcium Channel Blocker (Amlodipine) in Type 2 Diabetic hypertension patients: The OLCA study, Diabetes and Vascular Disease Research, 2012, 280-286.
18. Kunti D. Raskapur, et al, UV Spectrophotometric method development and validation for determination of Azelnidipine in pharmaceutical dosage form, International Journal of Pharmacy and Pharmaceutical Sciences, Volume-4, Issue 1, 2012, 238-240.
19. Raveendra Babu Ganduri, Stability indicating liquid chromatographic method for determination of Olmesartan Medoxomil and Azelnidipine in combined Tablet dosage form, International Journal of Pharma Science and Research, Volume-5, 2014, 275-282.
20. Jayvadan K. Patel Validated stability indicating RP-HPLC method for the Simultaneous determination of Azelnidipine and Olmesartan in their combined dosage form, Sci Pharm, 2014, 541-554
21. Rele Rajan V. Spectrophotometric estimation of Azelnidipine in bulk and pharmaceutical dosage form by second order derivative method, Journal of Chemical and Pharmaceutical Research, 2014, 198-202.
22. Selvadurai Muralidharan, et al. Simple validation of Azelnidipine by RP-HPLC Method, AIMST University, Malaysia, Volume-1, 2015, 43-45.
23. Rajan V. Rele, et al., Development and validation of Stability Indicating Reverse Phase Liquid Chromatographic method for the assay of Azelnidipine in bulk and pharmaceutical formulation , International Journal of Pharma and biosciences, 2016, 270-275.
24. Prabhakar D., et al., Development and Evaluation of Transdermal Patches of Azelnidipine, International Journal of Pharmacy and Pharmaceutical Sciences, Volume-5, 2013, 805.
    

    ---

    How to cite this article:

    Gore MG and Dabhade PS: RP-HPLC method development and validation of azelnidipine. Int J Pharm Sci Res 2016; 7(12): 5111-14.doi: 10.13040/IJPSR.0975-8232.7(12).5111-14.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---