• Nenhum resultado encontrado

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF SIMULTANEOUS ESTIMATION OF AMLODIPINE AND ATORVASTATIN BY RP-UPLC

N/A
N/A
Protected

Academic year: 2017

Share "ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF SIMULTANEOUS ESTIMATION OF AMLODIPINE AND ATORVASTATIN BY RP-UPLC"

Copied!
4
0
0

Texto

(1)

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF SIMULTANEOUS

ESTIMATION OF AMLODIPINE AND ATORVASTATIN BY RP-UPLC

Waghmare A. N, Muddukrishna B.S and, Vasantharaju S.G*

Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India Email:sg.vasanthraj@manipal.edu

Received -08-01-14; Reviewed and accepted -20-01-14

ABSTRACT

Objective: To develop and validate simple, sensitive, robust, rapid and specific isocratic RP-UPLC method for simultaneous estimation of Amlodipine and Atorvastatin in tablet dosage form. Methods: The developed method consisting the mobile phase of acetonitrile and 0.02 M Potassium dihydrogen phosphate (55:45) with isocratic programming, BEH C18 (100mm×2.1mm, 1.7μm) column as stationary phase with a flow rate of 0.3 mL/minute. Results and discussion: Proposed method was found to be

linear for Amlodipine and Atorvastatin in the concentration range of 0.5 to 40.0 μg/mL with r2 of 0.9999 for Amlodipine and 0.9997 for Atorvastatin respectively. Precision study showed that the percentage relative standard deviation was within the range of acceptable limits, and the mean recovery was found to be 100.79 % for assay of Amlodipine and 99.87% for Atorvastatin in tablet dosage form .The LOD and LOQ of Amlodipine and Atorvastatin were found to be 0.062 and 0.078µg/ml and 0.020 and 0.026 µg/mL.

Keywords: Amlodipine, Atorvastatin&UPLC method.

INTRODUCTION

Atorvastatin (fig-1) is chemically [R-(R*, R*0] [R-(R*, R*0] -2-(4-Fluorophenyl)-β,ϒ-dehydroxy- 5- (1-mehtylethyl)- 3- phenyl-4- [9phenyl amino)-carbonyl]-1H-pyrrole-1-heptanoic acid. Amlodipine (Fig. 2) is chemically 2-[(2-Aminoethoxy)methyl]-4-(2-chlorophenyl)-1, 4-dihydro-6-methyl-3, 5-pyridinedicarboxylic acid 3-ethyl 5-methyl ester act as a calcium channel blocker[1,2].

Fig 1: Atorvastatin

Amlodipine and Atorvastatin standards were obtained from Arene Life sciences, and DSM Sinochem Pharmaceutical India Pvt., Ltd., methanol, acetonitrile and potassium dihydrogen phosphate (HPLC grade) were obtained from RanChem. Atorvastatin and amlodipine tablets were purchased commercially. Milli-Q Water was used throughout the experiment. Other chemicals used were have analytical or HPLC grade.

Fig 2: Amlodipine

MATERIALS AND METHODS Chromatographic Conditions

A chromatographic system Waters UPLC Acquity H-Class with photodiode array detector, BEHC18 (2.1*100) mm, 1.7 µ column was used. A flow of 0.30 mL/min, injection volume of0.80 µl, detection wavelength of 242 nm for both the analytes. The peak purity was checked with the photodiode array detector [3].

Mobile Phase

20 mM phosphate buffer and acetonitrile in the ratio of 45:55(v/v), the pH of the buffer was adjusted to 3.5by (20%v/v) of ortho phosphoric acid in the milli- Q water. The mobile phase was mixed and filtered through a nylon filter and degassed.

Preparation of Amlodipine Besylate standard stock solution Accurately weighed and transferred about 10.0 mg of Amlodipine Besylate working standard to 100mL volumetric flasks, 30 mL of methanol was added to it and sonicated to dissolve. Volume was made up to the mark with methanol.

Preparation of Atovastatin Calcium standard stock solution Accurately weighed and transferred about 10 mg of Atovastatin Calcium working standard to a 100mL volumetric flasks, 30 mL of methanol was added to it and sonicated to dissolve for 5 min. Volume was made up to the mark with methanol.

Preparation of working standard solution

10 mL of the above prepared Atovastatin Calcium stock solution and 5 mL from Amlodipine Besylate stock was transferred to 100 mL volumetric flask and the volume was made with mobile phase and filtered through 0.22 μm nylon filter.

Preparation of sample solution

(2)

Vasanthraju et al Mintage journal of Pharmaceutical & Medical SciencesI22-25

Vol 3 Suppl 2, Feb 2014 www.mintagejournals.com 23

Method Validation [4-8] Linearity

The linearity of the calibration curves was determined for intra- and intraday precision on 3 different days

Primary stock solution

Weighed accurately 10 mg of each Amlodipine and Atorvastatin in 100 mL of A-grade volumetric flask separately added 70 ml of methanol to dissolve and sonicated for 10 min and volume was made up to mark with methanol(100 μg/mL ).

Intermediate stock solution

It was prepared in100 mL of A-grade volumetric flask by diluting 50 mL primary stock solution of Amlodipine and Atorvastatin with mobile phase to 100 mL to get concentration of 50 μg/mL.Linearity standards of concentration 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 40μg/mL were prepared in a separate set of 50 mL of A-grade volumetric flask by diluting 0.5,1,2,5,10,20, 40 mL intermediate working stock solution with mobile phase.

The calibration curves were constructed by plotting the absolute peak area(y) versus the concentration (x), by using linear regression analysis.

Precision

Repeatability of sample preparation and measurement of sample area were carried out using six injection of tablet sample of Amlodipine and Atorvastatin. The intermediate precision were calculated using different column and different system. The mean area and % assay for those injections were calculated.

System precision was the result of the method operating over a short time interval under the same conditions. Six injections of standard preparation were injected and compliance for system suitability test was checked.

Limit of detection (LOD) and Limit of Quantification (LOQ) The LOQ (defined as the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy) and the LOD (defined as the lowest absolute concentration of analyte in a sample that can be detected but not necessarily quantified) were calculated according to ICH guideline

Specificity

Specificity of the method was performed by analyzing drug and sample and interference from blank and placebo was checked using PDA detector and peak purity was confirmed.

Accuracy

The known amount of standard drug was spiked in triplicate to the placebo samples and the recovery of the drug was calculated. Accuracy was performed at 3 levels: 80%, 100%, 120% of sample concentration, in triplicate at each level, using the placebo spiked with drug. Samples were prepared by adding corresponding weight of Amlodipine and Atorvastatin in placebo and processes as per sample preparation.

Robustness

Robustness was tested by changing the following parameters of the method: a) Flow rate. (b) Column temperature (c) Variation of pH of buffer (d) Wavelength of the detector and the results were compiled and % RSD of area was calculated.

Application of proposed method to tablet formulation To determine the concentration of Amlodipine and Atorvastatin in tablets (label claim: 5 mg Amlodipine and 10 mg of Atorvastatin), the sample was prepared as per the earlier sample preparation procedure to get the concentration of 5µg/mL of Amlodipine and 10µg/mL of Atorvastatin. The analysis was repeated in triplicate. The possibility of excipient interference in the analysis was studied. Peak were found to be symmetric with good resolution as shown in figure 3.

Fig 3: Optimized chromatogram of Amlodipine and Atorvastatin

RESULT Calibration curve

The linear regression data for the calibration curve showed good linear relationship over the concentration range 0.5-40

µg/mL.Linear regression equation for Amlodipine and Atorvastatin were found to be y= 4670.8x-292.3, (r2=0.9999) and

y=5901.2x+778.99 (r2=0.9997) respectively.

Fig 4: Linearity of Atorvastatin

Fig 5: Linearity of Amlodipine

Precision

(3)

Vasanthraju et al Mintage journal of Pharmaceutical & Medical SciencesI22-25

Vol 3 Suppl 2, Feb 2014 www.mintagejournals.com 24

Table 1: Precision study

Parameter

Amlodipine Atorvastatin %RSD

(Area) %Assay

%RSD

(Area) % Assay Repeatability 0.25 101.23 0.22 99.43 Intermediate 0.62 101.47 0.28 99.01

System 0.26 - 0.28 -

LOD and LOQ

Limit of Detection and Limit of Quantification for Amlodipine and Atorvastatin were found to be 0.26 and 0.78µg/mL and 0.20 µg/mL and 0.62 µg/mL respectively based on signal to noise ratio method.

Accuracy

This parameter was evaluated by the recovery studies at concentration levels of 80, 100, and 120%, which consisted of

adding known amounts of Amlodipine and Atorvastatin reference materials to the placebo. The amount of drug added and determined and the % recovery are listed in table 2.

Specificity

The peak purity of Amlodipine and Atorvastatin was assessed by PDA detector which revealed that both peak were pure. There was no interference from blank and placebo at the retention time of Amlodipine and Atorvastatin.

Robustness

Results of robustness were compiled as indicated in Table 3. % R.S.D was calculated for each parameter and was found to be less than 2%.

Table 2: Accuracy study.

Drug Label claim

(mg/ tablets) Amount of standard added (%) %Drug recovered % RSD

Amlodipine 5 mg

80 99.16 0.75

100 100.54 0.11

120 99.36 0.67

Atorvastatin 10 mg

80 99.95 0.56

100 99.46 0.11

120 101.13 0.18

Table 3: Robustness study

Parameter Variation Amlodipine %R.S.D. for area Atorvastatin %R.S.D. for area Flow rate 0.285 mL 0.315 mL 0.59 1.33 0.08 1.1

Wavelength 240 nm 0.35 0.59

244 nm 1.19 0.16

Column temperature 0 C 0.63 0.24

0 C 0.39 0.33

pH 3.30 0.73 0.59

3.70 1.15 0.38

Analysis of marketed formulation

The combination of Amlodipine and Atorvastatin was assayed in the tablet which contains Amlodipine 5 mg and Atorvastatin 10 mg was present. Mean assay of Amlodipine and Atorvastatin was found to be 100.05and 100.63% w/w and % RSD of results of Amlodipine and Atorvastatin was found to be 0.413% and 0.65%w/w respectively.

CONCLUSION

A simple, sensitive and specific isocratic RP-UPLC method was developed for assay of Amlodipine and Atorvastatin in tablet dosage form. The proposed analytical method has been proved as rapid, simple, specific, accurate as well as cost effective and hence is considered to be reliable and suitable for the routine quality control analysis of Amlodipine and Atorvastatin.

REFERENCES

1. Drug bank, open data drug and drug target database Amlodipine. [Internet].[Cited 2012 Dec.13]. Available from URL: http://www.drugbank.ca/drugs/DB00381

2. Drug bank, open data drug and drug target database Atorvastatin.[Internet].[Cited 2012 Dec.13]. Available from URL: http://www.drugbank.ca/drugs/DB01076

3. Llyod Snyder R, Joseph Kirkland J, Joseph Glajch. Practical HPLC method development.2nd ed. New York. John Wiley & Sons, Inc. 1997. P.77- 80.

4. International conference on harmonization (ICH) Q2B. Validation of analytical procedures: methodology. [Internet] [Cited 2012 Nov 18]. Available from URL:

http://www.ich.org/products/guidelines/quality/quality- single/article/validation-of-analyticalprocedures-text-and-methodology.html

5. Ermer J, Miller JH, Method Validation in Pharmaceutical Analysis. A Guide to Best Practice. Wiley – Vch Verlag, GmbH & Co. 2005; p. 95.

6. Mohammad T.Zzaman, Sadaf J.Gilani,K.Nagarjun,Shdab A.siddiqui.HPLC Method development and validation for pharmaceutical analysis-A review.Internationale pharmaceutica scienceia Jul-Sep 2012;13-21

7. Masato Kazusaki, Shinji Ueda, Naoto Takeuchi, Yasutaka Ohgami. Validation of analytical procedures by high- performance liquid chromatography for pharmaceutical analysis, Chromatography 2012; Vol. 33 No. 2.

8. Azim Md. Sabir, Mitra Moloy, Bhasin Parminder.HPLC method development and validation A review. International Research Journal of Pharmacy 2013 April 11; 1-4.

9. Ministry of Health and Family Welfare, Government of India. Indian Pharmacopoeia 5th edn. The Indian Pharmacopoeial Commission, Ghaziabad; 2007.144-45.

10. Rang HP, Dale MM, Ritter JM, More PK, In; Pharmacology, 5th Edn, Elseiver SciencePublisher, 2003; 282-83, 310-11. 11. A.Mohammadi, N. Rezanour, M. Ansari Dogaheh, F.

Ghorbani Bidkorbeh, M. Hashem & R.B. Walker. A stability indicating high performance liquid chromatographic (HPLC) assay for the simultaneous determination of Atorvastatin and Amlodipine in commercial tablets.J.Chromatogrp. B, 2007; 846: 215-21.

(4)

Vasanthraju et al Mintage journal of Pharmaceutical & Medical SciencesI22-25

Vol 3 Suppl 2, Feb 2014 www.mintagejournals.com 25 13. DA Shah, KK Bhatt, MB Shankar, RS Mehta, TR Gandhi &

SL Baldania. RP-HPLC determination of Atorvastatin calcium and Amlodipine besylate combination intablets. Indian J. Pharm. Sci. 2006, 68:796-99.[Internet][ cited 2012 November 24].Available from: http://www.cda-adc.ca/jcda/vol-75/issue-3/201.html

14. V. Juyal, M. Chaudhary, P. Kumar, G. Gnanarajan & P. K. Yadav .Method development and its validation for simultaneous estimation of Atorvastatin and Amlodipine in combination in tablet dosage form by UV spectroscopy, using multicomponent mode of analysis. J.Pharmacy Research, 2008; 1: 182-187.

15. A.Kottai Muthu, Rattaiah Gupta, Shailesh Sharma, A.Anton Smith, R.Manavalan and N.Kannappan. Simultaneous estimation of Amlodipine and Atorvastatin in tablets using orthogonal function ratio spectrometry. International Journal of Chemtec Scince, 2008; 2233-41

16. Raja Rajeswari K, Sankar GG, Rao AL, Seshagirirao JVLN. RP-HPLC methods for the simultaneous determination of Atorvastatin and Amlodipine in tablet dosage form. Indian Journal of Pharmaceutical Sciences2006; 68: 275-77. 17. Samita ku.Acharjya, M.Mathrusri Annapurna and Sailaja

Koya. Liquid chromatographic methods for the simultaneous determination of Atorvastatin and Amlodipine in tablet dosage forms. International journal of pharma and bio scinces.2010; 161-70

18. Abdullah Al masud, Mohammad saydur rahman, moynul Hasan, Md.Kamal Hassain Ripon, Ahsanur Rahman Khan, Md.Rabiul Islam, Md.Monjurul Ahasan, Md.Rakib Uddin.Validated RP-HPLC methods for the simultaneous determination of Atorvastatin and Amlodipine in tablet dosage form. Bangladesh Research Publications journal.september-october, 2011; 52-6

19. Amit Kumar shrama and Abhay Dharmasi. Development and validation of RP-HPLC and spectrometric methods for the simultaneous determination of Atorvastatin and Amlodipine in pharmaceutical dosage forms. International journal of pharmaceutical sciences and research.30 March, 2012; 1202-07.

20. Manzoor Ahmed, Manohara Y.N, Ravi M.C. RP-HPLC methods for the simultaneous determination of Atorvastatin Calcium and Amlodipine besylate. International journal of chem.tech research. Jan-March, 2012; 337-45.

Referências

Documentos relacionados

The DD1, multivariate calibration technique (PLS) and HPLC methods enable the simultaneous determination of amlodipine and atorvastatin in their binary mixture with good

Commercial capsule formulation was successfully analyzed using the developed method and the proposed method is applicable to routine analysis of determination of

Then pipette out 10ml from the standard stock solution in other 100ml volumetric flask and diluted up to mark with Methanol to give a working standard solution having

A new UV- Spectrophotometric method has been developed for the simultaneous estimation of atenolol and amlodipine besylate in tablet dosage forms using 0.1N hydrochloric acid

There were no simple and reproducible methods so far reported for simultaneous determination of Methyl Sulphonyl Methane by GC in solid dosage form.It was essential to

A simple, accurate, precise, reproducible, highly sensitive, economic UV spectrophotometric method has been developed for the estimation of metaxalon in bulk and tablet dosage

The present manuscript describes new, simple, accurate, and precise high performance thin layer chromatography method for the simultaneous determination of Ambroxol Hydrochloride

Simultaneous estimation and validation of developed method of Nifedipine (NIF) and Atenolol (ATN) in combined dosage form as well as in laboratory mixture