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PHARM ANALYSIS AND QA
Year : 2011  |  Volume : 3  |  Issue : 1  |  Page : 55-59  

A validated densitometric method for analysis of atorvastatin calcium and metoprolol tartarate as bulk drugs and in combined capsule dosage forms


1 Department of Quality Assurance, A.I.S.S.M.S. College of Pharmacy, Kennedy Road, Near R.T.O., Pune - 411 001, India
2 Assistant Professor, Department of Pharmaceutical Chemistry, A.I.S.S.M.S. College of Pharmacy, Kennedy Road, Near R.T.O., Pune - 411 001, India

Date of Web Publication2-Feb-2011

Correspondence Address:
M C Damle
Assistant Professor, Department of Pharmaceutical Chemistry, A.I.S.S.M.S. College of Pharmacy, Kennedy Road, Near R.T.O., Pune - 411 001
India
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DOI: 10.4103/0975-1483.76420

PMID: 21607055

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   Abstract 

A simple, accurate and precise high-performance thin-layer chromatographic method has been developed for the estimation of Atorvastatin Calcium and Metoprolol Tartarate simultaneously from a capsule dosage form. The method employed Silica gel 60F 254s precoated plates as stationary phase and a mixture of Chloroform: Methanol: Glacial acetic acid (dil.) :: (9:1.5:0.2 ml %v/v) as mobile phase. Densitometric scanning was performed at 220 nm using Camag TLC scanner 3. The method was linear in the drug concentrations' range of 500 to 2500 ng/spot for Atorvastatin Calcium, also for Metoprolol Tartarate with correlation coefficient of 0.984 for Atorvastatin Calcium and 0.995 for Metoprolol Tartarate respectively. The retention factor for Atorvastatin Calcium was 0.45 ± 0.04 and for Metoprolol Tartarate was 0.25 ± 0.02. The method was validated as per ICH (International Conference on Harmonisation) Guidelines, proving its utility in estimation of Atorvastatin Calcium and Metoprolol Tartarate in combined dosage form.

Keywords: Atorvastatin calcium, high performance thin layer chromatography, metoprolol tartarate, validation


How to cite this article:
Patole S M, Khodke A S, Potale L V, Damle M C. A validated densitometric method for analysis of atorvastatin calcium and metoprolol tartarate as bulk drugs and in combined capsule dosage forms. J Young Pharmacists 2011;3:55-9

How to cite this URL:
Patole S M, Khodke A S, Potale L V, Damle M C. A validated densitometric method for analysis of atorvastatin calcium and metoprolol tartarate as bulk drugs and in combined capsule dosage forms. J Young Pharmacists [serial online] 2011 [cited 2014 Apr 24];3:55-9. Available from: http://www.jyoungpharm.in/text.asp?2011/3/1/55/76420


   Introduction Top


Atorvastatin Calcium is chemically known as (βR, 8R)-2-(4-fluorophenyl)-α, δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino) carbonyl] 1H-pyrrole-1-heptanoic acid trihydrate. [1] Metoprolol Tartarate is chemically known as(RS)-1-isopropylamino-3-p-(2-methoxyethyl)phenoxypropan-2-ol(2R,3R)-tartrate. [1] Atorvastatin Calcium is a member of the drug class known as statins, used for lowering blood cholesterol. It also stabilizes plaque and prevents strokes through anti-inflammatory and other mechanisms. Metoprolol Tartarate is an antihypertensive drug, It is a selective β1 receptor blocker used in the treatment of several diseases of the cardiovascular system, especially hypertension. Literature survey revealed that assay for Atorvastatin Calcium as bulk and its dosage form are official in Indian Pharmacopoeia 2007 [2] and for Metoprolol Tartarate is official in British Pharmacopoeia 2007. [3] RP-HPLC method has been reported for the estimation of Atorvastatin Calcium Fenofibrate. [4] Analytical methods reported for the estimation of Atorvastatin Calcium are HPLC, [5],[6],[7],[8] Stability indicating High Performance Liquid Chromatography (HPLC) [9],[10] High Performance Thin Layer Chromatography (HPTLC) [11],[12] and Spectrophotometry. [13],[14] Analytical methods reported for the estimation of Metoprolol Tartarate are Spectrophotometry [15] and HPLC. [16] Referring to the literature survey, there is no published HPTLC method for this combination. The present paper describes a simple, accurate and precise method for simultaneous estimation of Metoprolol Tartarate and Atorvastatin Calcium in combined capsule dosage form. The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) guidelines.




   Materials and Methods Top


Working standard of Atorvastatin Calcium and Metoprolol Tartarate was procured from Shreya Pharmaceuticals, Aurangabad, India as gift samples. Marketed formulation Betaone (Metoprolol Tartarate-25 mg and Atorvastatin Calcium-10 mg/capsule) was purchased from local market.

All other reagents used for experimentation were of analytical reagent (AR) grade. Chemicals used for this experiment were Acetonitrile, Methanol, Chloroform, Sodium hydroxide, Hydrochloric acid, and Hydrogen Peroxide.

Instruments

HPTLC system (Camag, Muttenz, Switzerland) comprising Linomat 5 sample applicator, twin-trough development chamber and TLC Scanner 3 with WinCATS evaluation ATS software (Version 1.4.3) was used in the studies. Electronic balance (Make SHIMADZU Model AY-120) was used for weighing purpose.


   Procedure Top


Preparation of standard stock solution

Ten mg each of Atorvastatin Calcium and Metoprolol Tartarate were weighed and transferred to 10-ml volumetric flask. Methanol (AR) was added to dissolve the drug and final volume was made with the same solvent to obtain a concentration 1000 μg/ml of each drug. Appropriate amount of stock solutions were diluted with methanol to obtain a concentration of 100 μg/ml of Atorvastatin Calcium and Metoprolol Tartarate.

Method development

Initially, trials were taken on TLC plates precoated with Silica gel 60F 254 . Each standard was applied as bands on TLC plates in five replicates. Plates were developed by linear ascending development using neat solvents like methanol, chloroform, acetic acid, acetone, etc. with chamber saturation. Based on the results of these initial chromatograms binary and ternary mixtures of solvents were tried to achieve optimum resolution with Atorvastatin Calcium and Metoprolol Tartarate respectively. Trials on TLC plates precoated with Silica gel 60F 254 showed movement of both drugs at nearly the same R f . After several trials, mixture of Chloroform: Methanol: Glacial acetic acid (dil.)  (9:1.5:0.2ml v/v) was chosen as the mobile phase for analysis which showed good separation for these two drugs. The linearity of the method was determined at five concentration levels ranging from 500 to 2500 ng/spot for Atorvastatin Calcium and the same for Metoprolol Tartarate.

Procedure for analysis of capsule formulation

Four capsules were weighed accurately and powder content was removed. Powder quantities equivalent to 10 mg of Atorvastatin Calcium and 25 mg of Metoprolol Tartarate were weighed separately and transferred to 25-ml volumetric flask and volume was made up. The resultant solution was further sonicated for 10 min. The volume was then made up to 10 ml with same solvent. Each solution was then filtered through whatmann filter paper no.41. From the filtrate, appropriate volumes were spotted to obtain final concentration of 320 ng/spot for Atorvastatin Calcium and 800 ng/spot for Metoprolol Tartarate. Spotting was done in the form of bands and plate was developed up to a distance of 90 mm, using the mobile phase Chloroform: Methanol: Glacial acetic acid (dil.)   (9:1.5:0.2ml v/v) in normal conditions of temperature and humidity. The peak areas of the spots were measured at 220 nm where both the drugs showed considerable absorbances [Figure 1] and concentrations in the samples were determined from the respective calibration curves. The amount of each drug present per capsule was calculated.
Figure 1 :Representative densitogram (linearity) of atorvastatin calcium and metoprolol tartarate

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Method Validation

As per the ICH guidelines, the method validation parameters checked were linearity and range, accuracy, precision, specificity, limit of detection, limit of quantitation and robustness.

Linearity

Different concentrations of Atorvastatin Calcium (500 ng to 2500 ng/band) and Metoprolol Tartarate (500 to 2500 ng/band) were applied on TLC plate and densitograms were developed. The data of peak area vs. drug concentration was obtained by linear least-square regression analysis.

Precision

Inter-day and intra-day precision was evaluated by analyzing sample preparations obtained from homogenous sample, six times and % RSD (Relative Standard Deviation) value obtained was calculated to determine any intra-day and inter-day variation.

Accuracy


To check accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 80, 100 and 120%. Mean percentage recovery was determined.

Limit of detection and limit of quantification

The limit of detection (LOD) and limit of quantification (LOQ) were obtained by calculations, using the standard formula as per the ICH guidelines,



where σ is Standard deviation of the response and S is slope of the calibration curve.

Specificity

The specificity of the method was ascertained by peak purity studies. Purity of the drug peaks was ascertained by analyzing the spectrum at peak start, max position and at peak end. The peak purity was determined by win CATS software.


   Results and Discussion Top


Linearity and Range

Linearity of the method was found in the range of 500 to 2500 ng/spot for Atorvastatin Calcium and 500 to 2500 ng/spot for Metoprolol Tartarate [Figure 2]. Rf for Atorvastatin Calcium and Metoprolol Tartarate were 0.45 and 0.25 respectively [Figure 3]. The linearity is indicated by regression equation. The linear regression equations obtained are:
Figure 2 :Representative densitogram of atorvastatin calcium with Rf 0.45 and metoprolol tartarate with Rf 0.25

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Figure 3 :Representative spectral comparison of atorvastatin calcium and metoprolol tartarate

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For Atorvastatin Calcium y = 2.209x + 556.3 (r 2 = 0.9991)

For Metoprolol Tartarate y = 1.853x + 250.9 (r 2 = 0.9950)

Accuracy

Excellent recoveries were obtained at each level of added concentration. The results obtained (n = 3 for each 80%, 100%, 120% level) indicated the recovery was within the range of 100 ± 2% which indicates accuracy of the method. Results are shown in [Table 1].
Table 2 :Validation parameters for Atorvastatin Calcium and Metoprolol Tartarate


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Precision

In the intra-day studies, three different concentrations of the mixed standard were analyzed in a day and percentage RSD was calculated and was found to be less than 1.5%. In the inter-day variation studies, three different concentrations of the mixed standard were analyzed on three consecutive days and percentage RSD was calculated which was found to be less than 1.5%. The results of the inter-day and intra-day studies are shown in [Table 2]. The data obtained indicates that the developed HPTLC method is precise.{Table 2}

Limit of Detection and Limit of Quantification

The LOD and LOQ values are shown in [Table 2].

Robustness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in procedure. Slight and deliberate changes were made to the following parameters like changing the mobile phase ratio and chamber size, and the effect on the Rf values and peak areas were noted. The method was found to be robust since the monitored parameters were not significantly affected.

Specificity

Win Cats software afforded automatic calculation of the peaks' purity by comparing the UV spectra, at the start, max and tail of the peak of each response. The correlation value r > 0.99 confirmed peak purity indicating that this HPTLC method is able to resolve and accurately measure two drugs in the presence of the matrix components. The peak purity values were found to be more than 0.9950 which show that the method is specific for the two drugs.


   Discussion Top


The method was found to be simple, fast and precise for the estimation of Atorvastatin Calcium and Metoprolol Tartarate simultaneously from a capsule dosage form. The accuracy of the method was found to be good. The method is also specific as confirmed by peak purity studies.


   Conclusion Top


The proposed HPTLC method for the simultaneous estimation of Atorvastatin Calcium and Metoprolol Taratrate in combined dosage forms was found to be sensitive, accurate, precise, simple and rapid. Hence the present HPTLC method may be used for routine analysis of these drugs and their formulations.


   Acknowledgments Top


The authors express their gratitude to the AISSMS College of Pharmacy, Pune, for providing the research facility and also to M/s. Shreya Pharmaceuticals, Ltd. Aurangabad, for providing the drug samples.

 
   References Top

1.Available form: http://www.wikipedia.com [last accessed on 2010 Jul 20].  Back to cited text no. 1
    
2.Indian Pharmacopoeia. Published by the Government of India, Ministry of Health and Family Welfare, the Indian Pharmacopoeia Commission, New Delhi, Vol 2 and 3, 2007. p. 749, 1574.  Back to cited text no. 2
    
3.British Pharmacopoeia. Vol. 1 - 4, London: The Stationary office Publications, 2009.  Back to cited text no. 3
    
4.Jain N, Raghuwanshi R, Jain D. Development and Validation of RP-HPLC method for simultaneous estimation of Atorvastatin Calcium and Fenofibrate in tablet dosage forms. Indian J Pharm Sci 2008;70:263-5.  Back to cited text no. 4
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5.Rajeswari RK, Sankar GG, Rao AL, Seshagirirao JV. RP-HPLC Method for the simultaneous determination of Atorvastatin and Amlodipine in tablet dosage form. Indian J Pharm Sci 2006;68:275-7.  Back to cited text no. 5
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6.Chaudhari BG, Patel NM, Shah PB, Patel LJ, Patel VP. Stability indicating reverse phase liquid chromatographic method for simultaneous determination of atorvastatin and ezetimibe from their combination drug products. J AOAC Int 2007;90:1539-46.  Back to cited text no. 6
    
7.Bhatt DS, Bhatt KK, Mehta RS, Baldania SL, Gandhi TR. Stability indicating RP-HPLC method estimation of atorvastatin calcium and Amlodipine Besylate in pharmaceutical formulations. Indian J Pharm Sci 2007;70:546-9.  Back to cited text no. 7
    
8.Stanisz B, Kania L. Validation of HPLC method for determination of Atorvastatin in tablets and for monitoring stability in solid phase. Acta Pol Pharm 2006;63:471-6.  Back to cited text no. 8
    
9.Mohammadia A, Rezanour N, Ansari M, Dogahehc F, Ghorbani BM. Hashemb RB, et al. A stability indicating HPLC assay for the simultaneous determination of atorvastatin and amlodipine in commercial tablets. J Chromatogr B Analyt Technol Biomed Life Sci 2007;846:215-21.  Back to cited text no. 9
    
10.Chaudhari BG, Patel NM, Shah PB, Modi KP. Development and Validation of HPTLC method for the simultaneous estimation of Atorvastatin Calcium and Ezetimibe. Indian J Pharm Sci 2006;68:793-6.  Back to cited text no. 10
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11.Deshpande P, Shridharan BG, Anandi L, Jadhav D, Damle MC, Gandhi SV. Validated method development for Estimation of Atorvastatin Calcium and Fenofibrate in fixed dose combination by HPTLC. The Pharma Review 2009;7(39) : 151-3.  Back to cited text no. 11
    
12.Patil UP, Gandhi SV, Sengar MR and Rajmane VS. Simultaneous determination of Atorvastatin calcium and Telmisartan in tablet dosage form by spectrophotometry. Int J Chem Tech Res 2009;1:970-3.  Back to cited text no. 12
    
13.Thamake SL, Jadhav SD, Pishawikar SA. Development and Validation of method for simultaneous estimation of Atorvastatin calcium and Ramipril from capsule dosage form by first order derivative spectroscopy. Asian J Res Chem 2009;1:52-3.  Back to cited text no. 13
    
14.Chitlange SS, Mohammed Imran, Sakarkar DM. RP-HPLC of Simultaneous Estimation of Metoprolol and Amlodipine in tablet dosage form. Asian J Pharmaceut 2008;2(4):232-4.  Back to cited text no. 14
    
15.Gupta KR, Tajane MR, Wadodkar SG. New spectrophotometric method for simultaneous determination of Metoprolol Tartarate and Hydrochlorothiazide in tablets. Indian J Pharm Sci 2008;70(4):511-3.  Back to cited text no. 15
    
16.Rao KV, Kumar BV, Rao ME, Rao SS. Determination of Metoprolol Tartarate by RP-HPLC. Indian J Pharm Sci 2003;65:204-6.  Back to cited text no. 16
  Medknow Journal  


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

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