High Performance Thin Layer Chromatographic Determination of Metoprolol Succinate and Olmesartan in Combined Capsule Dosage Form

 

Vandana S. Bhavnani, Padmanabh B. Deshpande*, Santosh V. Gandhi, Prajakta Pawar, Ashish K. Gaikwad

Department of Pharmaceutical Analysis, AISSMS College of Pharmacy, Kennedy Road, Near R. T. O., Pune–411001

*Corresponding Author E-mail: padmanabh77@yahoo.co.in

 

ABSTRACT:

This paper describes a new simple High Performance Thin Layer Chromatographic (HPTLC) method for determination of Metoprolol succinate and Olmesartan in combined capsule dosage form has been developed and validated. The mobile phase selected was n- butanol and Methanol (6:3, v/v) with UV detection at 225 nm. The retention factor for Metoprolol succinate and Olmesartan were found to be 0.23 ± 0.02 and 0.65 ± 0.05, respectively. The method was validated with respect to linearity, accuracy, precision and robustness as per the International Conference on Harmonisation (ICH) guidelines. Results found to be linear in the concentration range of 250-2500 ng band -1 for Metoprolol succinate and 100-1000 ng band -1 for Olmesartan. The method has been successfully applied for the analysis of drugs in pharmaceutical formulation. The % assay (Mean ± S.D.) was found to be 100.09 ± 0.97 for Metoprolol succinate and 100.54 ± 0.80 for Olmesartan.

 

KEY WORDS: Metoprolol Succinate, Olmesartan, HPTLC, Capsule dosage form.

 


 

INTRODUCTION:

Metoprolol Succinate (METO), chemically, (RS)-1-(Isopropylamino)-3-[4-(2-methoxyethyl) phenoxy] propan-2-ol is a selective β1 receptor blocker used in treatment of several diseases of the cardiovascular system, especially hypertension1. Olmesartan (OLME), Chemically, (5-methyl-2-oxo-2H-1,3-dioxol-4-yl)methyl 4-(2-hydroxypropan-2-yl)-2-propyl-1-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1H-imidazole-5-carboxylate,  it is an angiotensin II  receptor antagonist used to treat high blood pressure2.

 

Literature survey reveals that several spectrophotometric3-5, HPLC6-10, HPTLC11 methods have been reported for the determination of METO as a single drug and in combination with other drugs. Analytical methods reported for OLME includes spectrophotometric12-14, HPLC15-19, HPTLC20 as a single and in combination with other drugs.

 

To best of our knowledge no reports were found for the simultaneous estimation of METO and OLME in combined dosage form by HPTLC method. This paper describes a simple, sensitive, accurate, and validated High-performance Thin Layer Chromatographic (HPTLC) method for the simultaneous quantification of these compounds as a bulk drug and in capsule dosage forms. The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) guidelines21.

 
MATERIALS AMD METHODS:

Chemicals and reagents

Working standards of pharmaceutical grade METO and OLME were obtained as generous gifts from Emcure Pharmaceuticals Ltd, Pune, India. The pharmaceutical dosage form used in this study was Olsar- M capsule (Unichem Pvt Ltd. India) labelled to contain 50 mg of METO and 20 mg of OLME were procured from the local market. n - butanol (AR  grade) and Methanol (AR grade) purchased from Merck specialties Pvt. Ltd.  (Mumbai, India) were used for analysis.

 

Instrumentation and chromatographic conditions

The samples were spotted  in the form of bands of width of 6 mm with space between bands of 8.8 mm, with a 100 µL sample syringe (Hamilton, Bonaduz, Switzerland) on precoated silica gel aluminium plate 60 F254 (10 ×10) with 250 µm thickness (E. MERCK, Darmstadt, Germany) using a CAMAG Linomat 5 sample applicator (Switzerland). The slit dimensions 5 mm × 0.45 mm and scanning speed of 20 mm/sec was employed.

 

The linear ascending development was carried out in 10 cm × 10 cm twin trough glass chamber (CAMAG, Muttenz, Switzerland) using  n- butanol and Methanol  (6:3, v/v) as mobile phase. The optimized chamber saturation time for mobile phase was 10 min. The length of chromatogram run was 8 cm and development time was approximately 20 min. TLC plates were dried in a current of air with the help of a hair drier. Densitometric scanning was performed on CAMAG thin layer chromatography scanner at 225 nm for all developments operated by winCATS software version 1.4.2. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 200 to 400 nm.  

 

Preparation of standard stock solutions

Standard stock solutions of METO and OLME were prepared by dissolving 10 mg of each drug separately in 10 mLof methanol to get solution of concentration 1 mg mL-1  from which one millilitre of the solution was further diluted to 10 mL to get a working standard solution of concentration 100 ng µL-1 for both  the drugs.

 

Selection of Detection Wavelength

After chromatographic development bands were scanned over the range of 200-400 nm and the spectra were overlain. It was observed that both drugs showed considerable absorbance at 225 nm. So, 225 nm was selected as the wavelength for detection as shown in Figure 1.

 

Figure 1: Overlain spectra of METO and OLME

 

Analysis of Tablet formulation

Contents of twenty capsules were weighed accurately and powdered. A quantity of powder equivalent to 2.5 mg of METO  was weighed and transferred to a 10 ml volumetric flask containing approximately 6 ml of methanol, sonicated for 5 min and volume was made up to the mark with the methanol. The solutions were filtered through Whatman filter paper No. 41. Two microlitre volume of the solution was applied to a TLC plate to furnish final concentration of 500 ng band -1 for METO and 200 ng band -1 for OLME. After chromatographic development the peak areas of the bands were measured at 225 nm and the amount of each drug present in sample was estimated from the respective calibration curves. Procedure was repeated six times for the analysis of homogenous sample.

 

Method Validation

The method was validated for linearity, accuracy, intra-day and inter-day precision and robustness, in accordance with ICH guidelines 21.

 

Preparation of Calibration Curve

Aliquots 2.5, 5, 10, 15, 20, 25 µL of METO and 1, 2, 4, 6, 8 and 10 µL of OLME (100 ng µL-1 each) were applied by overspotting on HPTLC plate with the help of CAMAG 100 µL sample syringe, using Linomat 5 sample applicator. The plate was developed and scanned under above established chromatographic conditions. Each standard in six replicates was analyzed and peak areas were recorded. Calibration curves of METO and OLME were plotted separately of peak area vs. respective concentration.

 

Precision

Set of three different concentrations in three replicates of mixed standard solutions of METO and OLME were prepared. All the solutions were analyzed on the same day in order to record any intra day variations in the results. For Inter day variation study, three different concentrations of the mixed standard solutions in linearity range were analyzed on three consecutive days.

 

Limit of detection (LOD) and Limit of quantitation (LOQ)

LOD and LOQ for both the drugs were calculated by using the values of slopes and intercepts of the calibration curves.

 

Robustness Studies

In the robustness study, the influence of small, deliberate variations of the analytical parameters on peak area of the drugs was examined. Factors varied were mobile phase composition (± 2 %), mobile phase saturation (± 10 %), time from application to development (0, 10, 20, and 30 min) and from development to scanning (0, 30, 60, and 90 min) and development distance (± 10 %). One factor at a time was changed to estimate the effect. Robustness of the method was checked at a concentration level of 500 ng band-1 for METO and 200 ng band -1 for OLME.

 

Recovery Studies

To check the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 50, 100 and 150 %. Chromatogram was developed and the peak areas were noted. At each level of the amount, three determinations were carried out. The results of recovery studies were expressed as percent recovery and are shown in Table 1.

 

RESULTS AND DISCUSSION:  

Different mobile phases containing various ratios of n- butanol, Methanol, Chloroform and Toluene were examined (data not shown). Finally the mobile phase containing n- butanol: Methanol (6: 3, v/v) was selected as optimal for obtaining well defined and resolved peaks. The optimum wavelength for detection and quantitation used was 225 nm. The retention factors for METO and OLME were found to be 0.23 ± 0.02 and 0.65 ± 0.05, respectively. Representative densitogram of mixed standard solution of METO and OLME is shown in Figure 2.

 

Figure 2: Representative densitogram of mixed standard solution METO (500 ng/band, Rf = 0.23 ± 0.02) and OLME (200 ng/band, Rf = 0.65 ± 0.05)

Straight-line calibration graphs were obtained for METO and OLME in the concentration range 250-2500 ng band -1 and 100-1000 ng band -1 respectively with high correlation coefficient. The proposed method was also evaluated by the assay of commercially available Capsules containing METO and OLME. The % assay (Mean ± S.D.) was found to be 100.09 ± 0.97 for METO and 100.54 ± 0.80 for OLME. Robustness of the method checked after deliberate alterations of the analytical parameters showed that areas of peaks of interest remained unaffected by small changes of the operational parameters (% RSD < 2).

 

 

For METO, the recovery study results ranged from 100.02 to 100.78 % with % RSD values ranging from 0.26 to 0.85. For OLME, the recovery results ranged from 99.98 to 100.88 % with % RSD values ranging from 0.18 to 0.69. The method was found to be accurate and precise, as indicated by recovery studies as recoveries were close to 100 % and % RSD not more than 2. Intra-day variation, as RSD (%), was found to be in the range of 0.12–0.30 for METO and 0.41–0.50 for OLME. Interday variation, as RSD (%) was found to be in the range of 0.51–0.85 for METO and 0.35–0.44 for OLME. The summary of validation parameters of proposed method are given in Table 2.

 

Table 1: Recovery Studies of METO and OLME

Drug

Level of

% Recovery

Amount taken

(μg mL-1 )

Amount added

(μg mL-1 )

Amount found

(μg mL-1 )

% Recovery

Mean

± S.D.

% R.S.D.*

METO

50

500

250

750.20

100.02

100.40 ± 0.61

0.61

100

500

500

1007.80

100.78

150

500

700

1255.23

100.41

OLME

50

200

100

299.94

99.98

100.49 ± 0.39

0.39

100

200

200

403.52

100.88

150

200

300

503.06

100.61

a Average of three determinations

 

Table 2: Summary of validation parameters of proposed method

Parameters

METO

OLME

Linearity range (  ng band -1 )

250 - 2500

100 -1000

Correlation coefficient (r)

0.992

0.992

LODa ( ng band -1 )

46.08

11.51

LOQb ( ng band -1 )

139.64

34.90

Accuracy (% Recovery)

100.02-100.78

99.88-100.88

Precision (% RSD)c

Intra day (nd = 3)

0.12–0.30

0.41–0.50

Inter day (n = 3)

0.51–0.85

0.35–0.44

aLOD  = Limit of detection; bLOQ = Limit of quantitation; cRSD       = Relative standard deviation; dn  =  Number of determinations


CONCLUSION:

The validated HPTLC method employed here proved to be simple, fast, accurate, precise and robust, thus can be used for routine analysis of THIO and DEXKETO in combined capsule dosage form.

 

ACKNOWLEDGEMENTS:

The authors express their gratitude to Emcure Pharmaceuticals Ltd, Pune, India for the gift sample of pure METO and OLME. Thanks are also extended to Dr. Ashwini. R. Madgulkar, Principal, A.I.S.S.M.S. College of Pharmacy for providing necessary facilities and her constant support.

 

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Received on 05.10.2012                Modified on 11.10.2012

Accepted on 25.10.2012               © RJPT All right reserved

Research J. Pharm. and Tech. 5(11):November, 2012; Page 1461-1464