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DEVELOPMENT AND VALIDATION OF STABILITY INDICATING  RP-HPLC METHODS FOR ESTIMATION OF MANIDIPINE HYDROCHLORIDE


Alka Agarwal*, Indrajeet Singhvi, Ayush Garg 
Pacific College of Pharmacy, Udaipur India

ABSTRACT 

A simple, fast, precise and accurate high performance liquid chromatographic method has been developed for the quantitative estimation of Manidipine hydrochloride in bulk and tablet dosage form. The method was developed using acetonitrile: Phosphate buffer in the proportion of 40: 60 v/v at flow rate of 1.4 ml/min as mobile phase. The separation was carried out on inertsil ODS C18 (250 mm x 4.6 mm, 5 μm) column and the eluents were detected at 228nm. The linearity was observed in the concentration range of 20 to 150 μg/ml of manidipine hydrochloride. The LOD and LOQ of the method were 0.48 and 1.47 μg/ml respectively. The system suitability parameters and other validation studies were performed to ensure accurate and precise method for estimation of manidipine hydrochloride.

Keywords: Manidipine hydrochloride, RP-HPLC.

 

INTRODUCTION

Manidipine hydrochloride, chemically, 2-[4-(diphenylmethyl) piperazin-1-yl] ethyl methyl 2,6- dimethyl -4-(3- nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate. The molecular mass of manidipine hydrochloride is 610.69 g/mol. For the estimation of Manidipine hydrochloride from biological fluid, many HPLC methods has been reported. But in most of the method dimethylformamide (DMF) was used as mobile phase, which affect the life period of column, it also affects system suitability parameters of HPLC. So an attempt has been made in the present study to develop such a method, which exclude the use of DMF and prolong column life period and produce the good system suitability parameters1-4.

Material and method

For HPLC method development, inertsil ODS C18 (250 mm x 4.6 mm, 5 μm) column and phosphate buffer (pH-2.2): acetonitrile: 60: 40 as mobile phase were used. Instrumental conditions were: detection at 228 nm, flow rate 1.4 ml/min and run time was 12 min. The column temperature was kept at 40oC. The mobile phase was filtered through 0.45 μm Nylon 6, 6 membrane filter and degassed in ultrasonic water bath. The solvent used for analysis, consists acetonitrile: water (50:50).

Experimental work

Weighed quantity of 50 mg of Manidipine hydrochloride in to 50 ml volumetric flask and about 15 ml of solvent (ACN: Water, 50: 50) is added to it. Sonicated to dissolve completely and diluted to 50 ml with diluent. The final concentration was 1000 μg/ml of Manidipine hydrochloride. Injected the standard solution of Manidipine hydrochloride in to the stream of mobile system at a flow rate of 1.4 ml/min. The solution was injected 5 times into the column and the corresponding chromatograms were obtained. From these chromatograms the area under the peaks and respective retention time of the drug were noted. The retention time of Manidipine hydrochloride observed was 9.59 min.

A commercial brand of tablet for oral administration was chosen for testing suitability of the proposed method to estimate Manidipine hydrochloride in tablet for oral formulation. The label claim was 20 mg/ ml. 10 microlitre of the sample solution (100μg/ml Manidipine) was injected. Six replicates of the sample solution were applied. From the peak area of Manidipine amount of drugs in samples were computed.

Table 1: Linearity of Manidipine by RP-HPLC Method:

 

Level Amt. of drug

Added (mg)

Amt. of drug

Recovered(mg)

% Recovery Mean R.S.D
50% 25 24.9 99.6  

100.52

 

 

1.15

50% 25 25.03 100.13
50% 25 25.45 101.82
100% 50 50.86 101.71  

100.713

 

0.95

100% 50 50.32 100.63
100% 50 49.9 99.8
150% 75 74.25 99  

99.89

 

1.14

150% 75 75.8 101.18
150% 75 74.62 99.5

 


Table 2: Accuracy Study (Recovery)
 

Concentration (µg/ml) Mean area ± S.D % RSD
20 300546.3±3624.14 1.21
40 592104±23367.44 0.39
60 889920.3±6192.45 0.70
80 1186793±10258.64 0.87
100 1511226±15048.74 0.99
120 1774339±25681.2 1.45
150 2185589±11719.7 0.54

Table 3: Precision

Method of Precision
Concentration Area
 

 

100(μg/ml)

1507282

1507564

1506998

1508112

1509965

1512563

Mean 1508747.33
S.D 2147.25
R.S.D 0.14

To achieve sharp peaks with good resolution under isocratic conditions, mixture of buffer and acetonitrile in different proportion were tested as mobile phase on a C18 stationary phase. The mixture of buffer and acetonitrile in the proportion (60: 40 v/v) proportion was proved to be the most suitable for estimation. Since the chromatographic peaks were better defined, resolved, and free from tailing with this system, under the above mentioned chromatographic conditions, the retention time obtained for Manidipine hydrochloride was 9.59 min. The method was validated for specificity, linearity, accuracy, precision, as per ICH Q2 (R1) guidelines9, 10.

The recovery study was carried out by adding known amount of Manidipine hydrochloride and analyzing by the proposed HPLC method. Accuracy was determined in terms of recovery study and the recoveries were done at three levels i.e. 50%, 100% and 150%. Calculated amount of was added in placebo to attain 50%, 100% and 150% of sample concentration of Manidipine hydrochloride (1000 µg/ml). Each sample was prepared in triplicate at each level and injected each preparation in duplicate. The data shown that the proposed method was accurate. From the amount found, percentage recovery was calculated. The mean percentage recovery found was 100.52%, 100.713% and 99.89% and % RSD was 1.15, 0.95 and 1.14, respectively.

Linearity at seven levels over the range of 20% to 150% with respect to the sample concentration of Manidipine hydrochloride was performed.  Dilutions of the solution ranging from 100 µg/ml to 900 µg/ml for Manidipine hydrochloride were prepared and linearity was checked. The solutions were injected each in the stream of mobile phase, at a flow rate of 1.4 ml/min. Each of these dilutions of different concentration was injected in duplicate in to the column and corresponding chromatograms were obtained. From these chromatograms, the areas under the peaks of the drug were noted. Linearity has been obtained in the concentration range of 100 µg/ml to 900 µg/ml Manidipine hydrochloride against the absorbance. Based on the calibration curve, value of R2 is 0.9993, which is in acceptance range. Slope is 14637, intercept is 13824. LOD is 0.48 and LOQ is 1.47.

Placebo solutions (mixture of excipients), solvent used for preparation of standard solution and sample solution were injected in the chromatographic system and checked for interference at retention time corresponding to the retention time of Manidipine hydrochloride. There was no interference found from blank and placebo at retention time of Manidipine hydrochloride, so the method is specific.

The system suitability was carried out by injecting standard solution of Manidipine hydrochloride (1000 µg/ml) in to the chromatographic system to check reproducibility of peak areas (% RSD). The % RSD observed was 1.46 for Manidipine hydrochloride. 

RESULTS AND DISCUSSION  

In the present study, an attempt has been made to develop a simple, sensitive,accurate, precise HPLC method for estimation of Manidipine hydrochloride. The proposed method for determination of Manidipine hydrochloride from pharmaceutical dosage forms is specific, accurate, rapid, and precise. So, the proposed method can be used in routine analysis and have financial benefits, since it excludes the use of dimethylformamide (DMF) from the mobile phase, which affects the chromatographic parameters as well as column.

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