Development and Validation of UV Spectrophotometric Method for Simultaneous Estimation of Diazepam and Propranolol in Bulk Drug and Its Formulations

 

S. J. Daharwal*

University Institute of Pharmacy, Ravishankar Shukla University, Raipur (CG) India-492010

*Corresponding Author E-mail: daharwalresearch@rediffmail.com

 

ABSTRACT:

A simple, efficient, precise and accurate Vierodt’s method have been developed for the estimation of Diazepam and Propranolal in pure and in fixed dose Combination. In this method UV spectrum of Diazepam and Propranolal  were overlained which involves the formation of Simultaneous equation at maximum wavelength 241 nm and 289 nm. Both the drugs obeyed Beer’s law in the concentration range 2-25 μg/ml and 1-45 μg/ml for Diazepam and Propranolal , respectively. The accuracy of the method was determined by recovery studies and was found to be 100.14±0.219 % and 100.17±0.134 % for Diazepam and Propranolal , respectively. The method was validated as per ICH guidelines. The method was found to be simple, economic, accurate and reproducible and can be used for routine analysis of Diazepam and Propranolal  in pure and in fixed dose combination.

 

KEYWORDS: Vierodt’s method, Diazepam, Propranolal , ICH guidelines

 

 

INTRODUCTION:

Diazepam (DZ) is anxiolytic, sedative and anticonvulsant. Chemically it is 7-chloro-1,3-dihydro-1-methyl-5-phenyl-1,4-benzodiazepine-2-one. The molecular formula is C16H13ClN2O and molecular weight is 284.74. It is freely soluble in chloroform and ethanol, very slightly soluble in water. The wavelength maximum in aqueous acid is 241, 285 and 366 nm. DZ has been                              determined by potentiomentriaclly1, colorimetrically2-5, sectrophotometrically 6,7 and HPLC8.

 

Propranolal  hydrochloride (PL) is a β-adrenergic blockers used for the treatment of hypertension, angina pectoris and arrhythmia. Chemically is 1-[(1-methyl ethyl) amino-3-naphthalenyloxy-2-propranol. The molecular formula is C16H21NO2.HCl and molecular weight is 295.81. It is white or almost white crystalline powder, freely soluble in water and in alcohol. PL has been determined titrimetry9, colorimetry10-12, spectrophotometrically13-15, atomic spectrohpotometrically16-18, quantitative TLC19, HPLC20-21 and GC22.  

 

This combination is available in market in tablet dosage form. It is indicated for the treatment of hypertension, angina pectoris, cardiac arrythmias, post M.I., tachycardia, migrane, anxiety. Literature revealed that simultaneous estimation7 of diazepam and propranolol was available, in which methanol was used as solvent for analysis, but in the developed method the estimation was carried out in hydrochloric acid (0.1 M).

 

Hence the present work aims to develop a simple, precise, accurate and validated UV spectrophotometric method (Vierodt’s method) for the estimation of DZ and PL in pure and in fixed dose combination. Confirmation of the applicability of the developed method was validated according to the International Conference on Harmonization (ICH) guidelines for the determination of DZ and PL in pure and in fixed  dose combination.23

 

MATERIALS AND METHODS:

Pharmaceutically pure sample of DZ and PL were obtained as a generous gift samples from R. S. Spectra Lab. Ahmednagar, India. Hydrochloric acid AR grade was used as solvent in this study. Shimadzu 1800 Double Beam UV/Visible Spectrophotometer with a pair of 10 mm (1 cm) matched quartz cell was used for absorbance measurements.

 

The tablet formulation containing 20 mg of PL and 2.5 mg of   DZ.  All the chemicals and reagents used were obtained from Loba Chem. Mumbai, India.

 

Selection of solvent for analysis:

The UV spectra of DZ and PL, obtained from different solutions (methanol, isopropyl alcohol, water, 1 M HCl, 0.1 M HCl, and 0.1 M NaOH) were studied and 0.1 M HCl was chosen as a solvent for analysis.

 

Preparation of standard stock solutions:

The standard stock (1 mg/ ml) and working solutions (0.05 mg/ml) of DZ and PL were prepared in 0.1 M HCl.

 

Spectral characteristics of DZ and PL:

Aliquot portions equivalent to 25-µg/ml DZ and PL were accurately transferred into two 10-ml volumetric flasks and the volume was made up with 0.1 M HCl. The absorption spectra of solutions were recorded between 200- 400 nm and absorbance range from 0.00 to 2.00 at medium scanning speed. 

 

Selection of wavelengths for analysis:

The wavelengths selected were 241 nm (l max for DZ), 289nm (l max for PL) for simultaneous equation method .

 

Linearity for the spectrophotometric methods:

Aliquot portions (0.2, 0.4, 0.6, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ml) from the 0.05 mg/ml DZ and PL working solution were accurately transferred to 10 ml volumetric flasks, the volume was made up with 0.1 M HCl.  The absorbances of all the solutions were measured at 241nm and 289 nm. 

 

Preparation of laboratory mixtures for standardization of the developed methods:

Different mixtures of the two drugs were prepared by transferring different volumes of DZ and PL from working solutions into 10 ml volumetric flasks and diluting to volume with 0.1 M HCl. The concentrations of DZ and PL were determined by measuring the absorbance of the prepared mixtures at 241nm and 289 nm. 

 

Analysis of commercial tablet formulation:

Twenty tablets (Dizepax, Unimark Pharma (I) Ltd., Chandigrah; content:  diazepam-2.5 mg and propranolol- 20 mg) were weighed and average weight was calculated. The tablets were crushed to fine powder. The powder equivalent to 100 mg of propranolol was transferred to 100 ml volumetric flask. It was dissolved in 75 ml of 0.1 M HCl by intermittent shaking and volume was made up to 100 ml with the same solvent. The solution was then filtered through a Whatman filter paper (No. 41). 10 ml of the filtrate was accurately transferred to a 100 ml volumetric flask and the volume was made up with 0.1 M HCl. The solution was diluted further with 0.1M HCl to obtain 20 µg/ ml of propranolol and 2.5 µg/ ml of diazepam. The concentrations of both DZ and PL were determined by measuring the absorbance of the samples at selected wavelengths. The analysis procedure was repeated for six times.

 

Recovery studies:

The selectivity of the proposed procedure was examined by determining the recovery of the two drugs in different ratios. The standard addition technique has been carried out by spiking placebo (starch, lactose and magnesium stearate, which are common constituents of solid pharmaceutical formulations) with DZ and PL at 50% (1.25 mg /ml DZ: 10 mg /ml PL), 100% (2.5 mg /ml DZ: 20 mg /ml PL) and 150 % (3.75 mg /ml DZ: 30 mg /ml PL), of concentration level used in proposed method.

 

Validation of methods:

The methods were validated with respects to linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and repeatability.

 

Linearity: The calibration plots for each method were constructed after analysis of thirteen different concentrations and each concentration was measured for six times.

 

LOD (k=3.3) and LOQ (k=10) of the methods were established according to ICH definitions (C1= k S0/ s, where C1 is LOD or LOQ, S0 is mean standard deviation, s is the slope of the calibration curve and k is the constant related to the confidence interval).

 

The percentage recovery studies and bias value assessed the accuracy of the suggested methods. It was investigated by analyzing the tablets solution of DZ and PL in linear range in six independent replicates on the same day (intra-day precision) and on six consecutive days (inter-day precision).

 

Repeatability is based on the results of the methods operating over a short time interval under the same conditions.

 

RESULTS AND DISCUSSION:

Selection of solvent for analysis:

The selection of solvent for analysis was carried out by the effect of different solvents on the pure drug and tablet powder. In the methanol and isopropyl alcohol the drugs were soluble, while in water, DZ drugs were sparingly soluble and the tablet solution prepared for analysis was turbid and non filterable.  Keeping for long period of time, in 1 M HCl and 0.1 M NaOH the drugs stock solution were dark in color. At the end of these studies, 0.1 M HCl has been chosen for preparation of solution for analysis, because, the time gain while preparing working solutions and also cost saving by eliminating the purchase and disposal of organic solvents.

Spectral characteristics of DZ and PL:

The overlain spectra of DZ and PL (Fig.-1), shows overlap, that prevents the use of direct absorbance measurements for determination of both the drugs in their mixtures.  The spectra showed the wavelength maximum for DZ at 241 nm and for PL at 289 nm, respectively and this l max were chosen for simultaneous equation method.

 

PL
 

DZ
 
Fig.1- Absorption spectra of diazepam (DZ) and propranolol (PL). Concentration of each was 25 -µg / ml.

 

Simultaneous equations method:

The figure (Fig.1) shows the wavelength maximum for DZ at 241 nm and for PL at 289 nm, respectively and this l max were chosen for simultaneous equation method. The absorbance curves at the selected wavelengths were found to be proportional to the corresponding concentrations of the two drugs. The linearity ranges for DZ and PL were 2-25 µg/ ml and 1-45 µg/ ml, respectively and as shown by the small intercepts and correlation coefficients approaching unity in the regression equations (Table- 1).

 

For the computation of simultaneous equation the absorptivity values were required. The absorptivity values of the drugs were determined at the se­lected wavelength maximum. The absorptivity is the ratio of mean absorbance of the drugs at selected wavelength with the concentration of component in mg/ml. These absorptivity values were the mean of six independent deter­minations. Sets of two simultaneous equations obtained by using these mean absorptivity values are given below.

 

A1 =        87.2 CDZ    +          12.6 CPL -----------(at l 241)

A2 =        38.8 CDZ     +          20.1 CPL ----------   (at l 289)

 

Where, A 1 and A2 are absorbance of the tablet sample solution at 241 nm and 289 nm respectively. 87.2 and 38.8 are absorptivities of DZ at 241 nm and 289 nm, respectively. 12.6 and 20.1 are the absorptivities of PL at 241 nm and 289 nm, respectively. CDZ is the concentration of the DZ and CPL is the concentration of the PL in mg/ml. The result of tablet analysis was summarized in table- 2.

 

Validation of methods:

The regression equation and correlation coefficients of the mean of six consecutive calibration curves, LOD, LOQ and standard error of the pure drugs for the methods were given in table-1. The bias values for accuracy was expressed as bias (%) and were close to zero (Table-5). The intra-day and inter-day relative standard deviation (RSD) values (Table-5) and also the low RSD values obtained from the analysis of pharmaceutical formulations (Table-2) indicated that the intermediate precision of method was good.

 

The results of recovery study were found to be close to 100 % (Table-3) and the high percentage of recovery indicated no interference from ingredients and excipients that were used in formulation. The recovery from laboratory prepared mixtures (Table-3) and standard addition technique (Table- 4) indicated that the methods have a high accuracy.

 

Repeatability result of the methods has low RSD values of intra-day precision (Table 5), recovery (Table-4) and pharmaceutical preparations (Table-2) showed that the methods give a high repeatability.

 

Table 1: Data for calibration graph (n=6) for diazepam and propranolol for Simultaneous equation method.

Parameters

Diazepam

Propranolol

Slope*

0.0866±0.0005

0.0201±0.005

Intercept*

0.0173±0.0008

0.0012±0.0004

Correlation coefficient

0.9997

 

0.9999

 

Linearity range (µg/ ml)

2-25

 

1-45

 

LOD (µg/ ml)

0.77

0.39

LOQ (µg/ ml)

2.33

0.41

 *Mean ± standard error

 

Table 2: Determination of DZ and PL in tablet using Simultaneous equation method 

 

Label claim (%)a± S. D. (n=6)

DZ

PL

Tablet

100.21 ±0.159

100.23 ±0.264

RSD (%)

0.15

0.26

a: Mean, S. D.: Standard deviation, RSD: Relative standard deviation

 

Table 3: Results of the analysis of DZ and PL in the laboratory prepared mixtures.

Sr. no.

Con. 

(µg/ ml)

Recoverya (%)

DZ

PL

DZ

PL

Simultaneous equation method

Simultaneous equation method

1

30

00

99.4

--

2

25

05

99.5

98.98

3

20

10

99.9

99.8

4

15

15

99.1

99.6

5

10

20

99.3

99.81

6

05

25

99.2

99.65

7

00

30

--

99.92

Mean

99.4

99.75

Standard deviation

0.316

0.129

a: mean

 
 
Table 4: Results of the standard addition technique in tablet (n=6)

Amount taken (mg/ml)

Standard added (mg/ml)

Recovery of added standard (%)a ± S. D.

DZ

PL

DZ

PL

DZ

PL

Simultaneous equation method

Simultaneous equation method

2.5

20

1.25

10

99.92 ±0.075

100.23 ±0.148

2.5

20

2.5

20

100.23 ±0.185

99.89 ±0.156

2.5

20

3.75

30

100.26 ±0.395

100.39 ±0.098

Mean

100.14

100.17

Mean standard deviation

±0.219

±0.134

a: Mean, S. D.: Standard deviation


 

Table 5: Precision and accuracy of spectrophotometric method developed for analysis of tablet. (n=6)

 

For diazepam

For propranolol

 

Intra day

Amount found

(Mean%± S.D.)

Accuracy, Bias (%)

Precision, RSD (%)

 

 

100.18 ± 0.512

 

0.18

0.48

 

 

99.89 ± 0.357

 

-0.11

0.33

Inter day

Amount found

(Mean %±S.D.)

Accuracy, Bias (%)

Precision, RSD (%)

 

102.82 ± 0.628

 

2.82

0.58

 

102.78 ± 0.658

 

2.78

0.6

S. D.: Standard deviation, % Bias = [100(found- label claim)/ label claim], RSD: Relative standard deviation

 

CONCLUSION:

The proposed method is simple, accurate, precise and selective for the simultaneous estimation of PL and DZ in pure and in fixed dose combination. The method is economical, rapid and do not require any sophisticated instruments contrast to chromatographic method. Hence it can be effectively applied for the routine analysis of Propranolal  and DZ in pure and in fixed does combination.

 

ACKNOWLEDGEMENTS:

The authors wish to thank to Director, University Institute of Pharmacy, Ravishankar Shukla University, Raipur (CG) for their kind help and providing all necessary facilities. This analytical method was developed in year 2006 at University institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur. 

 

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Received on 11.02.2013          Accepted on 15.03.2013        

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