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Original Article
Simple RP-HPLC method for estimation of diazepam in tablet
dosage form
A. Sruthi, P. Tejaswi, N. Thanuja, D. Sudheer Kumar, P. Vivek Sagar*
Department of Pharmaceutical Analysis, Care College of Pharmacy, Warangal, Andhra Pradesh, India
article info abstract
Article history: A simple reverse phase HPLC method was developed and validated for the determination
Received 11 September 2012 of diazepam present in pharmaceutical dosage form. A Hypersil ODS C-18 column
Accepted 4 November 2012 (250 Â 4.6 mm, packed with 5 microns) is used as stationary phase. An isocratic mode with
mobile phase consisting of acetonitrile, methanol and 1% phosphate buffer (pH-3) in ratio
Keywords: of 18:58:24 (v/v/v) at a flow rate of 1 ml/min and effluent was monitored at 232 nm.
RP-HPLC Chromatogram showed a peak of DZP at retention time of 6.23 Æ 0.002 min. The linearity
Diazepam range was found to be 2e20 mg/ml. The method was validated for linearity, accuracy,
Validation precision, limit of detection, limit of quantitation, robustness and ruggedness. Recovery of
Pharmaceutical dosage form DZP was found to be in the range of 99.4e100.3%. The limit of detection and limit of
quantitation for estimation of DZP was found to be 0.898 mg/ml and 2.72 mg/ml, respectively.
Proposed method was successfully applied for the quantitative determination of DZP in
pharmaceutical dosage forms.
Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.
1. Introduction expensive. The present study focused on minimizing these
limitations and to develop a simple precise accurate and
Diazepam (7-chloro-1, 3-dihydro-1-methyl-5-phenyl-2H-1, economic method for estimation of diazepam in tablet dosage
4-benzodiazepin-2-one) is a benzodiazepine (BZD) generally form.
used as hypnotic, anxiolytic and muscle relaxant. Diazepam
(DZP) is also routinely prescribed as the standard first-line
treatment for acute convulsions and prolonged status epi-
lepticus.1 Several methods for the analysis of BZDs have been
reported.2 A number of chromatographic methods, such as
thin-layer chromatography (TLC)3 gas chromatography4e6 and
gas chromatographicemass spectrometry (GCeMS)7,8 have
been used in the analysis of diazepam and other 1,4-benzo-
diazopines. Several high-performance liquid chromato-
graphic (HPLC) methods have also been reported for the
determination of diazepam and other BZDs.9,10 However, all of
these methods have limitations such as long run times and/or
* Corresponding author.
E-mail address: viveksagar.p111@gmail.com (P. Vivek Sagar).
0974-6943/$ e see front matter Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jopr.2012.11.029

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j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 4 0 e1 4 4 141
2.4. Preparation of working standard solutions
2. Materials and methods
Working standard solutions ranging from 0.5 to 50 mg/ml were
2.1. Chemicals and reagents prepared by appropriate dilutions of the stock with distilled
water.
An analytically pure sample of diazepam was procured as
gift sample from Natco Pharma Ltd. (Hyderabad, India). 2.5. Preparation of sample solution
HPLC grade methanol was procured from E. Merck (Hyder-
abad). Liquid chromatographic grade water was obtained by Twenty tablets of diazepam hydrochloride were weighed and
double distillation and purification through Milli-Q water ground into a fine powder. A quantity of powder equivalent to
purification system. Potassium dihydrogen phosphate (AR 25 mg of diazepam was weighed and transferred into a 25 ml
grade, purity 99.5%) was procured from Qualigens. Tablet volumetric flask and was dissolved in 0.1 N HCl. The volume
formulations VALIUM (Nicholas Piramal India Ltd.) was was made up to the mark with the same. Above solution was
procured from a local pharmacy with labeled amount 5 mg suitably diluted with distilled water. From this stock, appro-
per tablet. priate dilution (10 mg/ml) was prepared. The solution thus
prepared was filtered through 0.45 m membrane filter and the
2.2. Instrumentation & chromatographic conditions resulting filtrate was sonicated for 10 min. After setting the
chromatographic conditions and stabilizing the instrument to
The HPLC analysis was performed on CYBERLAB HPLC obtain a steady baseline, the sample solution was loaded in
equipped with an LCP-100 reciprocating HPLC pump. A the 20 ml fixed e sample loop of the injection port.
manually operating Rheodyne injector with 20 mL sample
loop, a LC-UV 100 ultraviolet detector was used. Chromato- 2.6. Method development
graphic analysis was performed on a Hypersil reversed phase
C-18 column with 250 Â 4.6 mm i.d. and 5 mm particle size. Initial trial experiments were conducted, with a view to select
The mobile phase consist of acetonitrile, methanol, 1% a suitable solvent system for the accurate estimation of the
phosphate buffer (pH-3) in ratio of 18:58:24 (v/v/v) that was set drug and to achieve good retention time. The suitability of the
at a flow rate of 1 ml/min. The mobile phase was degassed mobile phase decided on the basis of the sensitivity of the
and filtered through 0.25 mm membrane filter before pumping assay, time required for the analysis, ease of preparation, and
into HPLC system. The eluent was monitored by UV detection use of readily available cost effective solvents. These include
at 232 nm. methanol-potassium dihydrogen phosphate, methanol-
ammonium acetate, acetonitrile-potassium dihydrogen phos-
2.3. Stock solutions and standards phate, acetonitrile-ammonium acetate, methanol-water. The
mobile phase consisting of acetonitrile, methanol, 1% phos-
Stock solution of diazepam (1 mg/ml) was prepared by phate buffer (pH-3) in ratio of 18:58:24 (v/v/v) that was set at
transferring 25 mg of drug in a 25 ml volumetric flask. The a flow rate of 1 ml/min was found to be optimum and further
drug is dissolved in sufficient amount of 0.1 N HCl and optimized by adjusting pH 3e4 by adding orthophosphoric acid.
finally the volume was made up to the mark with distilled The composition of acetonitrile, methanol, 1% phosphate
water. buffer in ratio of 18:58:24 (v/v/v) with pH-3 gave the best results.
Fig. 1 e Chromatogram of diazepam.

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142 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 4 0 e1 4 4
Table 1 e Linearity of diazepam. Table 2 e Precision.
S. No Concentration Peak area S. No Concentration Peak area
(ppm) (ug/ml)
1 2 7569.8 1 6 21709.4
2 4 17892.9 2 6 21627.7
3 6 23612.2 3 6 21939.6
4 8 34517.2 4 6 21710.5
5 10 40051.3 5 6 21337.9
6 12 47631.9 6 6 21929.5
7 14 54925.1 Mean 21709.1
8 16 65942.9 % RSD 1.02
9 18 72965.0
10 20 80440.3
triplicate.11 20 ml of each of standard solutions were injected
into the HPLC system to get the chromatograms. The retention
2.7. Solution stability
time, average peak areas were recorded. Calibration curve was
constructed by plotting average peak area against concentra-
In order to demonstrate the stability of both standard and
tion and regression equation was computed. The linearity
sample solutions during analysis, both solutions were analyzed
range was found to be 2e20 mg/ml. The results were shown in
over a period of 96 h at an interval of 24 h at room temperature.
Table 1. The results show that an excellent correlation exists
The results show that for solutions, the retention time and peak
between peak area and concentration of drug within the
area of diazepam hydrochloride remained unchanged and no
concentration range, regression graph is presented in Fig. 2.
significant degradation within the indicated period, this indi-
cates that both solutions were stable for 72 h.
3.2.2. Precision
The precision of method was ascertained from the peak area
response obtained by actual determination of six replicates of
3. Results and discussion a fixed amount of drug. The percent relative standard devia-
tions were calculated for diazepam and presented in the Table
3.1. Analysis of formulation 2. The precision of the method was found to be 1.02.
The sample solution was injected and a chromatogram was 3.2.3. Accuracy
recorded. The injections were repeated six times and the peak Accuracy of developed method was confirmed by doing
areas were recorded. The amount of drug present in the recovery study as per ICH norms. A known quantity of the
pharmaceutical formulation was calculated using standard pure drug was added to the pre-analyzed sample formulation
calibration curve (concentration in mg/ml was taken on X-axis (10 mg/ml) at three different concentration levels 80%, 100%
and average peak area on Y-axis). Percentage of drug present and 120% by replicate analysis (n ¼ 3). From the recovery study
in each tablet was found to be 100.2. A representative chro- it was clear that the method is very accurate for quantitative
matogram has been given in Fig. 1. estimation of diazepam hydrochloride in tablet dosage form
as all the statistical results were within the range of accep-
3.2. Validation of the assay method tance, 99.4e100.3%, which shows that there is no interference
with excipients. Percentage recovery values were calculated
3.2.1. Linearity and the results were shown in Table 3.
Different concentrations in the range of 0.5e50 mg/ml were
prepared. Each of the levels of concentration was prepared in 3.2.4. Ruggedness
Ruggedness is the degree of reproducibility of the results ob-
tained under a variety of conditions. From stock solution,
solutions containing 14 mg/ml of diazepam hydrochloride
was prepared and analyzed by two different analysts using
same operational and environmental conditions in different
Table 3 e Accuracy.
Amount of Amount of Average %
drug added drug recovered recovery
(ppm) (ppm)
8 7.592 99.4
10 9.98 99.8
12 12.096 100.3
Fig. 2 e Linearity of diazepam.

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j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 4 0 e1 4 4 143
Table 4 e Ruggedness. Table 7 e System suitability parameters.
Date % Recovery Drug RT Peak Peak USP plate USP
area height count tailing
Analyst-1 Day-1 100.2%
Analyst-2 Day-2 99.8% Diazepam 6.2266 31432.2 2588 5238.42 1.1
% Deviation 0.4%
accuracy and precision. The requirements for system suit-
ability are usually developed after method development and
Table 5 e Robustness. validation has been completed. The system suitability
parameters like Theoretical plates (N), Resolution (R), Tailing
S. No Wavelength 232 Wavelength 235
factor (T) were calculated and compared with the standard
1 52156.6 54973 values to ascertain whether the proposed RP-HPLC method for
2 52700.8 52955.4 the estimation of diazepam in pharmaceutical formulations
3 50662.6 52965.9 was validated or not. The results were shown in Table 7.
4 50905.7 54751.1
5 51418.4 54821.7
6 50911.8 52886.1
Mean 51459.32 53892.22 4. Conclusion
% RSD 1.57 1.94
A convenient, rapid, accurate, precise and economical RP-
HPLC method has been developed for estimation of diazepam
experimental periods. Percentage recoveries of the replicates
in bulk and tablet dosage form. The assay provides a linear
were calculated. It is checked that the results are reproducible
response across a wide range of concentrations and it utilizes
under differences in, analysts. The results are shown in Table 4.
a mobile phase which can be easily prepared and diluent is
economic, readily available. The proposed method can be
3.2.5. Robustness
used for the routine analysis of diazepam hydrochloride in
The method was found to be robust, although small deliberate
bulk preparations of the drug and, in pharmaceutical dosage
changes in method conditions did have a negligible effect on
forms without interference of excipients.
the chromatographic behavior of the solute. The results
indicate that changing the detector wavelength had no large
effect on the chromatographic behavior of diazepam hydro-
chloride. Even a small change of mobile phase composition
Conflicts of interest
(pH 3 Æ 0.2), did not cause a notable change in the peak area of
All authors have none to declare.
the used drug for this method. The results were presented in
Tables 5 and 6.
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LOD and LOQ for diazepam were estimated by injecting
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