| Research Article |
Open Access |
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| Development and Validation of an HPLC Method for Quantifying Dapiprazole in
Bulk Preparations |
| Jaya Prasanthi K and Syama Sundar B* |
| Department of Chemistry, Acharya Nagarjuna University, Guntur, India |
| *Corresponding author: |
B. Syama Sundar
Department of Chemistry,
Acharya Nagarjuna University
Nagarjuna Nagar, Guntur-522510, India
E-mail: profbsyamsundar@yahoo.co.in |
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| Received July 16, 2012; Accepted August 24, 2012; Published August 30, 2012 |
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| Citation:Jaya Prasanthi K, Syama Sundar B (2012) Development and Validation
of an HPLC Method for Quantifying Dapiprazole in Bulk Preparations. J Anal Bioanal Tech 3:143. doi:10.4172/2155-9872.1000143 |
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| Copyright: © 2012 Jaya Prasanthi K, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited. |
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| Abstract |
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| A simple, precise, rapid and accurate Reverse phase HPLC method was developed for the estimation of
Dapiprazole in bulk form. A Thermo hypersil C-18 column (250 mm×4.6 mm 5μm) with mobile phase consisting
of mixture of methanol: acetonitrile: and ortho-phosphoric acid in the ratio of 89: 9: 1 (v/v) at pH 5.8 adjusted with
ortho-phosphoric acid. The flow rate was 0.8 mL/min and the effluents were monitored at 243 nm. The retention
time was 3.725 min. The detector response was linear in the concentration of 20-120 μg/mL. The respective linear
regression equation being y=1956.4x+3409. The limit of detection and limit of quantification was 0.5 μg/mL and 1.6
μg/mL respectively. The percentage assay of Dapiprazole was 99.94%. This method has been validated and shown
to be specific, sensitive, precise, linear, accurate, rugged, robust and fast. Hence this method can be useful for the
routine determination of Dapiprazole in bulk drug and in its pharmaceutical dosage form. |
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| Keywords |
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| Dapiprazole; RP-HPLC; Bulk Drug |
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| Introduction |
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| Dapiprazole hydrochloride is an alpha-adrenergic blocking agent
[1]. Chemically, it is 5,6,7,8-tetrahydro-3-[2-(4- o-tolyl-1-piperazinyl)
ethyl]-s -triazolo[4,3-a] pyridine hydrochloride [2,3]. Dapiprazole
hydrochloride has the empirical formula C19H27N5
.HCl and a molecular
weight of 361.93. It is a sterile, white, lyophilized powder soluble in water.
Dapiprazole hydrochloride ophthalmic solution is indicated in the
treatment of iatrogenically induced mydriasis produced by adrenergic
(phenylephrine) or parasympatholytic (tropicamide) agents [4,5]. The
novel synthetic method has been developed by concerning the yields
and mild reaction conditions to synthesize Dapiprazole starting from
stable reactants such as ethyl 3-chloro propionate and o-tolyl piperzine,
which appears as impurity in the final product. In vivo evaluation was
performed for in ocular vehicle for long acting alfa adrenergic receptor
blocking activity of dapiprazole hydrochloride [6]. Following topical
instillation on the eye, it crosses the corneal epithelium reaching high
concentrations in the ocular tissue and producing a prompt mitotic
and hypotensive effect. The high concentration ratio between ciliary
bodies and iris versus aqueous humor suggests a peculiar affinity for
these structures containing adrenoceptors of the alpha type [7,8].
Also, a study was conducted to compare the cycloplegic and mydriatic
effects of Paremyd™, a formulation of 0.25% tropicamide and 1%
hydroxyamphetamine, to 0.5% tropicamide and 2.5% phenylephrine
[9]. The influence of dapiprazole on pupil size was compared with
brinzolamide and reported that pupil mydriasis at scotopic illumination
levels was reduced by both drugs in a similar fashion [10]. The aim
of this paper was to develop validate a simple and reliable HPLCUV
method for the determination of Dapiprazole in bulk drug. This
manuscript describes the development and validation, in accordance
with International Conference on Harmonization (ICH) guidelines, of
a rapid, economical, precise, and accurate stability-indicating isocratic
reversed-phase HPLC method for analysis of Dapiprazole in bulk
sample. To the best of our knowledge, literature survey reveals no
chromatographic methods for the estimation of Dapiprazole in bulk
samples. The availability of an HPLC method with high sensitivity and
selectivity will be very useful for the determination of Dapiprazole in
bulk and also in pharmaceutical formulations. The chemical structure
of Dapiprazole is represented in Figure 1. |
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Figure 1: Structure of Dapiprazole. |
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| Experimental |
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| Instrument |
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| Quantitative HPLC was performed on liquid Chromatograph,
Shimadzu LC 2010 dual λ detector equipped with automatic injector
with injection volume 20 μL. The HPLC system was equipped with LC
solution Software. The pH measurements were carried out with Elico,
model LI 120, pH meter equipped with a combined glass-calomel
electrode calibrated using standard buffer solutions of pH 4.0, 7.0 and
9.2. |
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| Materials/reagents |
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| Acetonitrile and methanol HPLC grade (Qualigens) and Water
HPLC grade (Milli-Q), ortho-Phosphoric acid (Rankem). Dapiprazole
working and reference sample is obtained as gift sample from M/s
Bioleo Analytical Labs India Pvt. Ltd, Hyderabad. |
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| HPLC conditions |
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| The contents of the mobile phase were methanol: acetonitrile:
and ortho-phosphoric acid in the ratio of 89: 9: 1 (v/v/v) at pH 5.8.
They were filtered before use through a 0.45 μm membrane filter, and
pumped from the respective solvent reservoirs to the column at a flow
rate of 0.8 mL/min. The run time was set at 8.0 min and the column
temperature was ambient. Prior to the injection of the drug solution,
the column was equilibrated for at least 30 min with the mobile phase
flowing through the system. The eluents were monitored at 243 nm. |
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| Preparation of standard stock solution |
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| A standard stock solution of the drug was prepared by dissolving 10 mg of Dapiprazole USP in 10 ml volumetric flask containing 5 ml of
methanol, sonicated for about 15 min and then made up to 10 ml with
mobile phase to get 1000 μg/ml of Dapiprazole. |
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| Working standard solution |
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| The primary standard solution was further diluted by taking 2 ml of
the stock solution with 25 ml of mobile phase to get the concentration
of 80 μg/mL. |
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| Preparation of sample solution |
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| A sample of the powder of Dapiprazole synthesized in in-house,
equivalent to 10 mg of the active ingredient, was mixed with 5 ml of
methanol in 10 ml volumetric flask. The mixture was allowed to stand
for 30 minutes with intermittent sonication for complete solubility of
the drug, and then filtered through a 0.45 μm membrane filter, followed
by addition of mobile phase up 10 ml to obtain a stock solution of 1
mg/ml. The resultant solution was further diluted by taking 2 ml of the stock solution with 25 ml of mobile phase to get the concentration of
80 μg/mL. |
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| Linearity |
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| Aliquots of standard drug solution of Dapiprazole 2.5, 5.0 and 7.5
ml (80 μg/ml) were taken and transferred into series of 10 ml volumetric
flasks and diluted up to 10 ml with mobile phase as dilute to get 20, 40
and 60 μg/ml. The working standard solution is directly used to get 80
μg/ml. Another set of aliquots of 1.0 and 1.2 ml (1000 μg/mL) were taken
and transferred into series of 10 ml volumetric flasks and diluted up to
10 ml with mobile phase as diluents to get 100 and 120 μg/ml. Each of
these drug solutions (20 μL) was injected three times into the column,
and the peak areas and retention times were recorded. Evaluation was
performed with UV detector at 243 nm and a Calibration graph was
obtained by plotting peak area versus concentration of Dapiprazole
(Figure 2). |
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Figure 2: Calibration curve of the Dapiprazole by RP-HPLC. |
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| Assay |
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| 20 μL of sample solution was injected into the injector of liquid
chromatograph. The retention time was found to be 3.724 minutes. The
amount of drug present in bulk sample was calculated by comparing
the peak area of the sample solution with that of the standard solution. |
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| Recovery studies |
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| Recovery is a useful way to assess how efficient an extraction
procedure is the closer the recovery value is to 100%, the better the
sensitivity will be. Recovery values have to be reproducible to prove
that they are accurate. Accuracy was determined by recovery studies
of Dapiprazole, known amount of standard was added to the preanalyzed
sample and subjected to the proposed HPLC analysis. Results
of recovery study are shown in Table 1. The study was done at three
different concentration levels. |
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Table 1: Results of HPLC Assay and Recovery studies |
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| Limit of Detection (LOD) and Limit of Quantification (LOQ) |
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| The LOD is defined as the analyte concentration that gave a
signal to noise (S/N) ratio of 3:1. The LOQ was defined as the analyte
concentration that gave an S/N ratio of 5:1. The limit of detection
(LOD) and limit of quantification (LOQ) for Dapiprazole were found
to be 0.5 μg/mL and 1.6 μg/mL respectively. The signal to noise ratio is
3 for LOD and 10 for LOQ. |
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| Results and Discussion |
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| For developing the method, a systematic study on the effect of
various factors was carried out by varying one parameter at a time and
keeping all other conditions constant, that is, OFAT (One Factor at a
Time) mode of study. Method development consists of selecting the
appropriate detection wave length and stationary and mobile phases. |
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| Proper wavelength was needed to determine maximum detector
response. From the spectrum, it is clear that Dapiprazole absorbs
maximum light between 235 nm to 245 nm. For the RP-HPLC analysis,
the UV/Vis detector was fixed at 243 nm as maximum wave length
(λmax) for determination of Dapiprazole. At this wavelength, we have observed a zero back ground noise and no absorption of light by mobile
phase solvent system. |
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| To explore the possibility of better separation a Cyano column
was tested with the same mobile phase ternary mixture of methanol:
acetonitrile: and ortho-phosphoric acid in the ratio of 89: 9: 1 (v/v/v) at
pH 5.8. The retention times were long with polar columns. Retention of
the analyte on the cyano columns was much weaker than on C18 columns,
resulting in unacceptable k’ (capacity factor) value of the column (<1)
for Dapiprazole. Further development trials have been performed with
octadecyl columns of different types and configurations from different
manufacturers. Under these chromatographic conditions, the analyte of
interest has exhibited poor peak efficiencies (N) and peak symmetries
(Tailing factor); and a partial resolution (Rs) between drug and mobile
phase components. Finally Thermo hypersil C18 column (250 mm×4.6
mm 5 μm) column was selected based on the peak shape and the
baseline separation from the other interfering peaks in the formulation
sample. |
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| Different mobile phases were tested to optimize analytical
performance. The optimum composition of mobile phase was
determined by comparing the influence of different binary mixtures
such as acetonitrile- methanol and acetonitrile -water and methanolwater
in different proportions. None of the solvent system in binary
phase has given good peak shape and theoretical plates. It is well
known that multiple-component mobile phases result in better
separation efficiency than binary mobile phases, because with these
solvent strength and selectivity can be varied simultaneously to obtain
the retention times desired. A third component, o-phosphoric acid,
was therefore included in the mobile phase and ternary mixtures of
methanol: acetonitrile: and ortho-phosphoric acid in the ratio of 89: 9: 1
(v/v) at pH 5.8. The present mobile phase system has been finalized for
the estimation of Dapiprazole in bulk drug in terms of peak symmetry,
optimum resolution, reasonable run time, and acceptable k values. |
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| System suitability |
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| The system suitability tests were carried out on freshly prepared
standard stock solution of Dapiprazole. The system was suitable for
use, the tailing factors for Dapiprazole were 1.15 and USP theoretical
plates were found to be significantly high around 21505 in number.
Parameters that were studied to evaluate the suitability of the system
are given in Table 2. |
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Table 2: Validation Summary. |
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| Specificity |
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| Specificity is the ability of a method to discriminate between the principal anlayte(s) of interest and additional excipients that are present
in the sample. The degree of specificity testing varies depending on the
method type and the stage of validation. The effect of wide range of
intermediates and other precursors, generally used in in-house synthesis
of Dapiprazole were investigated under optimized chromatographic
conditions. Specificity is the ability to assess unequivocally the analyte
in the presence of components which may be expected to be present.
Typically these might include impurities, degradants, matrix, etc. The
common reagents present in the synthetic route did not interfere with
the elution or quantification of the method. Acceptance criteria for
specificity, RSD should be less than 2%. |
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| Linearity and range |
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| The plot of peak area of each sample against respective concentration
of Dapiprazole was found to be linear in the range of 20-120 μg/mL with
correlation coefficient of 0.9999. Linear regression least square fit data
obtained from the measurements are given in Table 3. The respective
linear regression equation being y=1956.4x+3409. The regression
characteristics, such as slope, intercept, and %RSD were calculated for
this method and given in Table 3. |
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Table 3: Linear Regression Data for Calibration curves. |
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| Robustness |
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| Robustness is a measure of the performance of a method when
small, deliberate changes are made to the method conditions. The
intent of this validation parameter is to identify the most critical
method conditions to the successful performance of the method. The
robustness of the method was assessed by deliberate alteration of the
experimental conditions. One factor at a time was changed to study the
effect. Variation of the detection wavelength by ± 2 nm (241 nm and
245 nm), the amount of acetonitrile in the mobile phase was varied by
± 10%), and mobile phase pH (± 2 pH-units) had no significant effect
on the retention time and chromatographic response of the method,
indicating that the method was robust. When the chromatographic
conditions were deliberately altered, system suitability results remained
within acceptance limits and selectivity for individual substance was
not affected. |
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| Ruggedness |
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| Ruggedness is an older term that has been replaced by intermediate
precision, according to the USP, is the degree of reproducibility of the
results obtained under a variety of conditions, expressed as %RSD; which
is a measure of how well the method performs under normal conditions
form laboratory-to-laboratory, instrument-to-instrument, and analystto-
analyst. A ruggedness test is performed as part of the validation of
an analytical method. Ruggedness test was determined between two
different analysts, instruments and columns. Small differences in areas
and good constancy in retention times were observed. The high degrees
of reproducibility of detector responses and retention times indicate
that the method is fairly rugged. |
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| From the typical chromatogram of Dapiprazole as shown in Figure
3, it was found that the retention time was 3.724 min. A mixture of
methanol: acetonitrile: and ortho-phosphoric acid in the ratio of 89:
9:1 (v/v) at pH 5.8 as the mobile phase at a flow rate of 0.8 ml/min
was found to be most suitable to obtain a peak well defined and free
from tailing. In the present developed HPLC method, the standard and
sample preparation required less time and no tedious extractions were
involved. A good linear relationship (r2=0.9999) was observed between
the concentration range of 20-120 μg/mL. Low values of standard
deviation are indicative of the high precision of the method. The assay of
Dapiprazole in bulk sample was found to be 99.94%. From the recovery studies it was found that about 99.06% of Dapiprazole was recovered
which indicates high accuracy of the method. The absence of additional
peaks in the chromatogram indicates non-interference of the common
reagents and intermediates used in in-house synthesis of Dapiprazole
in bulk drug production. This demonstrates that the developed HPLC
method is simple, linear, accurate, sensitive and reproducible. |
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Figure 3: Typical Chromatogram of Dapiprazole by HPLC. |
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| Thus, the developed method can be easily used for the routine
quality control of Dapiprazole bulk sample within a short analysis time. |
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| Conclusion |
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| It can be seen from the results presented that the proposed
procedure has good precision and accuracy. Results of the analysis
of pharmaceutical formulations revealed that proposed methods are
suitable for their analysis with virtually no interference of the usual
intermediates and impurities formed during the in-house synthesis of
Dapiprazole. This demonstrates that the developed HPLC method is
simple, linear, accurate, sensitive and reproducible. Thus, the developed
method can be easily used for the routine quality control of bulk forms
of Dapiprazole within a short analysis time. |
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| Acknowledgements |
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| The authors are grateful to M/s M/s Bioleo Analytical Labs India Pvt. Ltd
Hyderabad for the supply of Dapiprazole as a gift sample. One of us (KJP) is highly
thankful to, Management of BCAS, Bapatla for giving her an opportunity to pursue
this project under FDP. The authors also thankful to Q.S.Labs, Hyderabad for
providing the necessary facilities to carry out the research work. |
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| References |
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- Buschmann W (1996) Dapiprazol - Erfahrungen in der Praxis. Augenspiegel 4: 14-18.
- Saettone MF, Alderigi C, Giannaccini B, Galli-angeli D (1989) Preparation and Evaluation of A Sustained-Release Ophthalmic Vehicle for Dapiprazole. Drug development and industrial pharmacy 15: 2621-2637.
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- Than TP (1996) Comparison of the cycloplegic and mydriatic effects of 0.5% tropicamide and 2.5% phenylephrine to Paremyd and the counteracting effects of dapiprazole. Clinical Eye and Vision Care 8: 209-213.
- Marx-Gross S, Krummenauer F, Dick HB, Pfeiffer N (2005) Brimonidine versus dapiprazole: Influence on pupil size at various illumination levels. J Cataract Refract Surg 31: 1372-1376.
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