Karazgi Kishwar Jahan et al / Int. J. Pharm. Phytopharmacol. Res. 2014; 3 (5): xxx-xxx
ISSN (Online) 2249-6084 (Print) 2250-1029
International Journal of Pharmaceutical and
Phytopharmacological Research (eIJPPR)
[Impact Factor – 0.7826]
Journal Homepage: www.eijppr.com
Research Article
Development and Validation of New HPLC Method for the Quantitative Estimation
of Naftopidil in Bulk and Pharmaceutical Formulation
Karazgi Kishwar Jahan1* and Malipatil SM2
1
JJT University, Vidyanagri, Churu-Bisau Rd, Jhunjhunu, Rajasthan, India.
HKE’s College of Pharmacy, Sedam Road, Gulbarga, India.
2
Article info
Abstract
Article History:
Received xx xxxx 2014
Accepted xx xxxx 2014
Keywords:
Naftopidil, isocratic, ICH
guidelines, validation
parameters.
A new, rapid, accurate, precise and economical method has been developed for the quantitative estimation of
Naftopidil in bulk as well as pharmaceutical formulation. Isocratic elution with Inspire C18 (4.6 x 150mm, 5 m)
column at ambient temperature was used. The mobile phase consist of a mixture of potassium dihydrogen
orthophosphate buffer: acetonitrile (35%: 65% v/v) which was pumped at a flow rate of 1 ml/ min. The eluent
was detected by UV detector at 230 nm. The retention time was 2.585 min. The method was linear between the
range of 2-10µg/ml with correlation coefficient of 0.9997. The proposed method was validated as per the ICH
guidelines for all the parameters. The %RSD for the precision, method precision and ruggedness was
calculated and was found to be 1.28, 1.076 and 0.17 respectively. Accuracy studies were carried out at three
different levels i.e. 50%, 100% and 150%. The results obtained between the range of 98.23%-100.07% with a
mean of 99.08%. LOD and LOQ for the proposed method was calculated and was found to be 3.05 and 9.95
respectively. Ruggedness studies were also carried out by altering the flow rate and organic composition of the
solvent. No significant changes were seen with the altered conditions. The results obtained by the present
investigation depicts that the developed method is precise, accurate, economical, rugged, and robust and can
be utilized for the routine analyses of Naftopidil in bulk and pharmaceutical formulation.
method and one spectrophotometric8 method for the estimation of
naftopidil in bulk and pharmaceutical formulation have been
developed. Naftopidil is a drug of choice for the treatment of sign
and symptoms of BPH. The objective of the present investigation
was to develop and validate a simple, accurate, precise, and
economical HPLC method for the quantitative estimation of
Naftopidil in bulk drug and pharmaceutical formulations.
1. INTRODUCTION
Naftopidil is a α1-adrenergic receptor antagonist (α1-blocker) used
to treat lower urinary tract symptoms (LUTS) suggestive of benign
prostatic hyperplasia (BPH)1. Different from tamsulosin
hydrochloride, in that it has higher and extremely higher affinity
respectively, for the α1A-adrenergic receptor subtype than for the
α1D type, naftopidil has distinct characteristics because it has a
three times greater affinity for the α1D-adrenergic receptor subtype
than for the α1A subtype. Naftopidil had a similar short-term
efficacy and adverse-effect profile compared to low-dose
tamsulosin, and better efficacy than phytotherapy (eviprostat).
Naftopidil is an antihypertensive drug which acts as a selective α 1adrenergic receptor antagonist or alpha blocker. Its chemical
formula is C24H28N2O3, with molecular weight 392. Chemical name
and structural formula (Fig.1) of naftopidil is: 1-[4-(2methoxyphenyl) piperazin-1-yl]-3-(1-naphthyloxy) propan-2-ol.
2. MATERIALS AND METHOD
2.1 Instrumentation and chemicals used
For the development of HPLC method High performance liquid
chromatography equipped with Auto Sampler and DAD or UV
detector was utilized. The reagents and chemicals utilized were of
HPLC grade, procured from various pharmaceutical companies.
All the reagents and chemicals required for the development of
HPLC method were procured from various standard pharmaceutical
companies as follows:
Sl.
No.
1
2
Fig. 1: Structure of Naftopidil
Materials/ Chemicals
Source
Acetonitrile
Potassium dihydrogen ortho
phosphate
Qualigens, Mumbai.
S.D.Fine Chem. Ltd.,
Mumbai.
2.2 Experimental work
Literature survey reveals that for the estimation of other BPH drugs
like tamsulosine 2,3, prazosine4 and vigabatrine5 number of
analytical methods are there but only one chiral HPLC6, RP-HPLC7
2.2.1 Preparation of Phosphate buffer
Weighed 6.8 grams of Potassium di hydrogen ortho phosphate into
a 1000 ml beaker, dissolved and diluted to 1000 ml with HPLC
water.
*Corresponding Author:
Karazgi Kishwar Jahan
JJT University, Vidyanagri, Churu-Bisau Rd,
Jhunjhunu, Rajasthan.
Email: jahan_kish@rediffmail.com
Contact No: +91-9448749715
2.2.2 Preparation of mobile phase: Mixture of above buffer 350ml
(35%) and 650 ml of Acetonitrile HPLC (65%) was taken and
degased in ultrasonic water bath for 5 minutes. The solution was
filtered through 0.45 µ filter under vacuum filtration.
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Karazgi Kishwar Jahan et al / Int. J. Pharm. Phytopharmacol. Res. 2014; 3 (5): xxx-xxx
2.2.3 Diluent: Mobile phase was used as diluent.
Accuracy:
2.2.4 Preparation of the Naftopidil Standard and Sample
Solution:
Standard Solution Preparation:
Accurately weighed and transferred 10mg of Naftopidil working
standard into a 100 ml volumetric flask, about 70 ml of diluent was
added and sonicated to dissolve it completely and volume was
made up to the mark with the same solvent. (Stock solution).
0.6 ml of the above stock solution was pipetted into a 10ml
volumetric flask and diluted up to the mark with diluent. Mixed well
and filtered through 0.45µm filter.
Sample Solution Preparation:
About 5 Naftopidil tablets were weighed and average weight was
calculated. Accurately weighed and transferred the sample
equivalent to 10 mg of Naftopidil into a 100 ml volumetric flask.
About 70 ml of diluent was added and sonicated to dissolve it
completely and volume was made up to the mark with diluent.
Mixed well and filtered through 0.45µm filter. Further pipetted 0.6 ml
of the above stock solution into a 10ml volumetric flask and diluted
up to the mark with diluent. Mixed well and filtered through 0.45µm
filter.
Preparation of stock solution:
Accurately weighed and transferred 10 mg equivalent of Naftopidil
working standard into a 100 ml volumetric flask about 70 ml of
diluent was added and sonicated to dissolve it completely and
volume was made up to the mark with the same solvent. (Stock
solution).
Preparation of Sample solutions:
a)For preparation of 50% solution (With respect to target Assay
concentration):
Accurately weighed and transferred 4.85 mg of Naftopidil API
sample into a 100 ml volumetric flask about 70 ml of diluent was
added and sonicated to dissolve it completely and volume was
made up to the mark with the same solvent. (Stock solution)
b)For preparation of 100% solution (With respect to target Assay
concentration):
Accurately weighed and transferred 10.0mg of Naftopidil API
sample into a 100 ml volumetric flask about 70 ml of diluent was
added and sonicated to dissolve it completely and volume was
made up to the mark with the same solvent. (Stock solution)
c)For preparation of 150% solution (With respect to target Assay
concentration):
Accurately weighed and transferred 15.25mg of Naftopidil API
sample into a 100 ml volumetric flask about 70 ml of diluent was
added and sonicated to dissolve it completely and volume was
made up to the mark with the same solvent. (Stock solution)
2.2.5 Optimized Chromatographic Parameters
Equipment
Column
Flow rate
Wavelength
Injection
volume
Temperature
Run time
:High performance liquid chromatography equipped
with Auto Sampler and DAD or UV detector.
: Inspire C18 (4.6 x 150mm, 5 m)
: 1.0ml per min
: 230 nm
Procedure:
Three volumetric flasks of 10ml were taken and labeled as
accuracy 50%,100% and 150%, to all the above flasks added 0.6ml
of stock solution and 0.6ml of accuracy 50%, 100% and 150%
solutions to the respective volumetric flasks and the volume was
made up to 10ml with diluent. The standard solutions, Accuracy -50%,
Accuracy -100% and Accuracy -150% solutions were injected. The
amount found and amount added for Naftopidil was calculated and
individual recovery and mean recovery values were also calculated.
For the determination of LOD and LOQ middle concentration
(6µg/ml) was selected.
: 20 l
: Ambient
: 6.0 min
About 20μl of the standard and sample were injected into the
chromatographic system and measured the area for the Naftopidil
peak and % assay was calculated by using the formulae. Figure No
2 and 3.
2.2.6 HPLC Method Validation
The developed method has been validated systematically for all the
validation parameters as per the ICH guidelines i.e. linearity, precision,
accuracy, LOD, LOQ and robustness as follows:
Limit of detection:
1.0ml of the above stock solution was pipetted into a 10ml
volumetric flask and diluted up to the mark with diluent. Mixed well
and filtered through 0.45µm filter.
Preparation of 0.15% solution At Specification level (0.01µg/ml
solution):
About 0.15mL of above solution was pipetted into a 10 ml of
volumetric flask and diluted up to the mark with diluent. LOD was
calculated from the data obtained and was found to be 0.01µg/ml.
Linearity:
Preparation of stock solution:
Accurately weighed and transferred 10mg of Naftopidil sample into
a 100 ml volumetric flask about 7 ml of diluent was added and
sonicated to dissolve it completely and volume was made up to the
mark with the same solvent. (Stock solution) From the above stock
solution 0.2, 0.4, 0.6, 0.8 and1.0 ml was pipetted out in different 10
ml of volumetric flasks and diluted up to the mark with diluent which
gives concentration ranging from 2-10 µg/ml.
Limit of Quantitation:
Preparation of 0.5% solution At Specification level (0.03µg/ml
solution):
From the stock solution of 6 µg/ml, 0.5 ml of solution was pipetted
into a 10 ml of volumetric flask and dilute up to the mark with
diluent, and LOQ was calculated which was found to be 0.03µg/ml.
Procedure:
Each level solution was injected into the chromatographic system
and measured the peak area. A graph of peak area versus
concentration was plotted and slope, intercept and the correlation
coefficient was calculated.
Robustness:
As part of the Robustness, deliberate changes in the flow rate and
mobile phase composition was made to evaluate the impact on the
method.
a) The flow rate was varied at 0.8 to1.2 ml/min.
Standard solution of 6µg/ml was prepared and analysed using the
varied flow rates along with method flow rate.
b) The Organic composition in the Mobile phase was varied from
55% to 65%.
Standard solution of 6µg/ml was prepared and analysed using the
varied mobile phase composition along with the actual mobile
phase composition in the method.
System precision:
The standard solution of middle concentration (6µg/ml) was injected for
six times and measured the area for all six injections in HPLC.
Ruggedness:
To evaluate the intermediate precision (also known as
Ruggedness) of the method, precision was performed on different
day by using different make column of same dimensions. The
standard solution of 6µg/ml was injected for six times and measured
the area for all six injections in HPLC.
3. RESULT AND DISCUSSION
A graph of peak area versus concentration was plotted and slope,
intercept and the correlation coefficient was calculated which was
found to be 117526, 18138 and 0.9997 respectively. Linearity
results are shown in table 1.
Repeatability:
The sample solution of middle concentration (6µg/ml) was injected for
six times and measured the area for all six injections in HPLC.
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Karazgi Kishwar Jahan et al / Int. J. Pharm. Phytopharmacol. Res. 2014; 3 (5): xxx-xxx
Precision studies were performed and the %RSD for the area of six
replicate injections was found to be within the specified limits. The
results are summarized in table 2.
Ruggedness Studies were carried out and the %RSD for the area of
six replicate injections was found to be within the specified limits. The
results are summarized in table 3.
Repeatability studies were performed and the %RSD for the area of six
replicate injections was found to be within the specified limits. The
results are summarized in table 4.
Accuracy studies were carried out by standard addition method at
three different levels and the results are summarized in table 5.
As part of the Robustness studies, deliberate changes in the flow
rate and mobile phase composition was made to evaluate the
impact on the method and the results are given in table 6 a and b:
a) The flow rate was varied at 0.8 to1.2 ml/min.
b) The Organic composition in the Mobile phase was varied from
55% to 65%.
pharmaceutical formulation. Isocratic elution with Inspire C18 (4.6 x
150mm, 5 m) column at ambient temperature was utilized. The
eluent was detected by UV detector at 230 nm. The retention time
was 2.585 min. The method was linear between the range of 210µg/ml with correlation coefficient of 0.9997. The proposed
method was validated as per the ICH guidelines for all the
parameters. The %RSD for the precision, method precision and
ruggedness was calculated and was found to be 1.28, 1.076 and
0.17 respectively. Accuracy studies were carried out at three
different levels i.e. 50%, 100% and 150%. The results obtained
between the ranges of 98.84%-100.65% with a mean of 100.04%.
LOD and LOQ for the proposed method were calculated and were
found to be 0.01μg/ml and 0.03μg/ml respectively. Ruggedness
studies were also carried out by altering the flow rate and organic
composition of the solvent. No significant changes were seen with
the altered conditions which show that the developed method was
rugged.
4. CONCLUSION:
From the above discussion it can be concluded that as the results
of the analysis were validated statistically and all the validation
parameters were found to be within the acceptable range, it proves
the precision, sensitivity, accuracy, ruggedness, robustness and
applicability of the proposed method for the routine quantitative
determination of Naftopidil in bulk drug and its dosage form.
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Figure 2: Typical chromatogram of standard Naftopidil
Figure 3: Typical chromatogram of sample Naftopidil
HPLC method has been developed and validated for the
quantitative estimation of Naftopidil in bulk as well as
15