A new simple, sensitive, rapid, accurate, precise and economical Derivative Spectrophotometric method for the simultaneous determination of Quinapril HCl

1. Int. J. Pharm. Res. Sci., 013, 01(1), 16-25.
.
ISSN: 2348 –0882
Development And Validation Of First Order Derivative Method
For Simultaneous Estimation Of Quinapril Hcl And
Hydrochlorothiazide In Combined Pharmaceutical Formulation
Reema Jaiswal*1, Pinak patel1
Department of Pharmaceutical Analysis, Indubhai Patel College of Pharmacy and Research
Centre, Dharmaj, Gujarat-388430, India
2
Department of Quality Assurance, Indubhai Patel College of Pharmacy and Research Centre,
Dharmaj, Gujarat-388430, India
1
====================================================================
ABSTRACT
A new simple, sensitive, rapid, accurate,
INTRODUCTION
Quinapril Hydrochloride (QUI), a 3precise
and
economical
Derivative
Isoquinolinecarboxylic
acid,2-[2-[[1Spectrophotometric
method
for
the
(ethoxycarbonyl)-3-phenylpropyl]amino]-1simultaneous determination of Quinapril
oxopropyl]-1,2,3,4-tetrahydroHCl
(QUI)
and
Hydrochlorothiazide
(HCTZ) in their combined pharmaceutical
,monohydrochloride [Figure 1a]. QUI is
dosage form was developed. The derivative
official in United State Pharmacopoeia
Spectrophotometric method was based the
(USP) but it is listed in Merck Index 1,
absorbance of the solutions were measured
Martindale and Complete Drug Reference2-7.
at 242.45 nm (λ1), and 257.17 nm (λ2) for
It is a ACE inhibitor and indicated in
the estimation of both the drugs.
The
treatment of symptomatic treatment of high
linearity was obtained in the concentration
blood pressure and used with some other
range of 80-240 μg/ml for QUI and 10-50
drugs in combination therapy. Literature
μg/ml for HSfl e mean recovery was
C
survey revealed that various8-12 , Capillary
99.93 – 100.33 % and 99.06-101.25% for
electrophoresis, Ion-pair HPLC and HPTLC
QUI and HCTZ respectively. The results of
methods have been reported for quantitative
analysis have been validated statistically as
estimation of QUI in pharmaceutical dosage
per ICH guidelines.
forms and biological fluids individually or in
combination
with
other
drugs.
KEYWORDS
Quinapril
Hydrochloride,
Hydrochlorothiazide (HCTZ) is chemically
Hydrochlorthiazide, Derivative method,
6-Chloro-3,4-dihydro-2H-1,2,4Methanol.
benzothiadiazine-7-sulfonamide 1,1-dioxide
[Figure 1b]. HCTZ is a thiazide diuretic
Corresponding Author
inhibits water reabsorption in the nephron by
Reema Jaiswal
inhibiting the sodium-chloride symporter
Email address: jreema42@yahoo.in
(SLC12A3) in the distal convoluted tubule,
Received: 18.12.2013
which is responsible for 5% of total sodium
Revised: 26.12.2013
reabsorption. It is used for the treatment of
Accepted: 29.12.2013
the treatment of blood pressure and in
16

2. Int. J. Pharm. Res. Sci., 013, 01(1), 16-25.
.
ISSN: 2348 –0882
management of oedema.13-15 HCTZ is
official in IP,BP16,17,USP18 and JP19 describe
HPLC method for its estimation. The review
of literature revealed that various analytical
methods involving Spectrophotometry,
HPLC20-26and HPTLC 27-29 have been
reported for HCTZ in pharmaceutical
dosage forms and biological fluids
individually or in combination with other
drugs.
is no published RP-HPLC method for this
combination. So, the present paper describes
a simple, accurate and precise method for
simultaneous estimation of QUI and HCTZ
in combined Pharmaceutical Formulation by
RP-HPLC method. The eveloped method
was validated in accordance with ICH
Guidelines30and successfully employed for
the assay of QUI and HCTZ in their
combined
dosage
form
To the best of our knowledge, there
.
Figure 1: Chemical structure of (a) QUI and (b) HCTZ
An accurately weighed quantity of
MATERIALS AND METHODS
QUI (100 mg) and HCTZ (100 mg) were
Reagents and Chemicals
Analytically pure QUI and HCTZ
transferred to a separate 100 ml volumetric
flask and 50 ml of methanol was added to
were kindly provided by Aurobindo
Pharmaceuticals Ltd.,Hyderabad
and
both volumetric flask and sonicated for 5
Chemyes Corporation Vadodara, Gujarat
minutes. Volume was adjusted up to the
India as gratis samples. Methanol was used
mark with methanol to obtain standard
as solvent. Tablet of QUI and HCTZ in
solution having concentration of QUI (1000
combined dosage form, i.e. Q-PRIL H-10
μg/ml) and HCTZ (1000 μg/ml). 12.5 ml
was procured from local market.11-13
solutions of QUI (1000 μg/ml) and 10 ml
HCTZ (1000 μg/ml) were transferred to a
INSTRUMENTS
A Shimadzu UV/Vis 1800 double
separate 25 ml volumetric flask and 100 ml
beam spectrophotometer with a wavelength
volumetric flask respectively and diluted up
accuracy (± 0.3 nm), 1 cm matched quartz
to concentration of QUI (500 μg/ml) and
cells and UV probe 2.32 software was used
HCTZ (50μg/ml) with methanol.
for all the spectral measurements and
First order Derivative method
Shimadzu UV/Vis 1601 double beam
80-240 µ g/ml solutions of QUI and 10-50
spectrophotometer with a wavelength
µ g/ml solutions of HCTZ were prepared in
methanol by appropriate dilution and
accuracy (± 0.3 nm) and 1 cm matched
spectrum was recorded between 200-400
quartz cells was used for reproducibility
nm. The absorbance of the solutions were
Study. Calibrated analytical balance K-EA
measured at 242.45 nm (λ1), and 257.17 nm
210 (K-Roy Instrument Pvt. Ltd) was used
(λ2), for the estimation of both the drugs by
for weighing purpose.
proposed
method.
The
quantitative
Preparation of standard stock solutions
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3. Int. J. Pharm. Res. Sci., 013, 01(1), 16-25.
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ISSN: 2348 –0882
determination of QUI is carried out by
measuring absorbance difference between
242.45 nm and 257.17 nm where HCTZ has
same absorbance at both the wavelengths.
For estimation of QUI and HCTZ using
derivative spectroscopy, zero crossing
method was decided to be used. In this
method two wavelengths are required. One
wavelength is selected at which QUI shows
zero absorbance while other drug HCTZ
shows considerable absorbance. The second
wavelength is selected such that HCTZ
shows zero absorbance while QUI shows
considerable absorbance.
The overlain derivative spectrum (first
order) of QUI and HCTZ at different
concentrations revealed that at 242.45 nm
different concentration of HCTZ possesses
zero D1 absorbance whereas QUI possesses
significant D1 absorbance. In a similar
manner,
at
257.17nm
different
concentrations of QUI possess zero D1
absorbance whereas HCTZ possesses
significant D1 absorbance. Considering
above facts, wavelength 242.45 nm and
257.17nm were selected for the estimation
of QUI and HCTZ respectively
Method validation
The proposed method has been extensively
validated in terms of specificity, linearity,
accuracy, precision, limits of detection
(LOD) and quantification (LOQ), robustness
and reproducibility. The accuracy was
expressed in terms of percent recovery of
the known amount of the standard drugs
added to the known amount of the
pharmaceutical dosage forms. The precision
(Coefficient of Variation - C.V.) was
expressed with respect to the repeatability,
intra-day and inter-day variation in the
expected drug concentrations. After
validation, the developed methods have been
applied to pharmaceutical dosage form.
Specificity
Commonly used excipients (starch,
microcrystalline cellulose and magnesium
stearate) were spiked into a pre weighed
quantity of drugs. The fundamental or
normal spectrum was recorded by
appropriate dilutions and the quantities of
drugs were determined.
Linearity
Appropriate volume of aliquot from
QUI and HCTZ standard stock solution was
transferred to volumetric flask of 10 ml
capacity. The volume was adjusted to the
mark with methanol to give solutions
containing 80-240 µ g/ml QUI and 1050µ g/ml HCTZ. All D0 Spectrum were
recorded for QUI and HCTZ respectively
(n=5).
Calibration curves were constructed by
plotting
average
absorbance
versus
concentrations for both drugs. Straight line
equations were obtained from these
calibration curves.
Accuracy
Accuracy
was
assessed
by
determination of the recovery of the method
by addition of standard drug to the prequantified placebo preparation at 3 different
concentration levels 80, 100 and 120 %,
taking into consideration percentage purity
of added bulk drug samples. Each
concentration was analyzed 3 times and
average recoveries were measured.
Precision
The repeatability was evaluated by
assaying 6 times of sample solution prepared
for assay determination. The intraday and
interday precision study of QUI and HCTZ
was carried out by estimating different
concentrations of QUI (120, 160, 200
µ g/ml) and HCTZ (20, 30, 40 µ g/ml), 3
times on the same day and on 3 different
days (second, third, fourth) and the results
are reported in terms of C.V.
Detection limit and Quantitation limit
ICH guideline describes several
approaches to determine the detection and
quantitation limits. These include visual
evaluation, signal-to-noise ratio and the use
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4. Int. J. Pharm. Res. Sci., 013, 01(1), 16-25.
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ISSN: 2348 –0882
of standard deviation of the response and the
slope of the calibration curve. In the present
study, the LOD and LOQ were based on the
third approach and were calculated
according to the 3.3σ/S and 10σ/S criterions,
respectively; where σ is the standard
deviation of y-intercepts of regression lines
and s is the slope of the calibration curve.
Robustness
The sample solution was prepared
and then analyzed with change in the typical
analytical conditions like stability of
analytical solution.
Reproducibility
The absorbance readings were
measured at different laboratory for sample
solution using another spectrophotometer by
analyst and the values obtained were
evaluated using t- test to verify their
reproducibility.
Determination of QUI and HCTZ in their
Combined Dosage
Twenty tablets were weighed and powdered.
A powder quantity equivalent to 10 QUI and
12.5 mg HCTZ was accurately weighed and
transferred to volumetric flask of 50 ml
capacity. 50 ml of methanol was transferred
to this volumetric flask and sonicated for 15
min. The flask was shaken and volume was
made up to the mark with methanol. The
above solution was filtered through
whatman filter paper (0.45µ ).The flask was
shaken and volume was made up to the mark
with methanol. From this solution 4 ml was
transferred to volumetric flask of 10 ml
capacity. Volume was made up to the mark
to give a solution containing 80 µ g/ml of
QUI using methanol(Solution A). This
solution was used for the estimation of QUI,
from (solution A) withdraw 1ml and dilute
to 10 ml to give 8 μg/ml QUI and 10 μg/ml
HCTZ and solution was used for estimation
of HCTZ(solution B). The resulting solution
was analyzed by proposed methods. The
quantitation was carried out by keeping
these values to the straight line equation of
calibration curve.
RESULTS AND DISCUSSION
In First order Derivative wavelength
method, For estimation of QUI and HCTZ
using derivative spectroscopy, zero crossing
method was decided to be used. In this
method two wavelengths are required. One
wavelength is selected at which QUI shows
zero absorbance while other drug HCTZ
shows considerable absorbance. The second
wavelength is selected such that HCTZ
shows zero absorbance while QUI shows
considerable absorbance. The overlain
derivative spectrum (first order) of QUI and
HCTZ at different concentrations revealed
that at 242.45 nm different concentration of
HCTZ
possesses zero D1 absorbance
whereas QUI possesses significant D1
absorbance. In a similar manner, at
257.17nm different concentrations of QUI
possess zero D1 absorbance whereas HCTZ
possesses significant D1 absorbance.
Considering above facts, wavelength 242.45
nm and 257.17nm were selected for the
estimation of QUI and HCTZ respectively .
Calibration data for QUI and HCTZ are
shown in Table 2 and 3 respectively.
Calibration curves for QUI and HCTZ were
plotted between D1 absorbance and
concentration. The following equations for
straight line were obtained for QUI and
HCTZ.
Linear equation for QUI, y = 0.002x -0.011
Linear equation for HCTZ, y = 0.030x 0.203
The linearity of the calibration curve was
validated by the high values of correlation
coefficient of regression. The C.V values for
QUI and HCTZ for repeatability was found
to be 0.54 and 0.51 %, respectively [Table
1]. The Coefficient of Variance(C.V) (less
than 2 %) indicates that the proposed
method is repeatable. The C.V values of
Intraday (0.52 – 0.89% and 0.10 – 0.48%)
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5. Int. J. Pharm. Res. Sci., 013, 01(1), 16-25.
.
ISSN: 2348 –0882
and Interday (0.25 – 0.43 % and 0.11 – 0.23
%) for QUI and HCTZ, respectively, reveal
that the proposed method is precise. LOD
values for QUI and HCTZ were found to be
1.88 and 0.18 μg/ml, respectively and LOQ
values for QUI and HCTZ were found to be
6.20 and 0.54 μg/ml, respectively [Table 1].
These data show that proposed method is
sensitive and precise for the determination
of QUI and HCTZ, respectively [Table 3, 4].
The recovery experiment was
performed by the standard addition method.
The percentage recovery was 99.93 –
100.33% and 99.06 – 101.25% for QUI and
HCTZ, respectively [Table 5, 6]. The results
of recovery studies indicate that the
proposed method is accurate. The proposed
validated method was successfully applied
to determine QUI and HCTZ in their
combined dosage form. The results obtained
for QUI and HCTZ was comparable with the
corresponding
labelled
amount.
No
interference of the excipients with the
absorbance of interest appeared; hence the
proposed method is applicable for the
routine simultaneous estimation of QUI and
HCTZ in pharmaceutical dosage forms.
Figure 2-Calibration Curve for QUI
Figure 3-Calibration Curve for HCTZ
Figure 4- Overlain D0 spectra of QUI (80-240)µg/ml in methanol
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6. Int. J. Pharm. Res. Sci., 013, 01(1), 16-25.
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ISSN: 2348 –0882
Figure 5- Overlain D0 spectra of HCTZ(10-50) µg/ml in methanol
Figure 6-QUI& HCTZ- first order ( D1) derivative overlain
Table 1: Summary of Validation Parameters of First order Derivative method
Parameters
QUI
HCTZ
% Recovery
99.70 – 101.10
99.80 – 101.62
Repeatability(RSD, n=6) 0.54
0.51
Precision(%RSD)
Intra-day (n=3)
0.52-0.89
0.10-0.48
Inter-day (n=3)
0.25-0.43
0.11-0.23
LOD
1.88
0.18
LOQ
6.20
0.54
Specificity
specific
specific
Solvent Stability
Stable for 24 hours Stable for 24 hours
Table 2: Statistical data QUI and HCTZ by First order Derivative method
Parameter
QUI
HCTZ
Analytical wavelength
242.45 nm 257.17 nm
Range (µ g/ml)
80– 240
10-50
Slope
-0.002
0.030
Intercept
-0.010
-0.202
Regression Coefficient (r2)
0.997
0.998
Standard deviation of intercept 0.00114
0.00164
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ISSN: 2348 –0882
Table 3: Precision data for QUI
Concentration
(μg/ml)
120
160
200
Intraday
C.V. Interday
-0.2333±0.0020 0.89
-0.3133±0.0024 0.64
-0.3934±0.0022 0.52
C.V.
-0.2321±0.0010 0.43
-0.3120±0.0012 0.30
-0.3921±0.0011 0.25
Table 4: Precision data for HCTZ
Concentration Intraday
C.V. Interday
C.V.
(μg/ml)
20
0.4333±0.0020 0.48 0.4320±0.0012 0.23
30
0.6836±0.0025 0.36 0.6820±0.0016 0.14
40
0.9960±0.0010 0.10 0.9981±0.0018 0.10
Table 5 :Reproducibility data for QUI (80 µg/ml)
Instrument
1
Mean ± S.D.
0.3153 ±
0.0030
Instrument
2
Mean ± S.D.
0.3166 ±
0.0025
Result of Value from
t-distribution
t
test *
table
0.5783
4.30
Inference
There is
difference
no
significance
no
significance
Table 6 :Reproducibility data for HCTZ( 10 µg/ml)
Instrument
1
Mean ± S.D.
0.2956 ±
0.0025
Instrument
2
Mean ± S.D.
0.2936 ±
0.0015
Result of Value from
t-distribution
t
test *
table
4.30
0.4380
Table 7: Specificity and Selectivity study
Study
QUI
Specificity Specific
HCTZ
Specific
Selectivity Selective Selective
22
Inference
There is
difference

8. Int. J. Pharm. Res. Sci., 013, 01(1), 16-25.
.
ISSN: 2348 –0882
Table 8: Accuracy data for QUI and HCTZ
Level
Amount
Amount
Std Amount
of
drug drug added
Recovered in
present
(µg/ml)
(µg/ml)
% Recovery ± S.D
US
80+10
QUI
80
80+10
100
120
HCTZ
QUI
HCTZ
QUI
HCTZ
80
16
79.95
15.85
99.93±0.58
99.06± 0.41
80+10
100
20
100.20
20.10
100.20±0.36
100.50±0.52
80+10
120
24
120.40
24.30
100.33±0.47
101.25±0.68
Table 9: Assay Results of Marketed Formulation
Tablet
Drug Labeled
Amount
claim
taken
(mg)
(µg/ml)
Q-PRIL
H-10 QUI
10
80
TAB
HCTZ 12.5
10
CONCLUSION
The proposed dual wavelength method
provides simple, specific, precise, accurate
and reproducible quantitative analysis for
simultaneous determination of QUI and
HCTZ in combined dosage form. The
methods were validated as per ICH
guidelines in terms of specificity, linearity,
accuracy, precision, limits of detection
(LOD) and quantification (LOQ), robustness
and reproducibility. The proposed methods
can be used for routine analysis and quality
control assay of QUI and HCTZ in
combined dosage form.
ACKNOWLEDGEMENT
The author is thankful to Indubhai
Patel College of Pharmacy and Research
Centre (Dharmaj, India) for providing the
necessary facilities for research work and to
all the staff members and friends for their
guidance and help throughout the research
work.
Amount
found
(µg/ml)
79.60
9.97
% Label
±S.D
claim
98.50±0.0152
99.70±0.0156
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