Journal of Food and Drug Analysis, Vol. 20, No.

3, 2012, Pages 577-587

577
doi:10.6227/jfda.2012200303
Validated High-Performance Liquid Chromatographic and
Chemometric Based Spectrophotometric Determination of
Ramipril and Atorvastatin in Pharmaceutical Dosage Forms
NAGARAJ GOWDA
1
*
,

RAJU TEKAL
1
, RADHASRI THANGAVELU
1
,
KALAMKAR VIPUL
2
AND MASHRU RAJASHREE
3
1.
Analytical Research Laboratory, Department of Pharmaceutical Analysis, PES College of Pharmacy, Hanumantanagar, Bangalore,
Karnataka, India
2.
Department of Statistics, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India
3.
Centre of Relevance and Excellence in Novel Drug Delivery System, Pharmacy Department, The Maharaja Sayajirao University of Baroda,
G. H. Patel Building, Donor's Plaza, Fatehgunj, Vadodara, Gujarat, India
(Received: July 1, 2011; Accepted: February 24, 2012)
ABSTRACT
A fast, simple RP-HPLC and three spectrophotometric methods based on classical least squares (CLS), principle component regres-
sion (PCR) and partial least squares (PLS) calibrations were developed for simultaneous estimation of ramipril (RAMP) and atorvastatin
calcium (ATOR) in formulations. The HPLC assay utilized Phenomenex - Luna RP C-18 (2) 250 4.6 mm 5 m column with mobile phase
composition of acetonitrile: 0.1 M sodium perchlorate (pH 2.5) (70 : 30, v/v), and flow rate of 1.5 mL/min with UV detection at 210 nm.
Chemometric calibrations were constructed by using absorption data matrix corresponding to concentration data matrix, with measure-
ments in the range of 201-270 nm ( = 1 nm) in their zero-order spectra using 25 samples in training set. The chemometric numerical
computations were realized using novel R-Software Environment (version 2.1.1). Reliability of predictions was validated for various ICH
regulatory parameters. Both the results of proposed chemometric methods and that of proposed chromatographic method were compared
and good agreement was found. Laboratory prepared mixtures and commercial tablet formulations were successfully analyzed by the
developed methods.
Key words: CLS, PCR, PLS, R-software environment, RP-HPLC
INTRODUCTION
Ramipril (RAMP) is a prodrug for the major metabo-
lite ramiprilat formed via ester hydrolysis, which is a highly
active inhibitor of angiotensin-converting enzyme (ACE-I)
(1)
. Atorvastatin (ATOR) calcium belongs to the category of
statins, which inhibit 3-hydroxy-3-methylglutaryl-coenzyme
A (3-HMG CoA) reductase
(2)
, thus resulting in a decrease in
intracellular cholesterol due to increased clearance of low-
density lipoproteins (LDL) cholesterol in plasma
(3)
.
Although there have been many advances in the manage-
ment of cardiovascular diseases (CVD) during the last
several years, these are still the main cause for morbidity and
mortality. The pathophysiology of CVD reveals that renin-
angiotensin-aldosterone system (RAAS) and dyslipidaemia
play an important role in the genesis and progression of
CVD risk that is endothelial dysfunction
(4)
. Intervention with
antioxidant agents such as 3-HMG-CoA reductase inhibitors
(statin), and ACE-I have been shown to counteract reactive
oxygen species production and improve endothelial function
in coronary artery disease. Treatment with RAMP plus ATOR
may act synergistically in the prevention and treatment of
atherosclerosis
(5,6)
.
An assortment of techniques has been described for the
quantifcation of ATOR and RAMP alone and in combination
with other drugs. Same authors have reported simultaneous
spectrophotometric estimation of ATOR in combination
with fenofbrate
(7)
. The other quantifcation methods for
ATOR samples in pharmaceutical formulations, bulk drug
and in plasma have also been reported along with ATOR
impurities, enantiomers, intermediates and metabolites
by HPLC
(8-14)
, LC-MS
(15,16)
, FT-Raman spectroscopy
(17)
and electrochemical methods
(18)
. Quantifcation of RAMP
alone and in combination with other drugs by HPLC
(19-22)
,
LC-MS
(23-25)
, GC-MS
(26)
, spectrophotometric
(27-29)
, fuori-
metric
(30)
, voltametric
(31,32)
, capillary electrophoresis
(33,34)

* Author for correspondence. Tel: (O) +91-9448380880;
Fax: +91-8026600113; E-mail: nraj_msubaroda@yahoo.co.in
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
578
and atomic absorption spectrophotometry
(35,36)
have already
been reported.
The purpose of this study was to develop and validate
HPLC and three chemometric techniques, viz classical least
squares (CLS), principle component regression (PCR) and
partial least squares (PLS) calibrations, for the determination
of title drugs. The employment of the developed methods
to determine the content of both the drugs in commercial
formulations was also demonstrated. The chemometric
methods proposed in this paper presumed that a linear rela-
tionship exists between absorbances and component concen-
trations. These methods have a calibration step followed by
the prediction step, in which the results of the calibration step
are used to estimate the component concentration from an
unknown sample spectrum. The methods do not require any
derivatisation, prior separation or sample pretreatment. These
methods have been successfully applied to the quantitative
analysis in spectrophotometric
(37-39)
, chromatographic
(40,41)

and electrochemical data
(42,43)
. Chemometric calibration
techniques were discussed in more detail elsewhere
(44-49)
.
MATERIALS AND METHODS
I. Apparatus and Software
The Shimadzu HPLC system consisting of gradient
pump (LC-10AT vp pump), mixer (SUS vp), rheodyne
injector, UV-VIS dual wavelength detector (SPD-10A vp),
Hamilton syringe (25 L) and analytical weighing balance
(AUX 200) (all from Shimadzu, Kyoto, Japan) were used.
The separations were achieved on a Phenomenex Luna C-18
(2) (250 4.6 mm, 5 m) column (UK) with UV detection
at 210 nm. Sonicator (SONICA 2200MH), vacuum pump
(model XI 5522050) and Millipore fltration kit for solvents
and sample fltration (Millipore, Bangalore, KRN, India)
were used throughout the experiment. The Spinchrom CFR
software-single channel was used for acquisition, evaluation
and storage of chromatographic data.
Shimadzu UV-1700 and UV-1601 double beam spectro-
photometers connected to a computer loaded with Shimadzu
UVProbe 2.10 software (Shimadzu, Kyoto, Japan) was used
for all the spectrophotometric measurements. The electronic
spectral bandwidth was 1 nm and the wavelength scanning
speed
(49)
was 2,800 nm/min. The electronic absorption
spectra of the reference and test solutions were acquired in a
1 cm quartz cells over the range of 201-350 nm. The chemo-
metric calculations on the resulting data were carried out in
R-software environment (version 2.1.1) (www.r-project.org
(website for the R software environment)) which is a GNU
implementation of S
(50)
language and environment which
was developed at Bell Laboratories.
II. Reagents and Pharmaceutical Preparations
RAMP and ATOR were donated by Dr. Reddys Labo-
ratories Limited (Hyderabad, AP, India) and Biocon, Ltd.
(Bangalore, KRN, India) and certifed to be 99.6% and 99.8%
pure respectively. The drugs were used without further purif-
cation. All the solvents used in analysis were of spectroscopic
and HPLC grade (Ranbaxy and SD-Fine Chemicals Limited,
New Delhi, India). Stator-R 2.5 tablets (label claim 2.5 mg
RAMP and 10 mg ATOR) batch No. 6004 of Accent Pharma
(Pondicherry, TN, India) and Rampitor*5 capsules (label
claim 5 mg RAMP and 10 mg ATOR) batch No. AF 70027
of Atoz Life Sciences, (Pondicherry, TN, India) were used in
analysis.
III. Standard Solutions
Stock solutions of RAMP and ATOR (1 mg/mL) in
acetonitrile: 0.1 M sodium perchlorate (70 : 30, v/v) were
used. The working solutions were 0.04 mg/mL prepared by
transferring 2.0 mL from the respective stock solution to a 50
mL volumetric fask and completing to volume with acetoni-
trile: 0.1 M sodium perchlorate (70 : 30, v/v).
IV. Preparation of Mobile Phase
A solution of 0.1 M sodium perchlorate was prepared by
dissolving 14.046 g in 1000 mL of HPLC grade water and the
pH of the resulting solution was adjusted to 2.5 0.2 using
85% orthophosphoric acid. HPLC experiments were carried
out using binary pump with acetonitrile in one solvent reser-
voir and 0.1 M sodium perchlorate in another one.
V. HPLC Method
The mobile phase acetonitrile: 0.1 M sodium perchlorate
(pH 2.5) (70 : 30, v/v) was chosen because it was found ideal
to resolve the peaks with retention time (R
t
) 2.267 0.003
min and 3.173 0.002 min for RAMP and ATOR respectively
(Figure 1). Detection wavelength 210 nm was selected by
scanning both standard ingredients over a wide range of 201
to 350 nm in spectrophotometer.
VI. HPLC Calibration
The HPLC calibration was performed individually for
both ingredients at seven different concentrations using either
ingredient as internal standard during calibration of the other.
Aliquots of standard RAMP working solutions were taken in
different volumetric fasks and 8 g/mL of ATOR was added
to each fask as internal standard and diluted with mobile
phase in the range of 4-32 g/mL (Figure 2). Similarly, ATOR
working solutions were taken in different volumetric fasks
and 8 g/mL of RAMP was added to each fask as internal
standard and diluted with mobile phase in the range of 4-22
g/mL. All stock and working solutions were sonicated for 5
min, and then fltered through a nylon membrane flter (0.45
) prior to use. Twenty microliter of sample was injected in
triplicate for each concentration and chromatographed under
specifed condition at ambient temperature (28C). Regres-
sion analyses were carried by plotting peak areas against the
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
579
corresponding concentrations and also by plotting peak area
response ratio of pure analytes concentrations to internal
standard. There was no signifcant difference in both the
regression analyses. Regression analysis of the calibration
data was then carried out (Table 1).
VII. Chemometric Calibration Sets
A calibration set of 25 sample mixtures was prepared
in acetonitrile: 0.1 M sodium perchlorate (pH 2.5) (70 : 30,
v/v), applying a multilevel multifactor design in which fve
levels of concentrations of RAMP and ATOR were intro-
duced. The levels were 4-32 and 4-22 g/mL for RAMP and
ATOR respectively. UV absorbance spectra were recorded
in the wavelength of 201-350 nm versus solvent blank and
digitized absorbances was recorded at 1 nm intervals. The
chemometric computation was made in R-software environ-
ment. CLS, PCR and PLS algorithms were applied to the UV
absorption data matrix between 201-270 nm of these binary
mixtures to determine calibration equations.
VIII. Pharmaceutical Sample Solution
Twenty Stator-R tablets and Rampitor* 5 capsules were
weighed accurately. An amount of the powder equivalent
to the content of one unit of tablet / capsule was dissolved
separately in 60 mL of mobile phase. The solutions were
sonicated for 10 min and fltered into a 100 mL volumetric
fask through 0.45 nylon membrane flter. The residue was
washed 3 times with 10 mL of mobile phase, and then the
volume was completed to 100 mL with the same solvent.
These solutions were further diluted 100 folds with mobile
phase. The proposed RP-HPLC and chemometric methods
were applied and the concentrations of each component in
both formulations were determined.
IX. Chemometric Theory
The programme required for chemometric methods and
written using software R version 2.1.1 is built in-house and is
available with the corresponding author.
(I) Classical Least Squares
This method assumes the Beers law model with the
absorbance at each frequency being proportional to the
component concentrations. In matrix notation, the Beers
law models for m calibration standards containing l chemical
components with UV spectra of n digitized absorbances is
given by
A = CK+E
A
(1)
Where A is the m n matrix of calibration spectra, C is the m
l matrix of component concentration, K is the l n matrix
of absorptivity-pathlength products, and E
A
is the m n
20
15
10
5
0
2
2
6
7
3
.
1
7
3
RAMP
ATOR
[mV]
V
o
l
t
a
g
e
[min.] Time
0 2 4 6 8 10 12 14
Figure 1. Chromatogram showing Retention Time (Rt) of 32 g/mL of
RAMP (2.267 min) and 22 g/mL of ATOR (3.173 min) in laboratory-
prepared mixture.
15
10
5
0
-5
-10
-15
[mV]
V
o
l
t
a
g
e
[min.] Time
0 2 4 6 8 10 12 14
Figure 2. HPLC 3-Dimensional Chromatograms set of seven stan-
dard dilutions RAMP (in triplicate) using 8 g/mL of ATOR as internal
standard.
Table 1. Characteristic parameters of calibration equation for the
proposed HPLC method for simultaneous determination of RAMP
and ATOR
Parameters
HPLC
RAMP ATOR
Calibration range (g/mL) 4-32 4-22
Detection limit (g/mL) 1.78 10
-2
1.98 10
-2
Quantitation limit (g/mL) 5.93 10
-2
6.59 10
-2
Regression equation
r
Slope
b
0.072 0.212
Standard deviation of the slope 0.0002 0.0040
Relative standard deviation of the slope (%) 0.279 1.653
Intercept
a
0.008 0.019
Standard deviation of the intercept 0.002 0.005
Relative standard deviation of the intercept
(%)
31.906 27.584
Correlation coefficient 0.999 0.999
Theoretical plates 2888 3308
Symmetry factor 1.274 1.244
Resolution 5.408
r
y = a + bc, where a is intercept, b is slope, c is the concentration of
compound in g/mL and y is the peak area.
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
580
matrix of spectral errors.
K, then represents the matrix of pure component spectra
at unit concentration and unit pathlength. The classical least
squares solution to equation (1) during calibration is,
K

= (C
T
C)
-1
C
T
A
Where K

indicates least-squares estimates of K.

And analysis based on the spectrum a, of unknown
components concentration (samples),
c
0
= (K

K
T
)
-1
K

a
Where, c
0
is vector of predicted concentrations and K

T
is
transpose of the matrix K

.
(II) Principal Component Regression and Partial Least
Squares
The PCR function of the R-package PLS (http://
mevik.net/work/software/pls.html (URL for pls package))
implements the well-known algorithm
(51,52)
based on singular
value decomposition. This function was used to ft PCR and
PLS model using the transformed absorbance data as input.
The leave-one-out (LOO) cross-validation method was
opted while ftting the PCR and PLS model.
RESULTS AND DISCUSSION
I. Chromatography
In order to effect the simultaneous elution of both
component peaks under isocratic conditions, the mobile
phase composition was optimized after several trials with
various organic solvents and buffers in different ratios. A
satisfactory separation was obtained with a mobile phase
consisting of acetonitrile: 0.1 M sodium perchlorate (70 : 30,
v/v); pH adjusted to 2.5 0.2 with orthophosphoric acid and
at a fow rate of 1.5 mL/min. Initial studies were performed
while the effuent was monitored at 210 nm. The wavelength
of detection between the runs was varied to achieve the best
response. Both components show reasonably good response
at 210 nm. Under the described chromatographic conditions,
the analyte peak was well defned, resolved and almost free of
tailing with the retention time of 2.267 0.003 min and 3.173
0.002 min for RAMP and ATOR, respectively. This allows
the determination of both drugs with reasonable responses
with the two well resolved peaks. For quantitative applica-
tion, linear calibration chromatograms were obtained with
correlation coeffcients 0.9999.
II. Chemometric Methods
Chemometric techniques are gaining wide application
for the resolution of the drug mixtures. A calibration set
consisting of 25 binary mixtures prepared within the stated
range was used. Absorption spectra for the calibration samples
shown in were recorded in the range 201-350 nm. The UV
absorbance data was obtained by measuring the absorbances
in the region of 201-270 nm and then chemometric calibra-
tions were carried within the CLS, PCR and PLS algorithms.
The numerical results of calibrations are shown in Table 2.
The quality of multicomponent analysis is dependent
on the wavelength range selected. CLS, PCR and PLS tech-
niques are designated as full spectrum computational proce-
dures, thus wavelength selection is seemingly unnecessary,
and so all available wavelengths are often used. However,
measurements from spectral wavelengths that are not infor-
mative in a model will degrade performance. Hence, UV
absorbance data above 270 nm were not used because RAMP
has no absorbance at the concentrations used in this wave-
length region. The spectral region which is best reconstructed
was considered. This entailed using 70 experimental points
per spectrum, as absorbance spectra were digitized at 1 nm
intervals.
III. Statistical Parameter
The predictive ability of a calibration model in chemo-
metric methods can be defned in various ways. The most
general expression is the standard error of calibration (SEC)
or prediction (SEP) which is given by the following equation,
n
C C
Found
i
N
i
Added
i
2
1
) (
SEC or SEP

=

=
Where C
i
Added
is the added concentration of drugs,
C
i
Found
the predicted concentration of drugs and n the total
number of the synthetic mixtures. The numerical values are
quoted in Table 3.
IV. Selection of Optimum Number of Factors for PCR and
PLS
For PCR and PLS methods, 25 calibration spectra were
used for the selection of the optimum number of factors by
using the cross validation technique. This allows modeling
Table 2. Statistical parameters of chemometric methods in calibration
step of Zero-order spectra
Component
CLS PCR PLS
SEC SEC PRESS
RSE
a

(%)
SEC PRESS
RSE
(%)
RAMP 1.572 0.014 0.004 0.068 0.013 0.004 0.066
ATOR 0.203 0.020 0.009 0.143 0.017 0.007 0.119
SEC: Standard error of calibration.
PRESS: Predicted error sum of squares.
a
Relative Standard Error of calibration of single component.
( )
( )
100 (%) RSE
1
2
1
2
a

=
=
N
i
Added
i
N
i
Found
i
Added
i
C
C C
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
581
regression, was applied. The predicted concentrations were
then compared with the actual ones for each of the calibration
samples and the mean squares error of prediction (MSEP)
was calculated. The MSEP was computed in the same manner
each time and a new factor was added to the PCR and PLS
of the system with the optimum amount of information and
avoidance of overftting or underftting. The cross-validation
procedure, LOO consisting of systematically removing
one of a group of calibration samples at a time and using
the remaining ones for the construction of latent factors and
Table 3. Statistical parameters of chemometric methods in prediction step of Zero-order spectra
Component
CLS PCR PLS
SEP a b r SEP a b r SEP a b r
RAMP 1.866 1.648 0.944 0.990 0.555 0.199 1.016 0.999 0.549 0.182 1.017 0.999
ATOR 0.354 0.482 0.984 0.999 0.188 0.024 1.012 0.999 0.177 0.034 1.012 0.999
SEP: Standard error of prediction.
a
: Intercept.

b
: Slope.
r
: Correlation coefficient.

5 10 15 20
No. of components
M
S
E
P
5 10 15 20
0.08
0.06
0.04
0.02
0.00
No. of components
M
S
E
P
Figure 3. MSEP plots of a calibration set obtained using LOO cross validation of PCR-model for (A) RAMP and (B) ATOR in zero-order absorp-
tion data.
(A) (B)
1.0
0.8
0.6
0.4
0.2
0.0
5
5
10
10
15
15
20
No. of components
No. of components
M
S
E
P
M
S
E
P
Figure 4. MSEP plots of a calibration set obtained using LOO cross validation of PLS-model for (A) RAMP and (B) ATOR in zero-order absorption
data.
0.08
0.06
0.04
0.02
0.00
(A) (B)
1.0
0.8
0.6
0.4
0.2
0.0
20
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
582
model. The selected model was that with the least factors
so that its MSEP values were not signifcantly greater than
that for the model, which yielded the lowest MSEP. Plots of
MSEP values against number of components shown in Figure
3 and Figure 4 indicated factor fve was optimum for the esti-
mation of title drugs by both PCR and PLS. At the selected
principal components of PCR and PLS, the concentrations of
each sample was then predicted and compared with known
concentrations and the PRESS (Prediction Error Sum of
Squares) was calculated by the equation,
) (
2
1
PRESS
Found
i
n
i
Added
i
C C =

=
V. Validation of Methods
To check the validity (predictive ability), the simulta-
neous analysis of the prediction set of 16 laboratory-prepared
binary mixtures containing various concentrations of RAMP
and ATOR (in triplicates) was carried out by HPLC and
chemometric methods. The mean recoveries, % errors and the
relative standard deviations of prediction sets were computed
and listed in Table 4. Their numerical values were completely
acceptable because of their good recoveries and hence found
satisfactory for the validation.
Another diagnostic test for chemometric methods with
prediction sets was carried out by plotting the concentration
residuals against the predicted concentrations. The residuals
appeared randomly distributed around zero, indicating good
prediction ability of the model.
(I) Linearity & Range
In this study a series of seven concentrations were
chosen, ranging 4-32 g/mL of RAMP and 4-22 g/mL of
ATOR. Each concentration was repeated three times and
information was obtained on the variation in peak area
response and absorbances at stated wavelength region in
HPLC and chemometric methods, respectively. The linearity
of the calibration graphs of proposed methods was validated
with the high value of correlation coeffcient, slope and the
intercept. The calibration range of the proposed methods was
established through wide consideration of the practical range
necessary, according to each ingredient concentration present
in pharmaceutical products of different manufacturers.
(II) Accuracy
The study was performed by increasing standard addi-
tion of known amounts of studied drugs to an unknown
concentration (constant volume)
(53)
of the commercial phar-
maceutical formulations. Standard addition accessed the
effect of a sample matrix changes on the analytical sensitivity
of the method.
A constant volume of the unknown solution is added
to each of six 10 mL volumetric fasks. Then a series of
increasing volumes of working standard solutions are added
and fnally, each fask is made up to the mark with solvent. The
concentration of the working standard solutions added should
be chosen to increase the concentration of the unknown by
minimum 30% in each succeeding fask.
The recovery of resulting mixtures were analyzed by the
proposed HPLC method, the response obtained was plotted
against the initial unknown concentration set at 0 (Figure
5) and chemometric recoveries were also determined. The
results are compared with expected results. The excellent
mean recoveries and standard deviation (Table 5) suggested
good accuracy of the proposed methods and no interference
from excipients in the formulation.
(III) Precision (Method Reproducibility)
Method reproducibility was demonstrated by repeat-
ability and intermediate precision measurements of % RSD
of peak area, peak asymmetry and retention time parameters
of HPLC and % recovery RSD in chemometric methods for
each title ingredient.
The repeatability (within-day in triplicates) and interme-
diate precision (for 3 days) was carried out at fve concentra-
tion levels for each compound. The obtained results within
and between days trials (Table 6) are in acceptable range
indicating good precision of the proposed methods.
(IV) Robustness
The robustness of the proposed HPLC method was
assessed for peak asymmetric and peak resolution factor by
purposely altering the HPLC conditions (Table 7):
Apparent pH of the mobile phase (0.3)
Mobile phase organic content (3%)
Mobile phase fow rate (0.1)
Detection wavelength (1)
In spectrophotometric methods Double-beam Shimadzu
(Japan) UV-VIS Spectrophotometers, model UV-1700
and 1601, were used to access the robustness. The digital
Figure 5. Plot of peak area versus concentration of RAMP with the
initial concentration set at zero.
1000
800
600
400
200
0
843.52
172.73
-200
283.21
407.36
-15 -10 -5 0 5 10 15 20 25 30 35
646.25
763.38
R
e
s
p
o
n
s
e

(
p
e
a
k

a
r
e
a
)
Concentration (in g/mL)
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
583
T
a
b
l
e

4
.

R
e
c
o
v
e
r
y

r
e
s
u
l
t
s

i
n

p
r
e
d
i
c
t
i
o
n

f
o
r

R
A
M
P

a
n
d

A
T
O
R

l
a
b
o
r
a
t
o
r
y

p
r
e
p
a
r
e
d

b
i
n
a
r
y

m
i
x
t
u
r
e
s

b
y

p
r
o
p
o
s
e
d

H
P
L
C

a
n
d

c
h
e
m
o
m
e
t
r
i
c

t
e
c
h
n
i
q
u
e
s
C
o
n
c
.

i
n

g
/
m
L
R
e
c
o
v
e
r
y

(
%
)
E
r
r
o
r

%
H
P
L
C
C
L
S

P
C
R
P
L
S
H
P
L
C
C
L
S
P
C
R
P
L
S
R
A
M
P
A
T
O
R
R
A
M
P
A
T
O
R
R
A
M
P
A
T
O
R
R
A
M
P
A
T
O
R
R
A
M
P
A
T
O
R
R
A
M
P
A
T
O
R
R
A
M
P
A
T
O
R
R
A
M
P
A
T
O
R
R
A
M
P
A
T
O
R
4
4
9
8
.
0
2
1
0
1
.
6
9
9
9
.
8
7
1
0
2
.
6
9
1
0
1
.
5
2
1
0
1
.
0
1
1
0
1
.
3
7
1
0
0
.
7
9
1
.
9
8
-
1
.
6
9
0
.
1
3
-
2
.
6
9
-
1
.
5
2
-
1
.
0
1
-
1
.
3
7
-
0
.
7
9
4
8
1
0
2
.
5
2
1
0
3
.
5
0
1
0
3
.
2
8
1
0
5
.
8
7
1
0
7
.
9
9
1
0
2
.
1
1
1
0
7
.
8
6
1
0
2
.
0
2
-
2
.
5
2
-
3
.
5
0
-
3
.
2
8
-
5
.
8
7
-
7
.
9
9
-
2
.
1
1
-
7
.
8
6
-
2
.
0
2
4
1
6
1
0
4
.
4
3
1
0
2
.
2
5
1
0
1
.
3
6
1
0
6
.
3
9
1
0
4
.
3
4
1
0
1
.
8
5
1
0
3
.
7
9
1
0
1
.
6
8
-
4
.
4
3
-
2
.
2
5
-
1
.
3
6
-
6
.
3
9
-
4
.
3
4
-
1
.
8
5
-
3
.
7
9
-
1
.
6
8
4
2
2
1
0
1
.
5
4
1
0
1
.
8
7
1
0
5
.
0
9
1
0
4
.
5
8
1
0
7
.
8
4
1
0
2
.
0
0
1
0
7
.
0
3
1
0
1
.
8
5
-
1
.
5
4
-
1
.
8
7
-
5
.
0
9
-
4
.
5
8
-
7
.
8
4
-
2
.
0
0
-
7
.
0
3
-
1
.
8
5
8
4
1
0
2
.
7
4
1
0
2
.
3
5
1
0
0
.
0
8
1
0
4
.
5
9
1
0
3
.
6
5
1
0
0
.
2
1
1
0
3
.
5
5
9
9
.
9
5
-
2
.
7
4
-
2
.
3
5
-
0
.
0
8
-
4
.
5
9
-
3
.
6
5
-
0
.
2
1
-
3
.
5
5
0
.
0
5
8
8
1
0
2
.
1
0
1
0
3
.
3
3
1
0
0
.
0
3
1
0
2
.
1
1
1
0
3
.
0
5
1
0
1
.
5
1
1
0
2
.
9
1
1
0
1
.
3
2
-
2
.
1
0
-
3
.
3
3
-
0
.
0
3
-
2
.
1
1
-
3
.
0
5
-
1
.
5
1
-
2
.
9
1
-
1
.
3
2
8
1
6
1
0
5
.
0
1
1
0
1
.
6
8
1
0
2
.
5
4
9
8
.
8
9
1
0
5
.
4
0
1
0
1
.
9
6
1
0
5
.
4
4
1
0
1
.
9
7
-
5
.
0
1
-
1
.
6
8
-
2
.
5
4
1
.
1
1
-
5
.
4
0
-
1
.
9
6
-
5
.
4
4
-
1
.
9
7
8
2
2
1
0
5
.
5
7
1
0
1
.
1
5
1
0
1
.
6
5
9
9
.
6
7
1
0
4
.
4
8
1
0
1
.
0
0
1
0
4
.
1
1
1
0
0
.
8
6
-
5
.
5
7
-
1
.
1
5
-
1
.
6
5
0
.
3
3
-
4
.
4
8
-
1
.
0
0
-
4
.
1
1
-
0
.
8
6
2
4
4
1
0
1
.
8
7
9
8
.
8
9
9
9
.
3
2
1
0
3
.
5
4
1
0
1
.
4
1
1
0
0
.
6
3
1
0
1
.
3
7
1
0
0
.
2
6
-
1
.
8
7
1
.
1
1
0
.
6
8
-
3
.
5
4
-
1
.
4
1
-
0
.
6
3
-
1
.
3
7
-
0
.
2
6
2
4
8
1
0
4
.
6
5
9
9
.
9
2
9
9
.
9
9
1
0
1
.
6
8
1
0
3
.
7
5
9
9
.
7
2
1
0
3
.
7
5
9
9
.
6
9
-
4
.
6
5
0
.
0
8
0
.
0
1
-
1
.
6
8
-
3
.
7
5
0
.
2
8
-
3
.
7
5
0
.
3
1
2
4
1
6
1
0
4
.
6
4
1
0
1
.
0
0
9
8
.
9
8
9
8
.
3
7
1
0
3
.
1
7
1
0
0
.
4
5
1
0
3
.
2
0
1
0
0
.
4
9
-
4
.
6
4
-
1
.
0
0
1
.
0
2
1
.
6
3
-
3
.
1
7
-
0
.
4
5
-
3
.
2
0
-
0
.
4
9
2
4
2
2
1
0
3
.
1
9
1
0
2
.
6
1
1
0
0
.
0
3
9
7
.
9
8
1
0
3
.
4
0
1
0
0
.
9
3
1
0
3
.
3
6
1
0
0
.
8
7
-
3
.
1
9
-
2
.
6
1
-
0
.
0
3
2
.
0
2
-
3
.
4
0
-
0
.
9
3
-
3
.
3
6
-
0
.
8
7
3
2
4
1
0
2
.
0
9
1
0
1
.
3
5
9
8
.
3
5
9
7
.
9
3
1
0
2
.
7
1
1
0
0
.
8
1
1
0
2
.
6
5
1
0
0
.
1
9
-
2
.
0
9
-
1
.
3
5
1
.
6
5
2
.
0
7
-
2
.
7
1
-
0
.
8
1
-
2
.
6
5
-
0
.
1
9
3
2
8
1
0
3
.
7
5
1
0
0
.
1
5
9
7
.
9
8
1
0
0
.
2
6
1
0
1
.
9
5
9
9
.
5
0
1
0
1
.
8
7
9
9
.
1
0
-
3
.
7
5
-
0
.
1
5
2
.
0
2
-
0
.
2
6
-
1
.
9
5
0
.
5
0
-
1
.
8
7
0
.
9
0
3
2
1
6
1
0
4
.
0
5
1
0
1
.
0
6
9
9
.
8
9
1
0
3
.
5
8
1
0
2
.
1
0
1
0
0
.
2
2
1
0
2
.
2
3
1
0
0
.
5
1
-
4
.
0
5
-
1
.
0
6
0
.
1
1
-
3
.
5
8
-
2
.
1
0
-
0
.
2
2
-
2
.
2
3
-
0
.
5
1
3
2
2
2
1
0
3
.
6
4
1
0
0
.
9
4
1
0
1
.
7
8
1
0
0
.
9
8
1
0
1
.
1
3
1
0
0
.
1
4
1
0
1
.
0
9
1
0
0
.
0
3
-
3
.
6
4
-
0
.
9
4
-
1
.
7
8
-
0
.
9
8
-
1
.
1
3
-
0
.
1
4
-
1
.
0
9
-
0
.
0
3
x
1
0
3
.
1
1
1
0
1
.
4
8
1
0
0
.
6
4
1
0
1
.
8
2
1
0
3
.
6
2
1
0
0
.
8
8
1
0
3
.
4
7
1
0
0
.
7
2
R
S
D
r
1
.
7
7
1
.
2
0
1
.
8
6
2
.
7
5
1
.
9
8
0
.
8
2
1
.
8
7
0
.
8
6
x
:

m
e
a
n

r
e
c
o
v
e
r
y

v
a
l
u
e
.
r
:

R
e
l
a
t
i
v
e

S
t
a
n
d
a
r
d

D
e
v
i
a
t
i
o
n
.
T
a
b
l
e

5
.

A
p
p
l
i
c
a
t
i
o
n

o
f

s
t
a
n
d
a
r
d

a
d
d
i
t
i
o
n

t
e
c
h
n
i
q
u
e

f
o
r

a
n
a
l
y
s
i
s

o
f

R
A
M
P

a
n
d

A
T
O
R

i
n

S
t
a
t
o
r

-
R

2
.
5

t
a
b
l
e
t
s
S
e
r
i
a
l

n
o
.
R
A
M
P
A
T
O
R
C
o
n
c
.

i
n

g
/
m
L
%

r
e
c
o
v
e
r
y
*

s
t
a
n
d
a
r
d

d
e
v
i
a
t
i
o
n
C
o
n
c
.

i
n

g
/
m
L
%

r
e
c
o
v
e
r
y

s
t
a
n
d
a
r
d

d
e
v
i
a
t
i
o
n
L
a
b
e
l

c
l
a
i
m
e
d
A
d
d
e
d
C
L
S
P
C
R
P
L
S
H
P
L
C
L
a
b
e
l

c
l
a
i
m
e
d
A
d
d
e
d
C
L
S
P
C
R
P
L
S
H
P
L
C
1
2
.
5
2
.
5

1
0
2
.
0

0
.
7
8
1
0
0
.
3

0
.
6
3
1
0
0
.
2

0
.
4
9
9
8
.
2

0
.
7
4
1
0
0
9
7
.
0

0
.
6
8
1
0
3
.
6

0
.
2
8
1
0
2
.
0

0
.
7
8
1
0
4
.
0

0
.
6
3
2
2
.
5
4
.
0
9
8
.
2

1
.
2
1
9
8
.
2

0
.
0
2
9
8
.
0

0
.
0
1
1
0
3
.
0

1
.
8
0
1
0
2
9
7
.
2

1
.
2
1
1
0
0
.
3

0
.
9
3
1
0
0
.
3

0
.
8
0
9
9
.
2

0
.
0
1
3
2
.
5
8
.
0
1
0
3
.
0

0
.
0
9
9
9
.
3

0
.
1
6
9
9
.
7

0
.
1
9
1
0
3
.
2

0
.
7
2
1
0
4
9
8
.
5

0
.
1
9
1
0
6
.
0

0
.
0
1
1
0
5
.
8

0
.
0
9
1
0
0
.
5

0
.
3
5
4
2
.
5
1
6
.
0
1
0
4
.
4

0
.
0
1
1
0
2
.
5

0
.
0
6
1
0
2
.
4

0
.
1
2
1
0
2
.
8

0
.
9
1
1
0
8
1
0
0
.
6

0
.
0
9
9
8
.
9

0
.
3
1
9
9
.
1

0
.
4
6
1
0
2
.
4

0
.
5
9
5
2
.
5
2
4
.
0
9
7
.
9

1
.
3
1
9
8
.
3

0
.
9
6
9
8
.
9

0
.
8
7
1
0
0
.
0

1
.
1
0
1
0
1
0
9
7
.
8

0
.
9
5
1
0
0
.
5

0
.
7
9
1
0
0
.
3

0
.
9
2
9
9
.
9

0
.
9
7
6
2
.
5
2
8
.
0
9
9
.
7

0
.
0
6
1
0
0
.
2

0
.
0
3
1
0
0
.
1

0
.
0
5
9
7
.
1

0
.
9
1
1
0
1
2
9
8
.
7

0
.
2
3
9
8
.
5

1
.
2
1
9
8
.
4

0
.
3
7
9
9
.
2

0
.
0
6
*
:

a
v
e
r
a
g
e

o
f

t
h
r
e
e

e
x
p
e
r
i
m
e
n
t
s
.

:

2
.
5

g
/
m
L

o
f

s
t
a
n
d
a
r
d

R
A
M
P

w
a
s

a
d
d
e
d

t
o

r
a
i
s
e

t
h
e

l
e
v
e
l

t
o

l
i
n
e
a
r

c
a
l
i
b
r
a
t
i
o
n

r
a
n
g
e
.
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
584
absorbances recorded by both instruments did not have
signifcant effect on the determination of title drugs.
(V) Limit of Detection (LOD) and Limit of Quantifcation
(LOQ)
The limit of detection (LOD) and limit of quantifcation
(LOQ) of HPLC are calculated according to ICH
(54)
recom-
mendations where the approach is based on the signal-to-
noise ratio. Chromatogram signals obtained with known low
concentrations analytes were compared with the signals of
blank samples. A signal-to-noise ratio of 3 : 1 and 10 : 1 is
considered for calculating LOD and LOQ respectively, and
values obtained are shown in Table 1. The LOD and LOQ
of spectrophotometric methods were calculated according to
Table 7. Robustness of chromatographic method
Parameter
Peak asymmetry* Resolution
between RAMP
and ATOR RAMP ATOR
Flow rate (mL/min)
1.4 1.284 0.068 1.208 0.059 5.310 0.000
1.5 1.274 0.000 1.240 0.108 5.340 0.050
1.6 1.249 0.043 1.283 0.006 5.348 0.001
Acetonitrile % in mobile phase
73 1.250 0.001 1.220 0.001 4.480 0.006
70 1.274 0.000 1.240 0.108 5.340 0.050
67 1.275 0.002 1.237 0.002 5.942 0.009
Change in pH
2.8 1.279 0.000 1.296 0.062 5.084 0.010
2.5 1.274 0.000 1.240 0.108 5.340 0.050
2.2 1.286 0.038 1.282 0.038 4.951 0.040
Change in detection wavelength
209 nm 1.309 0.062 1.298 0.090 4.79 0.030
210 nm 1.274 0.000 1.240 0.108 5.340 0.050
211 nm 1.316 0.060 1.303 0.025 5.210 0.076
*: Average of three experiments.
formula given by Miller
(55)
using the standard deviation of
UV response and slope of the calibration curve. The LOD
and LOQ were found to be 0.277 g/mL and 0.862 g/mL
for ATOR and 0.361 g/mL and 1.188 g/mL for RAMP,
respectively.
(VI) Application of the Developed Method for Analysis of
Commercial Formulations
Applicability of the proposed method was tested by
analyzing the commercially available tablet formulation
Stator-R 2.5 labeled to contain 2.5 mg of RAMP and 10 mg
of ATOR and Rampitor*5 capsules labeled to contain 5 mg
RAMP and 10 mg ATOR.
No published method has been reported for simultaneous
determination of these binary components in formulations.
So the results of the proposed CLS, PCR and PLS methods
were statistically compared between results of proposed
HPLC method at the 95% confdence level with the aid of
Students t-test and F-tests. The calculated t and F values
never exceeded the theoretical t- and F- values, at 0.05 level
of signifcant difference. The results of all methods were very
close to each other as well as to the label value of commercial
pharmaceutical formulations. Therefore, these statistical tests
denoted no signifcant difference in the results achieved by
the proposed methods.
CONCLUSIONS
For routine analytical purpose it is desirable to estab-
lish methods capable of analyzing large numbers of samples
in a short period of time with good accuracy and precision
without any prior separation step. The HPLC method and
spectrophotometric techniques coupled with multivariate
algorithms described in this paper meet these desires. The run
time of the HPLC procedure is only four minutes. Spectro-
photometric methods in general do not require sophisticated
instrumentation and large amount of solvents, which make
them more economical in comparison with HPLC procedure.
Good agreement was seen in the assay results of pharmaceu-
tical formulation as well as in laboratory prepared mixtures
Table 6. Precision study results of prepared binary mixture
Validation parameter
HPLC Chemometric
% RSD % recovery RSD
Repeatability
a
Peak area Peak asymmetry Retention time CLS PCR PLS
RAMP 0.734 0.563 0.105 2.085 1.814 1.834
ATOR 1.094 0.288 0.907 2.163 0.316 0.327
Intermediate precision
b
RAMP 1.522 0.771 0.495 2.446 1.087 1.597
ATOR 1.737 0.751 0.094 1.987 0.935 0.291
a
: Repeatability, three replicates of five concentration levels within-day.
b
: Intermediate precision, three replicates of five concentration levels between-days (3-days).
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
585
by developed methods. We concluded that all the proposed
methods are a good approach for obtaining reliable results
and were found to be suitable for the routine estimation of
RAMP and ATOR in pharmaceutical formulations.
ACKNOWLEDGMENTS
One of the authors Nagaraj gratefully acknowledges
the support for this research work from All India Council of
Technical Education (AICTE) sponsored, Quality Improve-
ment Programme (QIP).
REFERENCES
1. Campbell, D. J., Kladis, A. and Duncan, A. M. 1993.
Nephrectomy, converting enzyme inhibition and angio-
tensin peptides. Hypertension 22: 513-522.
2. Nawrocki, J. W., Weiss, S. R., Davison, M. H., Sprecher,
D. L., Schwarts, S. L., Lupien, P. J., Jones, P. H., Habert,
H. E. and Black, D. M. 1995. Reduction of LDL choles-
terol by 25% to 60% in patients with primary hypercho-
lesterolemia by atorvastatin, a new HMG-CoA reductase
inhibitor. Arterioscler. Thromb. Vasc. Biol. 15: 678-682.
3. Lea, A. P. and Tavish, D. M. 1997. Atorvastatin: a review
of its pharmacology and therapeutic potential in the
management of hyperlipidemias. Drugs 53: 828-847.
4. Chiong, J. R. and Miller, A. B. 2002. Renin-angiotensin
system antagonism and lipidlowering therapy in cardio-
vascular risk management. J. Renin Angiotensin Aldoste-
rone Syst. 3: 96-102.
5. Pizzi, C., Manfrini, O., Fontana, F. and Bugiardini, R.
2004. Angiotensin-converting enzyme inhibitors and
3-hydroxy-3-methylglutaryl coenzyme A reductase
in cardiac syndrome X: role of superoxide dismutase
activity. Circulation 109: 53-58.
6. Grothuson, C., Bley, S., Selle, T., Luchtefeld, M., Grote,
K., Tietge, U. J., Drexler, H. and Schiffer, B. 2005.
Combined effects of HMG-CoA-reductase inhibition
and reninangiotensin system blockade on experimental
atherosclerosis. Atherosclerosis 182: 57-69.
7. Nagaraj, G., Vipul, K. and Rajshree, M. 2007. Simul-
taneous quantitative resolution of atorvastatin calcium
and fenofibrate in pharmaceutical preparation by using
derivative ratio spectrophotometry and chemometric
calibrations. Anal. Sci. 23: 445-451.
8. Erturk, S., Onal, A. and Mugecetin, S. 2003. Analytical
methods for the quantitative determination of 3-hydroxy-
3-methylglutaryl coenzyme A reductase inhibitors in
biological samples. J. Chromatogr. B 793: 193-205.
9. Altuntas, T. G. and Erk, N. 2004. Liquid chromatographic
determination of atorvastatin in bulk drug, tablets and
human plasma. J. Liq. Chromatogr. Relat. Technol. 7:
83-93.
10. Bahrani, G., Mohammadi, B., Mizaeel, S. and Kiani, A.
2005. Determination of atorvastatin in human serum by
reversed-phase high-performance liquid chromatography
with UV detection. J. Chromatogr. B 826: 41-45.
11. Khadr, A. 2007. Stability-indicating high-performance
liquid chromatographic assay of atorvastatin with fluo-
rescence detection. J. AOAC Int. 90: 1547-1553.
12. Chaudhari, B. G., Patel, N. M., Shah, P. B., Patel, L.
J. and Patel, V. P. 2007. Stability- indicating reversed-
phase liquid chromatographic method for simultaneous
determination of atorvastatin and ezetimibe from their
combination drug products. J. AOAC Int. 90: 1539-1546.
13. Mohammadi, A., Rezanour, N., Dogaheh, M. A.,
Bidkorbeh, F. G., Hashem, M. and Walker, R. B. 2007.
A stability-indicating high performance liquid chromato-
graphic (HPLC) assay for the simultaneous determination
of atorvastatin and amlodipine in commercial tablets. J.
Chromatogr. B 846: 215-221.
14. Sivakumar, T., Manavalan, R., Muralidharan, C. and
Valliappan, K. 2007. An improved HPLC method with
the aid of a chemometric protocol: simultaneous analysis
of amlodipine and atorvastatin in pharmaceutical formu-
lations. J. Sep. Sci. 30: 3143-3153.
15. Nirogi, R., Mudigonda, K. and Kandikere, V. 2007.
Chromatography-mass spectrometry methods for the
quantitation of statins in biological samples. J. Pharm.
Biomed. Anal. 44: 379-387.
16. Miao, X. S. and Metcalfe, C. D. 2003. Determination
of cholesterol-lowering statin drugs in aqueous samples
using liquid chromatography-electrospray ioniza-
tion tandem mass spectrometry. J. Chromatogr. A 998:
133-141.
17. Skorda, D. and Kontoyannis, C. G. 2008. Identification
and quantitative determination of atorvastatin calcium
polymorph in tablets using FT-Raman spectroscopy.
Talanta 74: 1066-1070.
18. Erk, N. 2004. Development of electrochemical methods
for determination of atorvastatin and analytical appli-
cation to pharmaceutical products and spiked human
plasma. Criti. Rev. Anal. Chem. 34: 1-7.
19. Hogan, B. L., Williams, M., Idiculle, A., Veysoglu, T.
and Parente, E. 2000. Development and validation of a
liquid chromatographic method for the determination of
the related substances of ramipril in Altace capsules. J.
Pharm. Biomed. Anal. 23: 637-651.
20. Belal, F., Al-Zaagi, I. A., Gadkariem, E. A. and Abou-
nassif, M. A. 2001. A stability-indicating LC method for
the simultaneous determination of ramipril and hydro-
chlorothiazide in dosage forms. J. Pharm. Biomed. Anal.
24: 335-342.
21. Bhushan, R., Gupta, D. and Singh, S. K. 2005. Liquid
chromatographic separation and UV determination of
certain antihypertensive agents. Biomed. Chromatogr.
20: 217-224.
22. Bonazzi, D., Gotti, R., Andrisano, V. and Cavrini, V.
1997. Analysis of ACE inhibitors in pharmaceutical
dosage forms by derivative UV spectroscopy and liquid
chromatography (HPLC). J. Pharm. Biomed. Anal. 16:
431-438.
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
586
23. Gowda, K. V., Mandal, U. and Senthamil Selvan, P. et al.
2007. Liquid chromatography tandem mass spectrometry
method for simultaneous determination of metoprolol
tartrate and ramipril in human plasma. J. Chromatogr. B
858: 13-21.
24. Lu, X. Y., Shan-Tu, J. L. and Liu, J. 2006. High-
performance liquid chromatography mass spectrometric
analysis of ramipril and its active metabolite ramiprilat
in human serum: application to a pharmacokinetic study
in the Chinese volunteers. J. Pharm. Biomed. Anal. 40:
478-483.
25. Zhu, Z., Vachareau, A. and Neirinck, L. 2002. Liquid
chromatography-mass spectrometry method for determi-
nation of ramipril and its active metabolite ramiprilat in
human plasma. J. Chromatogr. B 779: 297-306.
26. Person, B. A., Fakt, C., Ervik, M. and Ahnoff, M. 2006.
Interference from a glucuronide metabolite in the deter-
mination of ramipril and ramiprilat in human plasma and
urine by gas chromatography - mass spectrometry. J.
Pharm. Biomed. Anal. 40: 794-798.
27. Al-Majed, A. A. and Al-Zehouri, J. 2001. Use of 7-fluoro-
4-nitrobenzo-2-oxo-1, 3-diazole (NBD-F) for the deter-
mination of ramipril in tablets and spiked human plasma.
Farmaco II. 56: 291-296.
28. Rahman, N., Ahmad, Y. and Azmi, S. N. 2005. Kinetic
spectrophotometric method for the determination of
ramipril in pharmaceutical formulations. AAPS Pharm-
SciTech. 6: 543-551.
29. Rontogianni, M. A., Markopoulou, C. K. and Koun-
dourellis, J. E. 2006. HPLC and chemometrically-assisted
spectrophotometric estimation of two binary mixtures
for combined hypertension therapy. J. Liq. Chromatogr.
Relat. Technol. 29: 2701-2719.
30. Abdellatef, H. E. 2007. Spectrophotometric and spectro-
fluorimetric methods for the determination of ramipril
in its pure and dosage form. Spectrochim. Acta A Mol.
Biomol. Spectrosc. 66: 701-706.
31. Al-Majed, A. A., Belal, F., Abadi, A. and Al-Obaid, A.
M. 2000. The voltammetric study and determination
of ramipril in dosage forms and biological fluids. IL
Farmaco 55: 233-238.
32. Prieto, J. A., Jimenez, R. M. and Alonso, R. M. 2003.
Square wave voltammetric determination of the angio-
tensin-converting enzyme inhibitors cilazapril, quinapril
and ramipril in pharmaceutical formulations. IL Farmaco
58: 343-350.
33. Gotti, R., Andrisanoa, V., Cavrinia, V., Bertuccib, C. and
Furlanettoc, S. 2000. Analysis of ACE - inhibitors by CE
using alkylsulfonic additives. J. Pharm. Biomed. Anal.
22: 423-431.
34. Hillaert, S., Grauwe, K. D. and Vanden, B. W. 2001.
Simultaneous determination of hydrochlorothiazide and
several inhibitors of angiotensin-converting enzyme by
capillary electrophoresis. J. Chromatogr. A 924: 439-449.
35. Abdellatef, H. E., Ayad, M. M. and Taha, E. A. 1999.
Spectrophotometric and atomic absorption spectrometric
determination of ramipril and perindopril through ternary
complex formation with eosin and Cu (II). J. Pharm.
Biomed. Anal. 18: 1021-1027.
36. Ayad, M. M., Shalaby, A. A., Abdellatef, H. E. and
Hosny, M. M. 2002. Spectrophotometric and AAS
determination of ramipril and enalapril through ternary
complex formation. J. Pharm. Biomed. Anal. 28: 311-321.
37. Dinc, E. 2002. Spectral analysis of benazepril hydrochlo-
ride and hydrochlorothiazide in pharmaceutical formula-
tions by three chemometric techniques. Anal. Lett. 35:
1021-1039.
38. Dinc, E. and Baleanu, D. 2004. Application of the wavelet
method for the simultaneous quantitative determination
of benazepril and hydrochlorothiazide in their mixtures.
J. AOAC Int. 87: 834-841.
39. Collado, M. S., Mantovani, V. E., Goicrechea, H. C.
and Olivieri, A. C. 2001. Simultaneous determination of
nicotinamide and inosine in ophthalmic solution by UV
spectrophotometry and PLS-1 multivariate calibration.
Anal. Lett. 34: 363-376.
40. Garcisa, M. D. G., Frenich, A. G., Vidal, J. L. M., Galera,
M. M., Pena, A. M. and Salinas, F. 1997. Resolution of
overlapping peaks in HPLC with diode array detection
by application of partial least squares calibration to cross-
sections of spectrochromatograms. Anal. Chim. Acta
348: 177-185.
41. Frenich, A. G., Galera, M. M., Vidal, J. L. M. and Garcisa,
M. D. G. 1996. Partial least-squares and principal compo-
nent regression of multi-analyte high-performance liquid
chromatography with diode-array detection. J. Chro-
matogr. A 727: 27-38.
42. Herrero, A. and Ortiz, M. C. 1997. Multivariate calibra-
tion transfer applied to the routine polarographic deter-
mination of copper, lead, cadmium and zinc. Anal. Chim.
Acta 348: 51-59.
43. Barthus, R. C., Mazo, L. H. and Poppi, R. J. 2005.
Simultaneous determination of vitamins C, B6 and PP in
pharmaceutics using differential pulse voltammetry with
a glassy carbon electrode and multivariate calibration
tools. J. Pharm. Biomed. Anal. 38: 94-99.
44. Haaland, D. M. and Thomas, E. V. 1988. Partial least-
squares methods for spectral analyses. 1. Relation to
other quantitative calibration methods and the extraction
of qualitative information. Anal. Chem. 60: 1193-1202.
45. Beebe, K. R. and Kowalski, B. R. 1987. An introduction
to multivariate calibration and analysis. Anal. Chem. 59:
1007A-1017A.
46. Geladi, P. and Kowalski, B. R. 1986. Partial least-squares
regression: a tutorial. Anal. Chim. Acta 185: 1-17.
47. Adams, M. J. 1995. Chemometrics in Analytical Spec-
troscopy. pp.216-260. Thomas, Graham House Science
Park. The Roy. Soc. of Chem. Cambridge.
48. Cowe, I. A., McNicol, J. W. and Cuthbertson, D. C. 1985.
A designed experiment for the examination of techniques
used in the analysis of near infrared spectra. Part 1. Anal-
ysis of spectral structure. Analyst 110: 1227-1232.
49. El-Gindy, A. 2005. HPLC and chemometric assisted spec-
trophotometric methods for simultaneous determination
Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012
587
of diprophylline, phenobarbitone and papaverine hydro-
chloride. IL Farmaco 60: 745-753.
50. Richard Becker, A., John, M. C. and Allan, R. W. 1988.
The New S Language. pp.52-99. Chapman & Hall. New
York.
51. Naes, T. and Martens, H. 1988. Principal component
regression in NIR analysis: viewpoints, background
details and selection of components. J. Chemometr. 2:
155-167.
52. Martens, H. and Naes, T. 1989. Multivariate Calibration.
pp.314-321. J. Wiley & Sons: Chichester, U.K.
53. Rubinson, K. A. 1987. Chemical Analysis. pp. 205-211.
Brown & Co Little. Boston.
54. Paper read at ICH Q2 (R1) 2006. Harmonised tripartite
guideline, Validation of analytical procedures text and
methodology. pp.11-12. Yokohama.
55. Miller, J. N. 1991. Basic statistical methods for Analytical
Chemistry. Part 2. Calibration and regression methods. A
review. Analyst 116: 3-14.