Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 369375

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Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Development and validation of multivariate calibration methods for

simultaneous estimation of Paracetamol, Enalapril maleate and
hydrochlorothiazide in pharmaceutical dosage form
Veena D. Singh , Sanjay J. Daharwal
University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Chhattishgarh, 492010, India

a r t i c l e

i n f o

Article history:
Received 7 January 2016
Received in revised form 7 August 2016
Accepted 16 August 2016
Available online 20 August 2016
Keywords:
Multivariate calibration methods
Paracetamol
Enalpril maleate
Hydrochlorothiazide

a b s t r a c t
Three multivariate calibration spectrophotometric methods were developed for simultaneous estimation of Paracetamol (PARA), Enalapril maleate (ENM) and Hydrochlorothiazide (HCTZ) in tablet dosage form; namely multilinear regression calibration (MLRC), trilinear regression calibration method (TLRC) and classical least square
(CLS) method. The selectivity of the proposed methods were studied by analyzing the laboratory prepared ternary mixture and successfully applied in their combined dosage form. The proposed methods were validated as per
ICH guidelines and good accuracy; precision and specicity were conrmed within the concentration range of 5
35 g mL1, 540 g mL1 and 540 g mL1of PARA, HCTZ and ENM, respectively. The results were statistically
compared with reported HPLC method. Thus, the proposed methods can be effectively useful for the routine quality control analysis of these drugs in commercial tablet dosage form.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Paracetamol (PARA) is chemically N-(4-hydroxy) acetanilide and it
is commonly used for analgesic, antipyretic and anti-inammatory activity [1,2]. Hydrochlorothiazide (HCTZ) is chemically 6-chloro-3, 4
dihydro-2H-1, 2, 4-benzothiadiazine-7-sulfonamide 1, 1-dioxide and it
is a potent diuretic by inhibiting reabsorption of chloride and other
ions [3,4]. Enalapril maleate (ENM) is chemically (S)-1-(N-(1(Ethoxycarbonyl)-3-phenylpropyl)-L-alanyl)-Lproline (Z)-2-butene
dioate an angiotensin-converting enzyme (ACE) inhibitor, use in management of hypertension [5]. Structures of PARA, HCTZ and ENM respectively were shown in Fig. 1.
The combinations of these drugs are frequently prescribed for treatment of blood pressure, uid retention and heart failure. Furthermore, it
is used to increase the pain threshold [6,7]. Several studies suggested
that use of NSAIDS is associated with cardiovascular risk i.e. increase
in blood pressure [8,9]. However, most of the hypertensive patients
who are suffering from osteoarthritis requiring chronic pain relief, particularly in case of increased risk of hypertensive and atherosclerotic
complications [10,11]. Though, Paracetamol belongs to NSAIDS hence,
it can also be associated with risk of increase in blood pressure but
there is no agreement on this issue [12]. Therefore, Paracetamol appears
to be one of the best options for hypertensive patients requiring
analgesia.
Corresponding author.
E-mail addresses: veena1806@gmail.com, daharwalresearch@rediffmail.com
(V.D. Singh).

http://dx.doi.org/10.1016/j.saa.2016.08.028
1386-1425/ 2016 Elsevier B.V. All rights reserved.

Present study involves with simultaneous estimation of marketed

formulation and laboratory prepared mixture of Paracetamol, Hydrochlorothiazide and Enalpril maleate. The tablet contains variable
amount of ingredients due to their recommended therapeutic dose;
325 mg Paracetamol, 25 mg Hydrochlorothiazide and 10 mg Enalpril
maleate. The variable amount and different chemical properties of
drugs in same formulation make the process tedious for the routine
analysis [13]. Furthermore, the resolution of the mixtures containing
two or more different analytes without prior chemical separation is
major problems of the conventional analytical techniques. Hence,
there is need for development of new methods for simultaneous estimation of multicomponent systems which have overlapping spectra.
The multivariate techniques such as classical least-squares, Tri-linear regression equation and Multi linear regression equation have been
extensively used due to some advantages, i.e. rapid data processing related to the concentrations and absorbance values of compounds
which have spectral interference [14]. Furthermore, minimize the errors
of calibration model by measuring the absorbance of the zero order
spectra at various points within the selected wavelength range. It
gives satisfactory resolution for multicomponent systems and eliminates the interference problems. Multivariate calibration spectrophotometric methods were found to increase the selectivity and sensitivity by
applying the mathematical algorithms methodology [15]. Hence, these
methods are a power full technique for the quantitative analysis of
these tablet formulations and laboratory prepared mixture.
Various methods have been adopted for estimation of Paracetamol
and its combinations in pharmaceuticals dosage form and biological
uids, which includes uorimetry [16], colorimetry [17], UV-

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V.D. Singh, S.J. Daharwal / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 369375

Fig. 1. Structure formulae of a) Paracetamol, b) Hydrochlorothiazide and c) Enalpril maleate.

spectrophotometry [18], quantitative thin-layer chromatography (TLC)

[19], high-performance liquid chromatography (HPLC) [2022], gas
chromatography (GC) [23] and chemometric methods [24,14]. Likewise, for estimation of hydrochlorothiazide in pharmaceutical formulations, spectrophotometric [25,26], HPLC [2728], HPTLC [29] and
chemometric method [3032] have been reported. Additionally, for
Enalpril maleate various methods such as UV-HPLC [33,34], LC-MS-MS
[35], UV [36,37] and chemometric method [32] have been reported. Beside this very few methods of derivative spectroscopy [38] and chromatography [39,40] were reported for the simultaneous estimation of
these drugs in combined dosage forms. These methods have ability to
give more resolved and precise results. In spite of that these methods required long time for preparation of samples, relied on tedious liquid
liquid extraction process, complex gradient elution and derivatization
of the spectra. Although no multivariate calibration methods have
been reported for simultaneous estimation of these three drugs. Therefore, there is need to develop new, fast and less expensive method for
the determination of PARA, HCTZ and ENM and improve the outcome
of analysis in multicomponent systems.
In the present study, multivariate calibration methods were developed for simultaneous estimation of these three drugs in tablet dosage
form. The proposed methods were validated as per ICH guidelines
[4142] and compared with reported HPLC [39] methods.
2. Theoretical background
2.1. Tri-linear regression-calibration (TLRC)
If the absorbance values of a mixture of three analytes (X, Y and Z)
are measured at a three wavelength set (i = 1, 2 and 3) [14]. The following equations (Eq.) can be written for a three-component analysis
as:
Amix1 bX1 C X bY1 C Y bZ1 C Z aXYZ1
Amix2 bX2 C X bY2 C Y bZ2 C Z aXYZ2
Amix3 bz3 C X bY3 C Y bZ3 C Z aXYZ3

The matrix b, corresponding to the slope values of linear regression

equations is called the matrix K:
0

1
bx1 by1 bz1
K @ bx2 by2 bz2 A
bz3 by3 bz3

In this case, the concentration of the analytes, X, Y and Z in ternary

mixture can be obtained by the matrix. (Amix aXYZ)3 1 is multiplied
by the inverse (K1)3 3of the matrix K3 3 and it can be written as:


C31 K1

33



 Amix axyz 31

This procedure is the mathematical basis of the TLRC method for

multi-component analysis. As given detail, this model can be applied
easily to improve resolution of the three-component mixtures. The
choice of selected wavelength set plays a signicant role for the application of this numerical method to a multi-mixture analysis. For this reason, Kaiser's technique [15] was applied for the selection of the
optimum wavelength set in order to provide the best sensitivity and selectivity in the application of the mathematical.
The sensitivity matrices K (square matrix) in Eq. (3) are formed by
taking each three-pair of pre-selected wavelengths for ternary mixtures. The matrices K of the slope values obtained in the linear regression functions of the individual analytes X, Y and Z at three selected
wavelengths are considered as the sensitivity parameter.
The sensitivity parameter is used for comparing different threewavelength sets. The sensitivity of a multicomponent analysis is dened
as the absolute value of the determinant of the sensitivity matrix K. The
calculated maximum determinant value permits to decide the optimum
wavelength set. The method is based on the nine linear regression functions with three linear regression lines for each compound at three selected wavelengths.

where, Amix1, Amix2 and Amix3 represent the absorbance of the mixtures
X, Y and Z analytes at the three-wavelength set. bX ( 1,2 and 3), bY( 1,2 and 3)
and bZ( 1,2 and 3) are the slopes of linear regression equations of X, Y and
Z, respectively; and aXYZ( 1,2 and 3) are the sums of intercepts of linear regression equations at the three wavelengths (aXYZ1 = aX1 + aY1 + aZ1,
aXYZ2 = aX2 + aY2 + aZ2 and aXYZ3 = aX3 + aY3 + aZ3).
Eq. (2) can be formulated in matrix notation as:
0

1 0
1 0 1
Amix1 axyz1
Cx
bx1 by1 bz1
@ Amix2 axyz2 A @ bx2 by2 bz2 A  @ Cy A
Amix3 axyz3
Cz
bz3 by3 bz3

In simple manner
Amix aXYZ 31 K33  C31

Fig. 2. Zero order overlain spectra of a) 15 g mL1 of Paracetamol, b) 15 g mL1 of

Hydrochlorothiazide, c) 15 g mL1 of Enalpril maleate and d) Mixture in 0.1 M HC1.

V.D. Singh, S.J. Daharwal / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 369375

371

Table 1
Statistical parameters for the analysis of PARA, HCTZ and ENM by TLRC method (n = 6).
Parameters

Paracetamol

Hydrochlorothiazide

Enalpril maleate

Wavelength range (nm)

Linearity range (g mL1)
Regression equation
Slopea (S.D.)
Intercepta (S.D.)
Regression coefcient (r2)
LODb (g mL1)
LOQc (g mL1)

242.8
535
0.065 + 0.004
0.065 0.0091
0.004 0.003
0.9998
0.015
0.045

271.4
540
0.014 + 0.013
0.014 0.0043
0.013 0.006
0.9996
0.14
0.42

202.4
540
0.071 + 0.007
0.071 0.0096
0.007 0.002
0.9996
0.011
0.033

n = 6 number of replicates.
a
Regression equation y = mx + c, where m-slope, c-intercept and x-concentration.
b
LOD- limit of Detection.
c
LOQ- Limit of Quantication.

the multi resolution of multi-component mixture system containing n

compound.

2.2. Multi-linear regression calibration (MLRC)

If the absorbance values of a mixture of three analytes (X, Y and Z)
are measured at n wavelengths (i = 1, 2, , n) [14]. The following
set of equations can be written for a three-component analysis:
Amix1 bx1 C x by1 C y bz1 C z axyz1
Amix2 bx2 C x by2 C y bz2 C z axyz2
Amixn bxn C x byn C y bzn C z axyzn

2.3. Classical least square method (CLS)

This method is based on the use the absorptivity values at the selected wavelengths of multi-component mixtures containing n compounds. Absorptivity values of three compounds, X, Y and Z are
calculated by using the absorbances measured at the selected wavelengths in the zero-order spectra for each of the compounds in ternary
mixture [14]. A system of equations with n unknowns was written
for the compounds in the ternary mixture as follows:

Same description as above mentioned Eq. (2).

In the matrix terms, the above multi-equation system (Eq. (5)) can
be formulated as:
0

1 0
1 0 1
Amix1 axyz1
Cx
bx1 by1 bz1
@ Amix2 axyz2 A @ bx2 by2 bz2 A  @ Cy A
Amixn axyzn
Cz
bzn byn bzn

A1 1 C x 1 C y 1 C z
A2 2 C x 2 C y 2 C z
An n C x n C y n C z

Where A1, A2An represent absorbance of solution of mixture Cx, Cy,

Cz are concentration and 1 2 n, 1 2 n, 1 2 n represent absorptivity values calculated for X,Y,Z at 1 2 3..... n wavelengths respectively. Matrix notation greatly simplies matters and easily solves the
system of equations with three unknowns as shown below:

In compact form




Amix axyz n1 K n3 C31

The matrices, (Amix a)n 1 and Kn 3, are multiplied by the

transpose (K)3 n of the matrix Kn 3. The concentration of the X, Y
and Z compounds in ternary mixture can be calculated by using the following formula.




 
C31 K03n Kn3 1 33  K03n Amix axyz n1

1 0
1 0 1
Amix1
1 1 1
Cx
@ Amix2 A @ 2 2 2 A  @ Cy A
Amixn
n n n
Cz

or

A EC

10

8
Using the similar procedure described in Section 2.2. This matrix was
solved and unknown concentrations were determined of X, Y and Z in
their mixture.

In this case, the MLRC model contains the use of linear algebra, also
known as matrix mathematics. This calibration model can be applied to
Table 2
Linear regression analysis of PARA, HCTZ and ENM by TLRC, MLRC CLS methods (n = 6).
TLRC method
Wave-length
(nm)

Paracetamol
Linear equation

202.4
0.071x + 0.007
242.8
0.065x + 0.004
271.4
0.014x + 0.013
MLRC method and CLS method
210
0.044x + 0.067
211.6
0.031x + 0.022
213.2
0.033x + 0.014
214.8
0.030x + 0.007
216.4
0.031x + 0.025
218
0.030x + 0.005
219.6
0.032x + 0.003
221.2
0.034x + 0.001
222.8
0.038x 0.013

Hydrochlorothiazide

Enalpril maleate

r2

SE(b)

SE(a)

SE(r)

Linear equation

r2

SE(b)

SE(a)

SE(r)

Linear equation

r2

SE(b)

SE(a)

SE(r)

0.9996
0.9998
0.9996

0.0096
0.0091
0.0043

0.0002
0.0003
0.0006

0.0094
0.0089
0.0042

0.032x + 0.024
0.009x + 0.011
0.062x + 0.019

0.9992
0.9997
0.9998

0.0054
0.0023
0.0095

0.0004
0.0006
0.0005

0.0051
0.0021
0.0094

0.044x 0.011
0.004x + 0.014
0.001x + 0.007

0.9997
0.9988
0.9998

0.0098
0.0017
0.0008

0.0007
0.0002
0.0008

0.0096
0.0015
0.0007

0.9996
0.9997
0.9998
0.9997
0.9989
0.9999
0.9998
0.9997
0.9994

0.0086
0.0054
0.0056
0.0053
0.0052
0.0049
0.0075
0.0078
0.0065

0.0005
0.0007
0.0006
0.0008
0.0004
0.0007
0.0003
0.0003
0.0002

0.0082
0.0051
0.0052
0.0046
0.0050
0.0047
0.0071
0.0075
0.0062

0.036x + 0.012
0.041x + 0.017
0.047x + 0.023
0.053x + 0.011
0.055x + 0.009
0.059x + 0.012
0.069x + 0.006
0.066x + 0.019
0.071x + 0.014

0.9998
0.9990
0.9979
0.9992
0.9998
0.9994
0.9997
0.9996
0.9997

0.0057
0.0089
0.0098
0.0102
0.0108
0.0115
0.0121
0.0128
0.0132

0.0003
0.0002
0.0005
0.0006
0.0005
0.0007
0.0006
0.0008
0.0009

0.0055
0.0086
0.0095
0.0099
0.0098
0.0102
0.0113
0.0122
0.0128

0.035x + 0.014
0.046x + 0.009
0.036x + 0.018
0.033x + 0.014
0.026x + 0.009
0.021x + 0.003
0.018x + 0.002
0.015x + 0.013
0.014x + 0.023

0.9991
0.9995
0.9993
0.9997
0.9997
0.9998
0.9998
0.9997
0.9997

0.0079
0.0101
0.0084
0.0077
0.0068
0.0063
0.0054
0.0049
0.0037

0.0003
0.0002
0.0004
0.0005
0.0004
0.0005
0.0002
0.0002
0.0004

0.0076
0.0098
0.0083
0.0075
0.0062
0.0060
0.0054
0.0047
0.0034

n = 6 number of replicates, r 2 correlation coefcient of regression equation, SE (b) = standard error of slop, SE (a) = standard error of intercept, SE(r) = standard error of correlation
coefcient.

372

V.D. Singh, S.J. Daharwal / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 369375

Table 3
The obtained sensitivity values () of PARA, HCTZ and ENM at twelve wavelengths.
i

202.4

210

211.6

213.2

214.8

216.4

218

219.6

221.2

222.8

242.8

271.4

PARA 103
HCTZ 103
ENM 103

71.08
33.7
43.9

43.9
36.5
36.1

32.0
41.0
46.0

33.2
47.1
36.1

30.1
54.1
32.0

31.1
55.6
26.1

30.7
59.3
21.7

32.8
69.3
18.1

34.6
66.2
16.1

40.1
70.4
14.1

64.9
9.1
4.0

14.5
63.2
1.1

3. Experimental

3.6. Preparation of standard stock solution

3.1. Standard samples

PARA, HCTZ and ENM were weighed accurately (100 mg) and dissolved separately with 0.1 M HCl in 100 mL volumetric ask. The solutions were further diluted to obtain 100 g mL 1 concentrations of
PARA, HCTZ and ENM, respectively. The solution were carefully diluted
with 0.1 M HCl to obtained the concentration range of 535 g mL1 of
PARA, 540 g mL1 of HCTZ and 540 g mL1 of ENM, respectively to
construct the calibration curves.

Pure samples of PARA, ENM and HCTZ were supplied as gift sample
from Zim Laboratories Limited, Nagpur (India). The percentage purity
was found to be 98.5, 98.5and 99.2 according to their ofcial methods
[43].

3.2. Market formulation

3.7. Preparation of laboratory prepared mixture

Invozide tablet formulation is manufactured by the Ranbaxy Laboratories Limited, India. Containing 325 mg Paracetamol (PARA), 25 mg
Hydrochlorothiazide (HCTZ) and 10 mg Enalpril Maleate (ENM) per
tablet were procured from the local chemist shop, Raipur, Chhattisgarh.

Accurate volume of PARA, HCTZ and ENM were taken from working
solutions and transferred into 10 mL volumetric ask and further diluted with 0.1 M HCl to prepare six synthetic mixtures.
3.8. Spectroscopic characteristics of PARA, HCTZ and ENM

3.3. Chemicals and solvents

1.5 mL of solutions (from 100 g mL1) was separately transferred

into three 10 mL volumetric asks and the volume was made up with
0.1 M HCl to obtain 15 g mL 1 concentrations of PARA, HCTZ and
ENM respectively. The absorbance spectra of solution were recorded
within the wavelength range of 200320 nm using 0.1 M HCl as a blank.

All chemicals and solvents were of analytical grade and solutions

were prepared by using distilled water (Mono quartz distillation unit,
Borosil) or Millipore water(Synergy Pak- ICW-3000,Bellerica). All
the solutions were prepared freshly and protected from light.

3.9. Sample preparation of commercial tablet formulation

3.4. Apparatus
Shimadzu (UV-1800) spectrophotometer (Japan) possessing a xed
slit width using 1-cm quartz cuvette. The spectrophotometer is connected to a computer installed with UV-probe 2.33 software. Scans
were carried out in the range of 220300 nm at 0.5 nm intervals. All
weights were taken on Denver electronic balance of 0.1 mg sensitivity
(Germany).

Twenty tablets were accurately weighed and powdered in mortar.

Average weight of each tablet was dissolved in methanol0.1 M HCl in
100 mL of volumetric ask with the aid of sonication for 15 min. The solutions were ltered in to 100 mL volumetric ask by using whatman
No.42 lter paper. The residues were washed three times with 0.1 M
HCl. The solutions were diluted to the same concentration of the appropriate working solutions and analysis was carried out by using TLRC,
MLRC and CLS method.

3.5. Software

4. Results and discussions

The regression and statistical analysis were achieved by using the

Excel 2010. MLRC, TLRC and CLS, matrix were created by MATLAB trail
version 6.5 (Natick, MA) for Windows.

The analysis of multi component mixture was quite difcult task in

terms of resolving the overlapped spectra without prior separation of
the constituents. The development of multivariate calibration

Table 4
Recovery results of PARA, HCTZ and ENM in the laboratory prepared mixtures by proposed methods.
Concentration
(gmL1)

Recovery (%) (n = 6)
TLRC

MLRC

CLS

Sr. no.

PARA

HCTZ

ENM

PARA

HCTZ

ENM

PARA

HCTZ

ENM

PARA

HCTZ

ENM

1.
2.
3.
4.
5.
6.
Mean
S.Da
RSDb

25
10
15
25
5
15

25
10
10
5
25
10

10
25
25
10
10
5

100.4
101.3
100.9
100.5
100.6
100.2
100.6
0.393
0.39

101.2
104.5
104.8
103.2
101.3
101.9
102.8
1.591
1.54

101.5
99.6
99.9
101.6
99.4
102.1
100.7
1.718
1.71

100.1
100.2
99.3
100.8
99.8
99.6
99.96
0.524
0.52

99.5
99.7
100.1
97.8
100.4
99.5
99.5
0.905
0.90

100.1
99.6
99.2
100.2
100.1
100.1
98.83
0.397
0.40

102.1
99.3
99.3
100.1
102.7
99.4
100.5
1.526
1.53

98.8
100.2
100.2
100.1
98.3
100.4
99.66
0.884
0.88

98.6
100.1
100.1
99.4
98.1
101.2
99.5
1.126
1.13

n = 6 number of replicates S.D.a = standard deviation RSDb = relative standard deviation.

V.D. Singh, S.J. Daharwal / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 369375

equations were constructed with absorbance values at twelve wavelength points by using standard series of each compound. All the calculated regression equations and their statistical parameter were shown
in Table 2. The sensitivity matrices were formed by the slope values of
the linear regression analysis of PARA, HCTZ and ENM in laboratory prepared mixture and were shown in Table 3. According to Kaiser's method
[15], absolute values of the determinant of the sensitivity matrices were
used to obtain the best sensitivity for construction of the TLRC model.
In this treatment, for the selection of optimum three-wavelength set,
220 three-pairs of the sensitivity matrices were possible. The results
were calculated by using formula given in [14]. An optimum threewavelength set, 242.8 nm, 271.4 nm and 202.4 nm was selected for
the TLRC method with highest determinant values of sensitivity matrices. The individual linear regression equations for each compound at
this selected three-wavelength set were shown in Table 2. All the data
matrices were arranged according to Eq. (1) as discussed in Section
2.1. The absorbance and concentration data matrices were calculated
for prediction of unknown concentration of PARA, HCTZ and ENM in
their laboratory prepared mixtures and tablet formulations. The results
were shown in Tables 4 and 8 respectively.

Table 5
Results of standard addition method applied to commercial tablet formulation.
Methods
TLRC

MLRC

CLS

Parameters

PARA HCTZ ENM PARA HCTZ ENM PARA HCTZ ENM

Mean (mg

323.9 24.3

9.3

323.2 24.6

9.6

323.7 24.4

9.4

mL1)
S.Da
R.S.Db
S.Ec

1.39
0.43
0.57

0.17
1.84
0.07

0.95
0.29
0.39

0.12
1.19
0.04

0.64
0.19
0.26

0.19
1.95
0.08

0.41
1.64
0.16

0.46
1.87
0.19

0.46
1.91
0.18

373

Label claim; 325 mg Paracetamol, 25 mg Hydrochlorothiazide and 10 mg Enalpril maleate,

n = 6 number of replicates S.D.a = standard deviation RSDb = relative standard deviation
S. Ec = Standard error.

spectrophotometric methods for the resolution of such mixtures has

shown signicant importance. Hence, spectrophotometric methods
are considered the most appropriate for analysis by using mathematical
algorithms methodology. In the present study, simultaneous estimation
of PARA, HCTZ and ENM in laboratory prepared mixture and tablet formulation were carried out by tri-linear regression calibration (TLRC),
multi-linear regression calibration (MLRC) and classical least square
(CLS). The determination of ENM in the formulation showed complexity
due to presence of their low concentration as compare to other components. Swamy and his co-workers [39] has been reported RP-HPLC
method for simultaneous estimation of PARA, HCTZ and ENM in bulk
form and in tablet formulation. The chromatographic separation was
achieved on BDS C8 Hypersil column (Hi-Q 250 4.6 mm ID; particle
size 5 m). The mobile phase was mixture of phosphate buffer pH 4.0
and acetronitrile in the ratio of 75:25. The max was set at 210 nm and
ow rate was 1.0 mL/min. Calibration curves were linear over the
range of 1060 g mL 1 for PARA, 530 g mL 1 for ENM and 4
24 g mL 1for HCTZ respectively. In this study % RSD of reported
HPLC method was compared with proposed method. Though, HPLC
method was used for analysis of this multicomponent mixture, it was
observed that % RSD of newly developed spectrophotometric methods
is found to be less than the reported method. Additionally, developed
multivariate calibration methods do not required chemical pre-treatment, cleanup steps, prior treatment for solvents like ltration and
degassing, high purity solvent and optimization of conditions such as
pH, temperature, and ow rate. Hence, the proposed multivariate calibration spectrophotometric methods were found to be sensitive and
yet very cheap for analysis of complex mixture. Furthermore, multivariate calibration methods allow the simultaneous inclusion of multiple
variables and improve the outcomes by reducing the possible interference in quantitative analysis [44,45].
In Fig. 2 zero order overlain absorbance spectra of each drug within
range of 200320 nm showed severe overlapping. The calibration
curves for TLRC, MLRC and CLS model(s) were constructed by using
535 g mL1of PARA, 540 g mL 1 of HCTZ and 540 g mL 1 of
ENM and their regression equations were shown in Table 1.

4.2. MLRC model

In this method nine wavelengths 210, 211.6, 213.2, 214.8, 216.4,
218.4, 219.6, 221.2, and 222.8 were selected for each drug within the
range of 200223 nm (in the = 1.6 nm interval). The selected wavelengths, regression equations and their statistical parameters were
shown in Table 3. All the data matrices were arranged according to Eq.
(5) as discussed in Section 2.2. The absorbance and concentration data
matrices were calculated for prediction of unknown concentration of
PARA, HCTZ and ENM in their laboratory prepared mixtures and tablet
formulations. The results were shown in Tables 4 and 8 respectively.
4.3. CLS model
This method was used as an alternative method for the MLRC. In this
method nine wavelengths (210, 211.6, 213.2, 214.8, 216.4, 218.4, 219.6,
221.2, and 222.8) were selected for each drug within the range of 200
223 nm (in interval = 1.6 nm). The selected wavelength and their
absorptivity values were shown in Table 3. All the data matrices were
arranged according to Eq. (9) as discussed in Section 2.3. The absorbance and concentration data matrices were calculated for prediction
of unknown concentration of PARA, HCTZ and ENM in their laboratory
prepared mixtures and tablet formulations. The results were shown in
Tables 4 and 8 respectively.
4.4. Method validation
The proposed methods were obey Beer's Law in the concentration
range of 535 g mL 1, 540 g mL1 for and 540 g mL 1 for
PARA, HCTZ and ENM, respectively. The regression coefcients (r2) for
TLRC method were found to be 0.9998, 0.9996 and 0.9996 for PARA,
HCTZ and ENM, respectively. Sensitivity of developed method for analyzing the mixtures by TLRC model were increased which were conrmed by low values of LOD and LOQ as shown in Table 1. The LOD
were found to be 0.015, 0.14 and 0.011 g mL1 and LOQ were found

4.1. TLRC model

In this method twelve wavelengths were selected within working
spectroscopic range of PARA, HCTZ and ENM. Linear regression
Table 6
Analysis of variance (ANOVA) of proposed methods in commercial tablet formulation.
SS*

MS***

f-test

Source of variations

PARA

HCTZ

ENM

Df**

PARA

HCTZ

ENM

PARA

HCTZ

ENM

Fcrit

Between the group

Within the group
Total

0.86
16.27
17.14

0.28
3.00
3.28

0.14
0.42
0.56

2
15
17

0.43
1.08

0.14
0.02

0.07
0.02

0.39

0.7

2.58

3.68

* sum square, ** degree of freedom (2,15), *** mean square, Fcrit (tabulated value = 3.68), P value (0.05).

374

V.D. Singh, S.J. Daharwal / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 369375

Table 7
Statistical comparison between proposed methods for PARA, ENM and HCTZ in commercial tablet formulation.
PARA

HCTZ

Method Mean S.D. t values

ENM

f value

Mean S.D.

t values

f value

Mean S.D.

t values

f value

25.15
0.37

TLRC MLRC =

TLRC MLRC =

TLRC MLRC =

TLRC MLRC =

24.65
0.46

1.06
TLRC CLS =
1.74

0.18
MLRC CLS =

9.88
0.13

0.87
MLRC CLS =

3.11
MLRC CLS =

25.03
0.36

TLRC CLS =
1.78

0.54
TLRC CLS =
0.81

1.07
TLRC CLS =
1.51

TLRC

324.96

TLRC MLRC =

TLRC MLRC =

MLRC

0.41
324.93

0.14
MLRC CLS =

0.54
MLRC- CLS =

CLS

0.53
324.11

1.23
TLRC CLS =

12.12
TLRC CLS =

0.26

1.32

1.47

1.31
TLRC CLS =
3.60

9.81
0.76
9.83
0.52

tcritical value = 2.01 and fcritical value = 5.05 at 95% condence limit.

to be 0.045, 0.42 and 0.033 g mL1 for PARA, HCTZ and ENM, respectively. The results suggested that the proposed method was more sensitive than reported HPLC method [39].
Validations of proposed method were done as per ICH guideline [41,
42]. Recovery studies were done by the quantitative analysis of laboratory prepared mixture of these three drugs. Result of the means, %recoveries and the standard deviation of TLRC, MLRC and CLS were computed
and summarized in Table 4. The percentages relative standard deviation
(%RSD) of TLRC, MLRC and CLS methods were found to be 0.39, 0.52 and
1.53 for PARA, 1.54, 0.90 and 0.88 for HCTZ and 1.71, 0.40 and 1.13 for
ENM respectively. The results were revealed that each calibration
model gave satisfactory results, particularly in the case of overlapping
spectra of PARA, HCTZ and ENM. MLRC showed the good recovery results as compared to TLRC and CLS methods.
According to the difference between added and predicted concentrations, the standard error of prediction (SEP) were found to be 0.35,
1.61 and 0.41 by using TLRC methods, 0.02, 0.29 and 0.67 by using
MLRC, and 0.28, 0.19 and 0.30 by using CLS method for PARA, HCTZ
and ENM respectively. Furthermore, the standard error of calibration
(SEC) were found to be 0.42, 1.18 and 0.58 by using TLRC methods,
0.03, 0.35 and 0.12 by using MLRC and 0.33, 0.24 and 0.34 by using
CLS method for PARA, HCTZ and ENM respectively. The acceptability
of the developed method may depends on the minimum values of SEP
and SEC [33]. The results suggested that the MLRC give better quantitative resolution of ternary mixtures than TLRC for these compounds. It
has earlier been found that MLRC methods could be used to improve
quantitative resolution of multicomponent mixtures [14].
Furthermore, the standard addition method was applied in six replicates for tablet formulation and their statistical results were summarized in Table 5. The ndings of developed methods were suggested
that TLRC, MLRC and CLS model gives precise and reliable results. The
Standard errors (SE) were calculated for TLRC MLRC and CLS methods
and it was found to be 0.57, 0.39 and 0.26 for PARA, 0.16, 0.19 and
0.18 for HCTZ and 0.07, 0.04 and 0.08 for ENM respectively. The results
were assured that the excipients in tablets do not interfere with the active compounds and good agreement was found in assay of tablet
formulation.
One-way ANOVA test was performed to analyze the results of the
proposed methods for analysis of tablets. The calculated f value was
found to be 0.61, 0.81 and 0.34 for PARA, HCTZ and ENM respectively,

which were below to the tabulated value in all cases (F = 3.40, n1 =

2 and n2 = 24 degree of freedom, at 95% condence limit). The experimental results were shown in Table 6. It was indicated that label claim
of tablets were shown good agreement.
These three proposed methods were statistically compared to each
other by applying t-test and f-test. The results obtained by comparing
TLRC, MLRC and CLS were summarized in Tables 7. The calculated tvalues and f values were found to be less than the tabulated value in
all the cases (t = 2.01 and F = 5.05 at 95% condence limit). The results
revealed that there is no signicant difference between three multivariate models. These three proposed methods were also compared with
reported HPLC method by applying t-test and f-test. The f values were
calculated for TLRC MLRC and CLS methods, it was found to be 2.16,
1.18 and 1.50 of PARA, 1.18, 2.52 and 1.13 of HCTZ and 1.69, 1.80 and
1.61 of ENM, respectively. The calculated f values were less than the tabulated value in all the cases (F = 5.05, at 95% condence limit). The result were revealed that there is no signicant difference between three
multivariate method and reported HPLC method.
The t values were calculated by TLRC MLRC and CLS methods, it was
found to be 1.36, 0.24 and 0.52, for PARA, 0.19, 1.54 and 0.34 for HCTZ
and 1.31, 1.58 and 1.79 for ENM, respectively. The calculated t values
were less than the tabulated value in all the cases (t = 2.01, at 95% condence limit). The statistical results were summarized in Tables 8. The
results were revealed that there is no signicant difference between
three multivariate models and reported HPLC method. Hence, the results were suggested that the proposed methods can be useful for simultaneous estimation of PARA, HCTZ and ENM in pharmaceutical
dosage form. Furthermore, these multivariate calibration spectrophotometric methods could be used as an alternative of HPLC method in quality control laboratories lacking the required facilities for those
expensive techniques.
5. Conclusion
A comparative study was conducted between proposed multivariate
calibration methods and reported RP-HPLC method for simultaneous
estimation of PARA, HCTZ and ENM in tablet dosage form and laboratory
prepared mixture. In this study, the proposed methods were proved to
be advantageous in terms of selectivity and sensitivity over the other
conventional spectrophotometric methods [15]. The proposed methods

Table 8
Statistical comparison of proposed methods with reported HPLC method for analysis of PARA, ENM and HCTZ in tablet formulation.
TLRC

MLRC

CLS

Reported method*

Parameters

PARA

HCTZ

ENM

PARA

HCTZ

ENM

PARA

HCTZ

ENM

PARA

Mean (mgmL1)
%Recovery
S.Da
R.S.Db
t (p value-0.05)
f (p value-0.05)

324.96
99.98
0.40
0.41
1.36
2.16

25.15
100.6
0.37
0.38
0.19
1.18

9.88
98.83
0.13
0.13
1.31
1.69

324.93
99.97
0.53
0.53
0.24
1.17

24.65
98.6
0.45
0.46
1.54
2.52

9.81
98.16
25.15
0.76
1.58
1.80

324.11
99.72
9.88
0.26
0.52
1.5

25.03
100.13
0.35
0.36
0.34
1.13

9.83
98.33
0.51
0.52
1.79
1.61

328.12
25.55
100.96
102.22
0.67
0.70
0.66
0.69
t critical value = 2.01
Fcritical value = 5.05

HCTZ

ENM
10.15
101.5
0.62
0.61

325 mg Paracetamol, 25 mg Hydrochlorothiazide and 10 mg Enalpril maleate Reported Method* - HPLC method [39] S.D.a standard deviation R.S.D.b.- relative standard deviation.

V.D. Singh, S.J. Daharwal / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 369375

were showing better regression parameters, lower values of LOD and

LOQ, good recovery results and lower SD values. Moreover, TLRC,
MLRC and CLS models have advantages over HPLC method because it
does not required chemical pre-treatment, cleanup steps, prior treatment for solvents like ltration and degassing, high purity solvent and
optimization of conditions such as pH, temperature, and ow rate
[46]. Result were also suggested that these methods are more reliable
with simple mathematical calculations than other spectrophotometric
methods.
The outcomes of proposed method encourage us to use multivariate
calibration method as an alternative of HPLC techniques for routine
analysis and quality control of commercial products containing
multicomponent.
Conict of interest
There is no conict of interest with any nancial organization regarding the material discussed in the manuscript. The authors alone
are responsible for the content and writing of the manuscript.
Acknowledgement
Authors are thankful to Zim Laboratories Limited, Nagpur (INDIA),
for providing the drugs as gift sample, and also gratefully to Director,
University Institute of pharmacy, Pt. Ravishankar Shukla, University,
Raipur (Chhattisgarh), for given that all the necessary facilities for this
work.
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