CHEMOMETRIC ASSISTED SPECTROPHOTOMETRIC TECHNIQUES FOR SIMULTANEOUS DETERMINATION OF AMLODIPINE BESYLATE AND TELMISARTAN IN MARKETED TABLET FORMULATION

Author : Khushboo Khatri, Prachi R. Bhamre and S. J. Rajput*

Page Nos : 57 - 69

Cite Article :

Khatri K, Bhamre PR, Rajput SJ. Chemometric Assisted Spectrophotometric and HPLC Methods for the Estimation of Amlodipine Besylate and Telmisartan Marketed Formulation. NUJPS. 2014;1(1):57–69.

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Introduction
Amlodipine besylate (AML) is chemically
described as (3-Ethyl 5-methyl (4RS)-2-
[(2-aminoethoxy) methyl] – 4 – (2-
chlorophenyl) – 6 – methyl – 1, 4 –
dihydropyridine – 3, 5 – dicarboxylate
benzene sulfonate). AML is a calciumchannel
blocking agent; a dihydropyridine
derivative with an intrinsically long
duration of action and can be given once
daily.AML is an antihypertensive and used
as a prophylaxis for angina.
Telmisartan (TEL) is an angiotensin II
receptor (type AT1) antagonist used in the
management of hypertension. TML
prevents the constriction (narrowing) of
blood vessels (veins and arteries). It is a
non-peptide molecule and chemically
described as 4′-[(1,7′-Dimethyl-2′-propyl-
1H,3’H -2,5′-bibenzimidazol-3′-yl)
methyl]-2-biphenyl carboxylic acid. The
combination of AML and TEL has been
reported to show substantial and sustained
24 hour blood pressure reduction and is
well-tolerated in a range of patients with
hypertension and at risk of cardiovascular
events. AML and TEL are available in
combined tablet dosage form for the
treatment of hypertension.
Literature review

The UV spectra of AML and TEL are
highly overlapping and hence very few
spectrophotometric methods are available
in the literature for their simultaneous
estimation. In the present study the
chemometric approach has been applied to
resolve the spectra and enable the
simultaneous determination of AML and
TEL in the marketed formulation
alongwith the HPLC method. The results
obtained by chemometric methods have
been statistically compared with the HPLC
method to confirm the results.
Materials and Methods

Instrumentation
Chemometric spectrophotometry
A double-beam Shimadzu UV-1700
spectrophotometer (Kyoto, Japan)
connected to a computer loaded with
Shimadzu UV Probe 2.10 software was
used for all the spectrophotometric
measurements. The absorbance spectra of
the reference and test solutions were
carried out in 1cm quartz cells over the
range of 200-400 nm. The numerical
calculations for ILS and CLS methods
were performed by using MATLAB
R2007a Software and Excel

HPLC
Chromatography was performed on
Shimadzu (Shimadzu Corporation, Kyoto,
Japan) chromatographic system equipped
with Shimadzu LC-20AT pump and
Shimadzu SPD-20AV absorbance detector.
Samples were injected through a Rheodyne
7725 injector valve with fixed loop at
20μL. Separation and quantitation were
made on a reversed-phase, Hypersil BDS

C column (250×4.6 mm i.d, 5 μm particle 18
size). Detector was set at 238nm. Data
acquisition and integration was performed
using Spinchrome software (Spincho
Biotech, Vadodara).
Materials and Reagents

AML and TEL (Bulk drugs) were kindly
gifted by Cadila pharmaceutical limited,
Ahmedabad. HPLC grade methanol
(Qualigens fine chemicals, Mumbai) was
used for HPLC and AR grade methanol
(Qualigens fine chemicals, Mumbai) was
used for spectrophotometry. Potassium
dihydrogen orthophosphate KH PO (AR 2 4
grade: Qualigens fine chemicals, Mumbai),
ortho phosphoric acid (AR grade:
Qualigens fine chemicals, Mumbai),
acetonitrile (HPLC grade, Qualigens fine
chemicals, Mumbai) were used for HPLC.
Double distilled water filtered through
nylon filter paper 0.2 μm pore size and 47
mm diameter (Pall Lifesciences, Mumbai,
India), was used throughout the analysis.
The marketed formulation of Telma-AM
tablet (Glenmark Pharmaceuticals Ltd.)
containing 5mg AML and 40mg TEL per
tablet was taken for the study.
Experimental conditions

Chemometric spectrophotometry

The UV absorption spectra of appropriate
solutions were recorded in the wavelength
range 200-400nm with the intervals of 4nm
(Δλ = 4nm) at 19 wavelength points. The
scanning range selected was 230-302nm.
HPLC
The mobile phase was prepared by mixing
acetonitrile and 0.01M phosphate buffer.

The phosphate buffer was prepared by
dissolving 0.27 g of potassium dihydrogen
phosphate in sufficient double distilled
water to produce 100 mL, 0.4 ml of
triethylamine (TEA) was added and the pH
was adjusted to 2.5 with orthophosphoric
acid and filtered through 0.2 μm nylon
filter paper. Acetonitrile and phosphate
buffer were mixed in a ratio of 45:55% v/v
and degassed by sonicating for 5 min in
ultrasonic bath. The flow rate was
maintained at 1.0 mL/min. All
determinations were performed at ambient
temperature. Quantitation based on peak
area was achieved with UV detection at
238nm. The injection volume was 20 μL.
Standard solutions and calibrations
AML stock solution (1000 ppm) was
prepared by dissolving 13.9mg of
Amlodipine Besylate (equivalent to 10 mg
of AML) in 10 mL methanol. TEL stock
solution (1000 ppm) was prepared by
dissolving 10 mg of TEL in 10 mL
methanol. The stock solutions were
ultrasonicated for 1 min. The working
solutions of AML and TEL (100 ppm)
were prepared by transferring 1 mL aliquot
from stock solution to a 10 mL volumetric
flask and making up the volume with
mobile phase in case of HPLC and making
up the volume with methanol in case of
chemometric spectrophotometry.

Preparation of solution mixture sets for
chemometric estimation

A calibration set of 24 mixtures was
prepared in methanol, applying a
multilevel multifactor design in which two
levels of concentrations of AML and TEL

Calibration Set and Validation Set

taken, from working stock solution, in 10
mL volumetric flask and diluted upto the
mark with the mobile phase to obtain the
final concentration of binary mixtures as
marketed available formulation. Triplicate
20 μL injections were made for each
concentration and chromatogram was
obtained under the specified
chromatographic conditions described
previously. The calibration graph was
constructed by plotting peak area versus
concentration of each drug and the
regression equation was calculated.
Sample preparation

Twenty tablets were weighed and finely
powdered. The powder equivalent to one
tablet (5 mg AML and 40 mg TEL) was
accurately weighed and transferred to
volumetric flask of 100 mL containing 50
mL of the methanol and sonicated for 15
min at cool temperature. The above
solution was carefully filtered through
whatman filter paper (No. 41) to 100mL
volumetric flask and residue were washed
thrice with methanol , the combined filtrate
was made up to the mark with methanol.

For chemometric spectrophotometry, an
aliquot of 0.2 mL was pipette out from
above prepared solution and transferred to
volumetric flask of 10 mL. Volume was
made up to the mark with methanol to give
a solution containing 1: 8 μg/mL of
AML:TEL. This solution was used for the
estimation of AML and TEL.

For HPLC, an aliquot of 1 mL was pipette
out from above prepared solution and
transferred to volumetric flask of 10 mL.
Volume was made up to the mark with

mobile phase to give a solution containing
5: 40 μg/mL of AML:TEL. This solution
was used for the estimation of AML and
TEL.
Results and Discussion
Chemometric spectrophotometry

Figure 1 shows the overlain zero-order
absorption spectra of AML, TEL and their
binary mixture solution in methanol in the
200-400nm absorption region.

The ratio of AML and TEL in commercial
tablet is 1:8. The zero-order absorption
spectra (Figure 1) of AML and TEL
completely overlap with each other making
it difficult to use conventional
spectrophotometric techniques for
simultaneous analysis. Thus the ILS and
CLS chemometric spectrophotometric
methods were found to be appropriate for
determination of AML and TEL in binary
mixture. A training set (calibration set) of
24 synthetic binary mixture solutions
(Table 1) and a validation set containing 17
synthetic binary mixture solutions (Table
1) in the possible combinations were
prepared with methanol (AR grade). The
UV absorption spectra of appropriate
solutions were recorded in scanning range
of 200-400nm and the wavelength range
selected was 230-302nm with the intervals
of 4nm (Δλ = 4nm) at 19 wavelength
points.

CLS method

CLS is one of the simplest methods, based
on a linear relationship between the
absorbance and the component

concentrations at each wavelength. In
matrix notation, the Beer’s law models for
m calibration standards containing l
chemical components with spectra of n
digitised absorbance is given by

A = C x K + EA ………………………… (Eq. 1)
where A is the m x n matrix of calibration
spectra, C is the m x l matrix of component
concentrations, K is the l x n matrix of
absorbance-concentration proportionality
constants (absorptivity-pathlength) and EA
is the in m x n matrix of spectral errors or
residuals not fit by the model.

In this method, the coefficient matrix (K)
was calculated by using the linear equation
system between the absorbance data and
training set. The observed absorbance
values of the compounds in the binary
mixture solutions, at the 19 wavelength
points with the interval of 4nm (Δλ = 4nm)
in the spectral region from 230nm to
302nm, were replaced in the equation 1
and the amount of AML and TEL in the
synthetic mixtures (validation set) were
calculated.

ILS method

In this method, the coefficient matrix (P)
was obtained from the linear equation
system using the absorbance data and the
calibration set. Introducing (P) into the
linear equation system we obtain the
calibration for ILS, as this method treats
concentration as a function of absorbance.
The inverse of Beer’s law model for m
calibration standards with spectra of n
digitised absorbance is given by

C = A x P + Ec …………………………. (Eq. 2)

Where C and A are as before, P is the n x l
matrix of unknown calibration co-efficient
relating the l component concentrations of
the spectral intensities and Ec is the m x l
vector of errors. Since in ILS the number
of wavelengths cannot exceed the total
number of calibration mixtures, stepwise
multiple linear regressions have been used
for the selection of wavelengths. The
observed absorbance values of the
compounds in the binary mixture solutions,
at the 19 wavelength points in the spectral
region from 230nm to 302nm, were
replaced in the equation 2 and the amount
of AML and TEL in the synthetic mixtures
(validation set) were calculated.<br/>
Validation of chemometric methods<br/>
The tested mixtures for ILS and CLS were
subjected to recovery studies. The results
obtained were in good agreement with the
true values. These results of percentage
recoveries and standard deviation are
shown in (Table 2) which determines the
accuracy of the chemometric methods. The
numerical values of the statistical
parameters (Table 3), such as plots of
predicted versus true value (their
regression coefficient), RMSEP (Root
mean square error of prediction) value
indicate that the proposed techniques are
suitable for the determination of these
drugs in the tablet formulation as
excipients do not interfere. Moreover the
plots of residual value versus predicted
concentration show that the residual values
for the model are close to zero and more
randomly distributed (Figure 2). The ILS
and CLS models were then applied to
estimate AML and TEL in commercial

tablet preparations. The results are shown
in (Table 4).

Selection and optimization of
chromatographic condition
Optimization of chromatographic
procedure

Different solvents in mixtures of varied
proportions, at different flow rate and
wavelength were tried to select the mobile
phase. The mobile phase with

water:acetonitrile 50:50 gave broad peaks.
Hence trials were carried out with different
pH of phosphate buffer and acetonitrile at
pH 2.5, 4.5 and 6. Among these trials the
most appropriate peak shape and resolution
alongwith appropriate system suitability
parameters (according to ICH guidelines)
were obtained with pH 2.5 with a ratio of
phosphate buffer:acetonitrile 55:45 with
0.4% TEA (Table 5). TEA was added
to minimise the tailing of the peaks. The
optimized chromatogram of the laboratory
sample solution has been shown in
Figure 3.

Selection of common wavelength

The sensitivity of HPLC method that uses
UV detection depends upon proper
selection of detection wavelength. An ideal
wavelength is the one that gives good
response for the drugs that are to be
detected. In the present study individual
drug solutions of 1μg/mL of AML and
8μg/mL TEL were prepared in methanol.
As the absorbance and concentration of
AML is less than TEL its absorbance
maxima 238nm (Figure 1) was selected as
the common wavelength for simultaneous
estimation of the two drugs.

The overlain zero-order absorption spectra of AML( 1 ppm) TEL(8 ppm) and their binary mixture

Figure 1: The overlain zero-order
absorption spectra of AML( 1 ppm),
TEL(8 ppm) and their binary mixture(
AML:TEL 1:8 ppm) in methanol
Concentration residuals vs actual concentration plot for AML and TEL for CLS and ILS
Figure 2: Concentration residuals vs
actual concentration plot for AML and
TEL for CLS and ILS

HPLC chromatogram of the formulation

Figure 3: HPLC chromatogram of the

formulation
Validation of HPLC method
Validation of the HPLC method was
carried out according to ICH guideline
[21].
Linearity and Range
The linearity study was carried out for both
drugs at ten different concentration levels.
The linearity of AML and TEL were in the

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Stability in sample solutions

Binary mixtures containing two different
concentrations of AML and TEL were
prepared from standard stock solution and
stored at room temperature for 24hrs. They
were then injected in to LC system. There
was no change in signal intensity and no
additional peak found in chromatogram
indicating the stability of AML and TEL in
the sample solution.
System suitability

The system suitability parameters
including resolution (Rs), asymmetric
factor (As) and theoretical plates were
established by 10 replicates of binary
mixture of AML and TEL (1:8 μg/mL,
respectively).
Analysis of commercial tablet preparation
The proposed multivariate ILS and CLS
spectrophotometric methods and HPLC
method were applied to the simultaneous
determination of AML and TEL in
commercial Telma-AM tablet preparation.
Satisfactory results were obtained for each
compound which were in good agreement
with the label claim. The assay results of
the proposed ILS and CLS
spectrophotometric methods were
compared to those of the proposed HPLC
method. The results obtained were
statistically analyzed by one way ANOVA
at 95% confidence level. The results are
shown in Table 6. The calculated values
did not exceed the theoretical ones;
indicating that there was no significant
difference between the methods compared.

Conclusion

RP-HPLC techniques are generally used
for separation and determination of
components in final pharmaceutical
preparations and are superior with regard
to identification and specificity. However,
the chemometric methods are less
expensive by comparison and do not
require sophisticated instrumentation nor
any prior separation step. The proposed
chemometric-assisted spectrophotometric
methods are applicable, prompt and
specific for the simultaneous determination
of AML and TEL in their synthetic
mixtures and commercial pharmaceutical
tablets. The results obtained were
compared with the proposed RP-HPLC
method and good coincidence in the means
of recovery was observed as there was no
significant difference between the methods
compared. The three proposed methods
were accurate, precise with good
reproducibility and sensitivity; hence can
be used for the routine analysis of AML
and TEL in their combined pharmaceutical
formulations.
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