American Journal of Analytical Chemistry, 2012, 3, 632-637 Published Online September 2012 (
Simultaneous Determination of Amlodipine with
H1-Receptor Antagonists by Reversed Phase High
Performance Liquid Chromatography and Application to
Interaction Studies
Muhammad Saeed Arayne1, Najma Sultana2, Saima Sher Bahadur1, Muhammad Nawaz3*
1Department of Chemistry, University of Karachi, Karachi, Pakistan
2Research Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
University of Karachi, Karachi, Pakistan
3Department of Chemistry, United Arab Emirates University, Al-Ain, UAE
Email: *
Received May 29, 2012; revised July 12, 2012; accepted July 27, 2012
A rapid, fast and precise method has been developed and validated for the simultaneous determination of amlodipine
with H1-receptor antagonists (cetirizine, fexofenadine, and buclizine) from dosage forms. The chromatography was
performed on a Purospher® Star, C18 (5 μm, 250 × 4.6 mm) column using acetonitrile: buffer (0.01 mM) (40:60, v/v, pH
adjusted to 3.0), as a mobile phase. The mobile phase was pumped at a flow rate of 1.0 mL·min1 and UV detection was
performed at 240 nm. The method was validated for linearity, accuracy, precision and specificity. The method was ap-
plied to study the interaction between amlodipine and H1-receptor antagonists. These interactions were carried out in
simulated gastric juice (pH 1), simulated full stomach (pH 4), blood pH (pH 7.4) and simulating GI (pH 9). The inter-
acting drugs were heated at 37˚C with intermittent shaking and the samples were withdrawn every thirty minutes for
three hours and drug contents were analyzed by RP-HPLC techniques. In most cases the in vitro availability of am-
lodipine was decreased. It was observed that the change in in vitro availability was pH dependent.
Keywords: Amlodipine; Cetirizine; Fexofenadine; Buclizine; Interactions; Reversed Phase High Performance Liquid
1. Introduction
Amlodipine (Figure 1) is a 1, 4-dihydropyridine-based
calcium antagonist, chemically it is R, S-2 [(2-amino-
ethoxy)methyl]-4-(2-chlorophenyl)-ethoxy carbonyl-5-me-
thoxycarbonyl-6-ethyl-1,4-dihydro pyridine or 2-[(2-ami-
noethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxy cabonyl-
5-methoxycarbonyl-6-methyl-1,4-dihydropyridine. It dif-
fers from other members of this group including the trial
product nifedipine, by the presence at 2-position of a
dihydropyridine ring of a side-chain which carries a basic
amino group [1]. According to Burges and his colleagues
[2] it is the presence of this side-chain with its basic
amino group which is primarily responsible for setting
this particular antagonist apart from other chemically
similar antagonists.
A number of HPLC methods are reported for the de-
termination of amlodipine in literature. Some of them quan-
titate amlodipine in pharmaceutical formulations [3,4],
while the others in human serum [5-11] or in combina-
tion with other formulations [12-15].
H1-receptor antagonists are the mainstay of treatment
for several allergic disorders, particularly rhinitis, conjunc-
tivitis, dermatitis, urticaria and asthma [16,17]. Antihyp-
ertensive drugs and H1-receptor antagonists can be co-
administered in a number of cases. Numerous HPLC
methods were reported for the quantitation of cetirizine
dihydrochloride or fexofenadine hydrochloride with pse-
udoephedrine in combined pharmaceutical dosage forms
[18], a method for cetirizine dihydrochloride and related
impurities is also reported [19]. Simultaneous quantifica-
tion of cetirizine or levocetirizine with cefpirome was
reported by Arayne et al. [20], and buclizine with pyri-
doxine and meclizine were reported by Arayne et al. [21].
The objective of our study was to develop a new method
for the simultaneous determination of amlodipine with
H1-receptor antagonists (cetirizine, fexofenadine hydro-
chloride and buclizine hydrochloride) since these drugs
are co-prescribed at high frequency. The proposed method
*Corresponding author.
opyright © 2012 SciRes. AJAC
Amlodipine Fexofenadine
Cetirizine Buclizine
Figure 1. Chemical structures of amlodipine and H1-receptor antagonists used in the present study.
was successfully applied for the determination of H1-rec-
eptor antagonists in commercial tablets. The method was
validated with respect to linearity, limit of detection and
quantification, precision, accuracy, specificity and applied
in interaction studies.
2. Experimental
2.1. Reagents
Reference standard of amlodipine (Sofvasc®, 5 mg) was
obtained from Wilson’s Pharmaceuticals. Cetirizine (Rigix®,
10 mg), fexofenadine (Fexet®, 60 mg) and buclizine (Lon-
gifene®, 25 mg) were obtained from Ali Gohar Pharma-
ceuticals, UCB Farchim SA, Switzerland, Getz Pharma
Pakistan and Ali Gohar Pharmaceuticals respectively.
Methanol and acetonitrile (HPLC grade) (TEDIA®, USA),
hydrochloric acid (11 N; Merck Marker), glacial acetic
acid, orthophosphoric acid (85%, from Merck Damstabt,
Germany), potassium chloride, potassium dihydrogen or-
thophosphate, disodium hydrogen orthophosphate, sodium
chloride, ammonium chloride, ammonia solution 26% were
obtained from Sigma Aldrich (Germany).
2.2. Preparation of Buffers
Hydrochloric acid (0.1 N) was prepared by diluting 9 mL
hydrochloric acid (36%, 11 N) in a liter volumetric flask
and the volume was made up to the mark with deionized
water. Buffer of pH 4 (chloride buffer) was prepared by
dissolving 3.725 g of potassium chloride in one liter de-
ionized water; pH was adjusted with 0.1 N hydrochloric
acid. For the preparation of buffer of pH 7.4 (chloride
buffer) 3.725 g of KCl was dissolved in 250 mL deion-
ized water and pH was adjusted with 0.1 N HCl. Buffer
of pH 9 (ammonia buffer) was prepared by dissolving
4.98 g of ammonium chloride in 100 mL of deionized wa-
ter and adjusted to pH 9 with 10% ammonia.
2.3. Instrumentation and Chromatographic
A liquid chromatographic system equipped with Shima-
dzu model LC-10AT VP pump, a Shimadzu model SPD-
10AT VP, variable wavelength UV-visible detector was
used. Chromatographic system was integrated via Shima-
dzu model CBM-102 Communication Bus Module to a
Pentium 4 PC. The chosen conditions were: mobile phase
acetonitrile: buffer (0.01 mM) (40:60, v/v), pH adjusted
to 3.0, with flow rate of 1.0 mL·min1. Column used was
Purospher® Star, C18 (5 μm, 250 × 4.6 mm). The absorp-
tion maxima of amlodipine, cetirizine, fexofenadine and
buclizine is 240 nm, 232 nm, 210 nm and 230 nm respec-
tively, however, 240 nm was selected for the quantifica-
tion as all these drugs gave good response at this wave-
2.4. Preparation of Stock and Working Standard
Stock solutions of amlodipine, and H1-receptor antago-
nists (cetirizine, fexofenadine and buclizine) (100 μg·mL1)
were individually prepared freshly by dissolving appro-
priate amount of each reference standard in their respec-
tive mobile phase ratios to yield final drug concentrations.
The stock solutions were diluted with mobile phase to
yield working standard solutions (5 - 50 μg·mL1) for
preparation of calibration curve for each drug.
Copyright © 2012 SciRes. AJAC
2.5. Analysis of Pharmaceutical Dosage Forms
Stock and working standard solutions of pharmaceutical
dosage forms were also prepared by the same procedure
as described above. For pharmaceutical dosage forms,
twenty tablets each of amlodipine 5 mg (Sofvasc®) and
interacting drugs, cetirizine (Rigix®, 10 mg), fexofenadine
(Fexet®, 60 mg) and buclizine (Longifene®, 25 mg) were
weighed and finely powdered in a mortar. An amount
equivalent to drug content in each tablet was weighed,
transferred to a 100 mL volumetric flask, dissolved by stir-
ring for 10 minutes and the final volume made up with
mobile phase. The primary stock solution was filtered thr-
ough 0.45 μm Millipore filter paper and the filtrate was
further diluted to prepare a secondary stock solution. Ali-
quots of the secondary stock solutions were diluted to their
respective concentration and the samples were analyzed
using proposed method.
2.6. Interaction Studies
Stock solutions (100 μg·mL1) of amlodipine, H1-receptor
antagonists (cetirizine, fexofenadine and buclizine) were
prepared in simulated gastric juice, buffers of pH 4, 7.4
and 9 individually. These solutions were mixed in 1:1 ratio
in flasks individually and kept on a water bath at 37˚C
for three hours with stirring. The samples were withdrawn
after 30 minutes time interval for 3 hours and drug con-
tents were filtered through a millipore filter (0.45 μ) and
analyzed by RP-HPLC. The % availability of each drug
was then calculated with respect their standard samples.
3. Results and Discussions
3.1. Linearity, Limit of Detection and
A representative chromatogram of amlodipine with H1-
receptor antagonists (cetirizine, fexofenadine and bucliz-
ine) is shown in Figure 2, indicating complete separation
of all these analytes. To evaluate the linearity of method,
different dilutions in the range 0.5 - 25 μg·mL1 were
analyzed. The limit of detection (LOD) for this assay for
amlodipine, cetirizine, fexofenadine and buclizine was
0.06, 0.12, 0.11 and 0.05 μg·mL1 respectively. While limit
of quantification (LOQ) was found to be 0.22, 0.41, 0.38
and 0.18 μg·mL1 for amlodipine, cetirizine, fexofenadine
and buclizine, respectively (Table 1).
3.2. Precision and Accuracy
To determine accuracy of the method, absolute recover-
ies at three different concentrations of amlodipine and
H1-receptors in placebo of respective dosage forms, were
determined by assaying the samples and comparing peak
areas of sample solution with respective standards. %RSD
was calculated by standard method. These results showed
that the method was precise (%RSD from 0.13% to 1.48%)
and accurate (accuracy from 98.58% to 102.01%). Recov-
ery tests were performed by adding known amounts of
stock solutions to samples with known contents. The per-
centage of recovery was calculated by comparing the de-
termined amount of these standards with the added amo-
unt (Table 2).
3.3. Specificity
No peak of excipients was found in chromatogram when
these drugs were tested in presence of excipients, which
proved that method can be applied successfully to dosage
formulation. Furthermore, the mean % recovery values
obtained also verified that this method could be applied
in pharmaceutical dosage formulations.
Figure 2. Chromatogram showing simultaneous determina-
tion of amlodipine with H1-receptor antagonists.
Table 1. Linear regression functions and their statistical parameters of amlodipine and H1-receptor antagonists.
Analyte Concentration range (μg·mL1) Regression equation r2 LOD
(μg·mL1) LOQ
Amlodipine 0.5 - 25 y = 10171x + 2473.3 0.9984 0.06 0.22
Cetirizine 0.5 - 25 y = 6128.7x 1950.0 0.9995 0.12 0.41
Fexofenadine 0.5 - 25 y = 4984.7x + 12743 0.9988 0.11 0.38
Buclizine 0.5 - 25 y = 14429x 12309.0 0.9996 0.05 0.18
Copyright © 2012 SciRes. AJAC
Table 2. Accuracy and precision of amlodipine and H1-receptor antagonists.
Mean measured
8 8.00 100.00 0.13
10 10.09 100.89 1.35
12 12.24 102.01 1.06
8 7.93 99.15 1.45
10 9.86 98.61 0.74 Cetirizine
12 11.59 98.58 1.07
8 7.95 99.37 0.21
10 10.80 100.08 1.81 Fexofenadine
12 11.84 99.54 1.18
8 7.96 99.55 0.93
10 10.09 100.90 1.48 Buclizine
12 12.05 100.42 1.27
Table 3. In vitro interactions studies of amlodipine with H1-receptor antagonists.
%Avail At pH 1 At pH 4
Time(min) Aml Cet Aml Fexo Aml Buc Aml Cet Aml Fexo Aml Buc
30 70 50 81 79 100 102 55 65 49 53 101 103
60 70 68 84 85 101 103 69 66 76 78 100 100
90 75 71 88 76 100 48 77 75 99 89 102 101
120 79 71 87 84 100 51 79 72 115 101 101 102
150 79 77 81 87 100 67 80 79 120 124 100 101
180 80 74 83 87 100 73 83 70 128 130 100 100
%Avail At pH 7.4 At pH 9
30 49 29 52 79 100 100 61 37 59 59 101 102
60 53 47 65 83 101 100 67 49 66 82 100 100
90 59 61 66 83 99 105 71 75 87 94 100 100
120 71 68 70 83 101 100 74 81 92 127 100 101
150 75 63 73 91 103 108 76 94 107 141 100 100
180 81 101 81 95 106 100 76 98 128 165 100 108
Aml: Amlodipine; Cet: Cetirizine; Fexo: Fexofenadine; Buc: Buclizine; %avail: %Availability.
3.4. Interaction Studies
The validated method as described above was used to mon-
itor drug interactions between amlodipine and H1-receptor
antagonists. These interactions were carried out at phys-
iological pHs, in simulated gastric juice (pH 1), simulated
full stomach (pH 4), blood pH (pH 7.4) and simulating
GI (pH 9). The results of these interactions are given in
Table 3. The % availability of all drugs was 100% at the
zero time point, but with time, the availability changed.
Amlodipine and cetirizine showed slightly reduced %
availability, showing the formation of charge transfer com-
plex of low molar absorptive values in buffers of pH 1, 4,
7.4 and 9. In amlodipine-fexofenadine interaction, a no-
ticeable change in % availability was observed which might
be due to formation of a complex between these two mol-
ecules, showing evidence of an interaction. On the con-
trary, there was no convincing change in availability %
of amlodipine in presence of buclizine, indicative of ab-
sence of an interaction; which is due to lack of electron
donating functionalities in buclizine.
4. Conclusion
We described the simultaneous determination of amlodip-
ine with H1-receptor antagonists in pharmaceutical dos-
age forms. The method was linear in the concentration
range of (5 - 50 μg·mL1).The method was applied to
study the interactions. The interaction results showed that
in most cases the availability of amlodipine was decreased
Copyright © 2012 SciRes. AJAC
in the presence of H1-receptor antagonists. This suggested
that for coadministration of amlodipine with H1-receptor
antagonists, a proper interval should be given to avoid such
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