Simultaneous Determination of Amlodipine with H<sub>1</sub>-Receptor Antagonists by Reversed Phase High Performance Liquid Chromatography and Application to Interaction Studies

doi:10.4236/ajac.2012.39082

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American Journal of Analytical Chemistry, 2012, 3, 632-637

http://dx.doi.org/10.4236/ajac.2012.39082 Published Online September 2012 (http://www.SciRP.org/journal/ajac)

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: *[email protected]

Received May 29, 2012; revised July 12, 2012; accepted July 27, 2012

ABSTRACT

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·min−1 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acting drugs were heated at 37˚C with intermittent shaking and the samples were withdrawn every thirty minutes for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

Chromatography

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.

M. S. ARAYNE ET AL. 633

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

Conditions

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·min−1. 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-

length.

2.4. Preparation of Stock and Working Standard

Solutions

Stock solutions of amlodipine, and H1-receptor antago-

nists (cetirizine, fexofenadine and buclizine) (100 μg·mL−1)

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·mL−1) for

preparation of calibration curve for each drug.

M. S. ARAYNE ET AL.

634

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·mL−1) 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

Quantification

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·mL−1 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·mL−1 respectively. While limit

of quantification (LOQ) was found to be 0.22, 0.41, 0.38

and 0.18 μg·mL−1 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·mL−1) Regression equation r2 LOD

(μg·mL−1) LOQ

(μg·mL−1)

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

M. S. ARAYNE ET AL. 635

Table 2. Accuracy and precision of amlodipine and H1-receptor antagonists.

Analyte

Spiked

concentration

(μg·mL−1)

Mean measured

concentration

(μg·mL−1)

Accuracy

Precision

%RSD

8 8.00 100.00 0.13

10 10.09 100.89 1.35

Amlodipine

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·mL−1).The method was applied to

study the interactions. The interaction results showed that

in most cases the availability of amlodipine was decreased

M. S. ARAYNE ET AL.

636

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

interactions.

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