Acta Chromatographica 25(2013)1, 59-77

DOI: 10.1556/AChrom.25.2013.1.3

Chemometrically Optimized Micellar Liquid

Chromatographic Method for the Simultaneous
Determination of Cetirizine Dihydrochloride in
its Combined Dosage Forms. Application to
Biological Fluids and Pharmacokinetic Studies
M.K. SHARAF EL-DIN, F. IBRAHIM, M.I. EID, AND M.E.K. WAHBA*
Department of Analytical Chemistry, Faculty of Pharmacy,
Mansoura University, Mansoura, 35516, Egypt
E-mail: marywahba2004@yahoo.com

Abstract. An accurate, simple, sensitive, and selective micellar liquid chromatographic

method has been developed for the simultaneous determination of cetirizine dihydro-
chloride in two of its combined pharmaceutical preparations with psudoephedrine hy-
drochloride and/or paracetamol. The chemometrics approach was applied for the opti-
mization of separation of the studied drugs to optimize their separation; the effect of six
experimental parameters on retention was investigated by means of multivariate analy-
sis. Separation was conducted using an ODS C18 (150 × 4.6 mm id) stainless steel column
at ambient temperature with UV-detection at 250 nm. A mobile phase composed of 0.135
M sodium dodecyl sulphate, 11% 1-propanol, 0.3% tri-ethylamine in 0.02 M phosphoric
acid, and adjusted to pH 3.3 has been used at a flow rate of 1 mL/min. Regression mod-
els were characterized by both descriptive and predictive ability (R2 ≥ 98.8% and R2cv ≥
94.5%) and allowed the chromatographic separation of the drugs with a good resolution
and a total analysis time within 6 min.
The calibration curves were rectilinear over the concentration ranges of 0.05–1.0,
0.07–4.0, and 1.0–10.0 μg/mL for cetirizine dihydrochloride, psudoephedrine hydrochlo-
ride, and paracetamol respectively; with detection limits of 0.01, 0.05, and 0.08 μg/mL,
and quantification limits of 0.04, 0.09, and 1.02 μg/mL, respectively. The results obtained
were in good agreement with those obtained by the comparison method. Furthermore,
the method was applied for the determination of the drugs in spiked human plasma and
used to reveal the pharmacokinetic characters in a healthy volunteer treated with oral
administration of the studied two combined dosage forms of cetirizine.

Key Words: micellar liquid chromatography, chemometric optimization, cetirizine, psu-

doephedrine, paracetamol, pharmacokinetics

Introduction
Most cold medicines contain multiple active ingredients that include anti-
pyretics, analgesics, antitussive agents, mucolytic agents, bronchodilators,
antihistamines, and several vitamins. Combinations of these compounds
were analyzed using RP-HPLC procedures. However, as it is difficult to
0231–2522 © 2012 Akadémiai Kiadó, Budapest

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60 M.K. Sharaf El-Din et al.

analyze simultaneously many different kinds of ingredients using a single

method, ingredients were often divided into several groups based on their
chemical properties, i.e., cationic compounds in one run and anionic or neu-
tral compounds in another run. Gradient elution was also required for si-
multaneous analyses [1].
Cetirizine dihydrochloride (CTZ), (±)-[2-[4-[(4-chlorophenyl) phenyl-
methyl]-1-piperazinyl]ethoxy]acetic acid [2] a piperazine derivative and me-
tabolite of hydroxyzine, is described as a long-acting non-sedating antihis-
tamine with some mast-cell stabilizing activity. It appears to have a low po-
tential for drowsiness in usual doses and to be virtually free of antimus-
carinic activity. It is used for the symptomatic relief of allergic conditions
including rhinitis and chronic urticaria [3].
Pseudoephedrine (PSU) (1S,2S)-2-(Methylamino)-1-phenylpropan-1-ol
hydrochloride [2] is a direct- and indirect-acting sympathomimetic. It is a
stereoisomer of ephedrine and has a similar action, but has been stated to
have fewer CNS effects. Pseudoephedrine is given by mouth for the symp-
tomatic relief of nasal congestion, it is commonly combined with other in-
gredients in preparations intended for the relief of cough and cold symp-
toms [3].
Paracetamol (PCA), N-(4-Hydroxyphenyl) acetamide [2], has analgesic
and antipyretic properties and weak anti-inflammatory activity [2]. The
chemical structure of the three drugs is presented in Fig. 1.

a)
CTZ
N OH

N O
Cl O

OH H
N
CH3

O
HN OH

PSU CH3 PCA

b) c)

Fig. 1. Structural formula of a) CTZ, b) PSU, c) PCA

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Simultaneous Determination of Cetirizine Dihydrochloride in its Combined Dosage Forms 61

Micellar liquid chromatography is an alternative to conventional

HPLC. The solubilizing ability of micelles is one of the most important
properties of this technique, and it allows direct injection of untreated sam-
ples, including biological fluids like serum and urine.
Several HPLC methods have been reported in literature for the deter-
mination of PCA with CTZ [4, 5], PCA with PSU [6–9], and CTZ with PSU
[10–12]. The three drugs were also determined in the presence of their deg-
radation products [13].
However, literature search revealed no report of any analytical method
for simultaneous determination of the three drugs as multi-component
preparations in biological fluids. Even the only reported method for their
simultaneous determination [13] applied a tedious and complicated gradi-
ent elution with relatively expensive solvents. Therefore, the present study
was involved in a research effort aimed at developing and validating a sim-
ple, selective, and accurate new micellar liquid chromatographic method for
the simultaneous determination of CTZ, PSU, and PCA in their combined
dosage form and in biological fluids.
The optimization of the experimental conditions is a complicated proc-
ess in MLC due to the large number of the variables, which must be simul-
taneously treated. These variables include pH of the mobile phases, concen-
tration of surfactant, type, and volume fraction of organic modifier, flow
rate, and column type. Variations of the experimental conditions affect both
the retention of the analytes and the extent of the surfactant monomers ad-
sorbed onto the stationary phase. As a result, the effect of these factors on
retention can be interdependent and nonlinear [14–16]. Therefore, methods
of multivariate analysis are usually used.
In order to optimize the time of the chromatographic analysis and the
resolution, chemometric methods of experimental design, multivariate
analysis, and multi-criteria decision-making (Pareto-optimality) [17] were
employed. The six experimental variables considered were SDS concentra-
tion; the organic modifier concentration, Cm; the alkyl chain length of or-
ganic modifier, N; pH of the mobile phase, flow rate Fl, and type of column.
The experiments for the optimization were performed according to the face
centered cube response surface experimental design. Subsequently, the method
of stepwise multiple linear regression (MLR) was used to select the most
important effects and to calculate the coefficients relating the effects to re-
tention time. The pareto-optimal approach was used for the evaluation of
the optimal points. To the best of our knowledge, no chemometric treatment
has been performed concerning the simultaneous study of six chroma-
tographic factors in micellar liquid chromatographic system for these com-
pounds.

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62 M.K. Sharaf El-Din et al.

Experimental
Materials and Reagents
• Cetirizine dihydrochloride (CTZ); of purity 99.87% was kindly pro-
vided by Pharco Pharmaceutical Company, Alexandria, Egypt.
• Psudoephedrine hydrochloride (PSU); of purity 99.65% as deter-
mined by the official method [2] was kindly provided by Sigma
Pharmaceutical Company, Cairo, Egypt.
• Paracetamol (PCA); of purity 100.12% as determined by the official
method [2] was kindly provided by Sedico Pharmaceutical Com-
pany, Cairo, Egypt.
• Ebastine (EBS) internal standard; of purity 99.94% was kindly pro-
vided by Meivo Pharmaceutical Company, Cairo, Egypt.
• Sodium dodecyl sulphate SDS (Sigma-Aldrich, Munich, Germany).
• 1-Propanol, methanol and acetonitrile (Sigma-Aldrich, Munich,
Germany), HPLC grade.
• Triethylamine (Riedel-deHäen, Sleeze, Germany), HPLC grade.
• Orthophosphoric acid (85% w/v); Prolabo, Fontenay-sous-Bois,
France.
• 1-Butanol and tetrahydrofuran (Merck, Darmstadt, Germany),
HPLC grade.
• Pharmaceutical preparations:
* Allercet cold® capsules (Batch #820834), labeled to contain 10 mg
Cetirizine dihydrochloride , 30 mg pseudoephedrine hydrochloride and 400
mg paracetamol/capsule, Global Napi Pharmaceuticals, 6th October city,
Giza, Egypt.
* Clearest® capsules (Batch #90380A), labeled to contain 5mg Cetiriz-
ine dihydrochloride and 120 mg pseudoephedrine hydrochloride/capsule,
ChemiPharm Pharmaceutical Industries, S.A.E. 6th Ocober, Egypt.
All were obtained from commercial sources in the local market.

Apparatus
• Separation was performed with a Shimadzu C-R6A Chromatopac
equipped with a Rheodyne injector valve with a 20-μL loop and a
UV/VIS detector.
• A Shimadzu UV 1601 PC Spectrophotometer equipped with a pair
of 1 cm matched cells, recording range: 0–2; wavelength: 200–400
nm; factor: 1; number of cells: 1; cycle time: 0.1 min was used.

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Simultaneous Determination of Cetirizine Dihydrochloride in its Combined Dosage Forms 63

• Software for chemometric study: all computations were performed

in Matlab for Windows™ version 6.5 Mathworks Inc., 2002 (MA,
USA). The (PLS) procedure was taken from PLS Toolbox 2.1, Eigen-
vector Research, Inc.

Columns and Mobile Phases

Separation was achieved on an EC nucleosil C18-SN: 4115568 column (150
mm × 4.6 mm id (5 μm)) combined with a guard column (Merck, Darm-
stadt, Germany). The columns were operated at ambient temperature. The
analytical system was washed daily with 60 mL of 1:1 mixture of water and
methanol to eliminate the mobile phase, this did not cause any change in
the column performance. The mobile phase was prepared by mixing 0.135
M SDS, 11% 1-propanol, and 0.3% tri-ethylamine in 0.02 M phosphoric acid,
and adjusting the pH to 3.3. The mixture was then sonicated for 30 min. The
resulting mobile phase was filtered through a 0.45-μm membrane filter (Mil-
lipore, Ireland).

Standard solutions
A stock solution containing either 1.0 mg/mL of CTZ, PSU, or PCA was
prepared in methanol and further diluted with the mobile phase. These so-
lutions were found to be stable for at least two weeks when kept in the re-
frigerator.
A stock solution containing 1.0 mg/mL of EBS internal standard was
prepared in methanol and further diluted with the mobile phase to obtain a
final concentration of 5.0 μg/mL.

Calibration Curve
Aliquots of CTZ, PSU, and PCA standard solutions covering the working
concentration ranges of 0.05–1.0, 0.07–4.0, and 1.0–10.0 μg/mL, respectively,
were transferred into a series of 10 mL volumetric flasks, mixed with 5
μg/mL of EBS internal standard, and diluted with the mobile phase to the
mark. Twenty-microliter aliquots were injected (in triplicates) and eluted
with the mobile phase under the reported chromatographic conditions. The
calibration curves were constructed by plotting the peak area ratio against
the final concentration of the drug (μg/mL). Alternatively, the correspond-
ing regression equation was derived.

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64 M.K. Sharaf El-Din et al.

Procedure for Analysis of Synthetic Mixtures

Aliquots of CTZ, PSU, and PCA standard solutions in the ratio of 1:3:40 or
aliquots of CTZ and PSU in the ratio of 1:24, respectively, were transferred
into a series of 10 mL volumetric flasks, mixed with 5 μg/mL of EBS (IS),
and diluted with the mobile phase to the mark. Then, the steps described
under “Calibration Curve” were preceded. The percentage recoveries were
calculated by referring to the calibration graphs previously prepared or by
applying the regression equations.

Procedure for Analysis of Co-Formulated Capsules

An accurately weighed quantity of the mixed capsular content of either Al-
lercet cold® capsules equivalent to 5 mg CTZ , 15 mg PSU and 200 mg PCA,
or Clearest® capsules equivalent to 5 mg CTZ and 120 mg PSU was trans-
ferred into a small conical flask, and extracted three successive times each
with 30 mL of methanol. The extract was filtered into 100 mL volumetric
flask. The conical flask was washed with few milliliters of methanol, the
wash was added to the filterate and completed to the mark with the same
solvent. The procedure was followed as described under “Calibration
Curve”. The nominal contents of the capsules were calculated using either
the calibration graphs or the corresponding regression equations.

Procedure for Spiked Human Plasma

One-milliliter aliquots of human plasma were transferred into a series of 10
mL volumetric flasks, spiked with increasing quantities of CTZ, PSU, or
PCA to give a final concentration range of 50–190, 70–400, and 1000–8000
ng/mL, respectively. The solutions were mixed with 5 μg/mL of EBS, and
diluted with the mobile phase to the mark. Then, the solutions were filtered
through Millipore filter. The procedure described under “Calibration
Curve” was followed. The nominal content of the drugs in plasma was de-
termined using the corresponding regression equations.

Procedure for Pharmacokinetic Study

The assay was applied to Phase-I pharmaceutics study in a healthy adult
female volunteer (35 years old), who received oral administration [10]. After
an overnight fast of 10 h, the volunteer took Allercet cold® orally with 200
mL of water. Regular standardized low-fat meals were not provided until

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Simultaneous Determination of Cetirizine Dihydrochloride in its Combined Dosage Forms 65

4 h after dose administration; water intake was allowed after 2 h. Following

the drug administration, venous blood samples (5 mL) were collected into
heparinized tubes according to the following schedule: immediately before
administration and 15, 30, 45, 60, 120, 150, 180, 210 min, 4, 6, 10, 15, 20 and
24 h after dosing. Blood samples were centrifuged at 1500g for 10 min to ob-
tain the plasma. The plasma samples were labeled and kept frozen at −20 °C
until analysis. Pharmacokinetic parameters were determined from the
plasma concentration–time curve.
Similar procedures were performed to follow the pharmacokinetics of
CTZ and PSU where Clearest® capsules was administered to the volunteer,
after which the steps described under “Procedure for spiked human
plasma” were performed.

Results and Discussion

The proposed liquid chromatographic method permits the separation and
quantitation of CTZ, PSU and/or PCA in their combined dosage forms and
in biological fluids. Under the described chromatographic conditions, clear
base line separation with satisfactory resolution between the peaks was
achieved in a short chromatographic run, less than 10 min. The proposed
method was assessed for selectivity, linearity, precision, accuracy, stability,
and recovery.
The different experimental parameters affecting the separation selectiv-
ity of the liquid chromatographic system have been investigated and opti-
mized. Hence, the method was applied to the determination of the studied
drugs in their combined capsules, and further for pharmacokinetic study.

Optimization of the Chromatographic Conditions

Well-defined symmetrical peaks with maximum separation parameters
were obtained upon measuring the response of the eluent under the per-
formance parameters after thorough experimental trials by using different
concentrations of SDS, various types of organic modifiers, different concen-
trations of 1-propanol, different pH ranges, several columns, and different
flow rates. After detailed investigation, separation of the studied drugs was
achieved using a mobile phase consisting of 0.135 M SDS, 11% 1-propanol
and 0.3% tri-ethylamine in 0.02 M phosphoric acid, at pH of 3.3.

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66 M.K. Sharaf El-Din et al.

Selection of Detector Wavelength

The UV detector response was studied from the range of 200–350 nm, and
the best wavelength was found to be 250 nm showing highest sensitivity
and appreciable absorbance of the studied drugs (Fig. 2).

0.80 ABS Smooth: 0 Deri.: 0

0.75 C
0.70

0.65

0.60

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15
A B
0.10

0.05

0.00 nm
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

Fig. 2 Absorption spectra of: A) 0.125 μg/mL CTZ, B) 0.375 μg/mL PSU,
C) 5.0 μg/mL PCA

Factorial Design
The experiments used for modeling and optimization of the studied drugs
were performed on three levels for each of the six factors using the face cen-
tered cube response surface experimental design. This design is one of the
experimental designs suitable for modeling and optimization. The feasible
region of the selected chromatographic factors in which the experimental
optimization could be carried out is listed in Table I. A full factorial design
for six variables and two levels would require 64 experiments. To reduce
the number of experiments, a two-level fractional experimental design con-
sisting of 26−3 experiments was used. This reduced design allows the first es-
timation of the principal factor effects confounded with the second-order in-
teraction; however, this leads to a partial loss of information. The experi-
ments 3–10 in Table II show fractional factorial design. The experiment in the

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Simultaneous Determination of Cetirizine Dihydrochloride in its Combined Dosage Forms 67

Table I. The six chromatographic factors and the corresponding three-level settings

Level pH [SDS] N Cm Flow rate Column type

– 3 0.075 1 6 0.6 C8
0 4.5 0.15 3 10 1 C18
+ 6 0.2 4 15 1.15 Phenyl column

Table II. Experimental conditions for central experiment replications, fractional factorial
design, fold-over, and star design

Flow Column
Experiment Design pH N [SDS] Cm
rate type
1 0 0 0 0 0 0
Central replication
2 0 0 0 0 0 0
3 −1 −1 −1 +1 +1 +1
4 +1 −1 −1 −1 −1 +1
5 -1 +1 −1 −1 +1 −1
6 Fractional factorial +1 +1 −1 +1 −1 −1
7 design −1 −1 +1 +1 -1 −1
8 +1 −1 +1 −1 +1 −1
9 −1 +1 +1 −1 −1 +1
10 +1 +1 +1 +1 +1 +1
11 0 0 0 0 0 0
Central replication
12 0 0 0 0 0 0
13 −1 −1 −1 −1 −1 −1
14 +1 −1 −1 +1 +1 −1
15 −1 +1 −1 +1 −1 +1
16 +1 +1 −1 −1 +1 +1
Fold over
17 −1 −1 +1 −1 +1 +1
18 +1 −1 +1 +1 −1 +1
19 −1 +1 +1 +1 +1 −1
20 +1 +1 +1 −1 −1 −1
21 Central replication 0 0 0 0 0 0
22 −1 0 0 0 0 0
23 +1 0 0 0 0 0
24 0 −1 0 0 0 0
25 0 +1 0 0 0 0
26 0 0 −1 0 0 0
27 0 0 +1 0 0 0
Star design
28 0 0 0 −1 0 0
29 0 0 0 +1 0 0
30 0 0 0 0 −1 0
31 0 0 0 0 +1 0
32 0 0 0 0 0 −1
33 0 0 0 0 0 +1
34 Central replication 0 0 0 0 0 0

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 68 M.K. Sharaf El-Din et al.

central point (experiments 1, 2, 11, 12, 21, and 34) was replicated to estimate
the pure experimental error and to check system reproducibility. The analy-
sis of these results showed that the retention of all the analytes is largely af-
fected by pH of the mobile phase and SDS concentration. A comparison be-
tween the experimental and predicted separation parameters is shown in
Table III.

Table III. A comparison of predicted and experimental retention data for optimized
separation of the studied compounds

Studied
CTZ PCA PSU
drug
Mathlab Mathlab Mathlab
Parameter Experimental Experimental Experimental
prediction prediction prediction
Rs 6.34 6.12 2.85 2.96 5.43 5.21
K’ 4.42 4.25 1.61 1.82 3.16 3.25
NTP 1588 1570 1260 1250 1423 1419
HETP 0.095 0. 096 0. 119 0. 12 0. 105 0.106

Fold-Over Fractional Factorial Design

For better understanding of the typical factors which govern the retention, a
complementary set of experiments as fold-over design was performed
(Table II, experiments 13–20). This represents a fold-over of the previous de-
sign and is obtained by changing the sign of the three last columns of the
fractional design. Fold-over design is used to separate the principal effects
from the confounded second-order interactions. The combined results (fold-
over and fractional factorial design) confirmed the previously observed re-
sults. For all the analytes, the largest effects on retention time (tR) were due
to pH and SDS concentration. Slight effect was observed due to the other
main factors.

Star Design
The existence of quadratic (or higher) significant effects was tested by
means of F-test that compares the difference between the experimental re-
tention time in the central point and factorial design [18]. The equation for
F-test is according to the following equation:

(y − y ) .
2
− −
0 f
F= 2
Spe ( 1/n − 1/n )
0 f

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Simultaneous Determination of Cetirizine Dihydrochloride in its Combined Dosage Forms 69

In this equation, y 0− , y −f , no, and nf are average response of the replicated

central, average response of the factorial design experiments, number of ex-
periment in the central point, and number of experiment in the factorial de-
2
sign, respectively. The Spe is the purely experimental variance. Since, for all
the compounds, the calculated F-values were greater than the critical F-
value, it was concluded that the quadratic (or higher) effects must be used
in the regression models. For this purpose, 13 experiments of a star design
(experiments 22–33 in Table II) were added to the factorial experimental de-
sign to provide a composite design. The best regression models were ob-
2
tained by a variable selection algorithm. The final models showed Rcv ≥
94.5% and R ≥ 98.8% clarifying the fitting of these models and that the
2

model prediction ability is quite satisfactory. In order to optimize the sepa-

ration of the studied drugs, a grid search program was applied. In this pro-
gram, the predicted retention times of all analytes are available for every
mobile phase composition within the feasible factor space.

Analytical Performance and Applications

The proposed HPLC method was tested for linearity, selectivity, accuracy,
and precision.

Sensitivity
Detection limit (LOD) is the lowest concentration of the drug that can be de-
tected, but not necessarily quantitated, under the stated experimental condi-
tions. The limit of detection is generally quoted as the concentration yield-
ing a signal-to-noise ratio of 3:1 [19] and is confirmed by analyzing a num-
ber of samples near this value using the following equation:

The signal to noise ratio = H/h

where H = height of the spectrum corresponding to the drug; h = absolute

value of the largest noise fluctuation from the baseline of the spectrum of a
blank solution.
While the limit of quantification (LOQ) is the lowest concentration of
the analyte that can be determined with acceptable precision and accuracy,
it is quoted as the concentration yielding a signal-to-noise ratio of 10:1 and
is confirmed by analyzing a number of samples near this value [19]. The
calculated values are listed in Table IV.

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 70 M.K. Sharaf El-Din et al.

Table IV. Performance and validation data of the proposed HPLC method

Value
Parameter
CTZ PSU PCA
Concentration range (μg/mL) 0.05–1.0 0.07–4.0 1.0–10.0
LOD (μg/mL) 0.01 0.05 0.08
LOQ (μg/mL) 0.04 0.09 1.02
Correlation coefficient (r) 0.9995 0.9999 0.9999
Slope 0.877 0.292 0.123
Intercept −0.0101 −1.19 × 10 −2 4.63 × 10−2
Sy/x, S.D. of the residuals 7.04 × 10−3 1.29 × 10−2 1.84 × 10−4
Sa, S.D. of the intercept of the regression line 2.36 × 10 −3 3.95 × 10 −3 1.26 × 10−4
Sb, S.D of the slope of the regression line 7.23 × 10−3 2.69 × 10−3 2.02 × 10−5
Validation parameters for determination of the studied drugs in pure and dosage forms
Preparation Repeatability, % found Intermediate precision, % found
CTZ pure
(0.5 μg/mL) (1.0 μg/mL)
form
X–± SD 100.62 ± 0.37 99.67 ± 0.58
PSU pure
(0.2 μg/mL) (3.0 μg/mL)
form
X– ± SD 100.45 ± 0.23 100.16 ± 0.83
PCA pure
(2.0 µg/mL) (5.0 μg/mL)
form
X–± SD 100.34 ± 0.59 99.56 ± 0.48
Allercet® CTZ 0.1 PSU 0.3 PCA 4.0 CTZ 0.2 PSU 0.6 PCA 8.0
tablets μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL
X–± SD 100.35 ± 0.13 100.42 ± 0.47 99.98 ± 0.46 99.29 ± 0.22 99.74 ± 0.86 99.77 ± 0.31
Clearest® PSU (2.4 PSU (3.6
CTZ (0.1 μg/mL) CTZ (0.15 μg/mL)
tablets μg/mL) μg/mL)
X– ± SD 100.45 ± 0.36 100.47 ± 0.12 99.67 ± 0.56 100.33 ± 0.64

Validation of the Method

The method was tested for linearity, accuracy, precision, and robustness ac-
cording to ICH guidelines [20]. By using the above liquid chromatographic
method, linear regression equations were obtained. The regression plots
showed that there was a linear dependence of the peak area ratios on the
concentrations of the drugs over the working concentration ranges. The va-
lidity of the proposed method was evaluated by statistical analysis of the
regression data (Table IV) regarding the standard deviation of the residuals

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Simultaneous Determination of Cetirizine Dihydrochloride in its Combined Dosage Forms 71

(Sy/x), the standard deviation of the intercept (Sa), and standard deviation of
the slope (Sb) [21]. The small values of the figures point to the low scattering
of the points around the calibration graph and high precision of the pro-
posed method.

Linearity
The linearity of an analytical procedure is its ability (within a given range)
to obtain test results which are directly proportional to the concentration of
analyte in the sample [20]. Linearity of the proposed method was assessed
by estimating the linear dependence of the obtained peak area ratios on the
concentrations of the drugs.
The peak area ratios of CTZ, PSU, and PCA varied linearly with the
concentration over the ranges mentioned in Table IV.

Accuracy

The accuracy of an analytical procedure expresses the closeness of agree-

ment between an accepted reference value and the value found [20]. The ac-
curacy of the proposed method was evaluated by analyzing standard solu-
tions of the studied drugs. The results obtained by the proposed method
were favorably compared with those obtained by the comparison method
[13].
Statistical analysis [21] of the results obtained by the proposed and
comparison methods using Student’s t-test and variance ratio F-test showed
no significant difference between the performance of the two methods, since
%recovery ± SD for CTZ, PSU, and PCA were found to be 100.09 ± 0.71,
100.08 ± 0.61, and 99.97 ± 0.53, respectively, with corresponding t values of
0.72, 0.64, and 0.43 and F values of 1.03, 1.11, and 1.08, keeping in mind that
tabulated t and F values are 1.83 and 19.38, respectively, at P = 0.05 [21].

Precision
The precision of an analytical procedure expresses the closeness of agree-
ment between a series of measurements obtained from multiple sampling of
the same homogeneous sample under the prescribed conditions. Precision
may be considered through repeatability and intermediate precision [20].

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72 M.K. Sharaf El-Din et al.

Repeatability
Repeatability expresses the precision under the same operating conditions
over a short interval of time. Repeatability is also termed intra-assay preci-
sion [20]. The repeatability was evaluated through the replicate analysis of
different concentrations of the drugs, either in pure or in dosage forms, the
mean percentage recoveries based on the average of three separate determi-
nations for pure and dosage forms are abridged in Table IV.

Intermediate Precision
Intermediate precision expresses within-laboratories variations: different
days, different analysts, different equipment, etc. [20]. It was performed
through replicate analysis of different concentrations of the drugs, either in
pure or dosage form on three successive days. The percentage recoveries are
based on the average of four separate determinations. The results are shown
in Table IV.

Robustness of the Method

The robustness of an analytical procedure is a measure of its capacity to re-
main unaffected by small, but deliberate variations in method parameters
and provides an indication of its reliability during normal usage [20]. The
robustness of the adopted method was demonstrated by the consistency of
the values of the peak area ratios with the deliberately minor changes in the
experimental parameters such as, 0.12–0.15 M of the surfactant, 10–12% of
1-propanol and pH 3–3.5 which did not greatly affect the peak area ratios.

Pharmaceutical Applications
Dosage Forms Analysis

The proposed method was successfully applied to the determination of CTZ

in its combined dosage forms. Typical chromatograms for the separation of
CTZ in Allercet cold® and Clearest® capsules are presented in Fig. 3. The av-
erage percentages found of different concentrations were based on the aver-
age of three replicate determinations. The results shown in Table V are in
good agreement with the comparison methods [10, 13].

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 Simultaneous Determination of Cetirizine Dihydrochloride in its Combined Dosage Forms 73

Table V. Determination of the studied drugs in their dosage forms using the proposed
method
Amount taken Amount found Comparison method [13],
Parameter % Recovery
μg/mL μg/mL % Recovery
CTZ PSU PCA CTZ PSU PCA CTZ PSU PCA CTZ PSU PCA
0.05 0.15 2.0 0.0496 0.1489 1.987 99.23 99.25 99.35 100.25 100.62 100.48
0.07 0.21 2.8 0.0697 0.2086 2.8229 99.56 99.35 100.82 99.58 100.48 99.58
0.09 0.27 3.6 0.0894 0.2685 3.6331 99.34 99.45 100.92 99.14 99.32 100.22
0.1 0.3 4.0 0.1005 0.3025 4.0288 100.54 100.84 100.72
Allercet®
0.12 0.36 4.8 0.1208 0.3606 4.8158 100.69 100.18 100.33
capsules
0.14 0.42 5.6 0.1406 0.4227 5.5619 100.44 100.64 99.32
0.16 0.48 6.4 0.1611 0.4811 6.3456 100.65 100.22 99.15
0.18 0.54 7.2 0.1807 0.5444 7.1798 100.39 100.82 99.72
0.2 0.6 8.0 0.1997 0.6022 7.9464 99.86 100.37 99.33
0.25 0.75 10.0 0.2491 0.7559 10.042 99.64 100.78 100.42
100.03 ± 100.19 ± 100.01 ± 99.66± 100.14± 100.09±
X¯ ± SD
0.57 0.63 0.71 0.56 0.71 0.46
t-test *0.59 0.08 0.76
F-test 1.04 1.27 2.38
Comparison method [10], %
0.05 1.2 0.0495 1.1953 99.06 99.61
Recovery
0.06 1.4 0.0599 1.3881 99.85 99.15 100.58 99.45
Clearest® 0.08 1.9 0.0795 1.8873 99.35 99.33 99.24 99.34
capsules 0.1 2.4 0.1008 2.4137 100.82 100.57 100.33 100.88
0.12 2.9 0.1204 2.9218 100.34 100.75
0.15 3.6 0.1527 3.6277 100.46 100.77
0.17 4.0 0.1714 4.0128 100.85 100.32
100.11± 100.07± 100.05± 99.89±
X¯ ± SD
0.71 0.69 0.71 0.86
t-test 0.22** 0.32
F-test 1.05 1.55

*1.83 and 19.38 are tabulated t and F values at P = 0.05 [21]

**1.94 and 19.38 are tabulated t and F values at P = 0.05 [21]

b
b e
d
4.087

6.094

a
6.142
2.742

0.775

c c
a d
5.114
0.815

5.057
4.112

Fig. 3a. Typical chromatogram of Fig. 3b. Typical chromatogram of CTZ:

CTZ:PSU:PCA in Allercet cold® capsules PSU in Clearest® capsules extract where:
extract where: a) Solvent front, a) Solvent front, b) 2.4 μg/mL PSU,
b) 8.0 μg/mL PCA, c) 0.6 μg/mL PSU, c) 0.1 μg/mL CTZ, d) 5.0 μg/mL EBS (I.S)
d) 0.2 μg/mL CTZ, e) 5.0 μg/mL EBS (I.S)

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 74 M.K. Sharaf El-Din et al.

Application to Spiked and Real Human Plasma

It has been reported that analysis of plasma samples following administra-

tion of an oral tablet formulation composed of 5 mg CTZ and 120 mg PSU to
healthy volunteers provided maximum concentrations of 167 ± 47, 350.36 ±
57.33 ng/mL reached within 0.7 ± 0.5, 4 ± 0.8 h and having t1/2 value of 6.76
± 1.82 h and 5.37 ± 0.52 for the two drugs, respectively [11].
Another study concerned with the pharmacokinetics of PCA men-
tioned that 7.56 ± 2.18 μg/mL is the maximum plasma concentration at-
tained within 0.77±0.45 h, and half life time of 2.91±0.45 h [22].
These values lie within the working concentration range of the pro-
posed method, thus it could be successfully applied to the determination of
the studied drugs in spiked human plasma over the working concentration
range. The within-day precision was evaluated through replicate analysis of
the drugs on three successive times, where the mean percentage recoveries
were based on the average of 3 separate determinations. The inter-day pre-
cision was evaluated through replicate analysis of plasma samples spiked
with 120, 200, and 4000 ng/mL of CTZ, PSU, and PCA, respectively, on
three successive days. The mean percentage recoveries based on the average
of 3 separate determinations are summarized in Table VI.

Table VI. Application of the proposed method to the determination of the studied drugs
in spiked human plasma

Amount taken Amount found

% Recovery
Parameter (ng/mL) (ng/mL)

CTZ PSU PCA CTZ PSU PCA CTZ PSU PCA

50 70 1000 48.63 67.54 964 97.25 96.48 96.44

Intra-day precision

70 100 2000 68.22 97.62 1925 97.45 97.62 96.25

100 150 3000 98.25 146.18 2927 98.25 97.45 97.58
120 200 4000 121.99 197.22 3955 101.66 98.61 98.88
140 250 6000 142.08 247.71 5944 101.48 99.08 99.07
170 300 7000 168.56 303.99 6821 99.15 101.33 97.45
190 400 8000 185.55 407.68 7860 97.66 101.92 98.25
X¯ ± SD 99.21 ± 1.87 98.93 ± 2.03 97.71 ± 1.11
Inter-day precision
1st day 120 200 4000 96.25 96.78 98.51
2 day
nd 120 200 4000 98.45 97.68 96.15
3rd day 120 200 4000 97.55 97.19 97.34
X¯ ± SD 97.42 ± 1.11 97.22 ± 0.45 97.33 ± 1.18

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Simultaneous Determination of Cetirizine Dihydrochloride in its Combined Dosage Forms 75

The aforementioned reported pharmacokinetic data are in accordance

with the results of the proposed method as illustrated from the pharma-
cokinetic parameters in Table VII.

Table VII. Pharmacokinetic parameters of the studied drugs applying the proposed
method

Dosage form Studied drug Parameter

Cmax
Tmax (h) t1/2(h) AUC
(ng/mL)

Allercet® tablets CTZ 190 0.75 6 991

PSU 400 3.5 6 3556

PCA 8000 0.5 3.5 35970

CTZ 220 1 7 1686

Clearest® tablets
PSU 390 3 6 2848

Typical chromatograms for the determination of CTZ in human plasma

after oral administration of its combined dosage forms Allercet cold® and
Clearest® capsules are presented in Figs. 4 and 5.

a e
b
0.758
2.748

0.789
6.017

cd
4.117
5.121

Fig. 4a. Typical chromatogram of CTZ, Fig. 4b. Chromatogram of drugs free
PSU, and PCA in human plasma after 1 h plasma
of administration of Allercet cold®
capsule where: a) Solvent front, b) PCA,
c) PSU, d) CTZ, e) EBS (I.S)

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76 M.K. Sharaf El-Din et al.

6.118

0.882
a
c
0.822

5.019

b
4.012

Fig. 5a. Typical chromatogram of CTZ Fig. 5b. Chromatogram of drugs free
and PSU in human plasma after 1 h of plasma
administration of Clearest ® capsule
where: a) Solvent front, b) PSU, c) CTZ,
d) EBS (I.S).

Conclusion
An accurate, simple, sensitive and selective micellar liquid chromatographic
method has been developed for the simultaneous determination of CTZ in
two of its combined pharmaceutical preparations with PSU and/or PCA.
The results obtained were in good agreement with those obtained by the
comparison method. The chemometrics approach was applied for the opti-
mization of separation of the studied drugs, by studying the effect of six ex-
perimental parameters on retention applying multivariate analysis. The
method was also applied to determine the drugs in spiked human plasma
and used to reveal their pharmacokinetic characters.

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Accepted by DA

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