Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 480–487

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Spectrochimica Acta Part A: Molecular and

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

Development and validation of a rapid stability indicating HPLC-method

using monolithic stationary phase and two spectrophotometric methods
for determination of antihistaminic acrivastine in capsules
Ayman A. Gouda a,b,⇑, Hisham Hashem c,d, Thomas Jira e
a
Chemistry Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt
b
Faculty of Public Health and Informatics, Umm AL-Qura University, Makkah, Saudi Arabia
c
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
d
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Jazan University, Saudi Arabia
e
Institute of Pharmacy, Pharmaceutical/Medicinal Chemistry, Ernst-Moritz-Arndt-University Greifswald, F.-L.-Jahn-Str. 17, D-17487 Greifswald, Germany

h i g h l i g h t s g r a p h i c a l a b s t r a c t
1
 This study includes rapid stability Separation of (200 lg mL ) acrivastine from its degradants after stress conditions in less than 3.0 min.
indicating HPLC determination of
acrivastine.
 As stationary phase monolithic
column was used at flow rate of
5.0 mL/min.
120
 Separation of acrivastine from its Acrivastine/UV
100
degradants took place in less than
Absorption (mAU)

3.0 min. 80
Acrivastine peak
 Also two spectrophotometric 60 Acrivastine/NaOH
methods were represented. 40
 Oxidation of acrivastine with excess
20
N-bromosuccinimide. Acrivastine/H2O2
0

0 1 2 3 4
Time (min.)

a r t i c l e i n f o a b s t r a c t

Article history: Simple, rapid and accurate high performance liquid chromatographic (HPLC) and spectrophotometric
Received 31 January 2014 methods are described for determination of antihistaminic acrivastine in capsules. The first method
Received in revised form 4 April 2014 (method A) is based on accurate, sensitive and stability indicating chromatographic separation method.
Accepted 7 April 2014
ChromolithÒ Performance RP-18e column, a relatively new packing material consisting of monolithic
Available online 18 April 2014
rods of highly porous silica, was used as stationary phase applying isocratic binary mobile phase of
ACN and 25 mM NaH2PO4 pH 4.0 in the ratio of 22.5:77.5 at flow rate of 5.0 mL/min and 40 °C. A diode
Keywords:
array detector was used at 254 nm for detection. The elution time of acrivastine was found to be
Acrivastine
Stability indicating LC-method
2.080 ± 0.032. The second and third methods (methods B and C) are based on the oxidation of acrivastine
Monolithic columns with excess N-bromosuccinimide (NBS) and determination of the unconsumed NBS with, metol–
Spectrophotometry sulphanilic acid (kmax: 520 nm) or amaranth dye (kmax: 530 nm). The reacted oxidant corresponds to
N-bromosuccinimide the drug content. Beer’s law is obeyed over the concentration range 1.563–50, 2.0–20 and 1.0–10 lg mL 1
Capsules for methods A, B and C, respectively. The limits of detection and quantitation were 0.40, 0.292 and
0.113 lg mL 1 and 0.782, 0.973 and 0.376 lg mL 1 for methods A, B and C, respectively. The HPLC
method was validated for system suitability, linearity, precision, limits of detection and quantitation,
specificity, stability and robustness. Stability tests were done through exposure of the analyte solution
for four different stress conditions and the results indicate no interference of degradants with

⇑ Corresponding author at: Chemistry Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt. Tel.: +20 552420204; fax: +20 552308213.
E-mail address: aymangouda77@gmail.com (A.A. Gouda).

http://dx.doi.org/10.1016/j.saa.2014.04.015
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
 A.A. Gouda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 480–487
HPLC-method. The proposed methods was favorably applied for determination of acrivastine in capsules
formulation. Statistical comparison of the obtained results from the analysis of the studied drug to those
of the reported method using t- and F-tests showed no significant difference between them.
Introduction
Acrivastine [(E)-3-(6-[3-pyrrolidino-1-(4-tolyl)-prop-1Eenyl]-
2-pyridyl)-acrylic acid] (Scheme 1), is second-generation,
non-sedating antihistamine derived from the first-generation
compound triprolidine. It is a potent competitive histamine
H1-receptor antagonist, which does not have significant anti-
cholinergic effects. The drug is indicated for the symptomatic relief
of allergic rhinitis and histamine-medicated dermatoses [1,2]. A
good guide to the mechanism of action, pharmacokinetics, adverse
effects and therapeutic uses of acrivastine is found in the
comprehensive review written by Slater et al. [3].
There is no official method for the determination of actrivatine
in any pharmacopoeia. A survey of the literature revealed that
there have been very few methods for its determination including
gas chromatography (GC–MS) [4] and radioimmunoassay [5] has
been reported. Simultaneous determination of acrivastine and
pseudoephedrine in capsules was carried out using liquid chroma-
tography-tandem mass spectrometry [6], HPLC–UV method [7]
and a derivative spectrophotometric procedure [8]. Quantification
of antihistamine acrivastine in plasma was done by solid-phase
extraction and high-performance liquid chromatography [9]. Spec-
trofluorimetric methods determination of acrivastine in spiked
human urine and pharmaceuticals [10,11]. Few spectrophotomet-
ric and colorimetric methods have been reported for determination
of acrivastine in urine and capsules [11–14] and derivative and
ratio spectra derivative spectrophotometry [15]. Electrochemical
methods [16,17] were discussed.
Monolithic columns can be operated at high flow rates of up
to 10 mL min 1 due to their high permeability leading to fast
separation without significantly reduced separation efficiency.
The monolithic system was found to be corresponding in the
pressure drop to column packed with 10 lm particles [18]. There
is an extremely small dependency of separation efficiency on
flow rate up to 10 mL/min; therefore, high separation efficiency
can be maintained at significantly increased flow rates [19].
Although monolithic HPLC columns are advantageous comparing
with conventional HPLC columns, there are few papers described
them for quantitative analysis purposes [20]. They were applied
for determination of some compounds in biological samples or
pharmaceutical dosage forms, such as fluphenazine [21], cefdinir
[22], Phenobarbital and Phenytoin [23], mianserin [24], cephalo-
sporin antibiotics in pharmaceuticals and body fluids [25], corti-
costeroids in tablets [26] glimepirid and glibenclamid [27],
OH
O
N was freshly prepared by dissolving 0.01 g of NBS (Aldrich, Ger-
N
Scheme 1. The chemical structure of acrivastine.
481
Ó 2014 Elsevier B.V. All rights reserved.
tetracyclines [28], levetiracetam [29] and famotidine [30]. Many
pharmaceuticals such as zidovudine [31], ziparsidone-HCl [32]
and pioglitazone-HCl [33] have previously been estimated spec-
trophotometrically using NBS as the oxidimetric reagent based
on charge-transfer complexation reaction with metol and sulph-
anilic acid.
The present study develop and validate for the first time a sta-
bility indicating HPLC-method using monolithic stationary phase
and two spectrophotometric methods using N-bromosuccinimide
(NBS) with metol and sulphanilic acid or with amaranth dye for
determination of antihistaminic acrivastine in bulk powder and
capsules formulation.
Experimental
Instrumentation
– HPLC: HP1090 II model equipped with a diode array detector
(DAD) system (Hewlett–Packard, Waldbronn, Germany).
– pH-meter: Knick Elektronische Meßgeräte GmbH & Co. (Berlin,
Germany).
– UV-lamp: Modell FMR 10 UV-Blutbestrahlungsgerät, 220 V,
50 Hz (VEK Präcitronik, Dresden, Germany).
– Kontron 930 (UV–Visible) spectrophotometer (Germany) with
scanning speed of 200 nm min 1, and band width 1.0 nm
equipped with 10 mm matched quartz cells.
Materials and reagents
Chemicals of analytical grade and doubly distilled waters were
used throughout the work. HPLC analytical grade ACN was pur-
chased from Mallinckrodt Baker B.V. (Deventer, Netherland).
Mobile phase components were degassed before use. NaH2PO4
was obtained from Merck KGaA (Darmstadt, Germany). Acrivastine
and SemprexÒ capsules (labeled to contain 8.0 mg acrivastine per
capsule) obtained from Glaxo-Wellcome Company (Egypt).
Stock standard and working solutions
– The stock solution of acrivastine (1.0 mg mL 1) was prepared by
dissolving 50 mg of acrivastine in 10 mL methanol in 50 mL cal-
ibrated flask and complete to the mark with the same solvent.
The standard solutions were kept in amber colored bottle and
stored in a refrigerator when not in use.
– The standard working solutions were prepared by diluting ali-
quots of the stock solution to obtain concentrations ranging
from 0.20 to 250 lg mL 1 and 0.20 to 100 lg mL 1 for HPLC
and spectrophotometric methods, respectively.
Reagents
All reagents used were of analytical grades.
– An aqueous solution of 0.01% (w/v) N-bromosuccinimide (NBS)
many) in hot water, filtered and diluted to 100 mL in a volumet-
ric flask. The solution was standardized iodometrically [34]. The
solution was kept in a refrigerator when not in use.
482 A.A. Gouda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 480–487
– Buffer pH 1.5 was prepared by mixing equal volumes of 1.0 N
HCl and 1.0 N sodium acetate and adjusting to pH 1.5 by varying
either of them.
– A freshly prepared 0.2% aqueous solution of metol was prepared
by dissolving 0.2 g of metol (Aldrich, Germany) in 100 mL bidis-
tilled water.
– Aqueous solutions of 0.2% sulfanilic acid was prepared by dis-
solving 200 mg of sulfanilic acid (El Naser, Company, Egypt) in
100 mL bidistilled water.
– The aqueous solution of amaranth (AM) 1.0  10 3 M was pre-
pared by dissolving an accurate weight of amaranth (Merck;
Germany) in the least amount of water and completed to the
mark in a 100 mL calibrated flask.
– Aqueous solutions of 1.0% (w/v) KBr was prepared by dissolving
1.0 g of KBr (Sigma) in 100 mL bidistilled water.
General procedures
Chromatographic method (method A)
Chromatography. The separations were achieved with an analytical
column ChromolithÒ Performance RP-18e, 100  4.6 mm I.D.
friendly supplied by (Merck KGaA Darmstadt, Germany) with an
isocratic binary mobile phase of ACN/NaH2PO4 pH 4.0 (22.5/77.5,
v/v) at a flow rate of 5.0 mL min 1 by 40 °C oven temperature.
Detection was achieved at 254 nm. The hold-up times (t0) were
determined for each mobile phase composition via uracil peak.
Intentional degradations. Forced degradation studies were per-
formed to provide an indication of the stability-indicating proper-
ties and specificity of the method. Intentional degradation was
attempted using acid, base, hydrogen peroxide and UV-radiation.
A degradation sample was prepared by dissolving of 50 mg of acri-
vastine in 50 mL methanol through shaking and sonication. Then
10 mL was transferred of this solution in three different 50 mL
round bottomed flasks to perform the first three degradation tests.
To the first flask, 10 mL of 1.0 N HCl was added for acidic degrada-
tion. To the second flask 10 mL of 1.0 N NaOH was added for basic
degradation. To the third flask 10 mL of 30% H2O2 was added for
oxidative degradation. All the three flasks were refluxed for about
1.0 h. After the completing of the degradation treatment, samples
were allowed to cool to room temperature and treated as follows:
The pH values of the first and second flasks were neutralized with
1.0 N NaOH and 1.0 N HCl, respectively. To the third flask 1.0 N
sodium bisulfite solution was added to destroy H2O2. The volume
of all the three flasks was adjusted to 50 mL methanol. Samples
were injected and analyzed against control samples (lacking of
degradation treatment). For degradation through UV-radiation,
2.0 mL of the sample solution was left in UV radiation for 1.0 h
then the radiated solution diluted with methanol to 10 mL, then
finally injected into LC and compared with control sample.
Spectrophotometric methods
Method B. An aliquot of the sample solution (0.2–2.0 mL) of a stan-
dard 100 lg mL 1 acrivastine solution were transferred into a ser-
ies of 10 mL calibrated flasks, to which 1.0 mL of buffer pH 1.5 and
1.0 mL of 0.01% (w/v) NBS were added successively; the solution
was kept aside with occasional shaking for about 10 min at 25 °C.
Then 1.0 mL of metol and after 1.0 min, 1.0 mL of sulphanilic acid
was added. The volume was diluted to the mark with water, mixed
well and absorbance measured against distilled water at 530 nm
against a reagent blank within the stability period 5.0–60 min. Cal-
ibration graph was prepared by plotting decreasing values of
absorbances (DA) against drug concentration. The concentration
of the unknown was read from the calibration graph or computed
from the regression equation derived using the Beer’s law data.
Method C. To each 10 ml calibrated flask containing 0.1–1.0 mL of a
standard 100 lg mL 1 acrivastine solution, 1.0 mL hydrochloric
acid (4.0 M), 1.0 mL of 0.01% (w/v) NBS, and 1.0 mL of 1.0% (w/v)
KBr were added successively and the solutions were diluted to
7.0 mL. The solutions were kept aside for about 5.0 min at 25 °C.
Then 1.0 mL of (1.0  10 3 M) of AM dye was added, mixed
throughout and completed to the mark with water. The absorbance
was measured at kmax of 520 nm against a reagent blank prepared
in the same manner without acrivastine and its absorbance was
measured against the distilled water. Calibration graph was pre-
pared by plotting decreasing values of absorbances (DA) against
drug concentration. The amount of acrivastine in any sample was
calculated from its calibration curve.
Procedures for SemprexÒ capsules
The contents of 20 SemprexÒ capsules were emptied and accu-
rately weighed and ground to a fine powder. An accurately
weighed portion of the powder equivalent 8.0 mg acrivastine was
transferred into 50 mL volumetric flask and extracted by shaking
in an ultrasonic bath with 50 mL methanol for 15 min, then the
solution was filtered and the first 5.0 mL was rejected and the
remain filtrate was diluted to 50 mL with methanol.
Results and discussion
Chromatographic method (Method A)
System suitability
The results of five runs indicate high system suitability (Table 1).
The tR-value of acrivastine was 2.08 ± 0.032 min. The RSD of peak
area was 0.03%.
Selectivity and specificity of the method. The selectivity of the pro-
posed methods was investigated by observing any interference
encountered from some common excipients of capsules such as
starch, lactose, talc, magnesium stearate, and microcrystalline cel-
lulose. The comparison between the chromatogram of the raw acri-
vastine and that of extracted acrivastine from capsules indicates
that the excipients in the formulation did not interfere with the
determination of acrivastine (Fig. 1). Each of twenty pharmaceuti-
cal substances was simultaneously injected with acrivastine
(Table 2) for examination of specificity of the method. This means
piroxicam and cefuroxime axetil only co-eluted with acrivastine
while the other 18 drugs are completely separated from acrivas-
tine. Hence acrivastine can be determined in presence of the 18
drugs but not in presence of piroxicam or cefuroxime-axetil.
Stability of the analytical solution and stability tests
Stability of the standard solution was studied by injection of the
prepared solution at periodic intervals into the chromatographic
Table 1
System suitability and regression data for HPLC method.
Parameters
System suitability
tR ± SD (min) 2.080 ± 0.032
N 12,655 (pro meter)
k 4.668
Linearity range (lg mL 1) 1.563–50
Detection limit (lg mL 1) 0.400
1
Quantitation limit (lg mL ) 0.782
Regression data
Slope (b) ± RSD 8.773 ± 0.058
Intercept (a) ± RSD 3.669 ± 0.613
Coefficient of determination (R2) 0.9997
 A.A. Gouda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 480–487 483

vastine after exposure to stress conditions show the method is sta-

bility-indicating.

Spectrophotometric methods
25
Acrivastine (from capsules) Of the several oxidizing agents employed for such study, N-
20 bromosuccinimide (NBS) was found to be convenient because of
Absorption (mAU)

its moderate oxidizing power. N-bromosuccinimide (NBS) has been

15
used widely as a brominating and oxidizing agent for pharmaceuti-
10 cal and organic compounds [31–33,35]. The experimental condi-
tions were established by varying one variable at a time [36] and
5 observing its effect on the absorbance of the colored species.
Acrivastine (raw material)
0
Method B
0 1 2 3 4 One of the sensitive methods developed for the determination
Time (min.) of primary aromatic amines involves the formation of purple-red
1
color when primary aromatic amines are made to react with metol
Fig. 1. Chromatograms of (40 lg mL ) acrivastine from raw material and capsules.
and an oxidizing agent [37]. In the method B, we describe the
application of NBS-metol–primary amine combination to the
determination of acrivastine. The method is based on the oxidation
Table 2 of the drug by a known excess of NBS in buffer medium and sub-
Compounds tested for interference with HPLC method. sequent determination of the unreacted NBS by interacting with
metol and the primary aromatic amine, sulphanilic acid at suitable
Not interfered Interfered
kmax 530 nm. Acrivastine, when added in increasing amounts to a
Theophylline Paracetamol Piroxicam
fixed amount of NBS, consumes NBS and consequently there will
Terbutaline sulfate Birprofen Cefuroxime axetil
Salbutamol Ketoprofen be a concomitant fall in the NBS concentration. This is observed
Pheniramine maleate Ciprofloxacin as a proportional decrease in the absorbance of the reaction mix-
Propylparabene Ofloxacin ture on increasing the concentration of drug.
Ephedrine Moxifloxacin-HCl The chemistry of the color reaction may be suggested on the
Pseudoephedrine Cefaclor
Acetyl salicylic acid Cefdinir
basis of a previously reported mechanism [38]. It is probable that
Clemastine hydrogen fumarate Cefixime trihydrate the p-N-methylbenzoquinone monoamine formed in situ from
the metol-NBS reaction, being a good electron acceptor, forms a
charge transfer complex with the amine as electron donor. The
probable reaction scheme is shown in Scheme 2.
system up to about 7 days. The results indicate that the RSD of the In the first stage for the oxidation of acrivastine, use of 1.0 mL of
peak area was within 1.02%. buffer pH 1.5, 1.0 mL of 0.01% (w/v) NBS and a waiting period
The results of stress degradation indicate that acrivastine is of 5.0–60 min, were found necessary. In the second stage, 1.0 mL
strongly affected with reflux with H2O2 or exposure to UV radiation of (0.2%) metol and 1.0 mL of (0.2%) SA solutions were found opti-
(Fig. 2). Reflux with 1.0 N NaOH and 1.0 N HCl leads to nearly the mal. The intermittent waiting period prior to the addition of SA
same degradation but the effect here is weaker than in case of was 1.0–3.0 min. In this part, two blanks were prepared. The
H2O2 and UV. The acidic, alkaline and oxidation degradations of reagent blank, which contained optimum concentrations of all
acrivastine were found to be stable in HCl, NaOH, H2O2 and UV reactants except drug, gave maximum absorbance. The other blank
with no degradation peaks. Although there are several degradants was prepared in the absence of NBS and drug to determine the con-
there is no interference from any of the degradants peaks with the tribution of other reactants to the absorbance of the system. Since
peak of the intact drug. The results from stress testing, including the absorbance of the second blank was negligible, the absorbance
separation of the degradation product and quantification of acri- of the developed color was measured against water. Incomplete
color development was observed when the order of addition was
changed. Therefore order of addition should be as in the general
procedure. Any delay in the addition of sulphanilic acid also results
in low and variable absorbance values. Addition of sulphanilic acid
should not be delayed beyond 3.0 min after adding metol.

120 Method C
Acrivastine/UV
This method involved two stages including oxidation of acrivas-
Absorption (mAU)

100
tine with excess freshly prepared NBS and estimation of excess oxi-
80 dant using a known excess of AM dye. The excess AM dye
Acrivastine peak
60 Acrivastine/NaOH remaining, is then measured colorimetrically at suitable kmax
520 nm. The probable reaction scheme is shown in Scheme 3.
40
The optimum acid of the examined acids (sulfuric, hydrochloric,
20 nitric, phosphoric and acetic acids) was found to hydrochloric acid
Acrivastine/H O
0 2 2 of 4.0 M concentration. Moreover, 1.0 mL of 4.0 M HCl was chosen
as optimum volume in 10 mL total volume. The reaction takes
0 1 2 3 4
place completely in presence of KBr after 5.0 min of mixing. Raising
Time (min.)
the temperature does not accelerate the oxidation process and
Fig. 2. Separation of (200 lg mL 1
) acrivastine from degradants after stress does not give reproducible results, so the optimum temperature
conditions. is the ambient (25 ± 1 °C). The effect of time after addition the
484 A.A. Gouda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 480–487

ACR Known excess of NBS Oxidized product of ACR Unreacted NBS

Scheme 2. Reaction scheme for spectrophotometric method B.

H
Unreacted NBS + Fixed amount of AM dye Absorbance measured at 520 nm [Method C]

Scheme 3. Reaction scheme for spectrophotometric method C.

dye indicated that shaken for 1.0 min is sufficient to give reliable
results. Drug-/acid-/oxidant-/KBr-(dye) is the optimum sequence
of addition, other sequences gave lower absorbance values under
the same experimental conditions. 1.0 mL of 1.0% (w/v) KBr was
chosen as optimum volume in 10 mL total volume to accelerate
the oxidation process. To establish the optimum concentration of
the reagent, different volumes (0.2–2.0 mL) of 1.0  10 3 M AM
dye were used. The optimum volume used for the production of
maximum and reproducible color intensity is 1.0 mL (Fig. 3). NBS
reacts with acrivastine with consumption of 4.0 mol of NBS per
each mole of acrivastine. The remaining oxidant reduces the inten-
sity of red color of AM dye through disruption of the conjugation
system in the dye.
Validation of the proposed methods
At described experimental conditions for high performance
liquid chromatographic (HPLC) and spectrophotometric methods
for determination of acrivastine, standard calibration curves were
constructed. The statistical parameters were given in the regres-
sion equation calculated from the calibration graphs.
Linearity
Ten concentrations of acrivastine solution ranging from 0.02 to
250 lg mL 1 were analyzed. The graph of the peak area against
concentration proved linear in the range of 1.563–50 lg mL 1
and the linearity equation is: A = 8.773C + 3.669 and regression
coefficient = 0.9997 (Table 1). The linearity of calibration graphs
for spectrophotometric methods was proved by the high values
of the correlation coefficient (r) and the small values of the y-inter-
cepts of the regression equations: A = 0.0052C + 0.0441 (r = 0.9995)
and A = 0.0042C + 0.0612 (r = 0.9992) for methods B and C,
respectively. The apparent molar absorptivities and Sandell sensi-
tivity of the resulting colored solution and relative standard devi-
ation of response factors for each proposed spectrophotometric
method were also calculated and recorded in Table 3.

0.5
Sensitivity
0.45
For chromatographic method (method A), the limit of detection
0.4
(LOD) defined as the injected quantity giving S/N of 3.0 (in terms of
0.35
peak area), was found to be 0.40 lg mL 1. The limit of quantifica-
Absorbance

0.3
tion (LOQ) is defined as the injected quantity giving S/N of 10 (in
0.25 terms of peak area), was found to be 0.782 lg mL 1.
0.2 The detection limits (LOD) for the proposed spectrophotometric
0.15 methods were calculated using the following equation [39]:
0.1 LOD = 3s/k, where s is the standard deviation of replicate determi-
0.05 nation values under the same conditions as for the sample analysis
0 in the absence of the analyte and k is the sensitivity, namely the
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2
slope of the calibration graph. In accordance with the formula,
Volume (mL) of AM dye
the detection limits were found to be 0.292 and 0.113 lg mL 1
Fig. 3. Effect of volume of (1.0  10 3
M) AM dye on absorbance of 8.0 lg mL 1
of for methods B and C, respectively. The limits of quantitation,
acrivastine. LOQ, defined as [39]: LOQ = 10s/k. According to this equation, the
 A.A. Gouda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 480–487 485

Table 3 Intra-day precision was assessed injecting of the standard solution

Statistical analysis of calibration graphs and analytical data for determination of at two concentrations five times during a day. The same was done
acrivastine using the proposed spectrophotometric methods.
for inter-day precision test except that the injection of the samples
Parameters Spectrophotometry was every day for 5 days.
Method B Method C While for spectrophotometric methods was carried out by five
Wavelengths kmax (nm) 530 520
determinations at four different concentrations. The results of
Beer’s law limits (lg mL 1) 2.0–20 1.0–10 accuracy and precision show, that the proposed methods have
Molar absorptivity e, (L/mol 1 cm 1
)  10 4
1.5698 2.1276 good repeatability and reproducibility. To confirm the accuracy
Sandell’s sensitivity (ng cm 2) 22.20 16.38 of the methods, recovery studies were performed by applying the
Regression equationa standard addition method [40]. This depends upon the addition
Slope (b) 0.0441 0.0612 of a known quantity of the standard ACR to a fixed amount of
Intercept (a) 0.0052 0.0046
the corresponding capsules formulation and then analyzing the
Correlation coefficient (r) 0.9995 0.9992
Detection limits LOD (lg mL 1) 0.292 0.113 resulting solution by the proposed methods. Results indicates high
Quantification limits LOQ (lg mL 1
) 0.973 0.376 accuracy with good recoveries (100.0 ± 0.385%) and
Recovery% ± RSD%b 100.38 ± 1.514 99.95 ± 1.17 (100.08 ± 0.386%), for methods B and C, respectively, proving the
Relative error (RE%) 1.589 1.229
lack of interference due to common excipients and, hence, indi-
a
A = a + bC, where A is the absorbance, a is the intercept, b is the slope and C is cates that the proposed methods are accurate (Table 5).
the concentration of drug in lg mL 1. LOD, limit of detection; LOQ, limit of quan-
tification; e, molar absorptivity coefficient.
b
Mean ± relative standard deviation (RSD%) of six determinations. Robustness and ruggedness
The robustness of the present HPLC method was evaluated in
1
limits of quantitation were found to be 0.973 and 0.376 lg mL the terms of temperature, flow rate, content of ACN in mobile
for methods B and C, respectively. phase, wavelength of detection, buffer pH-value and buffer salt
concentration and injection volume (Table 6). The slight variations
in the examined factors had no significant effect on the shape of
Reproducibility precision and accuracy the peak. The results indicate that the method is more sensitive
Results (Table 4) show that for chromatographic method there to changes in buffer salt concentration and in pH-value more than
were high intra- and inter-day precisions (both within 2.16%). to changes in the other factors. Compared with tR-values peak

Table 4
Reproducibility and precision of HPLC method (n = 5).
1
Injected amount (lg mL ) Intra-day (n = 5) Inter-day (n = 5)
a b
Observed amount (lg) ± SD C.V. (%) Accuracy (%) Observed amount (lg) ± SD C.V. (%)a Accuracy (%)b
6.25 6.49 ± 0.14 2.16 103.84 6.43 ± 010 1.56 102.88
25.00 24.49 ± 0.46 1.88 97.96 24.40 ± 0.36 1.48 97.60
a
Coefficient of variation (%) = SD  100/mean.
b
Accuracy (%) = observed concentration  100/used concentration.

Table 5
Intra-day and Inter-day accuracy and precision data for the proposed spectrophotometric methods on pure sample of acrivastine.
1
Method Taken (lg mL ) Intra-day (n = 5) Inter-day (n = 5)
Observed amount (lg) ± SD C.V. (%)a Accuracy %b Observed amount (lg) ± SD C.V. (%)a Accuracy %b
Method B 4.0 3.99 ± 0.11 2.76 99.75 4.02 ± 0.09 2.24 100.50
8.0 8.023 ± 0.23 2.87 100.40 7.99 ± 0.16 2.0 99.90
12 12.03 ± 0.25 2.08 100.25 11.98 ± 0.21 1.75 99.83
16 15.94 ± 0.37 2.32 99.60 15.92 ± 0.27 1.70 99.50
Mean ± SD 100.0 ± 0.385

Method C 2.0 2.006 ± 0.07 3.49 100.30 1.991 ± 0.06 3.01 99.55
4.0 3.988 ± 0.12 3.01 99.70 3.976 ± 0.13 3.27 99.40
6.0 6.03 ± 0.17 2.82 100.50 5.955 ± 0.16 2.69 99.25
8.0 7.984 ± 0.21 2.63 99.80 8.008 ± 0.19 2.37 100.10
Mean ± SD 100.08 ± 0.386
a
Coefficient of variation (%) = SD  100/mean.
b
Accuracy (%) = observed concentration  100/used concentration.

Table 6
Robustness of the HPLC method.

Slight changes Temp. (°C) Flow rate ACN (%) Wavelength of detection Injected vol. pH-value Buffer conc. (mM)
in (mL min 1) (nm) (lL)
38, 39 and 4.8, 4.9 and 5.0 21.5, 22.5 and 252, 254 and 256 23, 24 and 25 3.9, 4.0 and 12.5, 25.0 and
40 23.5 4.1 50.0

Affected parameter Peak area tR Peak area tR Peak area Peak area tR tR Peak area tR Peak area tR
C.V. (%) 2.52 1.86 2.44 1.52 0.89 2.25 0.33 0.93 0.01 2.73 2.68 1.39
486 A.A. Gouda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 480–487

were kept unchanged, and the recovery percentage was calculated

each time. The small variations in any of the variables did not sig-
nificantly affect the results. The ruggedness of the proposed spec-
trophotometric methods was assessed by applying the procedures
100
using two different instruments in two different laboratories at dif-
A ferent times. Results, obtained from laboratory-to-laboratory and
80
day-to-day variation, were found to be reproducible because the
Absorption (mAU)

60
RSD did not exceed 2.0%. This provided an indication of the reliabil-
Acrivastine peak
ity of the proposed methods during routine work.
B
40

20
C
0 recovery experiments applying standard addition technique. Anal-
0 1 2 3
Time (min.)
Fig. 4. Effects of small changes in chromatographic conditions on separation of
(200 lg mL 1) acrivastine from its UV-degradants. A – 21.5% ACN and pH 4.0. B –
23.5% ACN and pH 4.0. C – 22.5% ACN and pH 4.10.
areas were more affected with the slight changes in the chromato-
graphic conditions. Fig. 4 indicates also that the slight changes in
the chromatographic conditions did not lead to any interference
between peak of intact acrivastine and those of degradants. This
ensures that the method is robust and stability indicating.
Robustness of the spectrophotometric procedures was assessed
by evaluating the influence of small variation of experimental vari-
ables, i.e., concentrations of reagent and reaction time, on the ana-
lytical performance of the method. In these experiments, one
experimental parameter was changed while the other parameters
Application
The accuracy of the present HPLC method was determined by
ysis of acrivastine in capsules formulation showed high accuracy
4 with recovery of 101.90 ± 0.52%. Statistical comparison of the
results obtained from the analysis of the studied drug to those of
the reported method [7] using t- and F-tests as presented in
(Table 7). The values of f and t indicate that there is no significant
difference between both methods. Applying standard addition
technique for accuracy determination indicates that the method
is accurate.
The proposed spectrophotometric methods were successfully
applied to the determination of acrivastine in capsules formulation
(Table 8). The results were compared statistically, by applying the
t- and F-tests, with the results obtained by a reported method [13].
The results obtained by the proposed methods revealed that no
significant difference was found between the calculated and theo-
retical values of both the proposed and reference methods at 95%
confidence level. This indicated similar accuracy and precision in
the analysis by the proposed and reported methods. It is evident
from these results that the proposed methods are applicable to

Table 7
Accuracy of HPLC method applying standard addition technique.
1 1
Added (lg mL ) Found (lg mL ) Recovery (%)a ± SD C.V. (%) Reference method [7]
Method A 6.25 6.32 101.14 ± 0.95 0.94
25 25.67 102.67 ± 0.09 0.09
Mean ± SD 101.90 ± 0.52 101.53 ± 0.70
Student-t-test 1.039 (2.20)b
F-test 1.812 (4.12) b
a
Average of six determinations.
b
The figures in parenthesis are the theoretical values for t- and F-test at (P = 0.05).

Table 8
Spectrophotometric determinations of acrivastine in SemprexÒ capsules applying the standard addition technique and compared with the reference method [13].
1 1 1
Taken (lg mL ) Added (lg mL ) Found (lg mL ) Recovery (%)a ± SD C.V. (%) Reference method [13]
Method B 2.0 – 2.003 100.15 ± 0.05 2.496
4.0 5.976 99.60 ± 0.08 1.339
8.0 10.03 100.30 ± 0.12 1.20
12 13.916 99.40 ± 0.15 1.078
16 17.838 99.10 ± 0.17 0.953
Mean ± RSD 99.71 ± 1.413 99.90 ± 0.70
Student-t-test 0.295 (2.57)b
F-test 4.08 (5.05)b

Method C 2.0 – 1.98 99.00 ± 0.03 1.515

2.0 3.978 99.45 ± 0.05 1.257
4.0 6.042 100.70 ± 0.08 1.324
6.0 8.016 100.20 ± 0.10 1.248
8.0 9.88 98.80 ± 0.12 1.214
Mean ± RSD 99.63 ± 1.312 99.90 ± 0.70
Student-t-test 0.445 (2.57)b
F-test 3.512 (5.05)b
a
Average of six determinations.
b
The figures in parenthesis are the theoretical values for t- and F-test at (P = 0.05).
 A.A. Gouda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 480–487
the analysis of acrivastine in bulk form and in capsules formulation
with comparable analytical performance.
Conclusions
This study reports for the first time about a stability indicating
HPLC method for determination of acrivastine in bulk powder and
in capsules. Compared with the published HPLC-methods, this
method represents a strong reduction of analysis time and is a sta-
bility indicating at the same time. With the proposed HPLC method
a satisfactory separation of acrivastine from the degradation prod-
ucts with extended linear range and rapid analysis time was car-
ried out. A high recovery of acrivastine in capsules was achieved.
No interference from the excipients was noticed. Also in this study
two simple and sensitive spectrophotometric methods were
applied for quantification of acrivastine. These spectrophotometric
methods have not been used before for determination of acrivas-
tine. They depend on oxidation of acrivastine with excess NBS then
determination of the unreacted NBS either with metol–sulphanilic
acid or with KBr-AM dye. All three methods gave comparable
results concerning with a reported method for acrivastine.
Acknowledgements
The authors thank the above-mentioned companies for the
friendly supply of analyte and column.
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