Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxxxxx

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Bulletin of Faculty of Pharmacy, Cairo University

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Original Article

Simultaneous determination of Dimenhydrinate, Cinnarizine and

Cinnarizine impurity by TLC and HPLC chromatographic methods
Amal B. Ahmed a,, Nada S. Abdelwahab b, Maha M. Abdelrahman b, Fathy M. Salama c
a
Analytical Chemistry Department, Faculty of Pharmacy, Nahda University, Sharq El-Nile, 62514 Beni-Suef, Egypt
b
Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, AlshaheedShehata Ahmad Hegazy St., 62514 Beni-Suef, Egypt
c
Analytical Chemistry Department, Faculty of Pharmacy, EL-Azhar University, Yosief Abbas St., 11651 Cairo, Egypt

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

Article history: Two chromatographic methods have been established and validated for simultaneous determination of
Received 16 August 2016 mixture of Dimenhydrinate (DMH) and Cinnarizine (CIN) in their pharmaceutical formulation and in
Received in revised form 24 December 2016 presence of Cinnarizine impurity (1-(Diphenylmethyl) piperazine); CIN impurity. The first method was
Accepted 15 January 2017
TLC-densitometric one, depends on separation and quantitation of DMH, CIN and CIN impurity on TLC
Available online xxxx
silica gel 60 F254 plates, using chloroform:methanol:glacial acetic acid:ammonia solution
(9.5:0.5:0.1:0.1, by volume) as a developing system followed by densitometric measurement at
Keywords:
235 nm. Linear relationships were obtained in the range of 0.22, 0.41.6 and 0.11 lg/band for DMH,
Dimenhydrinate
Cinnarizine
CIN and CIN impurity, respectively. The studied components were well resolved from each other with sig-
1-(Diphenylmethyl) piperazine) nificantly different Rf values of 0.35, 0.52 and 0.04 for DMH, CIN and CIN impurity, respectively. The sec-
TLC ond method was RP-HPLC, separation on C8 column using 0.05 M KH2PO4 (pH = 3):methanol (35:65, v/v)
HPLC as the mobile phase at a flow rate of 1 mL/min and DAD detection at 240 nm. Linear relationships were
obtained in the ranges of 330, 220 and 110 lg/mL, with significantly different Rt values of 3.27, 6.95
and 2.87 min for DMH, CIN and CIN impurity, respectively. The developed methods were validated
according to ICH guidelines demonstrating good accuracy and precision. The results were statistically
compared with those obtained by reported HPLC method and no significant difference was obtained.
2017 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

1. Introduction BP [3] reported a titration method for determination of DMH

Dimenhydrinate (DMH); is chemically known as 8-chloro-3, 7-
dihydro-1, 3-dimethyl-1H-purine-2, 6-dione compound with 2-
(diphenylmethoxy)-N, N-dimethylethanamine (1:1), it is the
diphenhydramine salt of 8-chlorotheophylline [1], (Fig. 1a). DMH
has antihistaminic with antimuscarinic and significant sedative
effects. It is mainly used as an antiemetic drug in the prevention
and treatment of motion sickness [2]. Cinnarizine (CIN); chemi-
cally known as (E)-1-(Diphenylmethyl)-4-(3-phenylprop-2-enyl)
piperazine [1], (Fig. 1b), it is a piperazine derivative with antihis-
taminic, sedative and calcium-channel blocking activity. It is used
for the symptomatic treatment of nausea, vertigo, treatment of
motion sickness and cerebral vascular disorders [2]. British Phar-
macopeia (BP) [3] stated that 1-(Diphenylmethyl) piperazine (CIN
impurity) is Cinnarizine impurity, it is also known as ben-
zhydrylpiperazine [1], Fig. 1c.
Peer review under responsibility of Faculty of Pharmacy, Cairo University.
Corresponding author.
E-mail address: amal.badawy@nub.edu.eg (A.B. Ahmed).
with potentiometric detection, While EP [4] stated argentometric
titration and USP [5] describe a HPLC method using solution of
ammonium acetate:methanol (80:20 v/v) as mobile phase at a flow
rate1.5 mL/min with UV detection at 229 nm. Meanwhile BP [3]
and EP [4] developed a potentiometric titration method for estima-
tion of CIN. The combination of both DMH and CIN is not official in
any pharmacopeia.
Literature review shows that DMH can be determined by HPLC
method [6] and also CIN can be determined by TLC-Densitometric
method [711] and HPLC method [1014] either alone or in com-
bination with other drugs in pharmaceutical formulation or in bio-
logical fluids, few reports can be found in the scientific literature
for chromatographic determination of DMH and CIN. These reports
presented HPTLC using chloroform:hexane:methanol (8.5:0.8:0.7)
as mobile phase at 254 nm [15], TLC-Densitometric method
using ethyl acetate:methylene chloride (8:2 v/v) as mobile phase
at 254 nm [16] and two HPLC methods are available using
acetonitrile:water (90:10 v/v)as mobile phase at flow rate of
0.7 mL/min with UV detection at 265 nm, methanol:acetonitrile:
water:triethylamine (85:15:5:0.5%) at flow rate of 0.6 mL/min with

http://dx.doi.org/10.1016/j.bfopcu.2017.01.003
1110-0931/ 2017 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: A.B. Ahmed et al., Simultaneous determination of Dimenhydrinate, Cinnarizine and Cinnarizine impurity by TLC and
HPLC chromatographic methods, Bulletin Facult Pharmacy Cairo Univ (2017), http://dx.doi.org/10.1016/j.bfopcu.2017.01.003
2 A.B. Ahmed et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxxxxx
Fig. 1. Chemical structures of (a) Dimenhydrinate, (b) Cinnarizine, (c) Diphenyl-
methyl piperazine (Cinnarizine impurity).
UV detection at 254 nm and 0.2% Formic Acid and 0.2% tri ethyl
amine in water pH adjusted to 5.0 with formic acid and methanol
(40:60% v/v)) at a flow rate of 1.0 ml/min. The detection was car-
ried out at 260 nm [1618].
No method was reported for simultaneous determination of
Dimenhydrinate, Cinnarizine and Cinnarizine impurity. The previ-
ously reported chromatographic methods failed to resolve the
studied components. Therefore, the objective of work is to develop
two accurate, sensitive and reliable chromatographic methods for
simultaneous determination of Dimenhydrinate and Cinnarizine
in presence of Cinnarizine impurity with high sensitivity and selec-
tivity and to validate the developed methods according to ICH
guidelines [19].
2. Experimental
2.1. Instruments

For TLC-Densitometric method
The instruments used in this study included a TLC scanner 3
densitometer (Camag, Muttenz, Switzerland) controlled by WIN-
CATS software (version 3.15) (Camag, Muttenz, Switzerland); a
sample applicator for TLC Linomat V equipped with a 100 lL syr-
Please cite this article in press as: A.B. Ahmed et al., Simultaneous determination of Dimenhydrinate, Cinnarizine and Cinnarizine impurity by TLC and
HPLC chromatographic methods, Bulletin Facult Pharmacy Cairo Univ (2017), http://dx.doi.org/10.1016/j.bfopcu.2017.01.003
inge (Camag, Muttenz, Switzerland); an ultraviolet (UV) lamp with
short wavelength of 254 nm (VL-6.LC; Marne La Vallee, France);
TLC plates (20  10 cm) coated with 60 F254 silica gel (Merck,
Darmstadt, Germany) with 0.2 mm thickness. During TLC scanning
mode was absorbance, source of radiation was a deuterium lamp.
The slit dimensions were adjusted to 6  0.45 mm, and the scan-
ning speed was 20 mm/s.

For RP-HPLC method
HPLC instrument (Agilent 1260 Infinity, Germany) equipped
with an Agilent 1260 Infinity preparative pump (G1361A), Agilent
1260 Infinity DAD detector VL (G131SD), Agilent 1260 Infinity
Thermostated column compartment (G1316A) and Agilent 1260
Infinity preparative Autosampler (G2260A). Separation and quanti-
tation was performed on a C8 column (25 cm  4.6 mm i.d, 5 lm
particle size) (USA).
Sonix TV ss-series ultrasonicator (USA).
2.2. Materials
2.2.1. Pure samples
Pure samples of DMH and CIN were kindly supplied by Amoun
pharmaceutical company (Cairo, Egypt) with purity of 99.6 and
99.7%, respectively, according to analysis result from the reported
method [16]. While CIN impurity was obtained from (Sigma
Aldrich, Chemie GmbH, Germany) with claimed purity of 97%
according to the manufacturing certificates of analysis.
2.2.2. Pharmaceutical formulation
Amocerebral tablets (batch No. 6221025030658) were manu-
factured by Amoun pharmaceutical company. Each tablet is
claimed to contain 20 mg of DMH and 10 mg of CIN.
2.2.3. Chemicals
All chemicals and solvents used throughout this work were of
analytical grade and were used without further purification.

Chloroform, potassium dihydrogen Phosphate, glacial acetic
acid, orthophosphoric acid and ammonia solution (El-Nasr
Pharmaceutical Chemicals Co., Abu-Zabaal, Cairo, Egypt).

Methanol HPLC grade (SDS, France).

Deionized water (Otsoka Pharmaceuticals, Cairo, Egypt).
2.2.4. Standard solutions
a. Stock standard solutions of DMH, CIN and CIN impurity: 0.1 g
of each component was weighed into three separated 100-
mL volumetric flasks, 50 mL methanol was added to each
flask, dissolved well and then the volume was completed
to the mark with methanol.
b. Working standard solutions of DMH, CIN and CIN impurity:
10 mL of each component was transferred from their respec-
tive stock standard solutions (1 mg/mL) into three separate
100-mL volumetric flasks, then the volume was completed
to the mark with methanol for TLCdensitometric method
and with 0.05 M KH2PO4 (pH = 3 with orthophosphoric
acid):methanol (35:65, v/v) for the HPLC method.
3. Procedure
3.1. Chromatographic conditions
a. TLC-Densitometric method
Samples were applied as bands of 6 mm width on TLC plates
(20  10 cm with 250 lm thickness) using a Camag Linomat
 A.B. Ahmed et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxxxxx
IV applicator. The bands were applied at 5 mm intervals and
10 mm from the bottom edge of the plate. Linear ascending
development was carried out to a distance of 8 cm in a chro-
matographic tank previously saturated for 30 mins with a
developing system consisted of chloroform:methanol:glacial
acetic acid:ammonia solution (9.5:0.5:0.1:0.1, by volume) at
room temperature and scanned at 235 nm.
b. RP-HPLC method
Chromatographic separation was performed on C8 column
using isocratic elution of mixture of 0.05 M KH2PO4 pH = 3
with orthophosphoric acid:methanol (35:65, v/v) as a
mobile phase. The mobile phase was filtered using 0.45 lm
milipore membrane filter and delivered at a constant flow
rate of 1 mL/min. The injection volume was 20 lL of each
solution and detection was carried out at 240 nm. The run
time was 10 min and the column temperature was main-
tained at 25 C.
3.2. Construction of calibration curves
a. TLC-Densitometric method
Different aliquots equivalent to 0.22, 0.41.6 and 0.11 mg
of DMH, CIN and CIN impurity, respectively, were separately
transferred from their respective stock solutions (1 mg/mL)
into three separate series of 10-mL volumetric flasks and
the volume was completed using methanol till the mark,
10 lL of each solution was spotted as bands. The applied
bands were scanned at 235 nm and the calibration curves
were constructed by plotting the integrated peak area/104
versus the corresponding concentrations of each component
and the regression equations were computed.
b. RP-HPLC method
Different aliquots of DMH, CIN and CIN impurity equivalent
to 30300, 20200 and 10100 lg, respectively, were sepa-
rately transferred from their respective working solutions
(100 lg/mL) into three separate series of 10 mL volumetric
flasks, and the volumes were made up with the mobile
phase. Triplicate 20 lL injections were made for each con-
centration maintaining the flow rate at 1 mL/min and the
effluent was UV-scanned at 240 nm. The chromatographic
separation was performed following the procedure under
chromatographic conditions. The chromatograms were
recorded and the peak areas of DMH, CIN and CIN impurity
were determined and the calibration curves relating the
obtained integrated peak areas/104 to the corresponding
concentrations were constructed and the regression equa-
tions were computed.
3.3. Application to pharmaceutical formulation (Amocerebral tablets)
Ten tablets of Amocerebral tablets were weighed, powdered
and mixed well; an accurate weight of the powdered tablets equiv-
alent to 100 mg of DMH and 50 mg of CIN was transferred into
100 mL volumetric flask. 75 mL of methanol was added and ultra-
sonicated for 30 min; then filtered into 100-mL volumetric flask.
The residue was washed using methanol then the volume was
completed with methanol to obtain stock solution of 1 mg/mL.
From which sample working solution of 100 lg/mL was prepared
and then the procedure under linearity for each method was fol-
lowed. The concentrations of DMH and CIN were calculated using
the previously computed regression equations. Standard addition
technique has been carried out to assess validity of the method
by adding to the pharmaceutical formulation known amount of
standard drug powder. The recovery of the added standards was
then calculated after applying the proposed methods.
Please cite this article in press as: A.B. Ahmed et al., Simultaneous determination of Dimenhydrinate, Cinnarizine and Cinnarizine impurity by TLC and
HPLC chromatographic methods, Bulletin Facult Pharmacy Cairo Univ (2017), http://dx.doi.org/10.1016/j.bfopcu.2017.01.003
3
4. Results and discussion
The present work provides for the first time sensitive, accurate
and selective chromatographic methods for simultaneous determi-
nation of DMH, CIN and CIN impurity with application to their
pharmaceutical formulation.
Analytical monitoring of impurities and related substances in
new drug component is the key element of the recent guidelines
issued by the International Conference on Harmonization (ICH)
[19]. The developed chromatographic methods have the advantage
over the previously published chromatographic methods for deter-
mination of Dimenhydrinate and Cinnarizine of being highly selec-
tive and able to resolve and quantify Dimenhydrinate and
Cinnarizine in presence of Cinnarizine impurity with acceptable
accuracy and precision.
Different analytical methods have been found in the literature
for analysis of DMH and CIN in their binary mixtures. But these
methods were not able to resolve them in presence of CIN Impu-
rity. As previously mentioned no published method was found in
the literature for determination of DMH and CIN in presence of
CIN impurity. The work in this manuscript concerned with the
development and validation of two accurate, selective and precise
chromatographic methods for simultaneous separation of DMH,
CIN and CIN impurity with high resolution.
4.1. Methods development and optimization
4.1.1. TLC-Densitometric method
TLC-Densitometry is a useful method for the resolution and
determination of drug mixtures. There isa TLC-Densitometric
method in scientific literature available for estimation of both
DMH and CIN [16] using ethyl acetate:methylene chloride (8:2 v/
v) as mobile phase at 254 nm, which cannot be used to resolve
the studied components. Soto improve chromatographic separa-
tion, it was necessary to investigate the effect of different variables.
Determination of the optimum parameters for maximum separa-
tion was carried out as described below.
4.1.1.1. Developing system. Different developing systems of differ-
ent composition and ratios were tried including:chloroform:
methanol (5:5, v/v), chloroform:methanol (6:4, v/v), chloroform:
methanol (9:1, v/v), and chloroform:methanol:ammonia solution
(9:1:0.1, by volume), It was found that presence of ammonia in
the developing system is essential for separation and differentia-
tion between CIN and its impurity, Also chloroform:methanol:am-
monia solution:glacial acetic acid (9:1:0.1:0.05, by volume) and
chloroform:methanol:ammonia solution:glacial acetic acid
(9:1:0.1:0.1, by volume) were tried, It was found that presence of
glacial acetic acid provided good separation of DMH. The best
developing system was chloroform:methanol:ammonia solution:
glacial acetic acid (9.5:0.5:0.1:0.1, by volume). This selected devel-
oping system allows good separation between the studied compo-
nents with good Rf values without tailing of the separated bands,
Fig. 2.
4.1.1.2. Scanning wavelength. Different scanning wavelengths were
tried, such as 225, 230 and 235 nm to obtain good sensitivity with
minimum noise. It was found that the wavelength 235 nm was
found to be the best wavelength for scanning of all components
with good sensitivity and peaks shape with minimum noise, as
shown in Fig. 2.
This proposed method offers high sensitivity and selectivity for
analysis of DMH, CIN and CIN impurity using chloroform:metha-
nol:ammonia solution:glacial acetic acid (9.5:0.5:0.1:0.1, by vol-
ume) as developing system followed by densitometric
4 A.B. Ahmed et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxxxxx
Fig. 2. TLC chromatogram of a mixture of DMH, CIN and IMP using chloroform:
methanol:ammonia solution:glacial acetic acid (9.5:0.5:0.1:0.1, by volume) as a
developing system.
measurement of separated bands at 235 nm. The studied compo-
nents were well resolved from each other with significantly differ-
ent Rf values 0.35, 0.52 and 0.04 for DMH, CIN and CIN impurity,
respectively. The calibration curves for DMH, CIN and CIN impurity
were constructed by plotting the integrated peak areas/104 versus
the corresponding concentrations of each component in the range
of 0.22, 0.41.6 and 0.11 lg/band for DMH, CIN and CIN impu-
rity, respectively.
The concentration of each component was calculated from the
following regression equation:
A1 0:0596CDMH 0:0392; r 0:9998
A2 0:4867CCIN 0:072; r 0:9999
A3 0:1856Cimpurity 0:0387; r 0:9999
where A1, A 2 and A 3are the integrated peak areas/104 for DMH, CIN
and CIN impurity, respectively, C is the concentration in lg/band
and r is the correlation coefficient.
4.1.2. RP-HPLC method
A validated isocratic RP-HPLC method with DAD detection was
developed for the separation and determination of DMH and CIN in
presence of CIN impurity and it was applied to the pharmaceutical
formulation. In scientific literature there are two HPLC methods
available for estimation of DMH and CIN using mobile phases of
acetonitrile:water (90:10) at flow rate 0.7 mL/min with UV detec-
tion at 254 nm and the second method using mobile phase of me
thanol:acetonitrile:water:triethyl amine (85:15:5:0.5%) at flow
rate of 0.6 mL/min with UV detection at 254 nm [16,17] either of
these method can be used to resolve the studied components. Soto
optimize the proposed RP-HPLC method, it was necessary to test
the effects of different parameters that affect sensitivity, selectivity
and efficiency of the chromatographic separation.
4.1.2.1. Optimization of mobile phase. Different mobile phases with
different compositions and polarities were tested to achieve the
chromatographic separation. Initially, samples were analyzed
using a mobile phase consisting of acetonitrile:water (50:50, v/v)
at a flow rate of 1 mL/min. Under these conditions, the peaks were
very broad. Replacing acetonitrile with methanol improved the
peak broadening. Several trials were performed using methanol:
water (50:50, v/v), also in order to enhance the chromatographic
Please cite this article in press as: A.B. Ahmed et al., Simultaneous determination of Dimenhydrinate, Cinnarizine and Cinnarizine impurity by TLC and
HPLC chromatographic methods, Bulletin Facult Pharmacy Cairo Univ (2017), http://dx.doi.org/10.1016/j.bfopcu.2017.01.003
resolution and decrease peaks broadening 0.1% triethylamine was
tried. Unfortunately, neither the chromatographic resolution nor
the peak shapes were improved with increase analysis time.
The second step was to replace water with 0.05 M KH2Po4 buf-
fer adjusted to different pH values. It was noticed that changing the
buffer pH greatly affected the Rt values of DMH, CIN and CIN impu-
rity and hence the resolution. Extremely alkaline pH increased Rt
values of DMH, CIN and CIN impurity, which gives rise to a broader
peaks and increases the analysis time.
After extensive trials, it was found that the best separation and
resolution achieved using isocratic mixture of 0.05 M KH2Po4 buf-
fer (pH = 3 adjusted with orthophosphoric acid):methanol (35:65,
v/v) as mobile phase. This selected mobile phase provided good
separation with reasonable Rt values without tailing of the sepa-
rated peaks, as shown in Fig. 3. It was found that presence of
orthophosphoric acid in the mobile phase is essential for separa-
tion of DMH and CIN impurity giving sharp peaks and good
separation.
4.1.2.2. Detection wavelength. The DAD detector was set at different
wavelengths, including 225, 230 and 240 nm. Using 240 nm as a
detection wavelength provided the best results with respect to
sensitivity and peak shape.
4.1.2.3. Flow rate. Different flow rates values (0.8, 1 and
1.5 mL/min) were also tested to provide the required separation
within acceptable run time. The best flow rate used was 1 mL/
min. Finally a satisfactory separation was obtained upon using
the optimum experimental conditions.
This proposed method offers high sensitivity and selectivity for
analysis of DMH, CIN and CIN impurity using isocratic elution of
0.05 M KH2Po4 buffer (pH = 3):methanol (35:65 v/v) as mobile
phase with DAD detection at 240 nm using flow rate of
1 mL/min. The studied components were well resolved from each
other with significantly different Rt values of 3.27, 6.95 and
2.87 min for DMH, CIN and CIN impurity, respectively. The calibra-
tion curve for DMH, CIN and CIN impurity was constructed by plot-
ting the peak area/104 versus the corresponding concentrations of
each drug in the range of 330, 220 and 110 lg/mL for DMH, CIN
and CIN impurity, respectively.
The concentration for each component was calculated from the
following regression equation:
A1 0:0392CDMH 0:0822; r 0:9998
A2 0:0502CCIN 0:5875; r 0:9997
A3 0:0476Cimpurity 0:0325; r 0:9997
Fig. 3. RP- HPLC chromatogram of isocratic mixture of DMH, CIN and IMP using
0.05 M KH2PO4 buffer (pH = 3):methanol (35:65, v/v) as a mobile phase with DAD
detection at 240 nm.
 A.B. Ahmed et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxxxxx 5

where A1, A2 and A3 were the peak areas/104 for DMH, CIN and CIN
impurity, respectively, C is the concentration in lg/mL and r is the

100.26 0.689
correlation coefficient.
The proposed methods were applied for determination of DMH

101.24

100.21
99.64
99.98
and CIN in their pharmaceutical formulation (Amocerebral

CIN
tablets). Validity of the method was assessed by adding to the
pharmaceutical formulation known amount of standard drug pow-
der. The recovery of the added standards was then calculated,

100.44 1.182
% Recoveryb

Table 1. Regression equation parameters are given in Table 2. The

results obtained by applying the proposed methods were statisti-
100.38
101.50
101.07
98.80
DMH

cally compared with those obtained by applying the reported HPLC

method [16]. The values of the obtained t- and F-tests were less
RP-HPLC method

than the tabulated ones confirming that the difference between

CIN

10
Mean SD the developed methods and the reported one is insignificant
3
5
7
(lg/mL)

regarding to accuracy and precision, Table 3.

Added

DMH
3
5
7
9

4.2. Method validation

100.46 0.894

Method validation was performed according to the interna-

tional conference on harmonization (ICH) guidelines [19] for the
101.39
100.51
100.70
Determination of Dimenhydrinate and Cinnarizine in their pharmaceutical formulation by the proposed methods and application of standard addition technique.

99.25

proposed methods.
CIN

The linearity of the proposed methods was evaluated and it was

evident in the ranges of 0.22, 0.41.6 and 0.11 lg/band for DMH,
% Recoveryb

CIN and CIN impurity, respectively (for the TLC-densitometric

99.57 1.305

method) and 330, 220 and 110 lg/mL for DMH, CIN and CIN
100.58
100.78
98.79
98.15
DMH

impurity, respectively (for RP-HPLC method). The regression equa-

TLC-densitometric method

tions for the proposed methods were computed and given in

Table 2.
CIN
0.4
0.5
0.6
0.7

The accuracy of the proposed methods was checked by applying

the proposed methods for determination of pure samples of the
Mean SD

studied compounds. The concentrations were calculated from the

(lg/band)

corresponding regression equations and the results are shown in

Added

DMH

Table 2. Accuracy was further assessed by applying the standard

0.2
0.3
0.5
0.6

addition technique on Amocerebral tablets, and good recoveries

were obtained, revealing no interference from excipients and good
106.52 1.590

accuracy of the proposed methods Table 1.

The precision of the proposed methods provides acceptable
intra- and interday variation, indicating the good precision of the
CIN

method and revealing that it is suitable for the quality control of

RP-HPLC method

the suggested components,

103.89 1.174

Repeatability: Three concentrations 0.5, 0.8 and 1 lg/band of

each DMH, CIN and CIN impurity, for TLC-densitometric method
and (5,10 and 15 lg/mL of DMH), (5, 10 and 15 lg/mL of CIN)
DMH

and (5, 8 and 10 lg/mL of CIN impurity) for RP-HPLC method, were
analyzed three times intra-daily using the proposed methods.
105.03 0.918

Good results and acceptable %RSD values were obtained, Table 2.

Intermediate precision: The developed methods were repeated
TLC-densitometric method

inter-daily on three successive days for the analysis of the previ-

CIN

ously mentioned concentrations. Good results and acceptable %

RSD values were obtained, shown in Table 2.
% Recovery SDa

104.80 1.141

Detection and quantification limits: Low values of LOD and LOQ

shown in Table 2, proved high sensitivity of the developed meth-
ods, These approaches based on the SD of the response and the
DMH

slope were used for determining the detection and quantitation

limits (where LOD = 3.3 * SD/slope and LOQ = 10 * SD/slope),
average of three determinations.

Specificity of the proposed methods is evident from the TLC and

Amocerebral tablets Claimed to

average of six determinations.

contain 20 mg of DMH and

RP-HPLC chromatograms in Figs. 2 and 3, respectively. Also values

Pharmaceutical formulation

of the calculated parameters such as selectivity and resolution fac-

tors were within the acceptable limit, Table 4.
10 mg of CIN/ tablet

The robustness of the proposed methods was evaluated in the

development phase where the effects of different factors on TLC-
densitometric and RP-HPLC methods were studied to obtain the
optimum parameters for complete separation. Robustness of the
method was studied by deliberately varying parameters like flow
Table 1

rate (0.1 mL/min) and studying the effect of changing mobile

b
a

phase pH by (0.2) and methanol composition (5 mL).The low

Please cite this article in press as: A.B. Ahmed et al., Simultaneous determination of Dimenhydrinate, Cinnarizine and Cinnarizine impurity by TLC and
HPLC chromatographic methods, Bulletin Facult Pharmacy Cairo Univ (2017), http://dx.doi.org/10.1016/j.bfopcu.2017.01.003
6 A.B. Ahmed et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxxxxx

Table 2
Regression and analytical parameters of the proposed methods for determination of Dimenhydrinate,Cinnarizine and Cinnarizine impurity.

Parameters TLC-densitometric method RP-HPLC method

DMH CIN IMP DMH CIN IMP
Range 0.22 lg/band 0.41.6 lg/band 0.11 lg/band 330 lg/mL 220 lg/mL 110 lg/mL
Slope 0.0392 0.0720 0.0387 0.0822 0.0502 0.0325
Intercept 0.0596 0.4867 0.1856 0.0392 0.5875 0.0476
Correlation coefficient(r) 0.9998 0.9999 0.9999 0.9998 0.9997 0.9997
Accuracy 100.44 0.888 100.27 0.870 100.13 1.234 100.26 1.128 99.71 1.226 100.10 0.856
(mean SD)
Precision
Repeatabilitya 0.881 0.532 0.941 0.918 1.128 1.088
Intermediateb precision 1.732 1.344 1.036 1.462 1.598 1.598
LODc 0.038 0.109 0.025 0.866 0.527 0.285
LOQc 0.127 0.351 0.085 2.860 1.742 0.943
Robustness
Flow rate change (0.1 mL/min) 1.094 1.027 2.172
pH change of mobile phase (0.2) 0.749 1.949 1.023
Change methanolcomposition (0.5%) 1.025 0.825 0.129 1.624 2.014 0.931
a
The intraday (n = 3), average of three different concentrations repeated three times daily.
b
The interday (n = 3), average of three different concentrations repeated three times in three successive days.
c
limit of detection (3.3 * SD/Slope) and limit of quantitation (10 * SD/Slope).

Table 3
Statistical comparison of the results obtained by applying the proposed methods and the reported method for analysis of DMH and CIN.

Item TLC-densitometric method RP-HPLC method Reported method [16]

DMH CIN DMH CIN DMH CIN
Mean 100.44 100.27 100.26 99.71 101.04 101.00
SD 0.888 0.870 1.128 1.226 0.965 0.936
N 6 6 6 6 6 6
t-test (2.228) (a) 1.06 1.33 1.21 1.92
F-value (5.050) (a) 1.01 1.03 1.63 2.05
a
The values between parenthesis are corresponding to the theoretical values of t and F (P = 0.05).

Table 4
System suitability testing parameters of the developed methods.

Item TLC-densitometric RP-HPLC method Reference values

method
DMH CIN IMP DMH CIN IMP
Tailing factor 0.87 0.90 0.83 1 0.88 0.83 1
Resolution (Rs) 5.38 12 9.65 1.81 R greater than 2
Selectivity (a) 1.60 5.8 2.4 1.25 a less than 1.5
Capacity factor (K0 ) 5 12 4 110 acceptable
Column efficacy (N) 1.88  104 7.69  104 4  104 Increases with increasing efficiency of separation
HETP (height equivalent to theoretical plate 1.32  104 3.25  104 6.25  104 The smaller the value the higher the column
(cm/plate)) efficiency

values of the %RSD, as given in Table 2, indicated the robustness of
the two proposed methods.
System suitability testing for TLC-densitometric and RP-HPLC
methods was based on the concept that the equipment, electronics,
analytical operations and samples constitute an integral system
that can be evaluated as a whole. System suitability was checked
by calculating the capacity factor (K0 ), tailing factor (T), column
efficiency (N), selectivity (a) and resolution (Rs) factors. All calcu-
lated parameters were within the acceptable limits indicating good
selectivity of the methods and ensuring system performance,
Table 4.
out preliminary separation steps. The suggested TLC-densitometric
method provides good resolution between the studied components
with short analysis time as it runs simultaneously using a small
quantity of mobile phase making it cost effective. The RP-HPLC
method is more robust, accurate and specific than the previously
published ones.
Conflict of interest
None.

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Please cite this article in press as: A.B. Ahmed et al., Simultaneous determination of Dimenhydrinate, Cinnarizine and Cinnarizine impurity by TLC and
HPLC chromatographic methods, Bulletin Facult Pharmacy Cairo Univ (2017), http://dx.doi.org/10.1016/j.bfopcu.2017.01.003