Simultaneous determination of Dimenhydrinate, Cinnarizine and Cinnarizine impurity by TLC and HPLC chromatographic methods

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

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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 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 b

a r t i c l e

i n f o

Article history: Received 16 August 2016 Received in revised form 24 December 2016 Accepted 15 January 2017 Available online xxxx Keywords: Dimenhydrinate Cinnarizine 1-(Diphenylmethyl) piperazine) TLC HPLC

a b s t r a c t Two chromatographic methods have been established and validated for simultaneous determination of mixture of Dimenhydrinate (DMH) and Cinnarizine (CIN) in their pharmaceutical formulation and in presence of Cinnarizine impurity (1-(Diphenylmethyl) piperazine); CIN impurity. The first method was TLC-densitometric one, depends on separation and quantitation of DMH, CIN and CIN impurity on TLC 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 235 nm. Linear relationships were obtained in the range of 0.2–2, 0.4–1.6 and 0.1–1 lg/band for DMH, CIN and CIN impurity, respectively. The studied components were well resolved from each other with significantly different Rf values of 0.35, 0.52 and 0.04 for DMH, CIN and CIN impurity, respectively. The second method was RP-HPLC, separation on C8 column using 0.05 M KH2PO4 (pH = 3):methanol (35:65, v/v) 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 3–30, 2–20 and 1–10 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. Ó 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-ncnd/4.0/).

1. Introduction Dimenhydrinate (DMH); is chemically known as 8-chloro-3, 7dihydro-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); chemically known as (E)-1-(Diphenylmethyl)-4-(3-phenylprop-2-enyl) piperazine [1], (Fig. 1b), it is a piperazine derivative with antihistaminic, 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 Pharmacopeia (BP) [3] stated that 1-(Diphenylmethyl) piperazine (CIN impurity) is Cinnarizine impurity, it is also known as benzhydrylpiperazine [1], Fig. 1c. Peer review under responsibility of Faculty of Pharmacy, Cairo University. ⇑ Corresponding author. E-mail address: [email protected] (A.B. Ahmed).

BP [3] reported a titration method for determination of DMH 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 estimation 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 [7–11] and HPLC method [10–14] either alone or in combination with other drugs in pharmaceutical formulation or in biological 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/Ó 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

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A.B. Ahmed et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxx–xxx

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 scanning 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 quantitation 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 AmocerebralÒ tablets (batch No. 6221025030658) were manufactured 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.

Fig. 1. Chemical structures of (a) Dimenhydrinate, (b) Cinnarizine, (c) Diphenylmethyl 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 carried out at 260 nm [16–18]. No method was reported for simultaneous determination of Dimenhydrinate, Cinnarizine and Cinnarizine impurity. The previously 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 selectivity 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 WINCATS software (version 3.15) (Camag, Muttenz, Switzerland); a sample applicator for TLC Linomat V equipped with a 100 lL syr-


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 100mL 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 respective stock standard solutions (1 mg/mL) into three separate 100-mL volumetric flasks, then the volume was completed to the mark with methanol for TLC–densitometric 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

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

A.B. Ahmed et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxx–xxx

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 chromatographic 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 maintained at 25 °C. 3.2. Construction of calibration curves a. TLC-Densitometric method Different aliquots equivalent to 0.2–2, 0.4–1.6 and 0.1–1 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 30–300, 20–200 and 10–100 lg, respectively, were separately 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 concentration 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 equations were computed. 3.3. Application to pharmaceutical formulation (AmocerebralÒ tablets) Ten tablets of AmocerebralÒ tablets were weighed, powdered and mixed well; an accurate weight of the powdered tablets equivalent to 100 mg of DMH and 50 mg of CIN was transferred into 100 mL volumetric flask. 75 mL of methanol was added and ultrasonicated 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 followed. 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.

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4. Results and discussion The present work provides for the first time sensitive, accurate and selective chromatographic methods for simultaneous determination 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 determination of Dimenhydrinate and Cinnarizine of being highly selective 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 Impurity. 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 separation, it was necessary to investigate the effect of different variables. Determination of the optimum parameters for maximum separation was carried out as described below. 4.1.1.1. Developing system. Different developing systems of different 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 differentiation between CIN and its impurity, Also chloroform:methanol:ammonia 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 developing system allows good separation between the studied components 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:methanol:ammonia solution:glacial acetic acid (9.5:0.5:0.1:0.1, by volume) as developing system followed by densitometric

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

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A.B. Ahmed et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxx–xxx

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 components were well resolved from each other with significantly different 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 0.2–2, 0.4–1.6 and 0.1–1 lg/band for DMH, CIN and CIN impurity, respectively. The concentration of each component was calculated from the following regression equation:

A1 ¼ 0:0596CDMH þ 0:0392; A2 ¼ 0:4867CCIN þ 0:072;

r ¼ 0:9998 r ¼ 0:9999

A3 ¼ 0:1856Cimpurity þ 0:0387;

r ¼ 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 detection 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

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 buffer adjusted to different pH values. It was noticed that changing the buffer pH greatly affected the Rt values of DMH, CIN and CIN impurity 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 buffer (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 separated peaks, as shown in Fig. 3. It was found that presence of orthophosphoric acid in the mobile phase is essential for separation 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 calibration curve for DMH, CIN and CIN impurity was constructed by plotting the peak area/104 versus the corresponding concentrations of each drug in the range of 3–30, 2–20 and 1–10 lg/mL for DMH, CIN and CIN impurity, respectively. The concentration for each component was calculated from the following regression equation:

A1 ¼ 0:0392CDMH þ 0:0822; A2 ¼ 0:0502CCIN þ 0:5875;

r ¼ 0:9998 r ¼ 0:9997

A3 ¼ 0:0476Cimpurity þ 0:0325;

r ¼ 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.

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

101.24 99.64 99.98 100.21 100.26 ±0.689 100.38 101.50 101.07 98.80 100.44 ±1.182

DMH

3 3 5 5 7 7 9 10 Mean ± SD 99.25 101.39 100.51 100.70 100.46 ± 0.894 average of six determinations. average of three determinations. a

b

CIN DMH CIN

100.58 100.78 98.79 98.15 99.57 ± 1.305

DMH CIN

0.4 0.5 0.6 0.7 0.2 0.3 0.5 0.6 Mean ± SD

DMH CIN

106.52 ± 1.590 103.89 ± 1.174

DMH

4.2. Method validation

105.03 ± 0.918

CIN DMH

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 correlation coefficient. The proposed methods were applied for determination of DMH and CIN in their pharmaceutical formulation (AmocerebralÒ tablets). Validity of the method was assessed by adding to the pharmaceutical formulation known amount of standard drug powder. The recovery of the added standards was then calculated, Table 1. Regression equation parameters are given in Table 2. The results obtained by applying the proposed methods were statistically compared with those obtained by applying the reported HPLC method [16]. The values of the obtained t- and F-tests were less than the tabulated ones confirming that the difference between the developed methods and the reported one is insignificant regarding to accuracy and precision, Table 3.

104.80 ± 1.141 AmocerebralÒ tablets Claimed to contain 20 mg of DMH and 10 mg of CIN/ tablet

RP-HPLC method

% Recoveryb % Recoveryb

TLC-densitometric method

Added (lg/band) RP-HPLC method TLC-densitometric method

% Recovery ± SDa Pharmaceutical formulation

Table 1 Determination of Dimenhydrinate and Cinnarizine in their pharmaceutical formulation by the proposed methods and application of standard addition technique.

Added (lg/mL)

CIN

A.B. Ahmed et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxx–xxx

Method validation was performed according to the international conference on harmonization (ICH) guidelines [19] for the proposed methods. The linearity of the proposed methods was evaluated and it was evident in the ranges of 0.2–2, 0.4–1.6 and 0.1–1 lg/band for DMH, CIN and CIN impurity, respectively (for the TLC-densitometric method) and 3–30, 2–20 and 1–10 lg/mL for DMH, CIN and CIN impurity, respectively (for RP-HPLC method). The regression equations for the proposed methods were computed and given in Table 2. The accuracy of the proposed methods was checked by applying the proposed methods for determination of pure samples of the studied compounds. The concentrations were calculated from the corresponding regression equations and the results are shown in Table 2. Accuracy was further assessed by applying the standard addition technique on AmocerebralÒ tablets, and good recoveries were obtained, revealing no interference from excipients and good 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 method and revealing that it is suitable for the quality control of the suggested components, 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) and (5, 8 and 10 lg/mL of CIN impurity) for RP-HPLC method, were analyzed three times intra-daily using the proposed methods. Good results and acceptable %RSD values were obtained, Table 2. Intermediate precision: The developed methods were repeated inter-daily on three successive days for the analysis of the previously mentioned concentrations. Good results and acceptable % RSD values were obtained, shown in Table 2. Detection and quantification limits: Low values of LOD and LOQ shown in Table 2, proved high sensitivity of the developed methods, These approaches based on the SD of the response and the slope were used for determining the detection and quantitation limits (where LOD = 3.3 * SD/slope and LOQ = 10 * SD/slope), Specificity of the proposed methods is evident from the TLC and RP-HPLC chromatograms in Figs. 2 and 3, respectively. Also values of the calculated parameters such as selectivity and resolution factors were within the acceptable limit, Table 4. The robustness of the proposed methods was evaluated in the development phase where the effects of different factors on TLCdensitometric 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 rate (±0.1 mL/min) and studying the effect of changing mobile 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

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

TLC-densitometric method DMH

CIN

IMP

DMH

CIN

IMP

Range Slope Intercept Correlation coefficient(r) Accuracy (mean ± SD)

0.2–2 lg/band 0.0392 0.0596 0.9998 100.44 ± 0.888

0.4–1.6 lg/band 0.0720 0.4867 0.9999 100.27 ± 0.870

0.1–1 lg/band 0.0387 0.1856 0.9999 100.13 ± 1.234

3–30 lg/mL 0.0822 0.0392 0.9998 100.26 ± 1.128

2–20 lg/mL 0.0502 0.5875 0.9997 99.71 ± 1.226

1–10 lg/mL 0.0325 0.0476 0.9997 100.10 ± 0.856

0.881 1.732 0.038 0.127

0.532 1.344 0.109 0.351

0.941 1.036 0.025 0.085

0.918 1.462 0.866 2.860

1.128 1.598 0.527 1.742

1.088 1.598 0.285 0.943

0.129

1.094 0.749 1.624

1.027 1.949 2.014

2.172 1.023 0.931

Precision Repeatabilitya Intermediateb precision LODc LOQc Robustness Flow rate change (±0.1 mL/min) pH change of mobile phase (±0.2) Change methanolcomposition (±0.5%) a b c

1.025

RP-HPLC method

0.825

The intraday (n = 3), average of three different concentrations repeated three times daily. The interday (n = 3), average of three different concentrations repeated three times in three successive days. 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

Mean SD N t-test (2.228) (a) F-value (5.050) (a) a

TLC-densitometric method

RP-HPLC method

DMH

CIN

DMH

CIN

DMH

Reported method [16] CIN

100.44 0.888 6 1.06 1.01

100.27 0.870 6 1.33 1.03

100.26 1.128 6 1.21 1.63

99.71 1.226 6 1.92 2.05

101.04 0.965 6 – –

101.00 0.936 6 – –

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

Tailing factor Resolution (Rs) Selectivity (a) Capacity factor (K0 ) Column efficacy (N) HETP (height equivalent to theoretical plate (cm/plate))

TLC-densitometric method

RP-HPLC method

DMH

CIN

IMP

DMH

CIN

IMP

0.87 5.38 1.60

0.90

0.83 12 5.8

1 9.65 2.4 5 1.88  104 1.32  104

0.88

0.83 1.81 1.25 4 4  104 6.25  104

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 calculated parameters were within the acceptable limits indicating good selectivity of the methods and ensuring system performance, Table 4. 5. Conclusion The present work provides for the first time sensitive, accurate and selective analytical methods for simultaneous determination of Dimenhydrinate and Cinnarizine in presence of Cinnarizine impurity in bulk powder and in pharmaceutical formulation with-

12 7.69  104 3.25  104

Reference values

1 R greater than 2 a less than 1.5 1–10 acceptable Increases with increasing efficiency of separation The smaller the value the higher the column efficiency

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. References [1] S. Budavaried, The Merck Index: an encyclopedia of Chemicals, Drugs and biological, 14th ed., Merck & co. Inc, Whitehouse station, NJ, USA, 2012. [2] K. Profitt (Ed.), The extra pharmacopeia: the complete drug reference, 45th ed., Royal Pharmaceutical Society, London, UK, 2014. [3] British Pharmacopoeia, Vol. II, Stationary Office, Medicines and Healthcare Products Regulatory Agency, London, 2013.

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

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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