A Stability-Indicating RP-HPLC Method for the Quantitative Analysis of Meclizine Hydrochloride in Tablet Dosage Form

Journal of Chromatographic Science 2015;53:793 799 doi:10.1093/chromsci/bmu127 Advance Access publication February 1, 2015 Article A Stability-Indicating RP-HPLC Method for the Quantitative Analysis of Meclizine Hydrochloride in Tablet Dosage Form Ramalingam Peraman, Maheswari Manikala*, Vinod Kumar Kondreddy and Padmanabha Reddy Yiragamreddy Division of Pharmaceutical Analysis and Quality Assurance, Center for Pharmaceutical Research (CPR), Raghavendra Institute of Pharmaceutical Education and Research (RIPER), Anantapur, Andhra Pradesh 515721, India *Author to whom correspondence should be addressed. Email: mahi24.thaps@gmail.com Received 27 February 2013; revised 10 July 2014 A specific stability-indicating reversed-phase high-performance liquid chromatographic method was developed and validated for the estimation of meclizine hydrochloride (MEC) in tablet dosage form. The HPLC method has shown adequate separation of MEC from their degradation products. The separation was achieved on a C 8 (250 mm34.6 mm35 mm) column using a mobile phase composition of 0.2% triethylamine in water and methanol in the ratio of 65:35(pH adjusted to 3.0 with orthophosphoric acid) with a flow rate of 1 ml/min. The wavelength of a photo-diode array detector was kept at 229 nm. Stress studies were performed initially under milder conditions followed by stronger conditions so as to get sufficient degradation around 5 20%. There were six degradation products observed with adequate separation from the analyte peak. Among those detected degradation products, structures of four degradation products were verified by comparison with known impurities of meclizine analogs. The method was validated as per the International Conference on Harmonization (Q2) guidelines. The method was specific, selective, accurate and precise to quantify meclizine in the presence of degradation products. Introduction Meclizine (MEC) is chemically known as (RS)-1-[(4- chlorophenyl)(phenyl)methyl]-4-(3-methylbenzyl)piperazine (see Figure 1). Meclizine is available as tablet for the treatment of motion sickness and vertigo, but the safety and efficacy in children younger than 12 years have not been established. It has to be administered with caution in patients older than 65 years due to the risk of confusion and amnesia. The structure and biological behavior of meclizine is similar to buclizine, cetirizine, cyclizine and hydroxyzine (1, 2). Few analytical methods, such as UV spectrophotometric (3, 4), high-performance liquid chromatographic (HPLC) (5) and liquid chromatography tandem mass spectrometry (LC MS-MS) (6), were reported for the quantification meclizine alone or along with other drugs. A detailed literature survey on the stability-indicating assay method (SIAM) on meclizine revealed that a simultaneous stability-indicating method for meclizine and pyridoxine hydrochloride in dosage forms was reported. However, the method was not validated as per the requirement for SIAM and did not reveal structures for degradation products neither by comparison with known impurity nor by spectral identification (7). Based on the need for the development of new SIAM for meclizine, this study was designed to develop a sensitive and precise stability-indicating RP-HPLC method and validate as per International Conference on Harmonization (ICH) Q2 (R2) guidelines (8, 9). This study represents all possible degradation products likely to occur during stability testing of meclizine dosage form. In this study, attempts were made to detect and verify the structure of major impurities by retention comparison using available impurities in our laboratory. Two impurities (impurities 4 and 6) are common for buclizine, cyclizine, cetirizine, and meclizine as they differ structurally in the side chain at the piperazine residue while other impurities (impurities 1 and 5) were belong to related substances of meclizine. Impurity-5 was N-oxide derivative of meclizine, chosen based on the oxidative degradation profile of cetirizine. Experimental Materials and reagents Meclizine hydrochloride (99.89%) was kindly provided by Fleming Laboratories Limited, Hyderabad, India, as a gift sample. Meclizine hydrochloride tablets were procured from a local pharmacy, Anantapur, Andhra Pradesh, India. The placebo mixture used was prepared in the laboratory by mixing microcrystalline cellulose, lactose and magnesium stearate. All reagents and solvents were of HPLC grade and procured from Merck, India. Impurities were kindly provided by Analytical Research Laboratory, JSS College of Pharmacy, Nilgiris, India. Instrumentation The HPLC system (Agilent LC-1200 with EZchrom Elite Software) containing a C 8 (Qualisil BDS, 250 4.6 mm, 5 mm) column with a UV-PDA detector was used. The ph measurement was carried outwitheutechphtutor,equippedwithacombinedglasscalomel electrode. Preparation of sample solution The weight equivalent to 0.5 g dosage unit of tablet powder was transferred into a 100-mL volumetric flask and extracted with HPLC grade methanol using sonication and made up to the mark to obtain 1000 mg/ml. From this solution, 0.5 ml is diluted to 10 ml to get 50 mg/ml for the assay. The solution was filtered and then diluted immediately before use to appropriate concentration levels, by using mobile phase. The same procedure was repeated for inducing stress to tablet powder instead of methanol with appropriate stress agents. Forced degradation studies of meclizine hydrochloride Forced degradation of meclizine hydrochloride (MEC) was performed under neutral, acid, alkaline, oxidative, thermal and # The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

(0.01 1 N) and hydrogen peroxide (0.3 3%). Thermal degradation was carried out for both solid and solution states (in water) by heating the samples over a period in a hot air oven at 708C. Photodegradation was also carried on the solid sample. The blank and placebo mixtures were analyzed under the same condition and the same procedure to assess the method specificity. Hydrolysis Stock solutions of 1,000 mg/ml were prepared in 0.1 N NaOH (basic), 1 N HCl (acidic) and water (neutral) at the room temperature (RT). Then, 0.5 ml volume of the sample was withdrawn at different time points and made to 10 ml with mobile phase (50 mg/ml). The samples from acid hydrolysis were neutralized with 0.1 N NaOH, and the samples from base hydrolysis were neutralized with 0.1 N HCl. Oxidation In a 10-mL volumetric flask, 0.5 ml of the sample was withdrawn and made to 10 ml with mobile phase and injected under optimized conditions at various time intervals against the peroxide blank. Figure 1. Structure of meclizine hydrochloride and their impurities. photolytic stress conditions. In all stress conditions, degradation was induced at a concentration of 1000 mg/ml. At different time intervals, 0.5 ml of degraded solution was diluted with mobile phase (to achieve a concentration of 50 mg/ml), and if necessary ph was adjusted to 3 4 with diluted acid or base and then injected under the optimized condition with an appropriate blank. Blank solutions and laboratory placebo mixtures were prepared at the same time of preparation of stock solutions. The chromatograms of placebo, blank, dosage form and standard stress injection were compared to identify the degradation product. The percentage degradation and relative response factor were calculated based on the following formulae. The concentration of impurity used in the study was 5 mg/ml. Degradation (%) ¼ peak area (degraded) peak area (control) 100 Relative response factor (RRF) area (impurity) concentration (API) ¼ area (API) concentration (impurity) Preparation of stock solution for stress studies An accurately weighed 10 mg of drug substance was carefully transferred into a 10-mL volumetric flask, dissolved completely in water (for neutral degradation studies) and the volume was made up to get 1000 mg/ml. The same procedure was used to prepare stock stress solutions used for acid hydrolysis, base hydrolysis and oxidation, respectively, with HCl (0.1 1 N), NaOH Thermal degradation For solution state studies, 0.5 ml of the sample from the preheated stock solution was withdrawn into a 10-mL volumetric flask, made to 10 ml with mobile phase and injected. For the solid-state stability, it was performed on a thin layer of the preheated sample in the petri-dish at 708C. At various time intervals, 10 mg of the heated samples were weighed, suitably dissolved, then diluted with mobile phase to get a concentration of 50 mg/ml and analyzed. Photodegradation Photodegradation studies were conducted by exposing the powder sample in a photo-stability chamber (Thermolab 400G New Delhi India) at 1.2 million lux hour for a total period of 1 week. After degradation, stock solutions were diluted with mobile phase to achieve a concentration of 50 mg/ml and injected into the system. Preparation of sample solution Twenty tablets were weighed and finely powdered; a quantity of tablet powder equivalent to 25 mg of MEC was weighed accurately and transferred into a 100-mL volumetric flask. Tablet powder was extracted with 50 ml water with the aid of ultrasonication and made up to the mark with the same solvent and filtered through a 0.45 mm filter to obtain 250 mg/ml of MEC. From the stock solution, 2 ml was diluted to 10 ml with mobile phase to obtain a final concentration of 50 mg/ml and it was analyzed. The results of the assay are shown in Table I. Identification of impurities Impuritiesm-tolylmethanol (Impurity-1), (4-chlorophenyl)- phenyl-methanol (Impurity-4), meclizine N-oxide (Impurity-5), 1-((4-chlorophenyl)(phenyl)methyl)piperazine (Impurity-6) were suitably prepared in mobile phase at a concentration of 794 Peraman et al.

5 mg/ml and spiked with injection of standards under optimized chromatographic conditions. The retention time of the impurities was compared with the stress degradation products to identify the impurity. The relative response factor for that impurity at 229 nm was calculated based on the response factor and is reported in Table II. These impurities were selected based on the degradation profile of structurally similar drugs such as cetirizine, buclizine, cyclizine and from available related substances for meclizine. Results Optimization of the chromatographic conditions In preliminary experiments, MEC was subjected to separation by different trials by reversed-phase HPLC using increasing ratios of water in mobile phase composition using acetonitrile as organic phase. The drug was retained and its retention time increased with an increase in % aqueous, but the peak shape was not good. Then, the effect of ph of aqueous phase in mobile phase composition on peak shape and theoretical plate was analyzed. An improved peak shape was observed at ph 3.0, but the number of theoretical plates was still very less and the drug was eluted as void peak. In this method development phase, phosphate buffers were avoided to make this method as LC MS compatible. In order to retain the drug, acetonitrile was replaced with methanol as organic solvent. At ph 3.0 and methanol as organic phase in the mobile phase composition, the peak shape and theoretical plates of MEC were good but still there was a tailing effect when the retention of the drug was increased. Due to the basic nature of MEC and nitrogen atoms of piperazine nucleus in the structure of MEC, 0.2% triethyl amine (TEA) in water was chosen as aqueous phase to eliminate tailing effect. The various trials in optimization of chromatographic conditions are shownintableiii. The chromatogram obtained was better than all other experimental trials and is shown in Figure 2. Table I Assay Results for Marketed Formulations Brand name Labeled claim Amount found mean % Assay % RSD Antivert Meclizine-25 mg 24.5 + 0.12 98.0 0.48 Antivert Meclizine-50 mg 49.2 + 0.15 98.4 0.30 An LC system (Agilent HPLC Model-1200 with EZchrom Elite software) equipped with a C 8 (Qualisil BDS, 250 4.6 mm, 5 mm) column, UV-PDA detection with mobile phase of 0.2% TEAinwater(pHadjustedto3.0withOPA)andmethanolin the ratio of 65:35% v/v was used in stress studies and validation. All the time mobile phase and other solvents were filtered through a 0.45-mm filter using vacuum and degassed through a sonicator. The flow rate was kept at 1.0 ml/min. The injection volume was 20 ml, and the detection was performed at 229 nm using a PDA detector. The method has proven for its specificity by separating the degradation products under various stress conditions from MEC. It was observed that six major impurities were formed with retention times of 3.1 min (Impurity-1), 3.7 min (Impurity-2), 4.0 min (Impurity-3) 6.5 min (Impurity-4), 12.8 min (Impurity-5) and 24.6 min (Impurity-6). The resolution among the impurities was good and also the peak purity was satisfactory. The % of impurities and % degradation of the drug and the corresponding mass balances are detailed in Table II. The mass balance was calculated based on the % area normalization method. There were four impurities (Impurity 1, 4, 5 and 6), where structures were predicted using retention times of known substances. The predicted structures of four identified impurities are shown in Figure 1. Validation Parameters The method was validated as per the ICH (Q2) guidelines with respect to specificity, linearity, accuracy, precision, robustness, limit of detection (LOD) and limit of quantification (LOQ) (9). Specificity Forced degradation studies were used to support the specificity of the stability-indicating method. It was carried out by stress degradation of MEC by UV light exposure (for 7 days at RT), heat (708C for 3 days), acid hydrolysis (1 N HCl, at RT for 3 days), base hydrolysis (0.1 N NaOH, at RT for 1 day), water hydrolysis (at RT for 6 days) and oxidation (3% H 2 O 2, at RT for 1 day). The resolution between MEC and its degradation products was considered to assess the specificity of the method. Table II Stress Degradation Profile of Meclizine Hydrochloride (MEC) Stress conditions Duration (days) % Area Mass balance Imp Imp Imp Imp Imp Imp Total degradants MEC Assay 1 2 3 4 5 6 Water at RT 6 100 1 N HCl at RT 3 20.71 20.71 78.71 99.69 0.1 N NaOH at RT 1 6.08 2.73 2.14 5.37 16.32 81.66 96.98 b 3% H 2 O 2 at RT 1 16.26 16.26 80.71 98.97 Thermal-solution 3 23.12 12.17 35.84 63.18 99.34 Thermal-solid 7 99.92 Photolytic 7 10.94 11.06 22.12 76.85 98.91 b Retention time a (min) 3.1 3.7 4.0 6.5 12.8 24.6 10.5 RRF 0.54 0.22 0.78 0.86 RRF, relative response factor. a Mean value of retention time in all measurements; apparent mass balance. b Corrected mass balances. Stability-Indicating RP-HPLC Method for MEC 795

Linearity The linearity of detector response (at 229 nm) to different concentrations of MEC was studied in the range from 10 to 120 mg/ ml. The samples were analyzed in triplicate at eight concentrations, 10, 20, 30, 50, 60, 80, 100 and 120 mg/ml. The correlation coefficient (r 2 value) obtained was 0.9996 and indicated a linear response of MEC and suitability of the method for quantification. Accuracy Accuracy was performed by recovery studies using the standard addition method. Standard solutions of MEC in the range of 80, 100 and 120% of the sample concentration were added into the sample solution as per Table IV. Each concentration was analyzed in triplicate. The results of recovery studies were found to be in between 98.78 and 101.83% with % RSD of,2. Precision The data for intraday and interday precision studies were obtained for three different concentrations (30, 50 and 100 mg/ ml) in the linearity. The % RSD values for intraday and interday precision were,2 and the result is shown in Table V. Robustness test The robustness of the developed method was determined by analyzing the samples under a variety of conditions of the method Table III Trails Adopted in the Optimization of Chromatographic Conditions Trail number Mobile phase (% v/v) Retention time (min) Theoretical plates Tailing factor 1 Water ACN (20:80) 3.4 3,321 1.82 2 Water ACN (30:70) 5.1 3,008 1.98 3 Water ACN (20:80); ph 3.0 2.8 3,456 1.45 4 Water methanol (20:80); ph 3.0 3.1 4,704 1.32 5 Water methanol (15:85); ph 3.0 3.9 4,641 1.35 6 Water methanol (40:60); ph 3.0 4.6 4,373 1.42 7 0.2% TEA methanol (50:50); ph 3.0 7.1 6,313 1.09 8 0.2% TEA methanol (65:35); ph 3.0 10.9 6,083 1.11 ACN, acetonitrile. parameters, such as change in flow rate (+0.1 ml/min), ph of the buffer (+0.2 units), mobile phase composition (+2%) and wavelength (+2 nm). The results of robustness studies are shownintablevi. The method is robust for all parameters tested. Limit of detection and limit of quantification The LOD and LOQ were determined based on a signal-to-noise (S/N) ratio. The S/N ratio of 3:1 was taken as LOD and the S/N of 10:1 was taken as LOQ. The LOD was found to be 0.168 while the LOQ was 0.506 mg/ml. The same concentrations of LOD and LOQ were prepared and injected under the optimized conditions in triplicate and found to be the S/N of 4 for LOD and 12 for LOQ. The LOD and LOQ value for the impurity substances (impurities 1, 4, 5 and 6) were calculated based on the slope values obtained from their linearity curves of concentrations between 5 and 25 mg/ml. The respective LOD values for impurities 1, 4, 5 and 6 are 0.315, 0.763, 0.215 and 0.201 mg/ ml and the respective LOQ values are 0.955, 2.32, 0.711 and 0.649 mg/ml. Discussion Acid-induced degradation MEC was degraded to 20.71% with one degradation product (Impurity-1) at retention times of 3.1 min. The area percentage of Impurity-1 was 20.71. The assay MEC was 78.28% and the chromatogram is shown in Figure 3. Base-induced degradation MEC was degraded to 16.32% in 24 h at RT with four products (Impurities 1, 2, 3 and 4). The percentage area of for Impurity 1, Impurity-2, Impurity-3 and Impurity-4 are 6.08, 2.73, 2.14 and 5.37, respectively. The assay of MEC was found to be 81.66% and the chromatogram is shown in Figure 4. Neutral degradation MEC was not degraded at RT even after 7 days. Figure 2. Optimized RP-HPLC chromatogram for meclizine hydrochloride. 796 Peraman et al.

Oxidative degradation MEC showed negligible degradation in 0.3% H 2 O 2 for 3 days; hence, a severe stress condition of 3% H 2 O 2 was used. After 24 h, 16.26% degradation was observed at 24 h with one degradation product was formed. The percentage area of Impurity-5 was 16.26 whereas the MEC assay value was 80.71% and the chromatogram is shown in Figure 5. Thermal degradation Both solid and solution state MEC were exposed to dry heat in an oven at 708C for 7 days. There was no degradation product was observed for solid state, but the degradation product peaks were observed for solution state after 3 days. Two degradation products at 6.5 min (Impurity-4) and 24.6 min (Impurity -6) were detected with 63.18% assay value of MEC. The chromatogram is shown in Figure 6. Table IV Accuracy of the Method Amount (mg/ml) Amount added (mg/ml) Amount found (mg/ml) (mean + SD) % RSD % Recovery MEC 30 24 (80%) 54.9 + 0.39 0.11 101.7 30 30 (100%) 61.1 + 0.25 0.12 101.8 30 36 (120%) 65.2 + 0.23 0.14 98.7 Table V Precision of the Method Drug Amount (mg/ml) % RSD Intraday Interday MEC 30 0.28 0.60 50 0.22 0.42 100 0.28 0.30 Table VI Robustness Studies of the Method Robust parameter Change Retention time (in min) Tailing factor % Assay Flow rate (ml/min) 0.9 10.76 1.212 99.12 1.1 8.64 1.169 98.18 Water methanol ratio (% v/v) 33:67 7.99 1.216 98.92 37:63 11.6 1.178 98.13 Wavelength (nm) 227 10.49 1.109 101.01 231 10.52 1.106 98.35 Buffer (ph) 2.8 10.38 1.312 98.32 3.2 10.44 1.214 99.14 Photolytic degradation MEC was exposed to photolytic degradation at 1.2 million lux hours for a period of 1 week. At the end of seventh day, 22.12% degradation was observed with two degradation products (Impurity-1 and Impurity-2). The assay of the active substance in the photodegraded sample was 77.88% and the chromatogram is shown in Figure 7. With reference to the earlier report (7), the retention time of pyridoxine and meclizine was found to be 5.25 and 10.14 min, respectively, with the mobile phase of buffer acetonitrile trifluoroacetic acid (30:70:1). The mobile phase composition and ratio of this communication are more suitable for robust behavior of the method, and this method has not utilized any ion pair reagent. The number of degradation peaks detected in this study is six and among those, four impurities were verified. The reported simultaneous method has not verified the impurities for the respective analytes, hence a control study on stability for pure drugs always recommended to conclude the source of impurity in the simultaneous stability study. To consolidate the results, the drug was more susceptible to base hydrolysis than acid hydrolysis and oxidation. MEC was found to be more stable in neutral ph and unaffected by the solution phase. The mass balances between 97 and 99% indicate the suitability of method and the detector that could detect all possible impurity. Four degradation products were identified by comparing the retention time of impurity and related substances of meclizine and its analogs. They are m-tolylmethanol (Impurity-1), (4-chlorophenyl)-phenyl-methanol (Impurity-4), meclizine N-oxide (Impurity-5) and 1-((4-chlorophenyl)(phenyl) methyl)piperazine (Impurity-6). Among these, Impurity-1 and Figure 3. Acid degradation chromatogram for meclizine hydrochloride. Stability-Indicating RP-HPLC Method for MEC 797

Figure 4. Base degradation chromatogram for meclizine hydrochloride. Figure 5. Oxidative degradation chromatogram for meclizine hydrochloride. Figure 6. Thermal degradation chromatogram for meclizine hydrochloride. Impurity-5 are related substances of meclizine and the remaining two (Impurity 4 and 6) are common degradation products for diphenylmethyl piperazine core containing antihistamines. The predicted structures of impurities are shown in Figure 1. Impurity-5 was selected based on the expectation that the degradation pathway for oxidation would be the same for 798 Peraman et al.

Figure 7. Photolytic degradation chromatogram for meclizine hydrochloride. cetirizine and meclizine in which N-oxide impurity is more common. The relative response factor for all the impurity substance were.0.2, hence these substance can be used to identify the unknown impurities. Impurity-5 and Impurity-6 that have response factors of 0.78 and 0.86 indicate the structure resemblance of degradation product and active substance. Conclusion A new specific stability-indicating RP-HPLC method was developed for the estimation of meclizine hydrochloride (MEC) in pharmaceutical dosage form and validated according to the ICH guidelines. The method was found to be specific for the detection of all possible impurities in the dosage form under various conditions and accurate, precise and robust for the assay of MEC in dosage forms. Acknowledgments The authors are grateful to the Department of Pharmaceutical Analysis and Quality Assurance, Raghavendra Institute of Pharmaceutical Education and Research, Anantapur, India, for providing research facility to carry out the work. References 1. Kallen, B., Mottet, I.; Delivery outcome after the use of meclozine in early pregnancy; European Journal of Epidemiology, (2003); 18(7): 665 669. 2. Oishi, R., Shishido, S., Yamori, M., Saeki, K.; Comparison of the effects of eleven histamine H1-receptor antagonists on monoamine turnover in the mouse brain; Naunyn Schmiedebergs Archieves of Pharmacolology, (1994); 349(2): 140 144. 3. Shinde, P., Rai, C., Daswadhar, S., Pallavi, C., Kasture, P.V.; Development of UV spectrophotometric method of meclizine hydrochloride in bulk and pharmaceutical formulation; Research Journal of Pharmacy and Technology, (2012); 5(6): 857 859. 4. Ravalji, M.B., Shah, S.A., Shah, D.R., Daxina, K.L., Chauhan, R.S.; Spectroscopic methods for simultaneous estimation of Meclizine hydrochloride and Caffeine in their combined tablet dosage form; Asian Journal of Research in Chemistry, (2011); 4: 1249 1253. 5. Ramakanth Reddy, D., Padmanabha Reddy, P., Devanna, N., Amaranatha Reddy, B.; A new validated RP-HPLC-DAD method for simultaneous determination of meclizine and caffeine pharmaceutical dosage form; Inventirapid:Pharm Analysis & Quality Assurance, (2013) http://www.inventi.in/article/ppaqa/1099/13.aspx 6. Wang, Z., Qian, S., Zhang, Q., Chow, M.S.; Quantification of meclizine in human plasma by high performance liquid chromatography-mass spectrometry; Journal of Chromatography B, (2011); 879(1): 95 99. 7. Saddam Nawaz, Md.; A new validated stability indicating RP-HPLC method for simultaneous estimation of pyridoxine hydrochloride and meclizine hydrochloride in pharmaceutical solid dosage forms, Chromatography Research International, (2013); http://dx.doi.org/10.1155/2013/747060. 8. ICH Harmonized Tripartite Guidelines. Stability testing of new drug substances and products, Q1A(R2) (1995). 9. ICH Harmonized Tripartite Guidelines. Validation of analytical procedures: text and methodology, Q2A and Q2B (1995). Stability-Indicating RP-HPLC Method for MEC 799