Asian Journal of Chemistry; Vol. 25, No. 9 (2013), 4820-4824 http://dx.doi.org/10.14233/ajchem.2013.14114 Determination of Dapiprazole Hydrochloride in the Presence of Process and Degradation Impurities by a Validated Stability Indicating RP-HPLC Method * K. JAYA PRASANTHI and B. SYAMA SUNDAR Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur-522 510, India *Corresponding author: E-mail: [email protected] (Received: 27 May 2012; Accepted: 8 March 2013) AJC-13080 Dapiprazole hydrochloride is an α-adrenergic blocking agent. Chemically, it is 5,6,7,8-tetrahydro-3-[2-(4- o-tolyl-1-piperazinyl)ethyl]-s- triazolo[4,3-a]pyridine hydrochloride, which is widely used in ophthalmic practice. A stability-indicating RP-HPLC method has been developed and validated which can separate and accurately quantitate dapiprazole related compounds. The method was successfully validated in accordance to ICH guidelines acceptance criteria for system suitability, specificity, linearity, range, precision, accuracy, limits of detection and quantification for the impurities and robustness, following the ICH guidelines. Therefore, the proposed method was suitable for the simultaneous determination of dapiprazole and its process related and degreadation impurities. Finally, the applicability of the method was evaluated in commercial dosage form analysis as well as in stability studies. Key Words: Dapiprazole, Degradants, Validation, Stability indicating HPLC method. INTRODUCTION of dapiprazole in the drug substance. To the best of our know- ledge, no analytical methods are currently available to separate Dapiprazole hydrochloride is an α-adrenergic blocking and quantitate the known process related compounds of 1,2 agent . Chemically, it is 5,6,7,8-tetrahydro-3-[2-(4-o-tolyl- dapiprazole API. Furthermore, there is no stability-indicating 3 1-piperazinyl)ethyl]-s-triazolo[4,3-a] pyridine hydrochloride HPLC method reported in the literature that can adequately (empirical formula C19H27N5·HCl, m.w. 361.93). It is a sterile, separate and accurately quantitate dapiprazole and degradation white, lyophilized powder soluble in water. Dapiprazole hydro- compounds. Chemical structures of dapiprazole and it process chloride ophthalmic solution is indicated in the treatment of related impurity was shown below. (Fig. 1(a)) and the synthetic iatrogenically induced mydriasis produced by adrenergic route, in which the process related impurity has been formed, 4 (phenylephrine) or parasympatholytic (tropicamide) agents . is shown in Fig. 1(b). The aim of this paper was to develop validate a simple and reliable HPLC-UV method for the simultaneous determination EXPERIMENTAL of dapiprazole and its process related impurity-A. Besides the Waters Alliance 2695 separation module (Waters Corpo- simple route of synthesis for dapiprazole, we have observed a ration, Milford, USA) equipped with 2489 UV/visible detector potential impurity (Imp-A) in our synthetic process and some or 2998 PDA detector (for specificity and forced degradation unknown impurities in forced degradation. Hence, a stability- studies) with Empower 2 software was used for the analysis. indicating HPLC method for determination of dapiprazole in The chromatographic and the integrated data were recorded presence of process and degradation impurities was developed using HP-Vectra (Hewlett Packard, Waldbronn, Germany) and validated as per International Conference on Harmonization computer system. Chromatographic separation of dapiprazole (ICH) guidelines. This manuscript describes the development and its process related impurity was achieved on Inertsil ODS and validation, in accordance with ICH guidelines, of a rapid, C18-3V (250 mm × 4.6 mm, 5 µ particle size). The pH measu- economical, precise and accurate stability-indicating isocratic rements were carried out with Elico, model LI 120, pH meter reversed-phase HPLC method for analysis of dapiprazole in equipped with a combined glass-calomel electrode calibrated the presence of its process related impurity. This paper also using standard buffer solutions of pH 4.0, 7.0 and 9.2. deals with the forced degradation of dapiprazole under the All the reagents were of analytical-reagent grade unless stress conditions acidic and basic hydrolysis, oxidation, heat stated otherwise. Glass-distilled and deionized water (Nanopure, and light and validation of the method for accurate quantification Barnstead, USA), HPLC-grade acetonitrile, potassium dihydrogen Vol. 25, No. 9 (2013) Determination of Dapiprazole Hydrochloride in the Presence of Process and Degradation Impurities 4821 orthophosphate, sodium hydroxide, hydrochloric acid, hydrogen Forced degradation solutions: Weigh accurately 25 mg peroxide and orthophosphoric acid (SD Fine Chem, Mumbai, of dapiprazole sample and transfer into a 50 mL volumetric India) were used. Active pharmaceutical ingredient of flask and make up the volume up to 50 mL with mobile phase dapiprazole (reference standard) and its process related subs- to get the concentration of 500 µg/mL of dapiprazole standard tances viz. Impurity, 1-(o-tolyl)-piperizine were a kind of gift solution. from Hetero Drugs Pvt. Ltd., Hyderabad. Method validation: Validation of the developed method Chromatographic conditions: Separation was achieved for the determination of dapiprazole and its process related on a Inertsil-ODS-C18-3VS column (250 mm × 4.0 mm, 5 µm) mpurity was performed according to the ICH guidelines with using an isocratic mode by the mobile phase consists of a standards and API. Thus, system suitability along with method mixture of buffer and acetonitrile in the ratio of 85:15 v/v was selectivity, specificity, linearity, range, precision, accuracy, at pH-3.2 adjusted with 10 % orthophosphoric acid in water. limits of detection and quantification for the impurities, short The column was then re-equilibrated for 10 min with mobile term and long term stability of the analytes in the prepared phase. The flow rate of the mobile phase was 1 mL/min and solutions and robustness were demonstrated. the total elution time, including the column re-equilibration, System suitability: The system suitability was conducted was ca. 0.5 h. The UV detection wavelength was carried at using 1.5 % (w/w) of the impurity spiked to dapiprazole (3.0 205 nm. The injection volume was 20 µL and experiments mg/mL) and evaluated by making three replicate injections. were conducted at ambient temperature. The system was suitable for use if the tailing factors for Preparation of system suitability solution (standard dapiprazole and its impurity were < 1.55 (observed value is solution): Standards of dapiprazole and its process related 1.06) and the resolution was > 1.90 (observed value is 7.33). impurity (20 mg each) were accurately weighed, transferred Specificity: Specificity of a method can be defined as the into 100 mL volumetric flasks, dissolved in diluents and made absence of any interference at retention times of peaks of up to the mark with the mobile phase to get 200 ppm each of interest and is normally evaluated by observing the chromato- dapiprazole and its process related impurity solution. Transfer grams of blank samples and samples spiked with the API the 0.5 mL of the above stock solution and was adequately (active pharmaceutical ingredient) in the presence of all diluted up to 100 mL in volumetric flask with mobile phase to impurities in the mobile phase and the bulk drug. A separate get the concentration of 3 µg/mL (0.15 % level). solution of blank, standard and batch samples of dapiprazole Preparation of sample solutions: Samples of bulk drugs were evaluated along with impurity solutions. of dapiprazole (3 mg/mL) were prepared as by dissolving Linearity and range: The linearity of dapiprazole and 75 mg of dapiprazole in 25 mL volumetric flask containing its process related impurity was also studied by preparing diluents solution. standard solutions at seven different levels ranging from 0.75- N N CH3 N N .HCl NH N N Impurity-A: 1-(o-Tolyl)-piperizine Fig. 1. (a): Structures of dapiprazole and its impurity H3C O Cl + No2CO3 N CH3 O N CH3 ethyl 3-chloro-propionate NH O (I) 1-(o-tolyl)-piperozine O N Process related H3C Impurity-A ethyl 3-[4-(o-toly)-1-piperozinyl] propionate O Dapiprazole CH3 N H ·H Ob C H OH N CH I 2 4 2 2 5 N N 3 N CH Hydrozine hydrate 3 N N 6-methoxy-2,3,4,5-tetrohydropyridine N O HN N H2N 3-[4-(o-toly)-1-piperozinyl]propionate acid hydrazide Fig. 1. (b) Synthesis of dapiprazole 4822 Prasanthi et al. Asian J. Chem. 6 µg/mL. The data were subjected to statistical analysis using Forced degradation studies: Once the chromatographic a linear-regression model; the regression equations and coeffi- conditions were selected and the retention of dapiprazole was cients (r2) are given in calibration curves of dapiprazole and optimized, preliminary forced degradation studies were its process related impurity (o-tolyl piperizine) in Fig. 2. conducted using heat, acid and base hydrolysis, light and peroxide. The drug was exposed to extreme conditions to verify 450000 its stability under the conditions studied. To demonstrate the 400000 degradation of the sample in stress study, sample is prepared 350000 300000 and is evaluated at 0 h and after 48 h. 250000 Acid stress study: Weigh accurately and transfer ca. 300 200000 mg dapiprazole bulk sample in 20 mL of 0.1 N hydrochloric Y = 2113x + 3231 150000 R2 = 0.9998 acid. The 0 h-sample solution, has been prepared by taking 100000 Peak area/response immediately 5 mL of the above primary working standard 50000 0 solution and neutralize with 5 mL of 0.1 N sodium hydroxide 0.75 1.50 2.25 3.00 3.75 4.50 6.00 solution and makeup to 25 mL with diluents in volumetric Concentration (µµµg/mL) flask. The acid stress study sample after 48 h was prepared by taking the 5 mL of acid hydrolyzed sample solution in 25 mL 700000 volumetric flask and neutralize with 5 mL of 0.1 N NaOH 600000 solution and make up to 25 with diluent.