Development and Validation of an HPLC Method for Quantifying

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J Analytical & Bioanalytical Techniques ISSN: 2155-9872

Research Article Open Access Development and Validation of an HPLC Method for Quantifying Dapiprazole in Bulk Preparations Jaya Prasanthi K and Syama Sundar B* Department of Chemistry, Acharya Nagarjuna University, Guntur, India

Abstract A simple, precise, rapid and accurate Reverse phase HPLC method was developed for the estimation of Dapiprazole in bulk form. A Thermo hypersil C-18 column (250 mm×4.6 mm 5μm) with mobile phase consisting of mixture of methanol: acetonitrile: and ortho-phosphoric acid in the ratio of 89: 9: 1 (v/v) at pH 5.8 adjusted with ortho-phosphoric acid. The flow rate was 0.8 mL/min and the effluents were monitored at 243 nm. The retention time was 3.725 min. The detector response was linear in the concentration of 20-120 μg/mL. The respective linear regression equation being y=1956.4x+3409. The limit of detection and limit of quantification was 0.5 μg/mL and 1.6 μg/mL respectively. The percentage assay of Dapiprazole was 99.94%. This method has been validated and shown to be specific, sensitive, precise, linear, accurate, rugged, robust and fast. Hence, this method can be useful for the routine determination of Dapiprazole in bulk drug and in its pharmaceutical dosage form.

Keywords: Dapiprazole; RP-HPLC; Bulk drug Experimental Introduction Instrument Dapiprazole hydrochloride is an alpha-adrenergic blocking agent Quantitative HPLC was performed on liquid Chromatograph, [1]. Chemically, it is 5,6,7,8-tetrahydro-3-[2-(4- o-tolyl-1-piperazinyl) Shimadzu LC 2010 dual λ detector equipped with automatic injector ethyl]-s -triazolo[4,3-a] pyridine hydrochloride [2,3]. Dapiprazole with injection volume 20 μL. The HPLC system was equipped with LC . solution Software. The pH measurements were carried out with Elico, hydrochloride has the empirical formula C19H27N5 HCl and a molecular weight of 361.93. It is a sterile, white, lyophilized powder soluble in water. model LI 120, pH meter equipped with a combined glass-calomel Dapiprazole hydrochloride ophthalmic solution is indicated in the electrode calibrated using standard buffer solutions of pH 4.0, 7.0 and treatment of iatrogenically induced mydriasis produced by adrenergic 9.2. (phenylephrine) or parasympatholytic (tropicamide) agents [4,5]. The Materials/reagents novel synthetic method has been developed by concerning the yields and mild reaction conditions to synthesize Dapiprazole starting from Acetonitrile and methanol HPLC grade (Qualigens) and Water stable reactants such as ethyl 3-chloro propionate and o-tolyl piperzine, HPLC grade (Milli-Q), ortho-Phosphoric acid (Rankem). Dapiprazole which appears as impurity in the final product. In vivo evaluation was working and reference sample is obtained as gift sample from M/s performed for in ocular vehicle for long acting alfa adrenergic receptor Bioleo Analytical Labs India Pvt. Ltd, Hyderabad. blocking activity of dapiprazole hydrochloride [6]. Following topical HPLC conditions instillation on the eye, it crosses the corneal epithelium reaching high concentrations in the ocular tissue and producing a prompt mitotic The contents of the mobile phase were methanol: acetonitrile: and hypotensive effect. The high concentration ratio between ciliary and ortho-phosphoric acid in the ratio of 89: 9: 1 (v/v/v) at pH 5.8. bodies and iris versus aqueous humor suggests a peculiar affinity for They were filtered before use through a 0.45 μm membrane filter, and these structures containing adrenoceptors of the alpha type [7,8]. pumped from the respective solvent reservoirs to the column at a flow Also, a study was conducted to compare the cycloplegic and mydriatic rate of 0.8 mL/min. The run time was set at 8.0 min and the column effects of Paremyd™, a formulation of 0.25% tropicamide and 1% temperature was ambient. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase hydroxyamphetamine, to 0.5% tropicamide and 2.5% phenylephrine flowing through the system. The eluents were monitored at 243 nm. [9]. The influence of dapiprazole on pupil size was compared with brinzolamide and reported that pupil mydriasis at scotopic illumination Preparation of standard stock solution levels was reduced by both drugs in a similar fashion [10]. The aim A standard stock solution of the drug was prepared by dissolving of this paper was to develop validate a simple and reliable HPLC- UV method for the determination of Dapiprazole in bulk drug. This manuscript describes the development and validation, in accordance *Corresponding author: B. Syama Sundar, Department of Chemistry, with International Conference on Harmonization (ICH) guidelines, of Acharya Nagarjuna University, Nagarjuna Nagar, Guntur-522 510, India, E-mail: a rapid, economical, precise, and accurate stability-indicating isocratic [email protected] reversed-phase HPLC method for analysis of Dapiprazole in bulk Received July 16, 2012; Accepted August 24, 2012; Published August 30, 2012 sample. To the best of our knowledge, literature survey reveals no Citation: Jaya Prasanthi K, Syama Sundar B (2012) Development and Validation chromatographic methods for the estimation of Dapiprazole in bulk of an HPLC Method for Quantifying Dapiprazole in Bulk Preparations. J Anal Bioanal Techniques 3:143. doi:10.4172/2155-9872.1000143 samples. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the determination of Dapiprazole in Copyright: © 2012 Jaya Prasanthi K, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which bulk and also in pharmaceutical formulations. The chemical structure permits unrestricted use, distribution, and reproduction in any medium, provided of Dapiprazole is represented in Figure 1. the original author and source are credited.

J Anal Bioanal Techniques ISSN:2155-9872 JABT, an open access journal Volume 3 • Issue 4 • 1000143 Citation: Jaya Prasanthi K, Syama Sundar B (2012) Development and Validation of an HPLC Method for Quantifying Dapiprazole in Bulk Preparations. J Anal Bioanal Techniques 3:143. doi:10.4172/2155-9872.1000143

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stock solution with 25 ml of mobile phase to get the concentration of N 80 μg/mL. N

N Linearity N N Aliquots of standard drug solution of Dapiprazole 2.5, 5.0 and 7.5 ml (80 μg/ml) were taken and transferred into series of 10 ml volumetric H C 3 flasks and diluted up to 10 ml with mobile phase as dilute to get 20, 40 Figure 1: Structure of Dapiprazole. and 60 μg/ml. The working standard solution is directly used to get 80 μg/ml. Another set of aliquots of 1.0 and 1.2 ml (1000 μg/mL) were taken and transferred into series of 10 ml volumetric flasks and diluted up to Calibration Curve of Dapiprazole standard solution 10 ml with mobile phase as diluents to get 100 and 120 μg/ml. Each of 300000 these drug solutions (20 μL) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was 250000 performed with UV detector at 243 nm and a Calibration graph was obtained by plotting peak area versus concentration of Dapiprazole 200000 (Figure 2). 150000 Assay

Peak Areas Peak 100000 20 μL of sample solution was injected into the injector of liquid Y=1956.4x+3409 chromatograph. The retention time was found to be 3.724 minutes. The 50000 amount of drug present in bulk sample was calculated by comparing 2 R =0.9974 the peak area of the sample solution with that of the standard solution. 0 0 20 40 60 80 100 120 140 Recovery studies Concentration in micrograms/mL Recovery is a useful way to assess how efficient an extraction Figure 2: Calibration curve of the Dapiprazole by RP-HPLC. procedure is the closer the recovery value is to 100%, the better the sensitivity will be. Recovery values have to be reproducible to prove

565.00 that they are accurate. Accuracy was determined by recovery studies

mAU 3.70 Typical chromatogram of Dapiprazole of Dapiprazole, known amount of standard was added to the pre- standard solution contains analyzed sample and subjected to the proposed HPLC analysis. Results 452.00 =80 micrograms/mL of recovery study are shown in Table 1. The study was done at three different concentration levels.

339.00 Limit of Detection (LOD) and Limit of Quantification (LOQ)

226.00 The LOD is defined as the analyte concentration that gave a signal to noise (S/N) ratio of 3:1. The LOQ was defined as the analyte concentration that gave an S/N ratio of 5:1. The limit of detection 113.00 (LOD) and limit of quantification (LOQ) for Dapiprazole were found to be 0.5 μg/mL and 1.6 μg/mL respectively. The signal to noise ratio is 0.00 3 for LOD and 10 for LOQ. 0.000 0.800 1.600 2.400 3.200 4.000 4.800 5.600 6.400 7.200 8.000 Minutes Figure 3: Typical Chromatogram of Dapiprazole by HPLC. Results and Discussion For developing the method, a systematic study on the effect of 10 mg of Dapiprazole USP in 10 ml volumetric flask containing 5 ml of various factors was carried out by varying one parameter at a time and methanol, sonicated for about 15 min and then made up to 10 ml with keeping all other conditions constant, that is, OFAT (One Factor at a mobile phase to get 1000 μg/ml of Dapiprazole. Time) mode of study. Method development consists of selecting the appropriate detection wave length and stationary and mobile phases. Working standard solution Proper wavelength was needed to determine maximum detector The primary standard solution was further diluted by taking 2 ml of response. From the spectrum, it is clear that Dapiprazole absorbs the stock solution with 25 ml of mobile phase to get the concentration maximum light between 235 nm to 245 nm. For the RP-HPLC analysis, of 80 μg/mL. the UV/Vis detector was fixed at 243 nm as maximum wave length

Preparation of sample solution (λmax) for determination of Dapiprazole. At this wavelength, we have

A sample of the powder of Dapiprazole synthesized in in-house, Sample % found by the proposed % Recovery* equivalent to 10 mg of the active ingredient, was mixed with 5 ml of method methanol in 10 ml volumetric flask. The mixture was allowed to stand 1. 99.43 99.97 for 30 minutes with intermittent sonication for complete solubility of 2. 99.55 98.365 the drug, and then filtered through a 0.45 μm membrane filter, followed 3. 99.46 98.247 by addition of mobile phase up 10 ml to obtain a stock solution of 1 *Average of three different concentration levels mg/ml. The resultant solution was further diluted by taking 2 ml of the Table 1: Results of HPLC Assay and Recovery studies.

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Validation Parameter Results principal anlayte(s) of interest and additional excipients that are present System Suitability in the sample. The degree of specificity testing varies depending on the Theoretical Plates (N) 21505 method type and the stage of validation. The effect of wide range of Tailing factor 1.15 intermediates and other precursors, generally used in in-house synthesis Retention time in minutes 3.27 % Area 99.96 of Dapiprazole were investigated under optimized chromatographic LOD (μg/mL) 0.5 conditions. Specificity is the ability to assess unequivocally the analyte LOQ (μg/mL) 1.6 in the presence of components which may be expected to be present. Table 2: Validation Summary. Typically these might include impurities, degradants, matrix, etc. The common reagents present in the synthetic route did not interfere with Drug Dapiprazole the elution or quantification of the method. Acceptance criteria for Concentration range (μg/mL) 20-120 specificity, RSD should be less than 2%. Slope (m) 1956.4x Intercept (b) 3409 Linearity and range Correlation coefficient 0.9999 The plot of peak area of each sample against respective concentration % RSD 0.566 of Dapiprazole was found to be linear in the range of 20-120 μg/mL with Table 3: Linear Regression Data for Calibration curves. correlation coefficient of 0.9999. Linear regression least square fit data obtained from the measurements are given in Table 3. The respective observed a zero back ground noise and no absorption of light by mobile linear regression equation being y=1956.4x+3409. The regression phase solvent system. characteristics, such as slope, intercept, and %RSD were calculated for To explore the possibility of better separation a Cyano column this method and given in Table 3. was tested with the same mobile phase ternary mixture of methanol: Robustness acetonitrile: and ortho-phosphoric acid in the ratio of 89: 9: 1 (v/v/v) at pH 5.8. The retention times were long with polar columns. Retention of Robustness is a measure of the performance of a method when small, deliberate changes are made to the method conditions. The the analyte on the cyano columns was much weaker than on C18 columns, resulting in unacceptable k’ (capacity factor) value of the column (<1) intent of this validation parameter is to identify the most critical for Dapiprazole. Further development trials have been performed with method conditions to the successful performance of the method. The octadecyl columns of different types and configurations from different robustness of the method was assessed by deliberate alteration of the manufacturers. Under these chromatographic conditions, the analyte of experimental conditions. One factor at a time was changed to study the interest has exhibited poor peak efficiencies (N) and peak symmetries effect. Variation of the detection wavelength by ± 2 nm (241 nm and (Tailing factor); and a partial resolution (Rs) between drug and mobile 245 nm), the amount of acetonitrile in the mobile phase was varied by phase components. Finally Thermo hypersil C18 column (250 mm×4.6 ± 10%), and mobile phase pH (± 2 pH-units) had no significant effect mm 5 μm) column was selected based on the peak shape and the on the retention time and chromatographic response of the method, baseline separation from the other interfering peaks in the formulation indicating that the method was robust. When the chromatographic sample. conditions were deliberately altered, system suitability results remained within acceptance limits and selectivity for individual substance was Different mobile phases were tested to optimize analytical not affected. performance. The optimum composition of mobile phase was determined by comparing the influence of different binary mixtures Ruggedness such as acetonitrile- methanol and acetonitrile -water and methanol- Ruggedness is an older term that has been replaced by intermediate water in different proportions. None of the solvent system in binary precision, according to the USP, is the degree of reproducibility of the phase has given good peak shape and theoretical plates. It is well known that multiple-component mobile phases result in better results obtained under a variety of conditions, expressed as %RSD; which separation efficiency than binary mobile phases, because with these is a measure of how well the method performs under normal conditions solvent strength and selectivity can be varied simultaneously to obtain form laboratory-to-laboratory, instrument-to-instrument, and analyst- the retention times desired. A third component, o-phosphoric acid, to-analyst. A ruggedness test is performed as part of the validation of was therefore included in the mobile phase and ternary mixtures of an analytical method. Ruggedness test was determined between two methanol: acetonitrile: and ortho-phosphoric acid in the ratio of 89: 9: 1 different analysts, instruments and columns. Small differences in areas (v/v) at pH 5.8. The present mobile phase system has been finalized for and good constancy in retention times were observed. The high degrees the estimation of Dapiprazole in bulk drug in terms of peak symmetry, of reproducibility of detector responses and retention times indicate optimum resolution, reasonable run time, and acceptable k values. that the method is fairly rugged. System suitability From the typical chromatogram of Dapiprazole as shown in Figure 3, it was found that the retention time was 3.724 min. A mixture of The system suitability tests were carried out on freshly prepared methanol: acetonitrile: and ortho-phosphoric acid in the ratio of 89: standard stock solution of Dapiprazole. The system was suitable for 9:1 (v/v) at pH 5.8 as the mobile phase at a flow rate of 0.8 ml/min use, the tailing factors for Dapiprazole were 1.15 and USP theoretical was found to be most suitable to obtain a peak well defined and free plates were found to be significantly high around 21505 in number. from tailing. In the present developed HPLC method, the standard and Parameters that were studied to evaluate the suitability of the system sample preparation required less time and no tedious extractions were are given in Table 2. involved. A good linear relationship (r2=0.9999) was observed between Specificity the concentration range of 20-120 μg/mL. Low values of standard deviation are indicative of the high precision of the method. The assay of Specificity is the ability of a method to discriminate between the Dapiprazole in bulk sample was found to be 99.94%. From the recovery

Page 4 of 4 studies it was found that about 99.06% of Dapiprazole was recovered References which indicates high accuracy of the method. The absence of additional 1. Allinson RW, Gerber DS, Bieber S, Hodes BL (1990) Reversal of mydriasis by peaks in the chromatogram indicates non-interference of the common dapiprazole. Ann Ophthalmol 22: 131-133, 138. reagents and intermediates used in in-house synthesis of Dapiprazole 2. Allinson RW (1994) Rev Eyes: alpha blocker to reverse diagnostic mydriasis. in bulk drug production. This demonstrates that the developed HPLC Ocular Surgery News 19. method is simple, linear, accurate, sensitive and reproducible. 3. Bonomi L, Marchini G (1987) The potential interest of alpha-adrenergic blocking Thus, the developed method can be easily used for the routine agents in ophthalmology. Proceedings of second session of symposium 1-6. quality control of Dapiprazole bulk sample within a short analysis time. 4. Bucci MG, D'Andrea D, Bettini A, De Gregorio M (1987) Dapiprazole for the Conclusion reversal of mydriasis due to tropicamide. Glaucoma 9: 94-98. 5. Buschmann W (1996) Dapiprazol - Erfahrungen in der Praxis. Augenspiegel It can be seen from the results presented that the proposed 4: 14-18. procedure has good precision and accuracy. Results of the analysis of pharmaceutical formulations revealed that proposed methods are 6. Saettone MF, Alderigi C, Giannaccini B, Galli-angeli D (1989) Preparation and suitable for their analysis with virtually no interference of the usual Evaluation of A Sustained-Release Ophthalmic Vehicle for Dapiprazole. Drug Development and Industrial Pharmacy 15: 2621-2637. intermediates and impurities formed during the in-house synthesis of Dapiprazole. This demonstrates that the developed HPLC method is 7. Valeri P, Palmery M, Severini G, Piccinelli D, Catanese B (1986) Ocular simple, linear, accurate, sensitive and reproducible. Thus, the developed pharmacokinetics of dapiprazole. Pharmacol Res Commun 18: 1093-1105. method can be easily used for the routine quality control of bulk forms 8. Bianchi E, Segre G (1990) Ocular pharmacokinetics of radioactive dapiprazole of Dapiprazole within a short analysis time. after topical and parenteral administration. Euro J Pharmacol 183: 396.

Acknowledgements 9. Than TP (1996) Comparison of the cycloplegic and mydriatic effects of 0.5% tropicamide and 2.5% phenylephrine to Paremyd and the counteracting effects The authors are grateful to M/s M/s Bioleo Analytical Labs India Pvt. Ltd of dapiprazole. Clinical Eye and Vision Care 8: 209-213. Hyderabad for the supply of Dapiprazole as a gift sample. One of us (KJP) is highly thankful to, Management of BCAS, Bapatla for giving her an opportunity to pursue 10. Marx-Gross S, Krummenauer F, Dick HB, Pfeiffer N (2005) Brimonidine versus this project under FDP. The authors also thankful to Q.S.Labs, Hyderabad for dapiprazole: Influence on pupil size at various illumination levels. J Cataract providing the necessary facilities to carry out the research work. Refract Surg 31: 1372-1376.

J Anal Bioanal Techniques ISSN:2155-9872 JABT, an open access journal Volume 3 • Issue 4 • 1000143