The optimized reverse phase high performance liquid chromatographic method was developed for estimation of Brimonidine Tartrate in bulk drug and pharmaceutical dosage form. Chromatography was performed on Kromasil C 18 ( 250 mm X 4.6 mm i.d. , 5 Î¼m particle size) column with mobile phase citric acid monohydrate buffer:water:methanol (30:50:20 v/v/v) andÂ  pH 3 was maintained by using triethylamine. The flow rate was 1.0 ml/min. Elute was detected at 246 nm and it effectively separated at Retention Time of 5.96 min. The LOD and LOQ was 1.47 and 4.47 Î¼g/ml respectively. A linear response was observed over the concentration range 40-80 Î¼g/ml for Brimonidine Tartrate. Thus the proposed HPLC method was found accurate, specific, precise, robust and reproducible

Laddha, U D

Mandan, S S

Nerpagar, A V

Journal of Drug Delivery and Therapeutics (JDDT)

Mandan et al                                                                                                     Journal of Drug Delivery & Therapeutics. 2017; 7(7):146-149                       ISSN: 2250-1177                                                                             [146]                                                                             CODEN (USA): JDDTAO Available online on 25.12.2017 at http://jddtonline.info Journal of Drug Delivery and Therapeutics Open Access to Pharmaceutical and Medical Research © 2011-17, publisher and licensee JDDT, This is an Open Access article which permits unrestricted non-commercial use, provided the original work is properly cited Open  Access                                                                                                                     Research Article DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR ESTIMATION OF BRIMONIDINE TARTRATE AS BULK DRUG AND IN OPHTHALMIC FORMULATION S.S. Mandan, A.V. Nerpagar, U. D. Laddha, MET’s Institute of Nashik Shirpur, India (MH) E-mail address: ssmandan@yahoo.com  ABSTRACT The optimized reverse phase high performance liquid chromatographic method was developed for estimation of Brimonidine Tartrate in bulk drug and pharmaceutical dosage form. Chromatography was performed on Kromasil C 18 ( 250 mm X 4.6 mm i.d. , 5 μm particle size) column with mobile phase citric acid monohydrate buffer:water:methanol (30:50:20 v/v/v) and  pH 3 was maintained by using triethylamine. The flow rate was 1.0 ml/min. Elute was detected at 246 nm and it effectively separated at Retention Time of 5.96 min. The LOD and LOQ was 1.47 and 4.47 μg/ml respectively. A linear response was observed over the concentration range 40-80 μg/ml for Brimonidine Tartrate. Thus the proposed HPLC method was found accurate, specific, precise, robust and reproducible.  Cite this article as: Mandan S.S., Nerpagar A.V., Laddha U.D., Development and validation of HPLC method for estimation of brimonidine tartrate as bulk drug and in ophthalmic formulation, Journal of Drug Delivery and Therapeutics. 2017; 7(7):146-149  INTRODUCTION: Brimonidine Tartrate is used in the treatment of open-angle glaucoma or ocular hypertension. It is selective alpha-2 adrenergic receptor agonist. Chemically it is 5-bromo-6-(2-imidazolidinylideneamino) quinoxaline L-tartrate. No significant HPLC method reports were found for estimation of Brimonidine Tartrate in pharmaceutical formulation while few HPTLC, HPLC, LC-MS, HILIC (Hydrophilic interaction liquid chromatography) methods reported for the estimation of Brimonidine Tartrate in blood serum and in ocular fluids (Rahore, 2010, Phogat, 2011, Jiang, 2009, Sethi 2001). The aim of study was to develope and validates simple, specific, sensitive, accurate and precise HPLC method for determination of Brimonidine Tartrate in ophthalmic formulation as per International Conference on Harmonization (ICH) guidelines (ICH, 2005). MATERIAL AND METHODS: Apparatus  A Shimadzu RP-HPLC instrument ( LC -20 AD as Per 21 CFR) equipped with an photodiode array detector, manual injector with 20 μl loop, and Kromasil C18 column (250 mm × 4.6 mm id, 5 μm particle size) and LC- solution software was used. Contech CB-50 analytical balance and ultra sonic cleaner (Spetralab, UCB-40) were used during the study.  Figure 1: Structure of Brimonidine Tartrate Reagents and materials  Brimonidine Tartrate was received as gift sample from Cipla Ltd., (Mumbai, Maharashtra).  HPLC grade methanol (Qualigens), citric acid monohydrate buffer was of AR grade and water for RP-HPLC was prepared by double glass distillation and filtered through nylon membrane filter 0.45 μm (Pall Lab Sci). Chromatographic conditions Kromasil C18 Column (250 mm × 4.6 mm id, 5 μm particle size) was used at ambient temperature. The mobile phase consisted of citric acid monohydrate buffer: water: methanol (30: 50:20 v/v/v) and pH 3 was maintained by triethylamine.  Flow rate was 1.0 ml/min. The mobile phase was filtered through a 0.45 μm membrane filter and degassed before used. The elution was monitored at 246 nm and injection volume was 20 μl.  Mandan et al                                                                                                     Journal of Drug Delivery & Therapeutics. 2017; 7(7):146-149                       ISSN: 2250-1177                                                                             [147]                                                                             CODEN (USA): JDDTAO Preparation of solutions Citric Acid Buffer    Accurately weighed Citric acid monohydrate (1.05 gm) was transferred to a beaker (500 ml) and dissolved in double distilled water (500 ml). The mobile phase was citric acid monohydrate, pH 3: water: methanol (30: 50:20)  Preparation of Standard Stock Solution Standard stock solution of Brimonidine Tartrate was prepared by dissolving 10 mg of drug in10 ml of methanol to get the concentration of 1 mg/ml from which 1 ml was further diluted to 10 ml with methanol to obtain a working standard having a concentration of 100 μg/μl. Determination of wavelength of maximum absorbance The standard solutions of Brimonidine Tartrate were scanned in the range of 200-400 nm against buffer solution as a blank. Brimonidine Tartrate showed maximum absorbance at 246nm. So the wavelength selected for the determination of Brimonidine Tartrate was 246 nm. Method Validation (ICH, 2005) Calibration curve (Linearity) Calibration curve was plotted over concentration range of 40-80 ug/ml for Brimonidine Tartrate.  Accurately measured standard stock solution of Brimonidine Tartrate (4, 5, 6, 7 and 8 ml) was transferred to a series of 10 ml volumetric flask and volume in each flask was adjusted 10 ml with mobile phase. Resulting solution were injected into the column and the peak area obtained at flow rate of 1.0 ml per minute for Brimonidine Tartrate. Calibration curve was constructed for Brimonidine Tartrate by plotting peak area versus concentration at 246 nm. Each reading was average of three determinations. Accuracy (% Recovery) To check the accuracy of the method, recovery studies were carried out by addition of formulation to pre-analyzed sample solution at three different levels 80, 100 and 120 %. Chromatogram was obtained and the peak areas were noted. At each level of the amount, three determinations were carried out. Limit of Detection and Limit of Quantification The limit of detection (LOD) and limit of Quantification (LOQ) for Brimonidine Tratrate was derived by calculating signal-to-noise ratio (S/N, i.e. 3.3 for LOD and 10 for LOQ) and using following equation as per International Conference on Harmonization (ICH) guidelines. LOD = 3.3 × σ/S LOQ = 10 × σ/S Where σ = the standard deviation of the responses and S = Slope of calibration curve. Specificity and System Suitability Specificity is the ability to assess unequivocally the analyte in the presence of components, which may be expected to be present. Typically these might include impurities, degradants, matrix etc.  One blank and one standard preparation was injected and chromatograms were recorded which is further calculated for system suitability parameters. Robustness Robustness studies were carried out by examining the effect of small, deliberate variation of the analytical conditions on the peak areas of the drug. Factors varied were volume of mobile phase (± 3 ml), wavelength (±2 nm) and flow rate (± 0.2 ml/min). One factor at a time was changed to study the effect. Intra-day and Inter-day precision The intra-day precision was determined by analyzing standard solution of Brimonidine Tartrate at 60 ug/ml concentration for six replicates on the same day while inter-day precision was determind by analysing corresponding standard on two different days over a period of one week. RESULT AND DISCUSSION: To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry was found in a mixture of citric acid monohydrate buffer pH 3, methanol and water (30:20:50) at 1.0 ml/min flow rate. As it was shown in Figure 2, the optimum wavelength for detection was set at 246 nm at which much better detector responses for Brimonidine Tartrate was obtained.  The retention time was 5.962 min as reported in Figure 3.    Figure 2: Maximum detection wavelength of Brimonidine Tartrate Figure 3: Typical RP- HPLC Chromatogram Brimonidine Tartrate (40 ug /ml) with corresponding retention  time. Mandan et al                                                                                                     Journal of Drug Delivery & Therapeutics. 2017; 7(7):146-149                       ISSN: 2250-1177                                                                             [148]                                                                             CODEN (USA): JDDTAO Chromatographic conditions are outlined in Table 1. The calibration graph for Brimonidine Tartrate was constructed by plotting the peak area versus their corresponding concentrations; good linearity was found over the range 40-80 μg/ml. The calibration graph is shown in Figure 4.  Table 1:  Chromatographic conditions Parameter  Observation Mobile phase 0.01 Mol/L Citric acid monohydrate: Methanol : Water (30:20:50) pH   3 maintained by using triethylamine Column Kromasil – C 18 Column (250 mm × 6.5 mm × 5 um) Flow rate 1 ml/min Detection wavelength 246 nm Injection volume 20 µl Run time 10 Minutes Retention time 5.96  mins   Figure 4: Calibration Curve of Brimonidine Tartrate The proposed method has been applied to the assay of Brimonidine Tartrate in pharmaceutical dosage form. The results obtained indicate the additives present do not interfere with analysis of the studied formulation. System suitability test parameters for Brimonidine Tartrate for the RP-HPLC method are reported in Table 2. The optical and regression characteristics and validation parameters are reported in Table 3. Data of recovery study is shown in Table 4. The robustness study is reported in Table 5. On the basis of series of investigation the optimized method can be routinely use for analysis purpose. Table 2:  System suitability testing of the HPLC method System suitability Parameters Observation Acceptance Criteria Peak Area  3419885 - Tailing Factor 1.49 NMT 2 Column Efficiency  5861 NLT 2500 % R.S.D. 0.7425 NMT 2  Table 3:  The optical and regression characteristics and validation parameters of HPLC method for analysis of Brimonidine tartrate Parameter Observation Calibration range 40-80 μg/ml Detection limit       1.477 μg/ml Quantitation limit 4.476 μg/ml Slope 85830 Intercept 17081 Correlation coefficient 0.999 Intraday RSD, % 0.7425 Interday RSD, % Day 1 -0.7425 Day 2- 0.4202  Table 4:  Data of recovery study for Brimonidine Tartrate by HPLC method Amount taken (μg/ml) Amount added (μg/ml) Amount found % Recovery 25 20 45.17 100.39 25 25 49.48 98.96 25 30 54.18 98.52   Mandan et al                                                                                                     Journal of Drug Delivery & Therapeutics. 2017; 7(7):146-149                       ISSN: 2250-1177                                                                             [149]                                                                             CODEN (USA): JDDTAO Table 5:  Data of robustness study for Brimonidine Tartrate by HPLC method      REFERENCES: 1. Rathore K, Kumar R, Sisodia S, Timolol maleate a gold standard drug in glaucoma used as ocular films and inserts an overview, Int J Pharm Sci Rev Res, 2010, 3, 23-29. 2. Phogat A, Kumar M, Mahadevan N, Simultaneous estimation of Brimonidine Tartrate and Timolol Maleate in Nanoparticles Formulation by RP-HPLC, Int J Recent Adv Pharm Res, 2011, 3, 31-36.  3. Jiang S, Chappa A, Proksch J, A rapid and sensitive LC/MS/MS assay for quantitation of brimonidine in ocular fluid and tissue , J Chromatogr B Anal Technol Biomed Life Sci, 2009, 3, 107-114.  4. Sethi PD, Quantitative analysis of pharmaceutical formulation, HPLC, 2011, 1, 38-70.  5. ICH Harmonised Tripartite Guideline Validation of Analytical Procedures, Text and Methodology, 2005, Q2, R1.  Experiment % RSD Theoretical plates Tailing factor (+) Wavelength 0.244 5713 1.50 (-)  Wavelength 0.167 5992 1.49 (+)Flow rate 0.549 5760 1.40 (-)  Flow rate 1.300 6091 1.61 (+) Mobile phase  volume 0.303 7065 1.46 (-)  Mobile phase volume 0.653 5041 1.51 

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR ESTIMATION OF BRIMONIDINE TARTRATE AS BULK DRUG AND IN OPHTHALMIC FORMULATION

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DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR ESTIMATION OF BRIMONIDINE TARTRATE AS BULK DRUG AND IN OPHTHALMIC FORMULATION

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