International Journal of Research and Reviews in Pharmacy and Applied science www.ijrrpas.com Corresponding Author NARENDRA DEVANABOYINA* Ch.DURGAKUMARI, L.NAGARAJU, R.SRIDHARANI PRIYA, S.BHAVITHA Department of Pharmaceutical analysis, Lydia college of pharmacy, Ravulapalem-533238, India Email: narendra.kothapalli@gmail.com LIQUID CHROMATOGRAPHIC METHOD DEVELOPMENT AND VALIDATION FOR ANALYSIS OF DOXEPIN IN PHARMACEUTICAL DOSAGE ABSTRACT A new simple, rapid, selective, precise and accurate isocratic reverse phase high performance liquid chromatography assay has been developed and validated for the estimation of DOXEPIN in tablet formulation. The separation was achieved by using C-18 column (250x4.6mm, 5µm in particle size) at ambient temperature coupled with a guard column of silica in mobile phase, Acetonitrile: Methanol (80:20) with the ph value adjusted to 6.85.The flow rate of was 1.5ml/min and the drug was detected using UV detector at the wavelength of 220nm and the run time was 6min. The retention time was within 3.56minutes. The percentage of RSD for precision and accuracy of the method was found to be less than 2%. The method was validated as per ICH guidelines. The proposed method was found to be accurate, repeatability and consistent. It was successfully applied for the analysis of the drug in marketed formulation and could be effectively used for the routine analysis of formulation containing the drug without any alteration in the chromatography conditions. Keywords: DOXEPIN, RP HPLC, UV Detection, 220 nm, Validation, C18 column. INTRODUCTION 152
Doxepin is a drug used for the treatment of depression, anxiety, and insomnia. The medication is thought to work by allowing certain chemicals in the brain (serotonin and norepinephrine) to stay in the brain longer, which reduces depression symptoms and anxiety. Before taking doxepin, let your healthcare provider know if you are having Doxepin for long period for treating anxiety and depression. However, it has only recently been approved (in April 2010) for the treatment of insomnia. Only Silenor tablets (not generic doxepin products) are approved for insomnia treatment. Bipolar disorder, glaucoma, epilepsy, or any allergies. Figure: 1 STRUCTURE IUPAC NAME : dimethyl({3-[(2e)-9-oxatricyclo[9.4.0.0{3,8}]pentadeca-1(11),3(8),4,6,12,14-hexaen-2-ylidene]propyl})amine EXPERIMENTAL Working standard of DOXEPIN was obtained from well reputed research laboratories. Acetonitrile & Methanol used were of HPLC grade (E.Merck, Mumbai, India) 153
S.NO PARAMETER RESULT 1 Standard concentration 60µg/ml 2 Mobile phase Acetonitrile : methanol(80:20) 3 Wave length 220nm 4 ph 6.85 5 Flow rate 1.5ml/min. 6 Retention time 3.56 min. 7 Run time 6min. 8 Peak area 149591 9 Theoretical plates 8710 10 Pump pressure 4.5Psi Table 1: Chromatographic Conditions of DOXEPIN 154
Figure: 2 Sample chromatogram of DOXEPIN in HPLC 155
Different mobile phases were tried but the satisfactory separation, well resolved and good symmetrical peaks were obtained with the mobile phase Acetonitrile: Methanol (80:20).The retention time of DOXEPIN was found to be 3.56min with pressure 4.5psi which indicates a good base line. The system suitability and validation parameters are given in Table 1. The high percentage of recovery of DOXEPIN was found to be 100.52%indicating that the proposed method is highly accurate. Proposed liquid chromatographic method was applied for the determination of DOXEPIN in tablet formulation. The result for DOXEPIN was comparable with a corresponding labeled amount (Table 7). The absence of additional peaks indicates no interference of the excipients. Chemicals and reagents The Tablets of combined dosage form were procured from the local market. Other reagents used like Acetonitrile which are of HPLC grade water were purchased from E.Merck, Mumbai, India Analytical conditions The development and validation of the assay was performed on A Series HPLC system PEAK LC7000 isocratic HPLC with PEAK 7000 delivery system. Rheodyne manual sample injector with switch (77251), Analytical column zodiac C18. 250 4.6mm, manual injector Rheodyne valve with 20μL fixed loop, PEAK LC software was used. The mobile phase consisted of Acetonitrile: Methanol (80:20) Injections were carried out using a 20μl loop at room temperature and the flow rate was 1.5ml/min. Detection was performed at 220nm with 6min runtime. Instrumental Apparatus A Series HPLC system PEAK LC7000 isocratic HPLC with PEAK 7000 delivery system. Rheodyne manual sample injector with switch (77251), Analytical column zodiac C18. 250 4.6mm, Electronic balance-denver (SI234), a manual Rheodyne injector with a 20μl loop was used for the injection of sample. PEAK LC software was used. UV 2301 SPECTROPHOTOMETER was used to determine the wavelength of maximum absorbance. Determination of wavelength of maximum absorbance The standard solutions of DOXEPIN were scanned in the range of 200-400nm against mobile phase as a blank. DOXEPIN showed maximum absorbance at 220nm, so the wavelength selected for the determination of DOXEPIN was 220nm. 156
Preparation of Stock, working standard solutions and Sample solutions 10 mg amount of DOXEPIN reference substance was accurately weighed and dissolved in 10ml mobile phase in a 100 ml volumetric flask to obtain 1000ppm concentrated solution. From standard solution by the serial dilution we prepared required concentrations of 100 ppm. A composite of 20 tablets was prepared by grinding them to a fine, uniform size powder. 10 mg of DOXEPIN was accurately weighted and quantitatively transferred into a 100ml volumetric flask. Approximately 50ml mobile phase were added and the solution was sonicated for 15min. The flask was filled to volume with mobile phase, and mixed. After filtration, an amount of the solution was diluted with mobile phase to a concentration of 60μg/ml. Method validation procedure The objective of the method validation is to demonstrate that the method is suitable for its intended purpose as it is stated in ICH guidelines for linearity, precision, system suitability, specificity, limit of detection and limit of quantification and robustness. Range of linearity Standard curves were constructed daily, for three consecutive days, using nine standard concentrations in a range of 20, 40, 60, 80, 100 μg/ml for DOXEPIN. The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. The linear regression equation was y = 2389(r= 0.986) 157
S.NO Conc. ( ppm) Area 1 20µg/ml 61569 2 40µg/ml 104200 3 60µg/ml 149591 4 80µg/ml 194330 Slope:2386 Intercept:10115 CC: 0.986 5 100µg/ml 226495 Table: 2 Linearity Graph: 1 Calibration curve of DOXEPIN 158
Precision To study precision, six replicate standard solutions of DOXEPIN (100ppm) were prepared and analyzed using the proposed method. The percent relative standard deviation (%RSD) for peak responses was calculated and it was found to be which is well within the acceptance criteria of not more than 2.0%. Results of system precision studies are shown in table 3. S.NO CONCENTRATION AREA %RSD 1 60µg/ml 149330 2 60µg/ml 142119 3 60µg/ml 146530 4 60µg/ml 144503 1.89% 5 60µg/ml 149652 6 60µg/ml 148116 Table 3: Precision parameters of DOXEPIN 159
S.NO CONCENTRATION AREA %RSD 1 60µg/ml 140050.7 2 60µg/ml 144640.5 3 60µg/ml 143619.2 1.98% 4 60µg/ml 142522.3 5 60µg/ml 145431.8 6 60µg/ml 146802.3 Table:4 RUGGEDNESS System Suitability Having optimized the efficiency of a chromatographic separation the quality of the chromatography was monitored by applying the following system suitability tests: capacity factor, tailing factor and theoretical plates. The system suitability method acceptance criteria set in each validation run were: capacity factor >2.0, tailing factor 2.0 and theoretical plates >2000. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. A chromatogram obtained from reference substance solution is presented. System suitability parameters were shown in Table.1. Standard chromatogram was given in Figure.3 160
Figure: 3 Standard chromatogram of DOXEPIN 161
Specificity The specificity of the method was determined by comparing test results obtained from analysis of sample solution containing recipients with that of test results those obtained from standard drug. Limit of Detection and Limit of Quantification: To determine the Limit of Detection (LOD) sample was dissolved by using Mobile phase and injected until peak was disappeared. After 1.5ppm dilution Peak was not clearly observed, based on which 1.5ppm is considered as Limit of Detection and Limit of Quantification is 5ppm. Limit of detection (LOD): The Limit of Detection (LOD) is the smallest concentration that can be detected but not necessarily quantified as an exact value.lod can be calculated as: LOD = 1.5ppm S.NO CONCENTRATION RETENTION TIME (min.) AREA ABSORBANCE 1 3µg/ml 2.717 26229.4 Peak observed 2 1.5µg/ml.. No Peak Table: 5 Limit of detection 162
Limit of quantification (LOQ): The Limit of Quantization (LOQ) is the lowest amount of analyte in the sample that can be quantitatively determined with suitable precision and accuracy. LOQ = 3.3 x 1.5(LOD) LOQ=5ppm Typical variations in liquid chromatography conditions were used to evaluate the robustness of the assay method. In this study, the chromatographic parameters monitored were retention time, tailing factor and theoretical plates. The robustness acceptance criteria set in the validation were the same established on system suitability test describe above and the results are given in Table 6 S.NO PARAMETER CHANGE AREA % OF CHANGE 1. Standard 149591 2. Mobile phase ACN:Methanol( 85:15) 147300 0.83 3. Wavelength 224nm 148652 0.62 4. Flowrate 1.2ml/min 146174 1.59 Table: 6 Robustness parameters of DOXEPIN 163
Recovery Recovery test was performed at 3 different concentrations i.e. 30ppm, 60ppm, 90ppm. Results are given in Table 7 RECOVERY CONCENTRATION OF SAMPLE AMOUNT FOUND % DRUG RECOVERY AVERAGE 50% 30 µg/ml 30.34ppm 101.51 100% 60 µg/ml 59.97ppm 99.952 100.52 150% 90 µg/ml 90.08ppm 100.09 RESULT AND DISCUSSION Table: 7 Optimization of the chromatographic conditions The sample nature, its solubility and molecular weight confirms the correct selection of the stationary phase. The drug DOXEPIN being nonpolar is preferably analyzed by reverse phase columns and accordingly C18 column was selected. The elution of the compound from the column was influenced by polar mobile phase. The concentration of the acetonitrile and methanol were optimized to give symmetric peak with short run time based on asymmetric factor and peak area obtained. 164
TABLET FORMULATION Formulation Concentration Amount found % Assay SINEQUAN Tablet 60ppm 59.4ppm 99.00 Table: 8 Assay results of formulation CONCLUSION The proposed method for the assay of DOXEPIN in tablets or capsules is very simple and rapid. It should be emphasized it is isocratic and the mobile phase do not contain any buffer. The method was validated for linearity, precision, system suitability, specificity, LOD & LOQ and robustness. Although the method could effectively separate the drug from its products, further studies should be performed in order to use it to evaluate the stability of pharmaceutical formulations. ACKNOWLEDGMENT With profound sense of gratitude and humbleness we express our sincere thanks to our dedicated supervisor, Faculty members and management of Lydia College of pharmacy, Ravulapalem, E.G Dist., Andhrapradesh, India. REFERENCES 1. Hajak G, Rodenbeck A, Voderholzer U et al (2001). "Doxepin in the treatment of primary insomnia: a placebo-controlled, double-blind, polysomnographic study". J Clin Psychiatry 62 (6): 453 63. 2. "Phase 3 Data Show New Insomnia Drug, Silenor(R), Effective Without Side Effects". Medical News Today. 2006-04-12. Retrieved 2008-02-01. 3. Lippincot"nursing 2007 drug handbook" LWW press. 2007 4. Dilger C, High-performance liquid chromatographic determination of trans-doxepin and desmethyldoxepin, Arzneimittelforschung.(10):1525-8. 5. R. D. Faulkner, Comparative assays for doxepin and desmethyldoxepin using high-performance liquid chromatography and high-performance thinlayer chromatography, Journal of Pharmaceutical Sciences. Volume 72, Issue 10, pages 1165 1167, 6. Whall TJ, High-performance liquid chromatographic determination of (Z)- and (E)-doxepin hydrochloride isomers,j Pharm Sci. (11):1454-6. 165
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