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  1. 1. This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit:
  2. 2. Authors personal copy j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 4 0 e1 4 4 Available online at journal homepage: ArticleSimple RP-HPLC method for estimation of diazepam in tabletdosage formA. Sruthi, P. Tejaswi, N. Thanuja, D. Sudheer Kumar, P. Vivek Sagar*Department of Pharmaceutical Analysis, Care College of Pharmacy, Warangal, Andhra Pradesh, Indiaarticle info abstractArticle history: A simple reverse phase HPLC method was developed and validated for the determinationReceived 11 September 2012 of diazepam present in pharmaceutical dosage form. A Hypersil ODS C-18 columnAccepted 4 November 2012 (250 Â 4.6 mm, packed with 5 microns) is used as stationary phase. An isocratic mode with mobile phase consisting of acetonitrile, methanol and 1% phosphate buffer (pH-3) in ratioKeywords: of 18:58:24 (v/v/v) at a flow rate of 1 ml/min and effluent was monitored at 232 nm.RP-HPLC Chromatogram showed a peak of DZP at retention time of 6.23 Æ 0.002 min. The linearityDiazepam range was found to be 2e20 mg/ml. The method was validated for linearity, accuracy,Validation precision, limit of detection, limit of quantitation, robustness and ruggedness. Recovery ofPharmaceutical dosage form DZP was found to be in the range of 99.4e100.3%. The limit of detection and limit of quantitation for estimation of DZP was found to be 0.898 mg/ml and 2.72 mg/ml, respectively. Proposed method was successfully applied for the quantitative determination of DZP in pharmaceutical dosage forms. Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.1. Introduction expensive. The present study focused on minimizing these limitations and to develop a simple precise accurate andDiazepam (7-chloro-1, 3-dihydro-1-methyl-5-phenyl-2H-1, economic method for estimation of diazepam in tablet dosage4-benzodiazepin-2-one) is a benzodiazepine (BZD) generally form.used as hypnotic, anxiolytic and muscle relaxant. Diazepam(DZP) is also routinely prescribed as the standard first-linetreatment for acute convulsions and prolonged status epi-lepticus.1 Several methods for the analysis of BZDs have beenreported.2 A number of chromatographic methods, such asthin-layer chromatography (TLC)3 gas chromatography4e6 andgas chromatographicemass spectrometry (GCeMS)7,8 havebeen used in the analysis of diazepam and other 1,4-benzo-diazopines. Several high-performance liquid chromato-graphic (HPLC) methods have also been reported for thedetermination of diazepam and other BZDs.9,10 However, all ofthese methods have limitations such as long run times and/or * Corresponding author. E-mail address: (P. Vivek Sagar).0974-6943/$ e see front matter Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
  3. 3. Authors personal copy j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 4 0 e1 4 4 141 2.4. Preparation of working standard solutions2. Materials and methods Working standard solutions ranging from 0.5 to 50 mg/ml were2.1. Chemicals and reagents prepared by appropriate dilutions of the stock with distilled water.An analytically pure sample of diazepam was procured asgift sample from Natco Pharma Ltd. (Hyderabad, India). 2.5. Preparation of sample solutionHPLC grade methanol was procured from E. Merck (Hyder-abad). Liquid chromatographic grade water was obtained by Twenty tablets of diazepam hydrochloride were weighed anddouble distillation and purification through Milli-Q water ground into a fine powder. A quantity of powder equivalent topurification system. Potassium dihydrogen phosphate (AR 25 mg of diazepam was weighed and transferred into a 25 mlgrade, purity 99.5%) was procured from Qualigens. Tablet volumetric flask and was dissolved in 0.1 N HCl. The volumeformulations VALIUM (Nicholas Piramal India Ltd.) was was made up to the mark with the same. Above solution wasprocured from a local pharmacy with labeled amount 5 mg suitably diluted with distilled water. From this stock, appro-per tablet. priate dilution (10 mg/ml) was prepared. The solution thus prepared was filtered through 0.45 m membrane filter and the2.2. Instrumentation & chromatographic conditions resulting filtrate was sonicated for 10 min. After setting the chromatographic conditions and stabilizing the instrument toThe HPLC analysis was performed on CYBERLAB HPLC obtain a steady baseline, the sample solution was loaded inequipped with an LCP-100 reciprocating HPLC pump. A the 20 ml fixed e sample loop of the injection port.manually operating Rheodyne injector with 20 mL sampleloop, a LC-UV 100 ultraviolet detector was used. Chromato- 2.6. Method developmentgraphic analysis was performed on a Hypersil reversed phaseC-18 column with 250 Â 4.6 mm i.d. and 5 mm particle size. Initial trial experiments were conducted, with a view to selectThe mobile phase consist of acetonitrile, methanol, 1% a suitable solvent system for the accurate estimation of thephosphate buffer (pH-3) in ratio of 18:58:24 (v/v/v) that was set drug and to achieve good retention time. The suitability of theat a flow rate of 1 ml/min. The mobile phase was degassed mobile phase decided on the basis of the sensitivity of theand filtered through 0.25 mm membrane filter before pumping assay, time required for the analysis, ease of preparation, andinto HPLC system. The eluent was monitored by UV detection use of readily available cost effective solvents. These includeat 232 nm. methanol-potassium dihydrogen phosphate, methanol- ammonium acetate, acetonitrile-potassium dihydrogen phos-2.3. Stock solutions and standards phate, acetonitrile-ammonium acetate, methanol-water. The mobile phase consisting of acetonitrile, methanol, 1% phos-Stock solution of diazepam (1 mg/ml) was prepared by phate buffer (pH-3) in ratio of 18:58:24 (v/v/v) that was set attransferring 25 mg of drug in a 25 ml volumetric flask. The a flow rate of 1 ml/min was found to be optimum and furtherdrug is dissolved in sufficient amount of 0.1 N HCl and optimized by adjusting pH 3e4 by adding orthophosphoric acid.finally the volume was made up to the mark with distilled The composition of acetonitrile, methanol, 1% phosphatewater. buffer in ratio of 18:58:24 (v/v/v) with pH-3 gave the best results. Fig. 1 e Chromatogram of diazepam.
  4. 4. Authors personal copy142 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 4 0 e1 4 4 Table 1 e Linearity of diazepam. Table 2 e Precision. S. No Concentration Peak area S. No Concentration Peak area (ppm) (ug/ml) 1 2 7569.8 1 6 21709.4 2 4 17892.9 2 6 21627.7 3 6 23612.2 3 6 21939.6 4 8 34517.2 4 6 21710.5 5 10 40051.3 5 6 21337.9 6 12 47631.9 6 6 21929.5 7 14 54925.1 Mean 21709.1 8 16 65942.9 % RSD 1.02 9 18 72965.0 10 20 80440.3 triplicate.11 20 ml of each of standard solutions were injected into the HPLC system to get the chromatograms. The retention2.7. Solution stability time, average peak areas were recorded. Calibration curve was constructed by plotting average peak area against concentra-In order to demonstrate the stability of both standard and tion and regression equation was computed. The linearitysample solutions during analysis, both solutions were analyzed range was found to be 2e20 mg/ml. The results were shown inover a period of 96 h at an interval of 24 h at room temperature. Table 1. The results show that an excellent correlation existsThe results show that for solutions, the retention time and peak between peak area and concentration of drug within thearea of diazepam hydrochloride remained unchanged and no concentration range, regression graph is presented in Fig. 2.significant degradation within the indicated period, this indi-cates that both solutions were stable for 72 h. 3.2.2. Precision The precision of method was ascertained from the peak area response obtained by actual determination of six replicates of3. Results and discussion a fixed amount of drug. The percent relative standard devia- tions were calculated for diazepam and presented in the Table3.1. Analysis of formulation 2. The precision of the method was found to be 1.02.The sample solution was injected and a chromatogram was 3.2.3. Accuracyrecorded. The injections were repeated six times and the peak Accuracy of developed method was confirmed by doingareas were recorded. The amount of drug present in the recovery study as per ICH norms. A known quantity of thepharmaceutical formulation was calculated using standard pure drug was added to the pre-analyzed sample formulationcalibration curve (concentration in mg/ml was taken on X-axis (10 mg/ml) at three different concentration levels 80%, 100%and average peak area on Y-axis). Percentage of drug present and 120% by replicate analysis (n ¼ 3). From the recovery studyin each tablet was found to be 100.2. A representative chro- it was clear that the method is very accurate for quantitativematogram has been given in Fig. 1. estimation of diazepam hydrochloride in tablet dosage form as all the statistical results were within the range of accep-3.2. Validation of the assay method tance, 99.4e100.3%, which shows that there is no interference with excipients. Percentage recovery values were calculated3.2.1. Linearity and the results were shown in Table 3.Different concentrations in the range of 0.5e50 mg/ml wereprepared. Each of the levels of concentration was prepared in 3.2.4. Ruggedness Ruggedness is the degree of reproducibility of the results ob- tained under a variety of conditions. From stock solution, solutions containing 14 mg/ml of diazepam hydrochloride was prepared and analyzed by two different analysts using same operational and environmental conditions in different Table 3 e Accuracy. Amount of Amount of Average % drug added drug recovered recovery (ppm) (ppm) 8 7.592 99.4 10 9.98 99.8 12 12.096 100.3 Fig. 2 e Linearity of diazepam.
  5. 5. Authors personal copy j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 4 0 e1 4 4 143 Table 4 e Ruggedness. Table 7 e System suitability parameters. Date % Recovery Drug RT Peak Peak USP plate USP area height count tailing Analyst-1 Day-1 100.2% Analyst-2 Day-2 99.8% Diazepam 6.2266 31432.2 2588 5238.42 1.1 % Deviation 0.4% accuracy and precision. The requirements for system suit- ability are usually developed after method development and Table 5 e Robustness. validation has been completed. The system suitability parameters like Theoretical plates (N), Resolution (R), Tailing S. No Wavelength 232 Wavelength 235 factor (T) were calculated and compared with the standard 1 52156.6 54973 values to ascertain whether the proposed RP-HPLC method for 2 52700.8 52955.4 the estimation of diazepam in pharmaceutical formulations 3 50662.6 52965.9 was validated or not. The results were shown in Table 7. 4 50905.7 54751.1 5 51418.4 54821.7 6 50911.8 52886.1 Mean 51459.32 53892.22 4. Conclusion % RSD 1.57 1.94 A convenient, rapid, accurate, precise and economical RP- HPLC method has been developed for estimation of diazepamexperimental periods. Percentage recoveries of the replicates in bulk and tablet dosage form. The assay provides a linearwere calculated. It is checked that the results are reproducible response across a wide range of concentrations and it utilizesunder differences in, analysts. The results are shown in Table 4. a mobile phase which can be easily prepared and diluent is economic, readily available. The proposed method can be3.2.5. Robustness used for the routine analysis of diazepam hydrochloride inThe method was found to be robust, although small deliberate bulk preparations of the drug and, in pharmaceutical dosagechanges in method conditions did have a negligible effect on forms without interference of excipients.the chromatographic behavior of the solute. The resultsindicate that changing the detector wavelength had no largeeffect on the chromatographic behavior of diazepam hydro-chloride. Even a small change of mobile phase composition Conflicts of interest(pH 3 Æ 0.2), did not cause a notable change in the peak area of All authors have none to declare.the used drug for this method. The results were presented inTables 5 and 6. references3.2.6. LOD & LOQLOD and LOQ for diazepam were estimated by injectinga series of dilute solutions with known concentration. The 1. Crawley J, Smith S, Kirkham F, Muthinji P, Waruiru C,parameters LOD and LOQ were determined on the basis of Marsh K. Seizure activity and neurological sequelae inpeak response and slope of the regression equation. The LOD Ugandan children who have survived an episode of cerebraland LOQ of the drug were found to be 0.898 mg/ml and 2.72 mg/ malaria. QJM. 1996; respectively. 2. Rouini Mohammad-Reza, Ardakani Yalda H, Moghaddam Kambiz A, Solatani F. An improved HPLC method for rapid quantitation of diazepam and its major3.2.7. System suitability parameters metabolites in human plasma. Talanta. 2008;75:671.System suitability parameters can be defined as tests to 3. van der Merwe PJ, Steyn JM. Rapid and simpleensure that the method can generate results of acceptable chromatographic method for the determination of diazepam and its major metabolites in human plasma and urine. J Chromatogr. 1978;148:549. 4. Gjerde H, Dahlin E, Christophersen AS. Clinical impairment of Table 6 e Robustness. benzodiazepinesdrelation between benzodiazepine concentrations and impairment in apprehended drivers. S. No Mobile phase pH: 3.0 Mobile phase pH: 3.2 J Pharm Biomed Anal. 1992;10:317. 1 54247.2 52156.6 5. Fisher LE, Perch S, Bonfiglio MF, Geers SM. Simultaneous 2 53976.3 52700.8 determination of midazolam and flumazenil concentrations 3 53176.6 50662.6 in human plasma by gas chromatography. J Chromatogr 4 53315.8 50905.7 Biomed Appl. 1995;665:217. 5 53346.8 51418.4 6. Herraez-Hernandez R, Louter AJ, Van de Merbel NC, 6 53758.8 50911.8 Brinkman UA. Automated on-line dialysis for sample Mean 53636.92 51459.32 preparation for gas chromatography: determination of % RSD 0.669 1.43 benzodiazepines in human plasma. J Pharm Biomed Anal. 1996;14:1077.
  6. 6. Authors personal copy144 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 1 4 0 e1 4 47. Hold K, Crouch DJ, Rollins DE, et al. Quantitation of alprazolam performance liquid chromatography analysis. J Chromatogr B. and hydroxyalprazolam in human plasma by negative ion 2001;750:177. chemical ionization GC/MS. J Mass Spectrom. 1996;31:1033e1038. 10. Wilhelm M, Battista HJ, Obendorf D. HPLC with simultaneous8. Borrey D, Meyer E, Lambert W, Van Calenbergh S, Van UV and reductive electrochemical detection at the hanging Peteghem C, De Leenheer AP. Enzymatic hydrolysis improves mercury drop electrode: a highly sensitive and selective tool the sensitivity of emit screening for urinary benzodiazepines. for the determination of benzodiazepines in forensic J Chromatogr A. 2001;910:105. samples. J Anal Toxicol. 2001;25:250.9. Bolner, Tagliaro F, Lomeo A. Optimised determination of 11. Validation of Analytical Procedure: Methodology, ICH Harmonized clobazam in human plasma with extraction and high- Tripartite Guideline. 1996.