NEW VALIDATED RP - HPLC METHOD FOR THE ESTIMATION OF DIAZEPAM IN DOSAGE FORMS

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1 Page4054 Indo American Journal of Pharmaceutical Research, 2014 ISSN NO: NEW VALIDATED RP - HPLC METHOD FOR THE ESTIMATION OF DIAZEPAM IN DOSAGE FORMS K. Uma Maheswar, P.V.Lakshmana Rao, K.Balamurali Krishna and C.Rambabu Department of Chemistry, Acharya Nagarjuna University, Nagarjuanagar, A.P., India ARTICLE INFO Article history Received 09/10/2014 Available online 30/10/2014 Keywords Diazepam, RP-HPLC, UV Detection, Recovery, Precise. Corresponding author Prof.C.Rambabu, Department of Chemistry, Acharya Nagarjuna University, Guntur, India. rbchintala@gmail.com, ABSTRACT A simple reverse phase HPLC method was developed and validated for the estimation of diazepam in bulk and tablet dosage forms. Isocratic elution at a flow rate of 1.0 ml/min was employed on BDS Hypersil C18 (250 X 4.6) at ambient temperature. A mixture of 0.05M formic acid, methanol and acetonitrile in a ratio of 45:20:35 (v/v) was used as the mobile phase. The UV detection wavelength was 239nm and the sample size was 20µL. The retention time for diazepam in this method was 5.272min. Linearity was obtained in the range of µg/ml for diazepam. The mean recovery of diazepam was found to be 99.93%. The method was validated as per the ICH guidelines. The validated method was found to be precise and accurate for the estimation of diazepam in tablet dosage forms. Please cite this article in press as K. Uma Maheswar, et al. New Validated RP - HPLC Method For The Estimation of Diazepam in Dosage Forms. Indo American Journal of Pharm Research.2014:4(10). Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page4055 INTRODUCTION Diazepam(Fig.1) is a benzodiazepine antiepileptic drug. Like other drugs of this class, diazepam is used to treat anxiety disorders, alcohol withdrawal symptoms or muscle spasms[1]. Diazepam is sometimes used with other medications to treat seizures. The therapeutic activity of diazepam is rapid with high efficacy rate which is important in managing acute seizures, anxiety attacks and panic attacks. Like other Benzodiazepines, Diazepam also has a relatively low toxicity in overdose. Fig.1: Structure of Diazepam. Some instrumental methods of analysis like capillary electrophoresis[2], GCMS[3], HPTLC[4], UV[5] & Visible spectro photometry[6], HPLC[7-10] methods of determinations are so far reported. A few simultaneous determination methods[11,12] and a kinetic determination method on the basis of ligand exchange reaction[13] are also reported. We developed and validated a new RP- HPLC method to determine the quantity of diazepam individually in pure and tablet dosage forms. MATERIALS AND METHODS Chemicals and Instruments HPLC grade acetonitrile, methanol and formic acid were purchased from Merck Specialties Pvt. Ltd. The analysis of drug was carried out on a PEAK HPLC system running on PEAK Chromatographic Software version 1.06 equipped with a reverse phase C18 column (250x4.6mm, 5µm in particle size), a LC-P7000 isocratic pump, a 20µL injection loop and a LC-UV 7000 absorbance detector. Isocratic elution with Formic acid: Methanol : Acetonitrile in a ratio of 45:20:35 (V/V) (ph 5.8) was used at a flow rate of 1.0mL/min. The mobile phase was prepared freshly and used after sonication for 10 minutes. Stock and Working standard solutions 10mg of diazepam pure sample was accurately weighed and transferred into a 10mL volumetric flask and added some diluent. It was sonicated, dissolved completely and made the volume up to the mark with the same solvent. The contents were mixed well and filtered through 0.45 µm nylon filter paper to get the stock solution. The solutions of working standards were prepared by diluting the stock solution in the required contents. Each prepared solution is sonicated just before it is analyzed. The calibration curve(fig.3) was plotted with the six concentrations between ppm working standard solutions. Method Validation: 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. The method was validated for linearity, precision (repeatability and intermediate precision), accuracy, specificity, stability and system suitability. Standard plots were constructed with six concentrations in the range of ppm prepared to test linearity. The peak area of diazepam was plotted against the concentration to obtain the calibration graph. The linearity was evaluated by linear regression analysis that was calculated by the least square regression method. The precision of the assay was studied with respect to both repeatability and intermediate precision. Repeatability was calculated from five replicate injections of freshly prepared diazepam test solution in the same equipment at a concentration value of 100% (80ppm) of the intended test concentration value on the same day. The experiment was repeated by assaying freshly prepared solution at the same concentration additionally on two consecutive days to determine intermediate precision. Peak area of the diazepam was determined and precision was reported as %RSD. Method accuracy was tested (% recovery and %RSD of individual measurements) by analyzing sample of diazepam at three different levels in pure solutions using three preparations for each level. The results were expressed as the percentage of diazepam recovered in the samples. Optimization of the chromatographic conditions Proper selection of the stationary phase depends up on the nature of the sample, molecular weight and solubility. Among C8 and C18, C18 column was selected. Non-polar compound is very attractive with reverse phase columns. So the elution of the compound from the column was influenced by polar mobile phase. Mixture of formic acid, methanol and acetonitrile in the ratio 45:20:35 (v/v) was selected as mobile phase after the ph of it was adjusted to 5.8 with 0.05M formic acid and the effect of composition of mobile phase on the retention time of diazepam was thoroughly investigated. The concentration of the methanol and acetonitrile, formic acid were optimized to give symmetric peak with short run time (Fig.2).

3 Page4056 Fig. 2: Typical chromatogram of Diazepam standard. Assay of Diazepam tablets Twenty placidox(diazepam) tablets were weighed and the average weight was calculated. The working standard(80ppm concentration) was prepared in the same method as mentioned earlier in the case of calibration solutions. An aliquot of this solution was injected into HPLC system. Peak area of diazepam was measured for the determination and the results were reported in Table 8. RESULTS AND DISCUSSION Validation of the method Range of linearity Standard curves were constructed daily, for three consecutive days, using five standard concentrations in a range of 40, 50, 60, 70, 80 ppm for diazepam. The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. The linear regression equation was y = x (r= 0.999). Linearity values are shown in Table: 1 Table.1: Linearity results. Level Concentration in µg/ml Peak Area Level Level Level Level Level Level Level Range:40-100µg/mL Slope : Intercept :17636 Correlation coefficient: Fig. 3: Linearity Curve.

4 Page4057 Precision To study precision, six replicate standard solutions of diazepam (80 µg/ml) 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 well within the acceptance criteria of not more than 2.0%. Results of system precision studies are shown in Table.2 and Table.3. Table.2: Intraday Precision results. Sample Diazepam 80 Conc. (in µg/ml) Injection No. Peak Areas RSD (Acceptance criteria 2.0%) 0.50 Table.3: Inter day precision results. Sample Diazepam 80 Conc. (in µg/ml) Injection No. Peak Areas RSD (Acceptance criteria 2.0%) 0.78 System suitability The system suitability parameter like capacity factor, asymmetry factor, tailing factor and number of theoretical plates were also calculated. It was observed that all the values are within the limits (Table.4). The statistical evaluation of the proposed method revealed its good linearity, reproducibility and its validation for different parameters and let us to the conclusion that it could be used for the rapid and reliable determination of Diazepam in tablet formulation. The results are furnished in Table 4. Table 4: System suitability parameters. Mobile phase Formic acid: Methanol : Acetonitrile 45:20:35 (v/v) Pump mode Isocratic ph 5.8 Diluents Mobile phase Zodiac C18 column Column (250 X 4.6 mm, 5μ) Column Temperature Ambient Wavelength 239 nm Injection Volume 20 µl Flow rate 1 ml/min Run time 12 minutes Retention Time 5.27 minutes Area theoretical plates 4054 Tailing Factor 1.27 Pump pressure 6.2 MPa 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. Peak was not clearly observed on further dilution after 0.3 µg/ml. So, it is considered as Limit of Detection and the Limit of Quantification was observed to be 1.0 µg/ml.

5 Page4058 Table.5: LOD and LOQ results. Parameter Limit of Quantification Limit of Detection Measured Value 1.0 µg/ml 0.3 µg/ml Robustness 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, area, capacity factor, tailing factor and theoretical plates. The robustness acceptance criteria set in the validation were the same as established on system suitability test described above. Table.6: Robustness Results. S.NO Parameter Condition Mean area % difference 1 Unaltered FA: MeOH : ACN 45:30:25 (v/v) Mobile phase FA: MeOH :ACN 45:10:45 (v/v) Mobile phase ph Wavelength Recovery Recovery test was performed at 3 different concentrations by standard addition method. Results are given in Table.7 Table.7: Recovery results. % of Recovery 50% 100% 150% Diazepam Target Conc. (µg/ml) Spiked conc. (µg/ml) Final Conc. (µg/ml) Conc. Obtained % of Assay Formulation Analysis Table 8 : Formulation Analysis. S.NO Brand name Available form Label claim Concentration Amount found % Assay 1. Placidox Tab 10mg 80 µg/ml µg/ml 98.9 µg/ml CONCLUSION A validated RP-HPLC method has been developed for the determination of Diazepam in tablet dosage form. Previously, Rouini et al [9] reported a method for simultaneous determination of diazepam and its main active metabolites with conditions of RP- 18C 100 mm x 4.6mm column, using 10mM phosphate buffer (ph 2.5)-methanol-acetonitrile (63:10:27, v/v) as mobile phase. Snezana et al [13] reported a new kinetic-spectrophotometric method to determine diazepam in pharmaceutical formulations and human control serum. In this method the linearity range is µg/ ml The proposed method is simple, rapid, accurate, precise and specific. Its chromatographic run time is 10 min which allows the analysis of a large number of samples in short period of time. Therefore, it is suitable for the routine analysis of Diazepam in pharmaceutical dosage forms. The limit of detection for Diazepam was found to be 0.3 µg/ml and the limit of quantification was found to be 1.0 µg/ml which proved the sensitivity of the method. ACKNOWLEDGEMENT The authors acknowledge their sincere thanks to RV Labs, (Contract QC and Research Laboratory), Guntur for their gift sampling of the drug and technical support.

6 Page4059 REFERENCES 1. Mattson HR, Woodbury DM, Penry JK, Schmidt RP, Antiepileptic Drug, Raven Press, New York, 1982, Micke GA, Costa AC, Heller M, Barcellos M, Pioyezan M, Development of fast capillary electrophoresis method for the determination of propranolol total analysis time reduction strategies, J Pharmatogr A. 2009, 1216(45), Van T, Vu Fred P, Abramson, Quantitative Analysis of Propranolol and Metabolites by Gas chromatography and mass spectrometer computer Techniques. Biol. Mass Spectrom. 1978, 5(12), Vishali P, Michale, Avinash T Gatade, Dr. Ramesh T Sane, Validated HPTLC content uniformity test for the determination of diazepam in tablet dosage forms. International Journal of Pharmaceutical Research and Development. 2011, 3(6), Debabrata Ghosh, Dastidart, Biswanath SA, A comparative study of UV spectrophotometric and first order UV spectrophotometry methods for the estimation of diazepam in the presence of tween 20 and propylene glycols. AAPS Pharmscitech 2009, 10(4), Salem AA, Barsoum BN, Izake EL, Spectrochim. Acta Part A. 2004, 60(4), Modamio P, Lastra CF, Montejo O, Marifio EL, Development and validation of liquid chromatographic methods for the quantitation of propranolol, metoprolol, atenolol, bisoprolol. International J. Pharmaceutics. 1996, 130(1), Yuri kazakevich, Rosario lobrutto, High Performance liquid Chromatography for Pharmaceutical Scientist. john wiley and sons Inc. Hoboken New jersy Mohammad, Reza, Rouini Yalda, H, Ardakani, Kambiz A, Moghaddam F Solatani, An improved HPLC method for rapid quantitation of diazepam and its metabolites in human plasma. Talanta 2008, 75(3), Tulja Rani G, Gowri Shankar, Kadgapathi DP, Satyanarayana B, Development of an RP HPLC Method for the Simultaneous Estimation of Propranolol hydrochloride and Diazepam in combined Dosage forms. Indian Journal of Pharmaceutical Education and Research. 2011, 45(4), Samia M, El Gizawy, Simultaneous determination of diazepam, oxazepam, tenazepam, in spiked urine by HPLC. Analytical Letters. 2000, 33(4), Patel Satish A, Paresh U Patel and Natavarlal J, Development and validation of spectrophotometric method for the simultaneous estimation of diazepam and propranolol hydrochloride in combined dosage form. International Research Journal of Pharmacy. 2011, 2(8), Snezana S. Mitic, Aleksandra N. Pavlovic, Snezana B.Tosic, Emilija T. Pecev, Milan N. Mitic, Ruzica J. Micic, A kinetic method for the determination of diazepam based on ligand-exchange reaction. J. Chem Pharm Res. 2011, 3(2),