METHOD DEVELOPMENT AND VALIDATION BY RP-HPLC FOR ESTIMATION OF ZOLPIDEM TARTARATE
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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Ramalakshmi et al. SJIF Impact Factor Volume 7, Issue 2, Research Article ISSN METHOD DEVELOPMENT AND VALIDATION BY RP-HPLC FOR ESTIMATION OF ZOLPIDEM TARTARATE N. Ramalakshmi 1 *, C. N. Nalini 1, S. Sivaguru 1, A. Teja 1 and S. Arun Kumar 2 1 Department of Pharmaceutical Analysis, C. L. Baid Metha College of Pharmacy, Jyothi Nagar, Old Mahabalipuram Road, Thoraipakkam, Chennai Gulf medical University, Ajman, UAE. Article Received on 29 November 2017, Revised on 19 Dec. 2017, Accepted on 09 Jan DOI: /wjpps *Corresponding Author Dr. N. Ramalakshmi Department of Pharmaceutical Analysis, C. L. Baid Metha College of Pharmacy, Jyothi Nagar, Old Mahabalipuram Road, Thoraipakkam, Chennai analysis. ABSTRACT The objective is to develop and validate a new simple, rapid and sensitive isocratic RP-HPLC method for the determination of Zolpidem tartarate. The method employs Waters Alliance HPLC system on Novapak C18 (3.9 x 150 mm, 4.0 m) column. Best chromatographic separation was achieved by using buffer, Acetonitrile and methanol (59:18:23) as mobile phase with a flow rate of 1.5ml/min and isocratic elution with a total run time of 10 minutes. Detection of the compound was carried out at 254nm. The retention time of Zolpidem tartarate was found to be The linearity studies ranges from at the concentration range of µg/ml and this method was validated for accuracy, precision, linearity, ruggedness and robustness as per ICH guidelines. The present newly developed method was found to be accurate, precise and can be useful for routine Quality control KEYWORDS: Zolpidem tartarate, RP-HPLC, ICH, Analytical method validation. INTRODUCTION Zolpidem tartarate is a hypnotic drug, chemically N, N, 6-Trimethyl-2-p-tolylimidazo [1,2] pyridine-3-acetamide L-(+)-Tartarate. [1] It interacts with a GABA-BZ receptor complex and shares some of the pharmacological properties of the benzodiazepines. Zolpidem tartarate tablets are available in tablet dosage form as 5mg, 10mg. Literature survey reveals good numbers of analytical methods are available for the estimation of this drugs. Vol 7, Issue 2,
2 Various analytical methods including UV [2,3], HPTLC [4], and HPLC methods [5-7] are available for the estimation of Zolpidem tartarate in pharmaceutical dosage forms. We tried to develop a simple method for the estimation of Zolpidem tartarate by RP-HPLC in pharmaceutical dosage forms. The proposed method was optimized and validated as per the International Conference on Harmonization (ICH) guidelines. MATERIALS AND METHODS Instrumentation Waters alliance HPLC Novapak C18, 3.9 x 150 mm, 4.0 m particle size was used. The HPLC system is made up of 2695 separation module and 2998 photodiode array detector. The output was processed using Empower 2 software. Chemicals and solvents The working standard and samples of Zolpidem tartarate were obtained from PAR Formulations, Chennai, India. HPLC grade water, Acetonitrile (HPLC Grade), Orthophosphoric acid was obtained from Merck (India) Ltd, Mumbai, India. Chromatographic parameters The chromatographic separation was achieved by Novapak C18, 3.9 x 150 mm column. The mobile phase consists of Orthophosphoric acid buffer, Acetonitrile and Methanol in the ratio (59:18:23). The flow rate was maintained at 1.5 ml/min throughout the gradient program. The eluents were monitored at 254 nm. The column temperature was maintained at 30 o C. the injection volume was 20 µl. Mobile phase was also used as a diluent. Isocratic elution method was followed. Each sample was run for 10 min. Preparation of the Zolpidem tartarate Standard & Sample Solutions Preparation of Standard Stock Solution (100 µg/ml of Zolpidem Tartarate) Weigh accurately about 50 mg of Zolpidem tartarate WRS and transfer to a 50 ml volumetric flask. Add about 30 ml of diluent and sonicate to dissolve. Dilute to volume with diluent and mix well. Further, dilute 5 ml to 50 ml with diluent. Sample Preparation (For 10mg tablets) Weigh 20 tablets of Zolpidem Tartarate and transfer to a 200 ml volumetric flask. Add about 140 ml of diluent and sonicate for 10 minutes with intermittent shaking. Shake mechanically for 10 min. Dilute to volume with diluent and mix well. Centrifuge a portion of sample at Vol 7, Issue 2,
3 3500RPM for 10 min. Dilute 5 ml of supernatant liquid to 50 ml with diluent. Filter through 0.45 µm nylon filter. Sample preparation (For 5mg tablets) Weigh 20 tablets of Zolpidem Tartarate and transfer to a 100 ml volumetric flask. Add about 70 ml of diluent and sonicate for 10 minutes with intermittent shaking. Shake mechanically for 10 min. Dilute to volume with diluent and mix well. Centrifuge a portion of sample at 3500RPM for 10 min. Dilute 5ml of supernatant liquid to 50 ml with diluent. Filter through 0.45 µm nylon filter. Injection of Standards and Samples into the Chromatographic system 20 µl of each standard and sample solution were injected into the chromatographic system and the area of Zolpidem tartarate peaks was measured. The assay results, expressed as % of the label claim, are in Table 1 (Fig. 1). This indicates that the amount of each drug in the product meets the requirements. RESULTS Method Development The objective of the proposed work was to develop and validate an HPLC method for the estimation of Zolpidem tartarate in bulk and pharmaceutical dosage forms. Initially, various mobile phase compositions were tried to elute title ingredients. Mobile phase and flow rate selection was based on peak parameters (height, capacity, theoretical plates, tailing or symmetry factor, run time, resolution). In RP-HPLC method development, the estimation was carried out by Water alliance HPLC auto sampler separation module with UV detector and column used was Novapak C18, 3.9 x 150 mm, i.e. column with 4μ particle size. Injection volume of 20 μl is injected and eluted with the mobile phase (ph 5.5), Acetonitrile and methanol with the ratio of (59:18:23), which is pumped at the flow rate of 1.5 ml/min. Detection was carried out at 254 nm. Quantification was done by calibration curve method with the above mentioned optimized chromatographic condition. System Suitability System Performance parameters of developed HPLC method were determined by injecting standard solutions. Parameters such as number of theoretical plates (N), tailing factor, Vol 7, Issue 2,
4 resolution(r), retention time (RT) were determined. The results are shown in Table 2 (Fig. 3), it indicates good performance. Specificity Specificity was evaluated continually through the drug development process. The data is shown in Table 3. Blank/Placebo chromatogram did not show any disturbance peak at the retention times of Zolpidem tartarate. Linearity From the standard stock solutions, a suitably mixed standard solution was prepared to contain 50 to 150 % of the targeted level of the assay concentration of the standard drugs (containing internal standard). The solutions were examined by the assay procedure. The calibration curve was plotted using response factor (peak area ratio of the standard peak area and internal standard peak area) vs. concentration of the standard solution. From the calibration curve, the slope and intercept, correlation-coefficient was calculated. The calibration curves (Fig. 2) were constructed by plotting absorbance versus concentration and the regression equations were calculated. The results are shown in Table 4. The Correlation coefficient was found to be more than 0.99, which indicates that there was an excellent correlation between the peak area response and the concentration. Precision Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of the analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. The results obtained are tabulated as Table 5 (Fig. 4). The %RSD for the area of five standard injections results was found to be less than 2%. Accuracy The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and value found. The standard solution was injected with Accuracy 80%, 100% and 120% solutions. The amount found was calculated and amount added for Zolpidem tartarate were estimated. The individual recovery and mean recovery values were also calculated in Table 6. The % Recovery for each level was found to be between 98.0% and 102.0%. Vol 7, Issue 2,
5 Robustness As part of the Robustness, deliberate change in the flow rate, ph was made to evaluate the impact on the method. The typical variations are given below: Variation in flow rate was by ± 0.2ml/min. Variation in ph in the Mobile phase varied from ±10%. The results are shown in Table 7. Ruggedness The assay was performed in different condition, different analyst, and different dates. The results are given in Table 8. Limit of Detection (LOD) Loss of Detection was calculated from the formula; LOD = 3.3σ /S Where, σ = standard deviation of the response, S = slope of calibration curve. Limit of Quantification (LOQ) Loss of Quantification was calculated from the formula; LOQ = 10σ /S Where, σ = standard deviation of the response, S = slope of calibration curve The results for Limit of Detection and Limit of Quantification are shown in the Table 9. TABLES Table 1: Assay of Zolpidem tartarate. Injection Zolpidem area Amount of Zolpidem in tablet % Recovery Mean SD % RSD Table 2: System suitability. System Suitability Parameters of Theoretical Tailing Retention Zolpidem tartarate plates factor time Zolpidem min Table 3: Interference of Blank and Placebo. Name Interference Retention time (min) Blank Nil Nil Placebo Nil Vol 7, Issue 2,
6 Table 4: Linearity of Zolpidem tartarate. Sl. No. Conc. of Zolpidem tartarate Peak area of µg/ml Zolpidem tartarate Table 5: Precision of Zolpidem tartarate. Sl. No. Zolpidem tartarate Zolpidem tartarate Area Area System precision Method precision AVG STDEV % RSD Table 6: Accuracy and % Recovery of each analyte. Accuracy Level % Mean recovery % RSD Table 7: Robustness Parameters of Zolpidem tartarate. Drug name Parameters Retention time (min) Area (mm 2 ) Flow rate Flow rate RSD Temperature ZOLPIDEM Temperature TARTARATE RSD Buffer Buffer RSD Vol 7, Issue 2,
7 Table 8: Ruggedness of Zolpidem tartarate. Number of injection Retention time (min) Area (mm 2 ) Injection Injection Injection Injection Injection Mean SD RSD Table 9: L.O.D. & L.O.Q. of Zolpidem tartarate. Name of drug Zolpidem LOD LOQ Acceptance criteria Based on the standard deviation of the response and the slope Fig. 1: Assay of Zolpidem tartarate. Fig. 2: Linearity Curve for Zolpidem tartarate. Vol 7, Issue 2,
8 Fig. 3: Typical Chromatogram of Zolpidem tartarate. Fig 4: Precision. DISCUSSION This system produced symmetric peak shape, good resolution and reasonable retention time of Zolpidem tartarate was found to be The correlation coefficient(s) of Zolpidem tartarate was found to be respectively for which indicates excellent correlation the peak area response Vs concentration of standard solutions. Precision of the developed methods was studied under system precision, method precision. The %RSD values for precision was found to be within the acceptable limit, which revealed that the developed method was precise. The developed method was found to be robust. The %RSD values for recovery percentage of Zolpidem tartarate was found to be within the acceptable criteria. The result indicates satisfactory accuracy of method for simultaneous estimation of the above mentioned drugs. CONCLUSION From all results it was concluded that the developed RP - HPLC method for the estimation of Zolpidem tartarate in dosage forms are accurate, precise, linear, robust, simple and rapid. Vol 7, Issue 2,
9 Percentage recovery shows that the method is free from interference of excipients used in the formulation. ACKNOWLEDGEMENT 1. We would like to thank the management of C.L. Baid Metha College of Pharmacy, Chennai for their constant help & guidance and providing us a quality environment for completing our work. 2. We would like to thank PAR formulations, Chennai for providing us with the chemicals required for completing our work. REFERENCES 1. Sweetman S. Martindale: the complete drug reference. 36th ed. London: The Pharmaceutical Press; The Merck Index, Merck & Co, Inc, 14 th edn. White house station, NJ, Egginger G, Lindner W, Karh S, Stoschitzky K. Stereoselective HPLC bioanalysis of atenolol enantiomers in plasma: application to a comparative human pharmacokinetic study. Chirality. 1993; 5: Swarbrick JC, Boylan James. Suitable for preparative liquid chromatography on a much larger scale.encyclopedia of pharmaceutical technology, 1998; 1: Lindsay Sandy, HPLC by open learning., 1991; Lough WJ, Wainer IWW. HPLC fundamental principles and practices., 1991; Vol 7, Issue 2,
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