Airo International Research Journal ISSN : Volume : 7 October 2015

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1 Airo International Research Journal ISSN : Volume : 7 October 2015 METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF ESCITALOPRAM OXALATE AND ETIZOLAM IN TABLET DOSAGE FORM BY RP-HPLC Paul Richards M, Research Scholar Sun Rise University Alwar,Rajasthan Dr Vinesh Kumar, Department of Pharmacy, Sun Rise University, Alwar, Rajasthan ABSTRACT Present work was to develop a simple, fast, accurate, precise, reproducible and Reverse Phase High Performance Liquid Chromatographic method for simultaneous estimation of Oxalate and Etizolam in tablet dosage form marketed as Etizolam plus. Chromatographic separation was done using Hypersil BDS C 18 column having dimension of mm having particle size of 3.5µm, with mobile phase consisting of phosphate buffer ph 3 ±0.02 ph adjusted with ortho phosphoric acid and ACN in the ratio of 50:50% (v/v), flow rate was adjusted to 1.0 ml/min and detection wavelength at 254nm. The retention times of Oxalate and Etizolam was found to be and mints, respectively. The Proposed method has been validated for accuracy, precision, linearity, range and robustness were within the acceptance limit according to ICH Q2(R1) guidelines. Linearity for Oxalate and Etizolamwas in range of 20µg-120µg and correlation coefficient was found to be 0.998,% RSD for system precision was found to be , and for repeatability was 0.53, 0.403, % mean recovery for Oxalate and Etizolam was found to be 99.47% to %. The method was found to be robust even by change in the wavelength ±2% and in less flow condition.

2 DRUG PROFILE Name : (1S)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)- 1,3-dihydro-2-benzofuran-5-carbonitrile Molecular Formula : C 20 H 21 FN 2 O Molecular Weight : Physical Description Colour Solubility : Fine white to slightly yellow powder : Freely soluble in methanol and dimethyl sulfoxide Category : Antidepressant agent CAS No. : Brand Name : CIPRALAX, LEXAPROFOREST. 7-(2-chlorophenyl)- 4-ethyl- 13-methyl- 3-thia- 1,8,11,12- tetraazatricyclo ] trideca- 2(6),4,7,10,12- pentene Molecular Formula : C17H15ClN4S. Molecular Weight : Physical Description Colour Solubility M.P : White powder : Water insoluble. : C CAS No. : Category Brand Name : Anxiolytic, Anticonvulsant : Etilaam, Etizola.

3 Isobestic point of Oxalate & Etizolam The overlay spectrum of oxalate and Etizolam shows the isobestic point 254nm. Hence 254nm selected was maximum absorbance and the detection wavelength for the proposed method. Method Development Trail : 0.05M Potassium di hydrogen ortho phosphate ph-3 adjust with orthophosphoric acid: ACN (50:50v\v) column BDS Hypersil C18 1 ml/min. The retention Time found to be 2.83 and 4.0 min of resolution and theoretical plates 4437 and 7973 with good resolution and symmetry. Preparation of Solutions : Buffer Preparation : 3.4g of KH 2 PO 4 in 500 ml beaker dilute and make up with HPLC water addition of OPA to adjust ph 3 ± 0.01 Mobile Phase Preparation : Mix phosphate buffer 500 ml (50%) and 500 ml of Acetonitrile (50%) degas in sonicator for 5 min and filter through vaccum filtration. Sample stock Solution Weigh equivalent to 50mg of oxalate and 2.5 mg of Etizolam sample transfer into a 50 ml clean dry volumetric flask add 3 ml of mobile phase and sonicate to dissolve it

4 completely make volume up to the mark with mobile phase filter through 0.45 membrane filter.filter pipette out 1.0 ml of filtrate solution into three 10 ml volumetric flask make up the volume upto the mark with mobile phase. ASSAY Calculate the assay, in mg of Drug substance using the following formula: AT x W S x1x 50x10x P x Average Weight % Assay = X 100 AS x 10x WT x 5x100 X LA Where, AT = Average area of oxalate and Etizolam peak in test chromatograms AS= Average area of oxalate and Etizolam peak in standard chromatograms WS = Weight of oxalate and Etizolam working standard taken in mg WT = Weight of sample taken in mg P = Potency of oxalate and Etizolam working standard in % w/w Avg Wt = Average weight of oxalate and Etizolam tablets PR = Purity of working standard [%] LA = Labeled amount Observation: Retention time was found to be 2.8 and 4.0 mints and the % purity of the oxalate and Etizolam was found to be % w/w & % w/w respectively METHOD VALIDATION SPECIFICITY 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. Determination: Blank/Placebo are injected to check the interference from the peak of main components in control sample. Blank Preparation (Diluent): Mixed and filtered acetonitrile and phosphate buffer in the ratio 50: 50 v/v and degassed by sonication S. No. Blank injection Observations 01 Blank injection No interference 02 Blank injection No interference

5 LINEARITY The linearity of an analytical method is its ability to elicit test results that are directly well defined mathematical transformation, proportional to the concentration of analyte in sample within a given range. Linearity level Volume of stock solution(ml) Diluted to(ml) Linearity (µg/ml) conc of Linearity conc (µg/ml) of Etizolam Result: The linearity study was performed for range of µg/ml and 1-6 µg/ml. The correlation coefficient (R 2 ) was found to be and (NMT 0.999) 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 the value found. This is sometimes termed trueness.

6 Accuracy is performed in three different levels for oxalate and Etizolam. Spiked known quantity of oxalate and Etizolam at 80%,100% and 120% level into the placebo. Analysed samples in triplicate for each level. From the results, % recovery was calculated. Result: The accuracy study was performed for the range of 80 to 120. The % recovery of oxalate and Etizolam was found to be % and % respectively (NLT-98 & NMT-102). Drug Conc of sample(µg) Avg area of sample Amount recovery % recovery Mean % recovery oxalate Etizolam PRECISION The precision of an analytical procedure expresses the closeness of agreement, degree of scatter between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. System precision: Repeatability of standard preparation:

7 Repeatability expresses the precision under the same operating conditions over a short interval of time. Method Precision Reproducibility expresses the precision under the same operating conditions, from six preparations of a homogenous sample. ACCEPTANCE CRITERIA: %R.S.D. for 6 replicate injections of standard drug solutions not more than 2.0. Method precision The method precision was performed on oxalate and Etizolam formulation. The % RSD of the assay value for five determinations was calculated ACCEPTANCE CRITERIA Relative standard deviation of % Assay results should not more than 2.0%. System suitability results S. No. System Suitability Parameter Observations oxalate Etizolam Proposed Acceptance Criteria 1 % Relative Standard Deviation for five replicate injections of analyte peak in standard solution Should be not more than 2.0%

8 2 Tailing factor for analyte peak in standard solution 3 Plate count for analyte peak in standard solution Should be not more than 2.0 Should be not less than 2000 SUMMARY AND CONCLUSION The project summarizes a method development and validation for simultaneous estimation of oxalate and Etizolam in combined pharmaceutical table dosage form. An efficient high performance liquid chromatographic method was developed for oxalate and Etizolam. The HPLC method was developed by using BDS Hypersil C 18 column; ( µ) column at 254 nm, flow rate of 1.0 ml/min., Injection volume of 10 µl, column oven temperature of 25 0 C using equal volume of acetonitrile and 0.05M Potassium dihydrogen orthophosphate ph-3 adjust with orthophosphoric acid was used as mobile phase(50:50 v/v). The retention times were found to and mints. The % purity was found to be and 99.97% w/w respectively. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The correlation coefficient (r 2 ) was found to be respectively,% recovery was 99 %, % RSD for system precision was found to be , and for repeatability was 0.53, respectively. The HPLC method was found to be accurate, precise, economical and reproducible. The method can be suggested for routine analysis and method can be recommended for determination of relative substance. REFERENCES 1. Sharma B.K, Instrumental methods of chemical analysis, Introduction Analytical chemistry, Goel Publishing House, ed- 19 th, 2003, PP Kasture A.V,Mahadik K.R,Wadodker S.G,More HN,Instrumental methods of Pharmaceutical analysis, Nirali Prakashan Pune,, Volume -II, ed-14 th, 2006, PP Skoog DA, Holler FI, Niemen TA, Separation Methods, Principles of Instrumental Analysis, Thomson Asia Pte. Ltd, ed-5 th, 2005, PP Yogesh Gupth, Pharmaceutical Drug Analysis, New Age International (P) Ltd, New Delhi, India, ed-2 nd, 2003, PP Gurudeep R. Chatwal, Sham K. Anand, Instrumental Methods of Analysis, Shing Hanse, Mumbai, ed-4 th, 2006,.PP Stenlake JB, Backett AH, Practical pharmaceutical Chemistry, New Delhi: C. B.S, ed- 4 th,1997, PP-494.

9 7. Analytical Chemistry General Perspective. India: IGNOU, Available from: Graduate Diploma in Analytical Chemistry. 8. Beckett A.H, and Stenlake J.B, Pratical Pharmaceutical Chemistry, part i-ii, ed-4 th, 2005, PP H.H. Williard, L.L.Merit, F.A. Dean and F.A. Settle, Instrumental Methods of Analysis, CBS Publishers and Distributers, New Delhi, ed-7 th, 1986, PP-1, 592,

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