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ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF METHOCARBAMOL FOR ESTIMATION IN BULK AND PHARMACEUTICAL DOSAGE FORM BY UV SPECTROPHOTOMETRYBy Mr.D.S.KASIM VALIEmail: Kasimpharma786@gmail.com

VASAVI INSTITUTE OF PHARMACEUTICAL SCIENCESVASAVI NAGAR, PEDDAPALLI (V), SIDHOUT (M),NEAR BHAKARAPET RAILWAY STATION, KADAPA-5162472014

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Abstract Need for the studyReview literatureAim and objectivePlan of workMethod developmentMethod validationResults and discussionsConclusionBibliography CONTENTS

ABSTRACT:Pharmaceutical analysis occupied a pivotal role in determination of drugs in formulation and its combinations. The complexity of problems in existing methods in terms of achieving the selectivity, speed, cost, simplicity, sensitivity, precision and accuracy has been replaced by new methods of analysis. The present work attempts to minimize the time consumption and cost by simple spectrophotometric method based on use of acetone and 0.1N sodium hydroxide solution in which the drug is completely soluble, used as a solvent system. The drug has an absorption maximum at 267 nm and obeys Beers Lambert law. The absorbance was found to increase linearly with increasing concentration of Methocarbamol, which is corroborated by the calculated correlation coefficient value of 0.9999.The apparent molar absorptivity is 9215.368 L mol-1cm-1. The slope and intercept of the equation of the regression line are 3.3 10-2 and 5.4x10-2 respectively. The mean recovery obtained for Methocarbamol was found to be 100.56%. The optimum experimental parameters for the method have been studied. The validity of the elucidated method was assessed according to International Conference on Harmonization guidelines. Statistical analysis of the results has been carried out revealing high accuracy and good precision. The proposed method was successfully applied to the determination of methocarbamol in bulk and pharmaceutical dosage forms.Key words: Methocarbamol, UV spectrophotometry, Validation, International Conference on Harmonization.

NEED FOR THE STUDYSeveral methods have been reported using various instrumental techniques like LC-API-MS method, HPLC method, UV spectro photometric method, simultaneous estimation method in bulk & pharmaceutical dosage forms and also in biological fluids. Up to the best of our knowledge all these reported methods were time consuming, costly (LC-API-MS, HPLC) and requires skilled operator. Hence attempts have been made to develop and validate simple and sensitive Ultra Violet spectrophotometric analytical method for the estimation of Methocarbamol in bulk and pharmaceutical preparation (Tablets) by employing simple UV-spectro photometric method.

DRUG PROFILE:Methocarbamol, a carbamate derivative of guaifenesin, is a central nervous system (CNS) depressant with sedative and musculoskeletal relaxant properties. PHYSICO-CHEMICAL PROPERTIES OF THE DRUG:Description: Methocarbamol is white powder.Solubility: It is readily soluble in acetone, 0.1N Hydrochloric acid. Slowly soluble in 0.1N Sodium hydroxide solution, sparingly soluble in water and chloroform, soluble in alcohol (only with heating) and propylene glycol, and insoluble in benzene andn-hexane.Molecular formula: C16H20N4O3SMolecular Weight: 241.24.Chemical name: 3-(2-methoxyphenoxy)-l, 2-propanediol 1-carbamate.Molecular structure:

Storage: At20 to 25C (68 to 77F)

REVIEW LITERATURE

List of marketed products

S.NOBrand nameCompositionCompamy1FLEXINOL tabMethocarbamol 400mg, Paracetamol 325mgPROTEC2IBUGESIC-M tabMethocarbamol 750mg, Ibuprofen 400mgCIPLA3NEUROMOL-MR tabMethocarbamol 400mg, Paracetamol 500mgBIOSEARCH4ROBIFLAM tabMethocarbamol 750mg, Ibuprofen 200mgKHANDELWAL5ROBIFLAM tabMethocarbamol 750mg, Ibuprofen 400mgKHANDELWAL6ROBILILD tabMethocarbamol1000mg, Nimesulide 100mgKHANDELWAL7ROBINAX tabMethocarbamol 500mgKHANDELWAL8ROBINAX injMethocarbamol 100mg/10mlKHANDELWAL9ROBINAXOL tabMethocarbamol 350mg, Paracetamol 250mgKHANDELWAL10ROBINAXOL-D tabMethocarbamol 500mg, Paracetamol 325mg, Diclofenac 50mgKHANDELWAL

11METHOCARBAMOL USPMethocarbamol 500mgHetero drugs Pvt.Ltd

REPORTED METHODSS.NoTitleAuthors nameMethod Year 1Simple UV spectrometric for the determination of Methocarbamol in bulk and its formulation. Chaitanya Prasad MeherUV spectroscopy20132Simultaneous determination of methocarbamol and aspirin by RP-HPLC using fluorescence detection with time programming:its application to pharmaceutical dosage form.M Sharaf El-DinReverse Phase High-performance liquid chromatography20133RP-HPLC Method Development and validation for the simultaneous estimation of Ibuprofen and Methocarbamol in Ibuprofen-Methocarbamol CapletsNatraj K.SReverse Phase High-performance liquid chromatography20134Simultaneous UV-VIS spectrophotometric determination of aspirin and methocarbamol in tablets.A. SalehiUV spectroscopy20125Stability indicating HPLC method for simultaneous determination of methocarbamol and nimesulide from tablet matrixR. S. ManmodeHigh-performance liquid chromatography20116Simultaneous determination of Methocarbamol and Ibuprofen or Diclofenac potassium using mean centering of the ratio spectra methodNouruddin Wageih AliUV spectroscopy20127Simultaneous spectrophotometric estimation of ibuprofen and methocarbamol in tablet dosage formSATHEESHMANIKANDAN T. R. SUV spectroscopy20048Determination of Methocarbamol in Equine Serum and Urine by High-Performance Liquid Chromatography with Ultraviolet Detection and Atmospheric Pressure Ionization-Mass Spectrometric Confirmation*. Mohammed R. Koupai-AbyazaniHigh-performance liquid chromatography1997

AIM & OBJECTIVEThe main aim in this work is to develop a new simple, accurate, precise and efficient U.V Spectroscopic method for Methocarbamol for normal estimation of this drug present in the marketed dosage forms and to reduce the time of analysis, cost based on the literature survey have been made.In the present study, the main objective is to validate the developed analytical method for estimation of methocarbamol in bulk and pharmaceutical preparation with different parameters like linearity, Precision, Accuracy, Intermediate precision, Robustness, Limit of detection, Limit of quantification by employing UV-spectroscopic method.

PLAN OF WORKStep.1: Survey on literatureThe survey on literature performed for Methocarbamol for their physiochemical properties, solubility, pharmacology and analytical techniques. So this basic information gives notation for newer method development.Step.2: Method development.1) Determination of solubility of the drug.2) Development of a rapid and accurate UV method.3) Analysis of marketed formulations.4) Validation of developed analytical method.5) Statistical analysis of developed analytical methods.Step.3: Validation of developed methodThe developed method should be validated as per ICH guidelines. The parameters used to validate the developed method are Accuracy, Precision, Limit of Detection, Limit of Quantification, Linearity, Range, Robustness and Ruggedness.

EXPERIMENTAL WORKList of Chemicals and solvents used

List of Instruments used

S.No.Chemicals and reagentsGradeName of the company1.Acetone ARMerck Specialties Pvt.Ltd., Mumbai2.Sodium hydroxide pellets purifiedARMerck Specialties Pvt.Ltd., Mumbai3.Distilled waterNA--

S.No.Instrument usedModelSoftwareManufactured by1.Digital balance--Shimadzu2.UV - Visible double beam spectrophotometer2202-Systronics

Sr. No.Name of pure DrugSupplier1.MethocarbamolHetero drugs .ltd.

Sr. No.Brand NameContentMfg. Dt.Exp. Dt.Manufacturing Company1Methocarbamol USP500 mgApril 2014May 2016Hetero drugs ltd

Details of Gift Sample

Details of Marketed Formulation

METHOD DEVELOPMENT Solvent selectionScreening of various solvents was made by using a numeral of trials to come across the most advantageous solvent system for dissolving the drug methocarbamol. The drug was found to be soluble in acetone, methanol; strong base i.e., sodium hydroxide and strong acid i.e., hydrochloric acid.After careful observation of the solubility and spectrums of methocarbamol, the drug was found to be more soluble and showed maximum absorbance at 267 nm when acetone (AR Grade) and 0.1 N Sodium hydroxide (AR Grade) in the ratio of 1:9 was used as primary (Stock) solvent system and remaining dilutions were made by 0.1N sodium hydroxide solution. Based on the solubility studies and UV spectral data of the methocarbamol drug, combination of those solvents was selected as solvent system as those are easily available and used for the point of time saving.

Scanning and determination of max: In order to establish the wavelength of maximum absorption ( max) of the methocarbamol drug, solution of the drug was prepared in acetone and 0.1N sodium hydroxide solution in which the drug is completely soluble and scanned using UV spectrophotometer within the wavelength region of 200-400 nm against 0.1N sodium hydroxide solution as blank. The drug has an absorption maximum at 267nm.UV Spectrum of methocarbamol at 200-400 nm

Determination of Absorptivity Value, A (1%, 1cm), of Methocarbamol at Selected Wavelength

Absorbance data for A (1%, 1cm) determinationMolar absorbtivity = A1%1cm x molecular weight 10

From calibration vurve A1%1cm was calculated and used for the determination of molar absorptivity and it was found to be 9215.368 L mol-1cm-1

Sl.NoConcentration g/mlA-1A-2A-3A-4A-5Average Absorbance150.2210.2220.2220.2200.2210.2212100.3920.3920.3930.3930.3930.3923150.5200.5200.5230.5230.5250.5224200.7150.7150.7150.7160.7160.7155250.8850.8860.8860.8880.8860.886

METHOD VALIDATION LINEARITY:The linearity of an analytical procedure is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range.Procedure :Linearity solutions Preparation: From secondary stock solution i.e., 100g/ml different dilutions were made concentration ranging between 5-25g/ml and checked for uv absorbance and calibration curve was plotted by plotting graph between absorbance and concentration.Acceptance criteria for Linearity: Correlation coefficient should not be less than 0.999.

Calibration curve of Methocarbamol

Sl.NoConcentration (g/ml)AbsorbanceStatistical analysis150.221Slope=0.033Intercept =0.054Correlation coefficient=0.9992100.3933150.5364200.7155250.886

Linearity data for the proposed methodLinear UV spectrums of Methocarbamol

PRECISIONThe precision of the analytical method relates to the degree of agreement among individual test results and how individual results are scattered from the mean value usually expressed as standard deviation % RSD. Procedure :To check the method precision, standard solution containing 15g/ml of Methocarbamol was subjected to the proposed UV method of analysis. Acceptance criteria: %RSD should NMT 2%

Precision data of the proposed method

S.NoStandard concentrationAbsorbance115g/ml0.52215g/ml0.521315g/ml0.52415g/ml0.52515g/ml0.52615g/ml0.523Mean0.520S.D0.00121106% R.S.D0.23259% R.S.D limit2

INTRA DAY & INTER DAY PRECISION

To check the intra-day and inter-day variation of the method, solution containing 15 g/ml of Methocarbamol was subjected to the proposed UV method of analysis. The precision of the proposed method i.e. the intra and inter-day variations in the absorbances of the drug solutions was calculated in terms of % RSDAcceptance criteria: %RS D of 6 replicate preparations of assay should not be more than 2

Results from intra, inter day, analyst to analyst precision studies

Standard concentration 15g/mlAbsorbanceIntra dayInter dayMorning Evening Analyst-IAnalyst-IISample 10.5210.5150.5230.52Sample 20.520.520.5210.52Sample 30.520.5190.5240.52Sample 40.5230.5190.530.521Sample 50.5190.520.5250.521Sample 60.520.5190.5190.523Mean0.52050.5180.52360.5208S.D 0.0013780.001860.003770.00116% R.S.D0.2648230.358970.721280.22445% R.S.D limit2222

ACCURACY The accuracy of an analytical procedure is the closeness of test results obtained by that procedure to the true value. The accuracy of an analytical procedure is established across its range.The accuracy of the method was determined by preparing solutions of different concentrations that is 80%, 100%, 120% in which the amount of marketed formulation was kept constant and the amount of pure drug added was varied that is 12mcg/ml, 15mcg/ml, 18mcg/ml for 80%,100% and 120% respectively. The solutions were prepared in triplicates and accuracy was indicated by %recovery.Accuracy was demonstrated by preparing following solutions.S1 solution (80% of target concentration): Sample formulation at level of 80% - Marketed formulation which contain 15mcg/ml a known quantity of pure drug that is 12mcg/ml was added to 80% level sample solution. These solutions were prepared in triplicates.S2 solution (100% of target concentration):Sample formulation at level of 100% - Marketed formulation which contain 15mcg/ml a known quantity of pure drug that is 15mcg/ml was added to 100% level sample solution. These solutions were prepared in triplicates.S3 solution (120% of target concentration): Sample formulation at level of 120% - Marketed formulation which contain 15mcg/ml a known quantity of pure drug that is 18mcg/ml was added to 120% level sample solution. These solutions were prepared in triplicates.

Accuracy data of the proposed method Solut-ionConcentration(mcg/ml)Amount recovered(mcg/ml)%RecoveryMean% recoverySDRSDFormulationPureDrugS1151226.698.598.730.4040.4151226.899.2151226.698.5S2151529.9699.899.540.230.23151529.8599.5151529.799.33S3151832.8999.6699.670.070.07151832.9299.75151832.9099.6

LIMIT OF DETECTION (LOD)

Limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions.The detection limit (DL) may be expressed as: DL = 3.3 S Where, = standard deviation of the response S=slope of the calibration curve.The parameter LOD was determined on the basis of response and slope of the regression equation. The LOD for this method was found to be 0.121106 g/mL.

LIMIT OF QUANTIFICATION (LOQ)

LOQ is the smallest amount of the analyte in a sample matrix that can be quantified with acceptable accuracy and precision under the stated experimental conditions. It is expressed as the concentration of analyte (e.g. parts per million) in the sample. The Quantitation limit (QL) may be expressed as: QL = 10 S Where, = standard deviation of the response S=slope of the calibration curve.The parameter LOQ was determined on the basis of response and slope of the regression equation. The LOQ for this method was found to be 0.36698g/ml.

ROBUSTNESS:The robustness of analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.Robustness examines the effect of variation in operational parameters on the analysis results. For the determination of a methods robustness, parameters like variation in detector wavelength are varied within a realistic range and the quantitative influence of the variables is determined. If the influence of the parameter is within a previously specified tolerance, the parameter is said to be within the methods robustness range.Acceptance criteria: %RSD should not be more than 2%

Robustness data for the proposed method

Wave Length266 nm267 nm268 nmAbsorance-10.5160.520.514Absorbance-20.5150.5210.514Absorbance-30.5150.520.515Absorbance-40.5160.520.514Absorbance-50.5160.520.516Absorbance-60.5170.5230.516Mean0.51580.5200.5148Standard deviation0.000750.001211060.00098% R.S.D0.14590.232590.1909% R.S.D limit222

Estimation of Methocarbamol in Tablet Formulation.

Procedure : Twenty tablets were weighed and finely powdered. An accurately weighed tablet powder equivalent to 100 mg of Methocarbamol was transferred in 100 mL volumetric flask and 10 mL Acetone was added, after that 90 ml of 0.1 N sodium hydroxide i.e., 1:9 ratio was added. The volumetric flasks content was sonicated for 15 min. 10mL of above solution was diluted up to 100 mL with 0.1 N sodium hydroxide solvent system to obtain the concentration of 100 g/mL of Methocarbamol. 15mL of above solution was diluted up to 100 mL with 0.1 N sodium hydroxide solvent system to obtain the final concentration of 15 g/mL of Methocarbamol. The absorbance of final solutions was measured at 267 nm against solvent blank. The readings were taken in triplicate and performing the same experiment for dosage forms with different batches. The content of Methocarbamol was calculated by using following Equation.Drug % purity= At/AsDFAvg Wt/LC100Where,At= Sample absorbance of drugAs=Standard absorbance of drug Avg Wt = Average Weight DF= Dilution factorLC= Label Claim.

Assay results of Methocarbamol in Tablets:Acceptance criteria: % Assay should be between 90 to 110%. Since the %RSD of % assay was found to be below 2%.SampleLabel claimAmount takenAmount found%Assay%R.S.DMethocarbamol USP500 mg100mg100.01mg100.10.41100.07 mg100.7100.09 mg100.9

RESULTS & DISCUSSIONScreening of various solvents was made by using a numeral of trials to come across the most advantageous solvent system for dissolving the drug methocarbamol. The drug was found to be more soluble and showed maximum absorbance when Acetone (AR Grade) and 0.1 N Sodium hydroxide solution (AR Grade) in the ratio of 1: 9 used as primary solvent system and further dilutions by 0.1 N Sodium hydroxide solution (AR Grade). The max was found to be 267 nm. Based on the solubility studies and UV spectral data of the methocarbamol drug, combination of those solvents was selected as solvent system as those are easily available and used for the point of time saving.The calibration curve was constructed with concentrations of 5, 10, 15, 20 and 25g/ml. The absorbance of the drug was considered for plotting the graph. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. The correlation coefficient value was found to be 0.999.

The accuracy of the method was determined by standard addition method , by preparing solutions of different concentrations that is 80%, 100%, 120% in which the amount of marketed formulation was kept constant and the amount of pure drug added was varied that is 12mcg/ml, 15mcg/ml, 18mcg/ml for 80%, 100% and 120% respectively. The solutions were prepared in triplicates and accuracy was indicated by %recovery. The mean % recovery of the Methocarbamol at each spike level should be not less than 98.0 % and not more than 102.0 %. The mean % recovery of the Methocarbamol accuracy solutions i.e., S1, S2 and S3 was found to be 98.73%, 99.54% and 99.67% respectively and %R.S.D was found to be 0.4, 0.23 and 0.07. The accuracy results showed good recovery with percent relative standard deviation (% RSD) is below 2.0. The parameter LOD, LOQ was determined on the basis of response and slope of the regression equation. The LOD for this method was found to be 0.121106 g/mL. The LOQ for this method was found to be 0.36698g/mL.For the determination of a methods robustness, parameters like variation in detector wavelength are varied within a realistic range and the quantitative influence of the variables is determined. % R.S.D should not be more than 2. % R.S.D was found to be within limits.The proposed procedure was applied to the determination of Methocarbamol in commercially available tablets. The results obtained were satisfactory and good agreement as per the ICH guidelines.

Method validation data for the proposed method

S.NoPARAMETERVALUEACCEPTABLE LIMITS1Linearity Range5-25 g/ml------2Correlation Coefficient 0.9990.99993Regression EquationY= 0.33x+0.054------4Slope(m)0.033------5Intercept(c)0.054------6Molar Absorptivity9215.368 L mol-1cm-1NLT 50007Precision 0.23% RSD