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METHOD DEVELOPMENT, OPTIMIZATION AND VALIDATION BY USING INSTRUMENT (UV-VISIBLE SPECTRO PHOTOMETer)PRESENTED BY:T. LAKSHMI BHAVANI(2015MPH40023)Under the guidance of: Dr. A. SRIDEVI

SPMVV - TIRUPATHI

Pharmaceutical Analysis-1st year II-semester1M.Pharm .,Ph.DAugust 6, 2016

CONTENTS:

METHOD DEVELOPMENTOPTIMIZATIONVALIDATION OF INSTRUMENTATION (PHARMACEUTICAL DOSAGE FORMS,API,EXCIPIENTS)UV-VISIBLE SPECTROPHOTOMETERCONCLUSIONREFERENCESAugust 6, 20162

METHOD DEVELOPMENTDEFINITION: It is defined as a process of formulating the materials, conditions and protocol for measuring the analyte.

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STEPS INVOLVED IN UV SPECTROMETERSELECTION OF SOLVENT SYSTEMSELECTION OF ANALYTICAL WAVELENGTH.STUDY OF BEERS LAMBERTS LAWTO PERFORM ANALYSIS OF STANDARD LABORATORY MIXTURE & TABLET FORMULATIONS BY PROPOSED METHOD.TO VALIDATE THE DEVELOPED METHODS BY USING DIFFERENT VALIDATED PARAMETERS.

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STUDY OF BEERS-LAMBERTS LAW:The assay of single component sample, which contains other absorbing substances, is then calculated from the measured absorbance by using one of three principle procedures.They are, use of standard absorptive value,calibration graph &single or double point standardization.August 6, 20165

1. IN STANDARD ABSORPTIVE VALUE METHODThe use of standard A(1%,1cm) or E values are used in order to determine its absorptivity. 2.IN CALIBRATION GRAPH METHODThe absorbances of a number of standard solutions of the reference substance at concentrations encompassing the sample concentrations are measured and a calibration graph is constructed.August 6, 20166

3.The single point standardization:Procedure involves the measurement of the absorbance of a sample solution and of a standard solution of the reference substance.

The concentration of the substances in the sample is calculated from the proportional relationship that exists between absorbance and concentration.C test=(A test X C std)/A stdwhere, C test and C std are the concentrations in the sample and standard solutions respectively and A test and A std are the absorbances of the sample and standard solutions respectively.

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SELECTION OF ANALYTICAL WAVELENGTH:Proper wavelength selection of the methods depends upon the nature of the sample & its solubility.To develop a rugged & suitable spectro photometric method (UV), the analytical condition where selected after testing different parameters such as diluents, buffer & buffer concentration.Standard solution in UV spectrometer between 200 - 400nm on spectrum mode, using diluents as a blank.August 6, 20168

VALIDATION:It is defined as establishing documented evidences which provides a high degree assurance that a specific process will consistently produce a product meeting its pre-determined specification & quality characteristics.

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VALIDATION PARAMETERS:AccuracyPrecisionLimit of detectionLimit of quantitationSpecificity Linearity RangeRobustnessRuggedness

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ACCURACY:The closeness of agreement between the value which is accepted as a conventional true value or an expected reference value , and the value found.

PRECISION:It expresses the closeness of agreement between a series of measurements obtained from the multiple sampling of the homogenous sampling under the prescribed conditions.Precision may be considered at 3 levels: repeatability, intermediate precision, reproducability.

1.Repeatability : it expresses the precision under the same operating conditions over a short interval of time.

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2.Intermediate precision: It expresses with in laboratory variations : different days, different analyst, different equipment

3.Reproducability: It expresses the precision between laboratories.

DETECTION LIMIT: It is the lowest amount of analyte in a sample which can be detected but not quantitated as an exact value.

QUANTITATION LIMIT: It is the lowest amount of analyte in a sample which can be quantitatively determined which suitable accuracy and precision.

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SPECIFICITY: It is the ability to acsses the analyte in the presence of components which may be expected to be present.It includes impurities, degradants, matrix etc.This definition has following implications:

Identification: To ensure the identification of analytePurity test: To ensure an accurate content of impurities of an analyte i.e. heavy metals, residual solvent content.Assay: To provide an exact result this allows an accurate statement on content or potency of analyte in the sample.

LINEARITY: Its ability(with in a given range) to obtain test results which are directly proportional to the concentration(amount) of analyte in the sample.August 6, 201613

RANGE: It is the interval between upper and lower concentration of analyte in the sample.ROBUSTNESS: It is a measure of its capacity to remain unaffected by small variations in method parameters and provides an indication of its reliability during normal usage.RUGGEDNESS: Reproducibility under normal but variable laboratory conditions.August 6, 201614

DEVELOPMENT OF ANALYTICAL METHOD FOR RISPERIDONE(API) BY UV-SPECTROPHOTOMETRY: PREPARATION OF STOCK SOLUTION: 100mg of pure drug was weighed & transferred to 100ml volumetric flask, add 50ml of 0.1N HCl and make up the volume with 0.1 N HCl.August 6, 201615

SAMPLE SOLUTION: 2mg of RIS was weighed and transferred to 100ml volumetric flask.Add 20ml of 0.1 N HCl and sonicate for 5 min.Volume was made up with 0.1 N HCl the above solution was mixed well and filtered through Whattmanns filter paper.Dilutions were made with 0.1N HCl to attain a concentration of 4g /ml.6 replicates of analysis were carried out with sample weighed individually.August 6, 201616

VALIDATION PARAMETERS:ACCURACY & PRECISIONLINEARITYLOD & LOQSTABILITYCALIBRATION CURVEAugust 6, 201617

PRECISION & ACCURACY: Precision and accuracy were determined with standard quality control sample prepared in triplicate at different concentration levels. Precision of the assay was determined by repeatability and intermediate precision, which was studied by comparing the assays on 3different days, results are documented as SD & % RSD.Accuracy is the percentage of analyte recovered by assay from a known added amount.Data from 9determinations over 3concentration levels covering the specified range. August 6, 201618

Where s = the standard deviation of absorbance of the sample. m = the slope of the related calibration graphs. LOD: Lowest concentration of analyte that an analytical process can reliably differentiate from background levels.LOD = 3s/mLOQ: Lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability.LOQ = 10s/mAugust 6, 201619

LINEARITY:5 point calibration curve were generated.evaluated by least square regression method using un weighed data.STABILITY:

The stability of risperidone in 0.1 N HCl solution was studied by UV method.

Sample solutions were prepared in triplicate and stored at 4 & 250 C for 30,60,90 & 120 min.

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RECOVERY STUDY:To study the accuracy, precision and reproducability of the proposed method and dosage forms recovery experiments were carried out using standard method.

These studies were performed by addition of known amount of pure RIS to the pre analyzed tablet formulation & the mixtures analyzed using proposed techniques.

After parallel analysis the recovery results were calculated using the related calibration equations.August 6, 201621

VALIDATION PARAMETERS FOR RISPERIDONE:PARAMETERSVALUESLinearity range(g/ml)2-6Precision(%)2.0325+ 0.044Accuracy (%)1.02+ 0.188LOD(g/ml)1.012LOQ(g/ml)3.036Stability(h)2Std deviation(SD+)0.044

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DEVELOPMENT AND VALIDATION SPECTROSCOPY METHOD FOR ESTIMATION OF LACOSAMIDE IN PHARMACEUTICAL DOSAGE FORMS: DETERMINATION OF MAXIMUM WAVELENGTH:50 mg of lacosamide was weighed accurately and transferred into a 100ml vol, flask & dissolved in 50ml of acetonitrile : water(25:75) by sonication and made up to volume with the same solvent mixture to give a standard concentration of 500g /ml transfer 5ml of the above solution into 100ml vol. flask dilute and made up to volume with the solvent mixture to a standard concentration of 25g/ml.

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This solution was scanned against blank for the entire UV-visible wavelength 200-800nm.Based on the spectrum max of 230 nm was selected for further analysis.

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STANDARD PREPARATION: 50 mg of lacosamide was weighed accurately Dissolve in 50 ml of acetonitrile:water (25:75%v/v) by sonication

Make up the volume with solvent mixture to give a standard concentration 500g/ml

Transfer 2.5,3.5,5,6 &8ml ..of the standard solution into 100ml vol.flask make upto volume with the solvent mixture to get a series of standards 12.5,17.5,25,30 &40 g/ml

Absorbance of the these standards are measured at max of 230nm.

Standard curve was drawn by plotting concentration vs absorbance.

d

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SAMPLE PREPARATION: 50mg of Lacosamide in to 100 ml vol. flask sonicate for 30min. With shaking and made up to volume with acetonitrile: water(25:75% v/v) Filter the solution through 0.45 m membrane transfer 5ml of the above solution in to 100ml volume flask and dilute to volume with the same solvent.

The absorbance of the resulting solution was measured at 230nm.

The actual conc. Of the drug in the sample was determined from standard curve.

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METHOD DEVELOPMENT To develop a suitable UV method for the determination of lacosamide different compositions of water,methanol,acetonitrile were used as diluent.Finally good recoveries were found with diluent of acetonitrile:water(25:75 % v/v)On scanning the standard solution against diluent in entire UV- visible region of 200-800nm good response was found at 230nm for 25g/ml.There is no interference from dilute through the entire UV visible range of 200-800nm. August 6, 201627

METHOD VALIDATION:The developed UV method was validated as per ICH guidelines.ACCURACY:The method was determined by three conc.levels by recovery experiments . The recovery studies were carried out in triplicate preparation collected from 20 tablets of lacosamide and analyzed as per proposed method. The % recoveries : 98.4-99.8 with an overall %RSD of 0.5.August 6, 201628

Recovery studies for Lacosamide by proposed method% level recovery range % RSD at each level over all % RSD50 99.0-99.8 0.4 100 98.4-98.8 0.2 0.5150 98.6-99.2 0.3

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PRECISION: Evaluated by 3 independent assays of test Lacosamide sample. Intermediate evaluated by different analysts and different days in same day.

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METHOD PRECISION (INTER AND INTRA) FOR LACOSAMIDE:REPLICATESMETHOD PRECISIONINTER DAYINTRA DAY199.399.80298.4101.1399.199.9499.9100.35100.3100.8699.7100.3AVERAGE99.45100.37SD0.670.50% RSD0.670.50

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SPECIFICITY:To check for the interference of blank at the working wavelength blank was scanned from 200-800nm.As there is no blank interference observed at the working wavelength of 230nm.The UV spectroscopic method presented in this study is specific for Lacosamide.August 6, 201632

LINEARITY AND RANGE:The standard curve was obtained in the concentration range of 12-40g/ml.This method was evaluated by linear regression analysis.Slope, intercept and correlation coefficient of standard curve was