ANALYTICAL METHOD VALIDATION OF NIMESULIDE AND TIZANIDINE COMBINATION

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ANALYTICAL METHOD VALIDATION OF NIMESULIDE AND TIZANIDINE COMBINATION: 

ANALYTICAL METHOD VALIDATION OF NIMESULIDE AND TIZANIDINE COMBINATION UNDER THE GUIDANCE OF PROF. D.V.RAO M.Pharm BY: V.P.HIMABINDU, M.PHARM ROLL NO: 08021D2315

CONTENTS: 

CONTENTS Drug Profiles Method of Analysis Conclusion

Name of the product : NISE MR: 

Name of the product : NISE MR Composition

DRUG PROFILES: 

DRUG PROFILES NIMESULIDE TIZANIDINE

NIMESULIDE: 

NIMESULIDE Chemical name : N-(4-Nitro-2-fenossifenil) metansulfonamide. Molecular formula : C 13 H 12 N 2 O 5 S Molecular weight : 308.31 Characteristic nature Solubility

MOLECULAR STRUCTURE: 

MOA Nimesulide inhibits cyclo-oxygenase MOLECULAR STRUCTURE

PHARMACOKINETIC PARAMETERS: 

PHARMACOKINETIC PARAMETERS bioavailability of the drug is more influenced by the administration route Maximum plasma concentration (C max, 2.86-6.50 μ/ml) is achieved in 1-3 hours (t max) after oral administration of a single dose of 100 mg Nimesulide undergoes extensive biotransformation, mainly to 4-hydroxynimesulide only 6% of the dose is excreted as unchanged drug.

ADVERSE EFFECTS : 

ADVERSE EFFECTS Diarrhea Vomiting Skin rash Pruritis Dizziness Headache Bitterness in mouth

THERAPEUTIC USES: 

THERAPEUTIC USES Oral and suppositories formulations - the treatment of acute pain - the symptomatic treatment of painful osteoarthritis - primary dysmenorrhoea Gel 3% - Symptomatic relief of pain associated with sprains and acute traumatic tendinitis. DOSAGE/ADMINISTRATION Posology : Oral formulations: 100 mg b.i.d Suppositories: 200 mg b.i.d. Gel: 3 g ( 6-7 cm of gel) 2-3 times daily

TIZANIDINE: 

TIZANIDINE Chemical name: 5-chloro-N-(4, 5-dihydro-1H-imidazol-2-yl)-2, 1, 3-benzothiadiazol-4-amine Molecular Formula: C 9 H 8 Cl N 5 S Molecular Weight: 253.712 Nature: white to off-white, fine crystalline powder. Solubility: slightly soluble in water and methanol Category: Muscle relaxant, adrenergic agonists.

MOLECULAR STRUCTURE: 

MOA Tizanidine is an α2-adrenergic receptor agonist. MOLECULAR STRUCTURE

PHARMACOKINETIC PARAMETERS: 

PHARMACOKINETIC PARAMETERS Tizanidine was rapidly and almost completely absorbed. Maximum plasma concentration is achieved in 1.0 hour(Tmax) after dosing of drug. bioavailability was only 21%. It has a half-life of approximately 2 hours. Absorbed tizanidine was almost completely metabolized before excretion.

ADVERSE EFFECTS: 

Hypotension Risk of Liver Injury Sedation Hallucinations / Psychotic-Like Symptoms fainting, slow heart rate nausea, stomach pain, low fever, loss of appetite ADVERSE EFFECTS

THERAPEUTIC USES: 

THERAPEUTIC USES used to help in the treatment of multiple sclerosis and spinal cord injury. Decreasing spasms can reduce pain DOSAGE/ADMINISTRATION oral dose of 8 mg of tizanidine reduces muscle tone in patients The dose can be repeated at 6 to 8 hour intervals

METHOD FOR THE SIMULTANEOUS ESTIMATION OF NIMESULIDE AND TIZANIDINE IN TABLET DOSAGE FORM: 

METHOD FOR THE SIMULTANEOUS ESTIMATION OF NIMESULIDE AND TIZANIDINE IN TABLET DOSAGE FORM

PowerPoint Presentation: 

Instrument: Agilent technologies 1100 model HPLC. Reagents used for the Study: Monobasic potassium phosphate : AR grade Dibasic potassium phosphate : AR grade Water : Milli-Q grade Acetonitrile : HPLC grade

HPLC CHROMATOGRAPHIC CONDITIONS: 

HPLC CHROMATOGRAPHIC CONDITIONS Buffer solution : 1.99 g of monobasic potassium phosphate, 0.525 g of dibasic potassium phosphate, to a 1000 ml of volumetric flask. Mobile phase : filtered and degassed mixture of buffer solution and acetonitrile in the ratio 50: 50 (v/v). Column : C8, 250×4.6 mm ; 5µ Flow rate Wave length Temperature Load Run time

Standard stock preparation of tizanidine:: 

Standard stock preparation of tizanidine: 23mg of tizanidine HCL + in 100 ml volumetric flask + make up to volume with mobile phase and mix. Standard preparation : 20mg of nimesulide into 100 ml volumetric flask + 40 ml of mobile phase + 2 ml of standard stock solution of tizanidine + make up to volume with mobile phase and mix.

PowerPoint Presentation: 

sample preparation: weigh 20 tablets + grind to fine powder + transfer quantity equivalent to one tablet weight into 100 ml volumetric flask + add 70 ml of mobile phase + sonicate and shake for 30 mins + make up to volume with mobile phase and mix + filter through 0.45µ membrane filter + transfer 5ml of the filtrate to a 25 ml volumetric flask + make up to volume with mobile phase and mix

PowerPoint Presentation: 

Procedure: separately inject both the standard and sample preparations into the liquid chromatograph and record the peak area. Calculation : amount of nimesulide present in mg per average weight of tablet :-

PowerPoint Presentation: 

2. Calculation : amount of tizanidine present in mg per average weight of tablet :- Where, At = peak area due to tizanidine in sample preparation, As = peak area due to tizanidine in standard preparation, Sw = weight of tizanidine working standard taken in mg, Tw = weight of the sample taken in mg, Av.wt = average weight of the tablet, P = % purity of tizanidine working standard used.

Standard assay chromatogram: 

Standard assay chromatogram

PowerPoint Presentation: 

Sample assay chromatogram

PowerPoint Presentation: 

Robustness Specificity System suitability Precision Linearity Accuracy Ruggedness Method validation

SYSTEM SUITABILITY: 

SYSTEM SUITABILITY

PowerPoint Presentation: 

Injection number Peak areas for nimesulide Peak areas for tizanidine Acceptance criteria 01 7842.70801 325.03616 The % RSD of peak area of nimesulide and tizanidine five replicate injections should not be more than 2.0 02 7846.52148 325.07220 03 7847.90820 325.19739 04 7859.30908 325.84650 05 7854.83398 325.59235 mean 7850.25615 325.34892 %RSD 0.09 0.11 1. SYSTEM SUITABILITY VALIDATION DATA

SPECIFICITY: 

SPECIFICITY

2. SPECIFICITY: : 

Sample no. nimesulide tizanidine Peak found (yes/no) Peak found (yes/no) 1 no no 2 no no 2. SPECIFICITY: PLACEBO INTERFERENCE PLACEBO PLACEBO

LINEARITY: 

LINEARITY

3.LINEARITY: 

3.LINEARITY Spike level nimesulide tizanidine ‘mg/ml’ added ‘mg/ml’ found ‘mg/ml’ added ‘mg/ml’ found 50% 0.100702 0.100953 0.002378 0.00234 75% 0.151053 0.152537 0.003567 0.00356 100% 0.201404 0.202541 0.004756 0.00478 125% 0.251755 0.253018 0.005945 0.00598 150% 0.302106 0.304091 0.007134 0.00723 Coefficient of correlation.(r) 0.9999 0.9999

ACCURACY: 

ACCURACY

4.ACCURACY: 

4.ACCURACY SAMPLE NO. SPIKE LEVEL NIMESULIDE TIZANIDINE ‘mg/ml ’ added ‘mg/ml’ found Mean % recovery ‘mg/ml ’ added ‘mg/ml’ found Mean % recovery 1. 50% 0.100702 0.100967 100.3 0.002378 0.002328 98.4 2. 50% 0.100702 0.100838 0.002378 0.002330 3. 50% 0.100702 0.100959 0.002378 0.002348 4. 50% 0.100702 0.101214 0.002378 0.002344 5. 50% 0.100702 0.100899 0.002378 0.002346 6. 50% 0.100702 0.100844 0.002378 1.002339 1. 75% 0.151053 0.152275 101.0 0.003567 0.003552 99.8 2. 75% 0.151053 0.152473 0.003567 0.003555 3. 75% 0.151053 0.152864 0.003567 0.003574 1. 100% 0.201404 0.202769 100.6 0.004756 0.004770 100.5 2. 100% 0.201404 0.202335 0.004756 0.004789 3. 100% 0.201404 0.202519 0.004756 0.004783 1. 125% 0.251755 0.253719 100.5 0.005945 0.005983 100.7 2. 125% 0.251755 0.252787 0.005945 0.005992 3. 125% 0.251755 0.252547 0.005945 0.005976 1. 150% 0.302106 0.303171 100.7 0.007134 0.007188 101.4 2. 150% 0.302106 0.303238 0.007134 0.007188 3. 150% 0.302106 0.303577 0.007134 0.007204 4. 150% 0.302106 0.305204 0.007134 0.007247 5. 150% 0.302106 0.304627 0.007134 0.007294 6. 150% 0.302106 0.304727 0.007134 0.007267 4. ACCURACY:

PRECISION: 

PRECISION

5.PRECISION: 

5.PRECISION Sample no. % assay %assay nimesulide tizanidine 01 98.5 99.0 02 98.6 99.0 03 98.2 99.0 04 98.2 99.5 05 98.8 99.5 06 98.6 100.0 Average 98.5 99.2 %RSD 0.24 0.42

ROBUSTNESS: 

ROBUSTNESS

6.ROBUSTNESS: 

6.ROBUSTNESS System suitability parameters Organic phase composition in mobile phase Acceptance criteria 90% 100% 110% Resolution between nimesulide and tizanidine. 27.90 23.98 20.52 NLT 4.0 %RSD of five replicate injections of standard preparation for nimesulide. 0.20 0.12 0.31 NMT 2.0 %RSD of five replicate injections of standard preparation for tizanidine. 0.18 0.08 0.28 NMT 2.0 a). Effect of variation in mobile phase composition:

PowerPoint Presentation: 

System suitability parameters Flow rate Acceptance criteria 0.8 ml/min 1.0 ml/min 1.2 ml/min Resolution between nimesulide and tizanidine. 24.42 23.98 23.20 NLT 4.0 %RSD of five replicate injections of standard preparation for nimesulide. 0.18 0.12 0.27 NMT 2.0 %RSD of five replicate injections of standard preparation for tizanidine. 0.26 0.08 0.25 NMT 2.0 b). effect of variation in flow rate:

PowerPoint Presentation: 

System suitability parameters Column temperature Acceptance criteria 20⁰c 25⁰c 30⁰c Resolution between nimesulide and tizanidine. 26.74 23.98 21.81 NLT 4.0 %RSD of five replicate injections of standard preparation for nimesulide. 0.26 0.12 0.18 NMT 2.0 %RSD of five replicate injections of standard preparation for tizanidine. 0.34 0.08 0.13 NMT 2.0 c).Effect of variation in column temperature:

RUGGUDNESS: 

RUGGUDNESS

7.RUGGUDNESS: 

7.RUGGUDNESS parameter Analyst Analyst 1 Analyst 2 HPLC ID Number 34HPL-02 34HPL-01 Column ID DL015-01-03 DL014-01-02 System suitability Observed value Acceptance criteria nimesulide tizanidine %RSD of replicate injections of standard preparation. 0.15 0.31 NMT 2.0 Resolution between nimesulide and tizanidine. 31.50 NLT 4.0 a. Analyst to analyst/system to system/column to column.

PowerPoint Presentation: 

Sample no. % assay %assay nimesulide tizanidine 01 98.5 99.0 02 98.6 99.0 03 98.2 99.0 04 98.2 99.5 05 98.8 99.5 06 98.6 100.0 Average 98.5 99.2 %RSD 0.24 0.42 Sample no. % assay %assay nimesulide tizanidine 01 98.4 99.0 02 98.6 99.5 03 98.9 99.0 04 98.2 99.5 05 98.8 99.5 06 98.4 100.0 Average 98.6 99.8 %RSD 0.3 0.5 ANALYST-1, SYSTEM-1 AND COLUMN-1 RESULTS ANALYST-2, SYSTEM-2 AND COLUMN-2 RESULTS

PowerPoint Presentation: 

Time in days Similarity factor for standard. % assay of test preparation difference Test-1 Test-2 Test-1 Test-2 initial NA 98.5 98.6 NA NA 1 (24 hrs) 1.00 98.7 98.1 0.2 0.5 2 (48 hrs) 0.99 98.3 98.0 0.2 0.6 Time in days Similarity factor for standard. % assay of test preparation difference Test-1 Test-2 Test-1 Test-2 initial NA 99.0 99.0 NA NA 1 (24 hrs) 1.00 99.0 99.0 0.0 0.0 2 (48 hrs) 0.98 98.5 98.5 0.5 0.5 b). Bench top stability of nimesulide and tizanidine in test preparation and standard preparation: Bench top stability: Nimesulide Bench top stability: Tizanidine

PowerPoint Presentation: 

System suitability parameters Mobile phase stability Acceptance criteria initial 1-day 2-day The Resolution between nimesulide and tizanidine 25.68 25.65 25.21 NLT 4.0 %RSD of five replicate injections of standard preparation for nimesulide. 0.15 0.07 0.04 NMT 2.0 %RSD of five replicate injections of standard preparation for tizanidine. 0.13 0.15 0.15 NMT 2.0 C). Bench top stability of mobile phase :

CONCLUSION: 

CONCLUSION The proposed RP-HPLC method provides a simple, accurate, reproducible assay for quantitative analysis for determination of nimesulide and tizanidine without any interference with the excipients and degradative products

THANK YOU.: 

THANK YOU.