Assay method Development and Validation

1. PRESENTED BY
MR.VILAS H.KAMBLE
QUALITY ASSURANCE
TECHNIQUE
GIUDED BY
DR.MRS SONALI MAHAPARALE
ASST.PROFESSOR
DR.D.Y.PATIL COLLEGE OF PHARMACY AKURDI,PUNE.
DEVLOPMENT AND VALIDATION OF RP-HPLC ASSAY
METHOD FOR SIMULTANEOUS ESTIMATION OF MUSCLE
RELAXANT AND ANALGESIC DRUG IN PURE AND
PHARMACEUTICAL DOSAGE FORM
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2. CONTENT
 Literature survey
 Principle
 Need of work
 Aim and objective
 Plan of work
 Drug profile
 Experiment
 Outcome of study expected
 References
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3. LITRATURE SURVEY
1 2
JOURNAL
NAME
International journal of pharmacy and
pharmaceutical science
International journal of
pharmacy and
pharmaceutical science
AOUTHER
NAME
Nimila Carolin,Balan P. et. al. Patel Madhavi Bhawani
Singh et. al.
METHOD NAME RP HPLC of Tolperisone and
Paracetamol
UPLC of Tolperisone and
Paracetamol
COLUMN NAME Symmetry C18 250×4.6mm UPLC BEH C18
50mm×2.1mm
MOBILE PHASE Water:ACN (60:40) Water:ACN (30:70)
WAVE
LENGTH
258 nm 254 nm
FLOW RATE 0.7 per min 0.20ml per min
RT Tolperisone=2.25,Paracetamol=3.29 Tolperisone=1.396,Paraceta4/17/2017 3

4. LITRATURE SURVEY
3 4
JOURNAL NAME Pelagia Research Library
International journal of
pharmacy and pharmaceutical
science
AOUTHER NAME
Satyanarayana M. V
Anuradha. V. et al
Thippeswamy M. Somanna P.
at al
METHOD NAME
RP HPLC of Tolperisone and
Paracetamol
RP HPLC of Paracetamol
COLUMN NAME
Hypersil BDS, 150 x
4.6mm C18
C18 colum (250 mm X 4.6 mm
i. d, 5 μm particle size)
MOBILE PHASE
Buffer:ACN (70:30) pH 3 with
TEA
ACN;Phospahte Buffer(15:85)
WAVE
LENGTH
267 nm. 210 nm
FLOW RATE
1.0 Per Min 1.0 ml/min
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5. CHROMATOGRAPHY
Principle-
 When a mixture of components are introduced into a HPLC
column, they travel according to their relative affinities towards
the stationary phase.
 The component which has more affinity towards stationary
phase travel slower.
 The component which has less affinity towards stationary
phase travel faster.
 Since no two component have the same affinity towards the
stationary phase, the component are separated.
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6. CHROMATOGRAPHY
FIG.SEPARATION OF COMPONENT BY HPLC
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7. NEED OF WORK
 Simultaneous estimation of muscle relaxant and analgesic
drug combination in pharmaceutical dosage form.
 Checking the purity of API
 Separation of muscle relaxant and analgesic drug.
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8. AIM AND OBJECTIVE
 To develop stability profile including RP-HPLC stability
indicating assay method for muscle relaxant and analgesic
drug in pure and pharmaceutical dosage form.
 To perform force degradation study for muscle relaxant and
analgesic drug by using RP-HPLC.
 To validate the developed method as per ICH guidelines.
 To study stability indicating method of drug.
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9. PLAN OF WORK
Literature
survey
Selection of
drug
Study of
physicochemical
properties of
drugs
Optimisation of
analytical
method
Validation of
developed
method
Force
degradation
study
Result and
discussion
Summary and
conclusion
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10. DRUG PROFILE
SR
NO
PARAMETER MASCLE RELAXANT
DRUG
ANALGESIC DRUG
1 Chemical
Structure
2 Chemical name 2-methyl-1-(4-methyl
phenyl)-3-(1-piperidinyl)-
1-propsnone
N-(4-hydroxyphenyl)-acetamine
3 Molecular
formula
C16H23NO.HCl C8H12 NO2
4 Category Muscle relaxant Antipyretic analgesic
5 Melting range Litrature-176-177 Litrature-169-170
6 Dissociation
constant /pKa
value
9.4 9.5
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11. EXPERIMENT
 Solubility studySR NO SOLVENT /BUFFER
MUSCLE RELAXANT
DRUG
ANALGESIC DRUG
1 Water Very soluble Sparingly soluble
2 Acetonitrile Very soluble Freely soluble
3 Methanol Very soluble Very soluble
4 Acetone Soluble Freely soluble
5 Chloroform Very soluble Practically insoluble
6 Tetra hydro furan Soluble Soluble
7 Ethyl acetate Very slightly soluble Soluble
8 Isopropyle alcohol Freely soluble Freely soluble
Solubility in solvent
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12. EXPERIMENT
 Solubility in buffer
SR
NO
NAME OF BUFFER pH SOLUBILITY OF DRUG
MUSCLE RELAXANT
DRUG
ANALGESIC
DRUG
1 0.1M Ammonium acetate buffer 6.50 Soluble Sparingly soluble
2 0.1M Potassium dihydrogen
phosphate buffer
4.75 Freely soluble Sparingly soluble
3 0.1M Sodium dihydrogen phosphate
buffer
4.66 Freely soluble Freely soluble
4 0.1M Dipottasium hydrogen
phosphate
9.76 Sparingly soluble Soluble
5 0.1 M Disodium hydrogen
phosphate
9.82 Sparingly soluble Soluble
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13. EXPERIMENT
 Melting point determination
SR NO NAME OF DRUG OBSERVED
MELTING POINT
1 Muscle Relaxant
drug
176.5-177.7
2 Analgesic Drug 170.3-173.5
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14. EXPERIMENT
 λmax determination
By preparing 10 ppm solution of muscle relaxant drug and
analgesic drug individually and scanning in UV at 200nm-400nm
• Preparation of diluents for λmax determination
 Selected diluents ratio-70:30
SR
NO
DILUENT RATIO
(WATER:ACN)
SOLUBILITY OF MUSLE
RELAXANT DRUG
SOLUBILITY OF
ANALGESIC DRUG
1 90:10 Freely Soluble Very Slightly Soluble
2 80:20 Freely Soluble Soluble
3 70:30 Freely Soluble Freely Soluble
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15. EXPERIMENT
 Muscle relaxant drug
 λmax of Muscle relaxant drug was found to be 260nm
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16. EXPERIMENT
 Analgesic Drug
 λmax of Analgesic Drug was found to be 246nm
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17. EXPERIMENT
• pH determination
By preparing 10% solution of each drug in water and
observed pH on pH meter
SR NO DRUG CATEGORY OBSERVED pH
Of 10% solution
1 Muscle relaxant
drug
4.70
2 Analgesic drug 6.35
3 Water 6.50
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18. EXPERIMENT
Selection of mobile phase
• Selected mobile phase-Water:ACN
• Ratio-50:50
• Chromatographic condition
SR NO PARAMETER DISCRIBTION
1 Mode Isocratic
2 Column Hypersil BDS, C18,150×4.6,5Micrion
3 Wavelength 254nm
4 Flow Rate 1ml Per Min
3 Column Oven Temperature 30 Degree Celsius
4 Injection Volume 20 Micro Litter
5 Run Time 60 Mins
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19. EXPERIMENT
 Blank
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20. EXPERIMENT
 Muscle relaxant drug standard
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21. EXPERIMENT
 Analgesic drug standard
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22. WORK DONE
 Observation
Sr
No
Drug
Category
Retention
Time
Area Theoretical
Plates
Asymmetry
1 Blank 1.60 1725 2844 1.34
2 Muscle
Relaxant
Drug Std
- - - -
3 Analgesic
Drug Std
0.95 34933940 784 1.35
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23. OUTCOME OF STUDY EXPECTED
 The accurate and precise method for simultaneous estimation of
both the drug
 Simple method
 Cost effective method
 Less time consuming method
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24. REFRENCES
 Carolin N. Balan P. Development and Validation of A Reverse
Phase HPLC Method of Simultaneous Estimation of Tolperisone
Hydrochloride and Paracetamol in tablet dosage form,
International Journal of Pharmacy and Pharmaceutical
Science,2012,4(5),84-88.
 Patel M. Parmar R. The Simultaneous Estimation of Paracetamol
and Tolperisone Hydrochloride in Tablet by UV Spectrophotometric
Methods, Jouranl of Pharmaceutical Science and Bioscientific
Research, 2012, 2(2),63-67.
 Chatwal G. Anand S. Instrumental Method of Chemical Analysis,
Himalaya Publishing House, 2012, fifth edition, 2.624-2.639
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25. REFRENCES
 Thippeswamy M. Somanna P. A New Method Development and
Validation for Estimation of Paracetamol In Pharmaceutical dosage
form by RP-HPLC, International Journal of Pharmacy and
Pharmaceutical Sciences, 2015, 7(8), 190-194.
 Beckett A . Stenlake J . Practice Pharmaceutical Chemistry. CBS
Publisher, 4th Edition, Part II. New Delhi,285-288.
 Sharma K. Patel P. First Derivative Spectrophotometric Method for
the Simultaneous Estimation of Tolperisone and Paracetamol in their
Combined Dosage Form, Journal of Pharmaceutical Sciences and
Bioscientific Research, 2012,2(2),92-96.
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26. REFRENCES
 Mathew M. Puthusseri S. Validated HPTLC Method for
Simultaneous Estimation of Tolperisone Hydrochloride And
Paracetamol In Tablet Dosage Form, World Journal of
Pharmaceutical Research, 2012,3(10), 513-522.
 Patel M. Singh B. Stability Indicating UPLC Method for
Quantification of Tolperisone HCl and Paracetamol from muscle
relaxant combination tablet, International Journal of Pharmacy
and Pharmaceutical Sciences, 2015,7(8),300-304.
 www.pelagiaresearchlibrary.com
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