TO DEVELOP AND VALIDATE

DERIVATIVE SPECTROSCOPICMETHOD FOR ESTIMATION OF

SERRATIOPEPTDASE AND DICLOFENAC SODIUM IN BULK AND IN TABLET

BYROHIT BIYANI

GUIDEMRS. MANSI. J. WAGARIKAR

AIMS AND OBJECTIVES To develop Derivative Spectroscopic

method for UV visible spectrophotometric qualitative and quantitative estimation of Serratiopeptidase and Diclofenac Sodium in bulk and in tablet.

  To validate the developed analytical

method for Linearity, Accuracy and Precision as per the ICH guidelines.

LITERATURE SURVEY Serratiopeptidase: A proteolytic enzyme.

Diclofenac Sodium: Analgesic.

MATERIALS AND METHOD Instrument: The instrument used is

SCHIMADZU UV-1700 Solvents: All the chemicals used in

this work were of AR grade.

Drug: Pure drug sample was obtained from Alkem Pharmaceuticals limited.

INTRODUCTION TO UV VISIBLE SPECTROPHOTOMETER

Principle

Beer-Lamberts Law

Different methods for multicomponent analysis

Simultaneous Equation Method Area Under Curve Method Derivative Spectroscopic Method Multicomponent mode Method Q-Analysis Method

Instrumentation Instruments for measuring the absorption

of Ultraviolet and Visible radiation are made up of Following parts-

1. Radiation Source 2. Wavelength selectors 3. Sample containers 4. Radiation transducers 5. Signal processors and Read-out device.

INSTRUMENTS SINGLE BEAM SPECTROPHOTOMETER

DOUBLE BEAM SPECTROPHOTOMETER

DERIVATIVE SPECTROPHOTOMETRY

PRINCIPLE Derivative spectrophotometry involves the conversion of a normal spectrum

(fundamental,zero ,D spectrum) to its first ,second or higher derivative spectrum by differentiating absorbance of a sample w.r.t wavelength λ for high accuracy

From fig above, [A]=f(λ):zero order [dA/d λ]=f(λ):first order [d2A/d λ2 ]=f(λ):second order. The strong positive &negative bands with max and min at same wavelength of an

absorption band as inflection point in absorbance band governs the odd (first and third ) of an absorption band as inflection point in absorbance band governs the odd (first and third )derivative spectrum whereas the strong positive &negative band with min or max at same wavelength as λ max of absorbance band governs the even (second and fourth) derivative spectrum.

No of bands=Derivative order+1 The amplitude (D) is Directly proportional to the conc of analyte provided beer law is

obeyed by D0spectrum. In First Order Derivative spectroscopy ,zero crossing point for both drugs is found and

wavelength are selected in a manner such tht at zero crossing of one drug .the other drug should show substantial absorbance.

GAUSSIAN CURVE

PREPARATION OF SOLUTIONS Preparation of stock solution

Preparation of standard solution

Preparation of sample solution: BULK and TABLET

WAVELENGTH OF MAXIMUM ABSORPTIONFOR DICLOFENAC SODIUM

WAVELENGTH OF MAXIMUM ABSORPTIONFOR SERRATIOPEPTIDASE

Derivative spectroscopy curve for serratiopeptidase

Derivative spectroscopy curve for diclofenac

CALIBRATION CURVE FOR DICLOFENAC SODIUM

Calibration curve equation ABS = K3C3 + K2C2 + K1C + Kθ K3 = 0.0000 K2 = 0.0000 K1 = 0.0297 Kθ = 0.0000 r2 = 0.9978

CALIBRATION CURVE FOR SERRATIOPEPTIDASE

Calibration curve equationABS = K3C3 + K2C2 + K1C + KθK3 = 0.0000K2 = 0.0000K1 = 0.0005Kθ = 0.0000r2 = 0.9340

DERIVATIVE SPECTROSCOPY

STANDARDS

Derivative spectroscopy

Absorbance 1

264.5

Absorbance 2

234 Std. SERRATIOPEPTIDASE(X)

0.003 0.015

Std.

DICLOFENAC SODIUM(Y)

1.023 1.224

Derivative spectroscopy

Bulk TabletMixture No.

264.5nm 234nm 264.5nm 234nm1 0.963 1.155 1.216 1.441

2 1.003 1.197 1.1881 1.414

3 0.881 1.053 1.085 1.291

4 1.010 1.210 1.147 1.355

5 0.964 1.159 1.069 1.268

Derivative spectroscopy of standard solutions:

Derivative spectroscopy of sample solutions:

RESULT:Derivative spectroscopy

Bulk Tablet

 MixtureNo.

Cx Cy Cx Cy

1 17.27 48.23 16.16 55.55

2 17.59 49.60 19.30 56.93

3 19.58 49.38 18.69 60.72

4 15.83 49.14 17.90 57.92

5 14.88 45.79 10.94 57.00

VALIDATION Linearity

Accuracy

Precision

 PRECISION

Method Drug Standard deviation RSD COV

Bulk Tablet Bulk Tablet Bulk Tablet

derivative SER

1.41231 2.69733 0.029161 0.04680 2.9161074 4.68078

DIC

0.32548 3.05189 0.095560 0.18384 9.5560775 0.18384

CONCLUSION UV Visible spectrophotometric derivative spectroscopy

Method was developed for qualitative and quantitative estimation of Serratiopeptidase and Diclofenac Sodium in tablet formulation and in bulk.

The developed method was found to be simple, rapid and precise for routine estimation of Serratiopeptidase and Diclofenac Sodium in tablet dosage form. The standard deviation was satisfactorily low indicating precision of methods. Method was found to be accurate, precise.

Thus, developed UV Visible spectrophotometric method can be satisfactorily used for determining the content of Serratiopeptidase and Diclofenac Sodium in bulk and tablet dosage form.

REFERENCES Indian Pharmacopoeia, 2010, Volume-2, Published By The Indian Pharmacopoeia Commission, Pg No 2097-

2099. A.H.Beckett, J.B.Stenlake, Practical Pharmaceutical Chemistry, Part-2, 4 th Edition 2004, Cbs Publication, Pg No

255. Holler,Skoog,Crouch, Principles Of Instrumental Analysis, 6 th Edition, Thomson Publication Pg 335. Gurdeep R Chatwal, Sham K Anand, Instrumental Methods of Chemical Analysis, Himalaya Publishing House, 5th

Edition, Pg No 2.7-2.28 Martindale, the Complete Drug Reference 34th Edition Published By Pharmaceutical Press Pg No 32.1 and 1743.2 The Merck Index, An Encyclopedia Of Chemicals, Drugs & Biologicals, Published By Merck Research Laboratories

Division Of Merck & Co, Inc, Whitehouse Stn, Nj, 13 th Edition, Pg No 1937.  Sweetman S.C., Martindale, the Complete Drug Reference. 32nd Edition. Pharmaceutical Press London, 1999;

Pg No 252.  Sandeep Kumar, Asim k Jana,,Isha Dhamija European journals of Pharmaceutics and Biopharmaceutics.  Roshni A Patel, Smita Joshi, World Journal of Pharmacy Amd Pharmaceuticals Sciences, Vol. 3, Pg 1279-1291. Ekta J Pandya, Pankaj Kapupara and Ketan V Shah, Journal of Chemical And Pharmaceuticals Research, 2014.  www.medindia.net  http://www.centaurpharma.com/pdf/Infladase-Forte.pdf

THANK YOU