This document outlines the development and validation of a derivative spectroscopic method for estimating serratiopeptidase and diclofenac sodium in bulk and tablet form. The method involves using UV-visible spectrophotometry and derivative spectroscopy to qualitatively and quantitatively analyze the two drugs. The method was developed using the Shimadzu UV-1700 instrument. Standard solutions of the drugs were used to generate calibration curves and determine linearity, accuracy, and precision of the method as per ICH guidelines. The developed and validated method was found to be simple, rapid, precise, and accurate for routine analysis of serratiopeptidase and diclofenac sodium in tablet dosage forms.

1. TO
DEVELOP AND VALIDATE
DERIVATIVE SPECTROSCOPIC
METHOD FOR ESTIMATION OF
SERRATIOPEPTDASE AND DICLOFENAC SODIUM
IN BULK AND IN TABLET
BY
ROHIT BIYANI
GUIDE
MRS. MANSI. J. WAGARIKAR

2.  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.

3.  Serratiopeptidase: A proteolytic enzyme.
 Diclofenac Sodium: Analgesic.

4.  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.

5.  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

6.  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.

7. SINGLE BEAM SPECTROPHOTOMETER

9.  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.

11.  Preparation of stock solution
 Preparation of standard solution
 Preparation of sample solution: BULK and
TABLET

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

17. Calibration curve equation
ABS = K3C3 + K2C2 + K1C + Kθ
K3 = 0.0000
K2 = 0.0000
K1 = 0.0005
Kθ = 0.0000
r2 = 0.9340

18. 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 Tablet
Mixture No.
264.5nm 234nm 264.5nm 234nm
1 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:

19. Derivative spectroscopy
Bulk Tablet
Mixture
No.
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

20.  Linearity
 Accuracy
 Precision

21. 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

22.  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.

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24. THANK YOU
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