EXPERIMENT NO.

AIM: To perform analytical method validation (specificity, linearity, LOD &LOQ)

of Diclofenac sodium using UV visible spectrophotometer.

REFERENCE:
 ICH Guidelines Q2R1; Validation of analytical procedure; Textbook of
methodology; international conference of Organization (2005).
 Government of India, Ministry of Health and Family welfare; INDIAN
PHARMACOPOEIA-2018,8th edition, Ghaziabad; The Indian
Pharmacopoeia Commission; 2018;Volume 2 ; page no. 2858.

REQUIREMENTS:
Chemicals: Diclofenac, 0.1N NaOH
Apparatus: Volumetric flask, beaker, pipette etc
Instrument used: UV-visible spectrophotometer.

MONOGRAPH:
DICLOFENAC SODIUM TABLET
Diclofenac sodium contains tablet contain NLT 90% and NMT 110% of the
stated amount of Diclofenac sodium C14H10Cl2NNaO2.
IUPAC Name: Sodium 2-[(2,6-dichlorophenyl)amino]phenylacetate
Molecular Weight: 318.1
STRUCTURE:

STORAGE:
Store protected from light
PROCEDURE:
1. To prepare standard paracetamol solution:
Weigh accurately 10mg of pure diclofenac sodium Transfer it into
100ml of volumetric flask and 50ml of 0.1 M NaOH. Dissolve it and make
up the volume with NaOH (1000ppm).

2. To prepare test solution of paracetamol:

Weigh accurately a quantity of tablet powder equivalent to 10mg of
diclofenac sodium and transfer it to 100ml of volumetric flask. Add 50ml of
0.1M NaOH and shake it for 15 minutes (sonicate the solution) and make up
the volume with the same (NaOH). Filter the solution and concentration of
this solution is 100ppm.

3. Procedure to determine specificity :

From the stock solution of standard and test, pipette out 1ml of
solution in 10ml of volumetric flask and record the spectra separately.
Report the λ value and check the interference (10ppm).
max

4. Procedure for linearity :

From the standard stock solution (100ppm). Prepare the solutions
containing 5, 10,15, 20 ,25ppm.
Pipette out 0.5, 1.0, 1.5, 2.0 and 2.5 ml in 10ml volumetric flask and record
the absorbance in triplicate at 275nm.

5. Procedure for LOD :

Obtain the value of standard deviation of intercept from the regression
line and slope. It is calculated as:

LOD = 3.3 σ/S

Where,
σ = standard deviation of intercept
S = mean of slope
6. Determination of LOQ:
It can be calculated as:

LOQ = 10 σ/S

Where,
σ = standard deviation of intercept
S = mean of slope

OBSERVATION AND CALCULATION:

Concentration(ppm Absorbance
)
X y1 y2 y3
5 0.2137 0.2133 0.2130
10 0.4062 0.4082 0.4058
15 0.5890 0.5920 0.5903
20 0.7353 0.7348 0.7361
25 0.9309 0.9317 0.9326

For absorbance 1:
xi yi xi2 yi2 xiyi
5
10
15
20
25
 Ʃxi = Ʃyi= Ʃxi2 = Ʃyi2 = Ʃxiyi =
x̄I = ȳi = x̄i2 = ȳi2 = x̄iȳI =

ƩXi2= Ʃxi2 -n(x̄i)2

ƩYi2 = Ʃyi2 -n(ȳi)2

ƩXiYi = Ʃxiyi - n(x̄i) (ȳi)

m1= ƩXiYi ⁄ ƩXi2

c1 = yi - mx̄i
r21 = m√ ƩXi2 ⁄ ƩYi2

For absorbance 2:
xi yi xi2 yi2 xiyi
5
10
15
20
25
Ʃxi = Ʃyi= Ʃxi2 = Ʃyi2 = Ʃxiyi =
x̄I = ȳi = x̄i2 = ȳi2 = x̄iȳI =

ƩXi2= Ʃxi2 -n(x̄i)2

 ƩYi2 = Ʃyi2 -n(ȳi)2

ƩXiYi = Ʃxiyi - n(x̄i) (ȳi)

m2= ƩXiYi ⁄ ƩXi2

c2 = yi - mx̄i
r22 = m√ ƩXi2 ⁄ ƩYi2

For absorbance 3:
xi yi xi2 yi2 xiyi
5
10
15
20
25
Ʃxi = Ʃyi= Ʃxi2 = Ʃyi2 = Ʃxiyi =
x̄I = ȳi = x̄i2 = ȳi2 = x̄iȳI =

ƩXi2= Ʃxi2 -n(x̄i)2

ƩYi2 = Ʃyi2 -n(ȳi)2

ƩXiYi = Ʃxiyi - n(x̄i) (ȳi)

M3= ƩXiYi ⁄ ƩXi2

C3 = yi - mx̄i

r23 = m√ ƩXi2 ⁄ ƩYi2

 TABLE 1 TABLE 2 TABLE 3
m1 m2 m3
c1 c2 c3
r1 r2 r3

S = slope
S = m1 + m2 + m3
3

Cavg = c1 +c2 + c3
3
 σ = standard deviation of intercept

σ = √ (c1 – (cavg)2 + c2 – (cavg)2 + c3 – (cavg)2

3

RESULT:
Sr Parameter Observed Standard Inference
No: value value
Std= 0.441 Abs of test and Complies
1. Specificity Test=0.441 std should be with
near standard
value
0.9952 Regression Complies
2. Linearity coefficient with
near 1 standard
value
0.310 Complies
3. Detection limit - with
standard
value
0.9403 Complies
4. Quantification - with
limit standard
value