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

of paracetamol 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: Paracetamol, 0.1N NaOH
Apparatus: Volumetric flask, beaker, pipette etc
Instrument used: UV-vis spectrophotometer.

MONOGRAPH:
PARACETAMOL TABLETS
Acetaminophen Tablets.
Paracetamol tablet contain NLT 95% and NMT 105% of the stated amount of
paracetamol C8H9NO2.

STRUCTURE:
STORAGE:
Protect from light and moisture.
USUAL STRENGTH: 500mg, 650mg

PROCEDURE:
1. To prepare standard paracetamol solution:
Weigh accurately 10mg of pure paracetamol 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
PCM 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 2, 4, 6, 8,10ppm.
Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml in 10ml volumetric flask and record
the absorbance in triplicate at 257nm.

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 Absorbance
x y1 y2 y3 Mean

For absorbance 1:
 xi yi xi2 yi2 xiyi

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

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

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
Abs of test and
1. Specificity std should be
near
Regression
2. Linearity coefficient
near 1

3. Detection limit -

4. Quantification -
limit