ANALYTICAL METHOD VALIDATION PROTOCOL

No.: AMVP / ACP – 001

FOR
QUANTITATIVE ESTIMATION
OF

ACETAMINOPHEN
BY
HPLC
FOR
ASSAY
OF

ACETAMINOPHEN
(ACETAMINOPHEN TABLETS USP 500mg)

CONTENTS

S. No Section Title Page No

 1.0 Protocol Approval 3
2.0 Overview 4
a) Objective
b) Purpose
c) Revalidation
3.0 Instruments / Equipments required 4
4.0 Chemicals Required 4
5.0 References 4
6.0 Method of analysis 5-6
7
7.0 Validation Parameters
7.1 Accuracy
8-9
i) Assay over 3 concentrations / 3 replicates
9-10
7.2 Precision
i) Repeatability
ii) Intermediate Precision / Ruggedness
10-11
7.3` Specificity
i) Selectivity
11-12
7.4 Linearity & Range
13
7.5 Robustness
13
7.6 System Suitability

8.0 Overall Acceptance Criteria 14-15

1.0 PROTOCOL APPROVAL

This is to certify that the Analytical Method Validation Protocol No. AMVP / ACP –
001, described here is approved to validate the analytical method for the quantitative
estimation of Acetaminophen by HPLC for its assay in Acetaminophen Tablets
500mg as per Method of Analysis and ICH Guideline number: Q2 (R1) of CPMP /
ICH / 281 / 95

Designation Signature Date

Prepared by Executive – QC Analytical

Checked by &
Manager – QC Analytical
Approved by

Authorized by Manager – QA

2.0 OVERVIEW
2.1 Objective: To validate analytical method for Assay of Acetaminophen in Acetaminophen
Tablets 500mg by HPLC as per Method of Analysis.
2.2 Purpose: To establish documentary evidence that the method meets the acceptance criteria,
as the method given for assay is a pharmacoepial method.
2.3 Revalidation: Analytical method needs to be revalidated, if there is any change in
a) Synthesis of Active Substance
b) Composition of Drug Product
c) Analytical Procedure
3.0 INSTRUMENTS / EQUIPMENTS REQUIRED
3.1 High performance liquid chromatograph, with UV detector
3.2 HPLC Analytical column of C 18 (3.9 X 30 cm)
3.3 Analytical weighing balance
3.4 Laboratory accessories
4.0 CHEMICALS REQUIRED
4.1 Acetaminophen working standard
4.2 Acetaminophen Tablets 500mg
4.3 Placebo or Excipient mixture
4.4 Methanol – HPLC Grade
4.5 Distilled Water

5.0 REFERENCE
5.1 ICH Guideline number: Q2 (R1) of CPMP / ICH / 281 / 95

Quantity of Excipient
S. No. Excipients
(mg/Tablet)

1 Microcrystalline Cellulose USP 0.0445

2 Gelatin USP 0.011

3 Topical Starch 0.0220

4 Magnesium Stearate 0.0020

5 Croscarmellose Sodium USP 0.0100

6.0 METHOD OF ANALYSIS

6.1 Quantitative estimation of Acetaminophen in Acetaminophen Tablets 500mg by HPLC

method.
6.1.1 Mobile Phase: Water: Methanol Ratio: 750: 250.
6.1.2 Placebo preparation: Weigh accurately about 120 mg of placebo in to 200 ml volumetric
flask, dissolve and make up to the mark with mobile phase. Transfer 5 ml of this solution in
to a 250 ml volumetric flask and make up to the mark with mobile phase.
6.1.3 Standard Preparation (10 ppm): Weigh accurately about 100 mg of Acetaminophen
(W.S.) into a 200 ml volumetric flask, dissolve and make upto the mark with Mobile phase.
Transfer 5 ml of this solution into a 250 ml volumetric flask and make upto the mark with
Mobile phase
6.1.4 Sample Preparation (10 ppm): Weigh 20 tablets and make fine powder Transfer the
powder into a 200ml volumetric flask which is Equivalent to 100 mg Acetaminophen , add
50ml of mobile phase shake for 10 minutes and sonicate and make up to the mark with
mobile phase. Transfer 5 ml 0f this solution to 250 ml volumetric flask and make up he
mark with mobile phase.
6.1.5 Chromatographic System:

HPLC : HPLC - UV detector

Analytical Column : C 18 (3.9 X 30 cm)

Flow rate : 1.5 ml/minute

Detector Wavelength : 243 nm
Injection volume : 20 µl
Temperature : Ambient
6.1.6 System Suitability: Chromatograph the Standard preparation and record the peak
responses as directed under procedure. The column efficiency as number of theoretical
plates for Acetaminophen peak should not be less than 1000 and the peak symmetry as
tailing factor for Acetaminophen peak should not be more than 2.0, and the relative
standard deviation for replicate injections of standard preparation should not be more than
2.0%.
6.1.7 Procedure: Separately inject 20 l of Standard preparation and Sample preparations into
the chromatograph, record the chromatograms and measure the responses for
Acetaminophen peak. Calculate the content of Acetaminophen per tablet in mg using the
following expression.

AS WS. 200 P.
------------- X ----------- X ---------- X -------------- X [Link]. =
AT 200 Sp. W 100

= _____________ mg of Acetaminophen per tablet.

Where,
AS : Average Peak area of Acetaminophen for Standard
preparation
 AT : Peak area of Acetaminophen for sample preparation
WS : Weight of Acetaminophen WS in mg
Sp.W : Weight of Acetaminophen Spl in mg
P : Purity of Acetaminophen WS on dried basis
Avg. Wt : Average weight of 20 tablets.

7.0 VALIDATION PARAMETERS

The following parameters are selected for validation.

S. No. Validation Parameter Assay

+
1. Accuracy

Precision
2. i) Repeatability +
ii) Intermediate precision (Ruggedness) +

3. Specificity +
i) Selectivity

+
4. Linearity & Range of Acetaminophen
i) In Sample preparation from 80% to 120% of Test Conc.

5. Robustness +

6. System suitability +

‘+’ To be performed, ‘-’ not to be performed

7.1 ACCURACY
7.1.1 Objective: To establish the accuracy of the analytical method using the following method.
a) Assay from various sample concentrations: Establish the accuracy by measuring the
3 assay determinations over 3 different test concentrations (3 concentrations / 1
replicates) i.e. 80%, 100% and 120% of test concentration. Report RSD for the assay of
3 concentrations, the variance between mean value of assay of these 3 determinations
and the mean assay value obtained under Precision (for Repeatability).
7.1.2 Preparation of Analytical Solutions
[Link] For Various Sample concentrations
a) Preparation of Acetaminophen Standard Solution: Weigh accurately about 100 mg
of Acetaminophen (W.S.) into a 200 ml volumetric flask, dissolve and make upto the
mark with Mobile phase. Transfer 5 ml of this solution into a 250 ml volumetric flask
and make upto the mark with Mobile phase.
b) Preparation of 80% sample Solution: Weigh 20 tablets and make fine powder
Transfer the powder into a 200ml volumetric flask which is Equivalent to 80 mg
Acetaminophen , add 50ml of mobile phase shake for 10 minutes and sonicate and
make up to the mark with mobile phase. Transfer 5 ml 0f this solution to 250 ml
volumetric flask and make up he mark with mobile phase.
c) Preparation of 100% sample Solution: Weigh 20 tablets and make fine powder
Transfer the powder into a 200ml volumetric flask which is Equivalent to 100 mg
Acetaminophen , add 50ml of mobile phase shake for 10 minutes and sonicate and
make up to the mark with mobile phase. Transfer 5 ml 0f this solution to 250 ml
volumetric flask and make up he mark with mobile phase.
d) Preparation of 120% sample Solution: Weigh 20 tablets and make fine powder
Transfer the powder into a 200ml volumetric flask which is Equivalent to 120 mg
Acetaminophen , add 50ml of mobile phase shake for 10 minutes and sonicate and
make up to the mark with mobile phase. Transfer 5 ml 0f this solution to 250 ml
volumetric flask and make up he mark with mobile phase.

7.1.3 Procedure: Inject separately 20µl of each preparations prepared under [Link] into the
chromatograph and measure the response for Acetaminophen peak. Calculate the
 %recovery of Acetaminophen in recovery preparations and assay of Acetaminophen in
solutions of Various Sample concentrations respectively using the following calculations.

7.1.4 Calculations

AS WS. 200 P.
------------- X ----------- X ---------- X -------------- X [Link]. =
AT 200 Sp. W 100

= _____________ mg of Acetaminophen per tablet.

Where,
AS : Average Peak area of Acetaminophen for Standard
preparation
AT : Peak area of Acetaminophen for sample preparation
WS : Weight of Acetaminophen WS in mg
Sp.W : Weight of Acetaminophen Spl in mg
P : Purity of Acetaminophen WS on dried basis
Avg. Wt : Average weight of 20 tablets.

[Link] Recovery Calculation:

mg of Acetaminophen recovered
% of Recovery = -------------------------------------------- X 100
mg of Acetaminophen added
7.1.5 Acceptance Criteria
The variance between the mean value of assay obtained for the various sample
concentrations and the mean value of the assay obtained under Precision should not be
more than 2.0% of the absolute assay value. The RSD of the assay of various sample
concentrations should not be more than 2.0%.
7.2 PRECISION
7.2.1 Objective: To establish the precision of the analytical method using the following two
methods.
a) Repeatability: Establish the repeatability of the analytical method by estimating the
assay for six different sample preparations of the same batch of Acetaminophen Tablets.
Calculate the assay for all six-sample preparations and report the RSD for the same.
b) Intermediate Precision (Ruggedness): Establish the ruggedness of the analytical
method by estimating the assay for six different sample preparations of the same batch
 of Acetaminophen Tablets 500mg by different analyst on a different day. Calculate the
assay for all six-sample preparations and report the RSD for the same.
7.2.2 Repeatability
a) Preparation of Standard Solution: Prepare as described under 6.1.3.
b) Preparation of Sample Solutions: Prepare as described under 6.1.4. Repeat this
procedure for another five more sample preparations.
c) Procedure: Inject separately 20 µl of standard preparation and sample preparations
into the chromatograph and measure the peak responses for the major peaks. Calculate
the content of Acetaminophen in the individual solutions using the expression described
under method of analysis.
7.2.3 Intermediate Precision (Ruggedness)
A different analyst using a different HPLC system with a different similar column on a
different day should carry out this experiment.
a) Preparation of Standard Solution: Prepare as described under 6.1.3.
b) Preparation of Sample Solutions: Prepare as described under 6.1.4. Repeat this
procedure for another five more sample preparations.
c) Procedure: Inject separately 20 µl of standard preparation and sample preparations into
the chromatograph and measure the peak responses for the major peaks. Calculate the
content of Acetaminophen in the individual solutions using the expression described
under method of analysis.
7.2.4 Acceptance Criteria
[Link] For Repeatability: The RSD of the assay of all six-sample preparations of same batch of
Acetaminophen Tablets 500mg should not be more than 2.0%.
[Link] For Intermediate Precision (Ruggedness)
a) The relative standard deviation for the assay values of six sample preparations of
same batch of Acetaminophen Tablets 500mg should not be more than 2.0%.
b) The difference in the assay of same batch of Acetaminophen Tablets 500mg
between two analysts should not be more than 2.0% of the absolute assay value.
7.3 SPECIFICITY
7.3.1 Preparation of analytical solutions
[Link] For Selectivity
a) Blank solution: Mobile phase as blank solution.
b) Placebo Preparation: Weighed accurately about 125 mg of placebo into a 200 ml
volumetric flask, dissolve and make up to the mark with Mobile phase. Transfer 5 ml of
this solution into a 250 ml volumetric flask and make up to the mark with Mobile phase
c) Preparation of Standard Solution: Prepare as described under 6.1.3.
b) Preparation of Sample Solution: Prepare as described under 6.1.4.
7.3.2 Procedure: Inject separately 20 µl of each of all the above solutions described under
[Link] into the chromatograph and continue the chromatography 2.5 times the retention
time of Acetaminophen peak. Measure the responses for all the peaks for all the solutions.
7.3.3 Observations: Record the retention times of all the peaks obtained for all the solutions,
measure the resolution between the peaks and peak purity for Acetaminophen peak for all
solutions.
7.3.4 Acceptance Criteria: The interference of the peaks obtained for all the above-
described solutions from Acetaminophen peak should be NIL.

7.4 LINEARITY & RANGE

7.4.1 Objective: To establish the linearity of the analytical method for assay using the following
method.
a) Linearity & Range for Acetaminophen in Samples of different
Concentrations: Demonstrate the linearity of the analytical method for assay by
injecting the various concentrations of Sample preparations prepared in the range of
80% - 120% of test concentration, into the chromatograph, covering 5 different
concentrations. Draw a plot between the concentrations in ppm Vs. Peak response of
Acetaminophen in sample preparations. Report the slope, intercept and regression
coefficient from the plot obtained for Concentration Vs. Peak response of
Acetaminophen in sample preparations.
7.4.2 Preparation of Analytical Solutions
[Link] For Linearity & Range for Acetaminophen:
a) Preparation of Acetaminophen WS solution: Prepare as described under 6.1.3.
b) Preparation of 80% sample Solution: Weigh 20 tablets and make fine powder
Transfer the powder into a 200ml volumetric flask which is Equivalent to 80 mg
Acetaminophen , add 50ml of mobile phase shake for 10 minutes and sonicate and
make up to the mark with mobile phase. Transfer 5 ml 0f this solution to 250 ml
volumetric flask and make up he mark with mobile phase.
c) Preparation of 90% sample Solution: Weigh 20 tablets and make fine powder Transfer
the powder into a 200ml volumetric flask which is Equivalent to 90 mg
Acetaminophen , add 50ml of mobile phase shake for 10 minutes and sonicate and
make up to the mark with mobile phase. Transfer 5 ml 0f this solution to 250 ml
volumetric flask and make up he mark with mobile phase.
d) Preparation of 100% sample Solution: Weigh 20 tablets and make fine powder
Transfer the powder into a 200ml volumetric flask which is Equivalent to 100 mg
Acetaminophen , add 50ml of mobile phase shake for 10 minutes and sonicate and
make up to the mark with mobile phase. Transfer 5 ml 0f this solution to 250 ml
volumetric flask and make up he mark with mobile phase.
e) Preparation of 110% sample Solution: Weigh 20 tablets and make fine powder
Transfer the powder into a 200ml volumetric flask which is Equivalent to 110 mg
Acetaminophen , add 50ml of mobile phase shake for 10 minutes and sonicate and
 make up to the mark with mobile phase. Transfer 5 ml 0f this solution to 250 ml
volumetric flask and make up he mark with mobile phase.
f) Preparation of 120% sample Solution: Weigh 20 tablets and make fine powder
Transfer the powder into a 200ml volumetric flask which is Equivalent to 120 mg
Acetaminophen , add 50ml of mobile phase shake for 10 minutes and sonicate and
make up to the mark with mobile phase. Transfer 5 ml 0f this solution to 250 ml
volumetric flask and make up he mark with mobile phase.
.
7.4.3 Procedure: Inject separately 20µl of Acetaminophen standard solution prepared under
[Link].a into the chromatograph and measure the responses for the Acetaminophen peak.
Calculate RSD for the 5 replicate of standard preparation. Continue the injections of 20µl in
duplicate for other Standard preparations described from [Link].b to [Link].f into the
chromatograph and record the chromatograms. Measure the peak responses for the major
peaks for all the solutions.
7.4.4 Calculations: Draw a plot between the concentrations vs. the average peak responses of
Acetaminophen peak for all the above studies described under [Link]. Calculate slope,
intercept and regression coefficient from the plot. using statistical equations or suitable
programs like Excel, Lotus, etc…
7.4.5 Acceptance Criteria: The regression coefficient for all the various plots should not be less
than 0.999.

7.5 ROBUSTNESS

7.5.1 Objective: To determine the robustness of the analytical method by estimating the assay of
Acetaminophen in Acetaminophen Tablets 500mg under deliberately modified
chromatographic conditions and report the RSD of results.
7.5.2 Deliberately Modified Chromatographic Conditions: Deliberately modify the actual
chromatographic conditions specified under the method like flow rate, composition of
mobile phase, on lower and higher side of the actual value. Determine the assay of
Acetaminophen in Acetaminophen Tablets 500mg under these deliberately modified chromatographic
conditions.

Chromatographic Conditions
S. No. Chromatographic Parameter
Lower Actual Higher

1 Flow Rate (ml/min) 1.4 1.5 1.6

2 composition in Mobile Phase 875:125 750:250 625:375

7.5.3 Preparation of Analytical Solutions
a) Preparation of Acetaminophen Standard Solution: Prepare as described under 6.1.3
b) Preparation of Sample Solution: Prepare as described under 6.1.4
7.5.4 Procedure: Inject separately 20l of standard, sample and placebo preparations into the
chromatograph set under deliberately modified chromatographic conditions and record the
chromatograms. Measure the response for the Acetaminophen peak and calculate assay of
Acetaminophen in Acetaminophen Tablets 500mg using the expression described under
method of analysis.
7.5.5 Acceptance Criteria: The RSD for the assay values Acetaminophen in Acetaminophen
Tablets 500mg obtained under deliberately modified chromatographic conditions should
not be more than 2.0%. The difference between the assay value obtained under deliberately
modified chromatographic conditions and the assay value obtained under Precision should
not be more than 2.0% of the absolute assay value.
7.6 SYSTEM SUITABILITY
7.6.1 Objective: To determine the suitability of chromatographic system described for the
method of analysis by establishing system suitability parameters like Peak tailing factor,
number of theoretical plates and the RSD for replicate injections of Acetaminophen
standard preparation on daily basis.
7.6.2 Procedure: Inject 20l of 5 replicate of standard preparation into the chromatograph and
record the chromatogram. Measure the responses for the Acetaminophen peak.
7.6.3 Calculations: Calculate Peak symmetry as tailing factor, column efficiency as number of
theoretical plates for Acetaminophen peak and RSD for replicate injections of standard
preparation.
7.6.4 Acceptance Criteria:
a) Peak symmetry as tailing factor for Acetaminophen peak for standard preparation
should not be more than 2.0.
b) Column efficiency as number of theoretical plates for Acetaminophen peak for
standard preparation should not be less than 1000.
c) RSD for five replicate injections of standard preparation should not be more than
2.0%.
8.0 OVERALL ACCEPTANCE CRITERIA
8.1 ACCURACY
8.1.1 Assay over different sample concentrations: The variance between the mean value of
assay obtained for the various sample concentrations and the mean value of the assay
obtained under Precision should not be more than 2.0% of the absolute assay value. The
Relative standard deviation of the assay of various sample concentrations should not be
more than 2.0%.
8.2 PRECISION
8.2.1 Repeatability: The RSD of assay of all six-sample preparations should not be more than
2.0%.
8.2.2 Intermediate Precision (Ruggedness)
a) The RSD of assay of all six-sample preparations of same batch of Acetaminophen
Tablets 500mg analyzed by different analyst using different HPLC instrument and
column should not be more than 2.0%.
b) The difference in the assay of the same batch of Acetaminophen Tablets 500mg
between two analysts should be less than 2.0% of the absolute assay value.
8.3 SPECIFICITY
8.3.1 Selectivity: The interference between the peaks (if any) obtained for blank, placebo,
Acetaminophen standard and Sample preparation should be NIL.

8.4 LINEARITY & RANGE

8.4.1 Linearity of Acetaminophen in different concentrations of Sample preparations in the
Range of 80% to 120% of test concentration: The relationship between the concentration
and the response (peak area) of Acetaminophen should be linear in the specified range and
the regression coefficient should not be less than 0.999.
8.5 ROBUSTNESS
The RSD for the assay of Acetaminophen under deliberately modified chromatographic
conditions should not be more than 2.0%.
8.6 SYSTEM SUITABILITY
8.6.1 Column Efficiency: The Column efficiency as number of Theoretical plates (N), should
not be less than 1000 for Acetaminophen peak obtained for Standard preparation.
8.6.2 Peak Symmetry: The peak symmetry as tailing factor should not be more than 2.0 for
Acetaminophen peak obtained for Standard preparation.
8.6.3 Relative Standard Deviation: The relative standard deviation for the peak response of
Acetaminophen for 5 replicate injections of Standard preparation should not be more than
2.0%.