Pseudo tablets assay validation protocol

ANALYTICAL METHOD VALIDATION PROTOCOL FOR ASSAY OF PSEUDOEPHEDRINE
HYDROCHLORIDE IN CETIRIZINE HYDROCHLORIDE 5 mg AND PSEUDOEPHEDRINE
HYDROCHLORIDE 120 mg EXTENDED RELEASE TABLETS BY HPLC
Validation Protocol No.:FP/001
INDEX
Section No./
Table No.
I
II
III
IV/Table 1
V
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Table 8
Table 9
Table 10
Table 11
Table 12
Table 13
Table 14
Table 15
Table 16
Table 17
Table 18
Table 19
Table 20

Table 21
Table 22
Table 23
Table 24
VI
VII

Contents
Protocol Approval Page
Introduction
Scope
Objective
Acceptance Criteria
Experimental Plan & Data Evaluation
Sample Sequence for Selectivity Study
Preparation of Solutions for Forced Degradation Studies (API)
Preparation of Solutions for Forced Degradation Studies (Excipient blend)
Preparation of Solutions for Forced Degradation Studies (Cetirizine Hydrochloride 5 mg
and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets)
Sample Sequence for Forced Degradation Studies
Dilutions for Linearity
Sequence for Linearity
Sequence for System Precision
Sequence for Method Precision
Sequence for Intermediate Precision
Dilutions for Accuracy
Sequence for Accuracy
Sample Sequence for Filter Validation
Sample Sequence for change in Column Lot
Sample Sequence for change in Flow Rate (1.3 mL/minute)

Sample Sequence for change in Flow Rate (1.7 mL/minute)
Sample Sequence for change in Wavelength (213 nm)
Sample Sequence for change in Wavelength (217 nm)
Sample Sequence for change in Buffer pH (3.8)
Sample Sequence for change in Buffer pH (4.2)
Sample Sequence for change in Temperature (45°C)
Sample Sequence for change in Temperature (35°C)
Sequence for Solution Stability
Validation Report
Annexure – 1 : Analytical Method

I. INTRODUCTION:
Pseudoephedrine Hydrochloride is a fine, white to off-white crystals or powder. Pseudoephedrine Hydrochloride is
freely soluble in Water, Methanol and in Hydrochloric acid. Pseudoephedrine Hydrochloride is an orally active
sympathomimetic amine and exerts a decongestant action on the nasal mucosa. Pseudoephedrine Hydrochloride is
recognized as an effective agent for the relief of nasal congestion due to allergic rhinitis.
Cetirizine Hydrochloride 5 mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets are White to
off white round, coated, biconvex, bilayer tablets debossed with “106” on one side and “EM” on
other side.


Structure:

PSEUDOEPHEDRINE HYDROCHLORIDE
Chemical name:
[S-(R*,R*)]-a-[1-(methyl amino) ethyl]-benzene Methanol Hydrochloride
Molecular formula
: C10H15NO·HCl
Molecular weight
: 201.7

The limits for assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride 5 mg and Pseudoephedrine
Hydrochloride 120 mg Extended Release Tablets is not less than 90.0% and not more than 110.0% of the labeled
amount.
II. SCOPE:
A reverse phase HPLC method for the assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride 5 mg
and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets is considered for validation.
This protocol is intended for the validation of HPLC method for the Assay of Pseudoephedrine Hydrochloride in
Cetirizine Hydrochloride 5 mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets.

III. OBJECTIVE:
To validate the HPLC method for the Assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride 5 mg
and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets.
(Method No.:CPH/ASY-02/0)
For Analytical method (wherever indicated) refer to Annexure 1.
Instruments to be used:
A Waters Alliance HPLC system equipped with quaternary gradient pump, autosampler and a UV / Photo Diode
Array detector.
Column to be used: C18, 150 mm x 4.6 mm, 5 μm (Zorbax SB or Waters symmetry or equivalent)
Working Standard to be used:
Pseudoephedrine Hydrochloride USP : WS/PEH-USP/03 with Assay: 99.86% (on as is basis)
To be used before: 13/05/2008
Impurities to be used for validation:
Name
Ephedrine Hydrochloride
Norpseudoephedrine Hydrochloride

Batch No.

Expiry
Date

Potency
(in % on as is basis)
99.40
99.73


Norephedrine Hydrochloride

99.80
99.85 (By HPLC)

Pseudoephedrine Hydrochloride API to be used for validation:
Pseudoephedrine Hydrochloride USP B.No.:
Mfg. Date:
Retest Date:
Sample to be used for validation:
Cetirizine Hydrochloride 5 mg and Pseudoephedrine Hydrochloride 120 mg Extended Release tablets : B. No.:
Mfg. Date: Apr-2007
Excipient Blend sample to be used for validation will be prepared in house as per the manufacturing formula
except the active pharmaceutical ingredient.
Parameters to be validated are as follows:
1 Specificity
1.1
Selectivity
1.2
Forced Degradation
2 Linearity and Range
3 Precision
3.1

System Precision
3.2
Method Precision
3.3
Intermediate Precision
4 Accuracy (% Recovery)
5 Filter Validation
6 Robustness
7 Stability of Analytical Solution
IV. ACCEPTANCE CRITERIA:
Table 1
Validation
Parameter
Specificity -

Specification

Range of Study

Acceptance Criteria

----

By injecting diluent blank
solution, excipient blend
solution,
all
known
impurities, test solution and
test solution spiked with

impurities.
The forced degradation
studies should be performed
using 10N HCl, 10N NaOH
and 50% H2O2. Also effect
of temperature, humidity
and photolysis should be
checked. The studies shall
be performed on API,

The Pseudoephedrine Hydrochloride peak and all
known impurities should be well resolved from any
other peak and from each other.
The diluent blank, excipient blend and known
impurities should not show any peak at the retention
time of the Pseudoephedrine Hydrochloride.
The Pseudoephedrine Hydrochloride peak should
be well resolved from any other peak.
The intent is to create approximately 10% – 30%
degradation in at least one condition.
The peak purities will be demonstrated by a Photo
Diode Array (PDA) detector.

Selectivity

Specificity –
Forced
degradation
study

----


excipient blend and tablets
separately.

Linearity and
Range

----

50% to 150%

System
precision

----

----

Method
precision
Intermediate
precision

----

Accuracy
(%Recovery)
Filter

validation

----

Robustness –
change in
column lot
Robustness –
change in
flow rate
(± 0.2 mL/
minute)
Robustness –
change in
column oven
Temperature
(± 5°C)
Robustness –
change in pH
of buffer
(± 0.2 unit)
Stability of
analytical

----

Correlation coefficient should be greater than or
equal to 0.999.

% RSD of peak areas of ten replicate injections of

system suitability solution should not be more than
2.0% and system suitability criteria should pass as
per analytical method.
---% RSD of the results of six test solutions should not
be more than 2.0%.
---% RSD of the results of twelve test solutions (six of
Method Precision and six of Intermediate Precision)
should not be more than 2.0%.
50%, 100% & 150% of Mean recovery at each concentration level should
assay concentration
be between 98.0% and 102.0%.
Unfiltered and filtered test There should not be any significant difference
solution.
between filtered and unfiltered test solution.

0.45 µm
Nylon Filter
Paper.
C18, 150 mm Same make, different serial System suitability criteria should pass as per
x 4.6 mm,
no.
analytical method and the % RSD between results
5 µm
obtained with changed condition and average result
of method precision, should not be more than 2.0%.
1.5 mL/
1.3 mL/minute and
System suitability criteria should pass as per
minute
1.7 mL/minute

analytical method and the % RSD between results
obtained with changed condition and average result
of method precision, should not be more than 2.0%.
40°C

35°C and 45°C

4.0

3.8 and 4.2

----

System suitability criteria should pass as per
analytical method and the % RSD between results
obtained with changed condition and average result
of method precision, should not be more than 2.0%.

System suitability criteria should pass as per
analytical method and the % RSD between results
obtained with changed condition and average result
of method precision, should not be more than 2.0%.
Evaluate the stability upto 7 The analyte is considered stable if there is no
days for standard and test significant change in % assay.


solution

solution.

V. EXPERIMENTAL PLAN AND DATA EVALUATION:
The analytical method validation will be executed as per the following plan:
 The experiments may be done as sequential or simultaneous operations.


Sample sequence of each experiment may be run together or independently with necessary alteration of

sample sequence.
 The system suitability parameters should be monitored throughout the validation study.

1.

Any deviation from the plan shall be evaluated and approved by protocol approving authority.
Specificity – Selectivity and Forced Degradation:

Selectivity:
Prepare the diluent blank solution, system suitability solution, test solution as per the analytical method.
Preparation of excipient blend solution:
Weigh and transfer a quantity of excipient blend of about 187.2 mg to a 100 mL of volumetric flask add 70 mL
diluent and sonicate for 15 minutes. Dilute upto mark with diluent. Centrifuge this solution at 8000 rpm for 10
minutes. Decant the supernatant solution into another test tube and transfer 10 mL of supernatant solution into
another 50 mL volumetric flask and make up the volume with diluent. Filter the solution through 0.45 µm nylon
membrane filter.
Preparation of known impurity sample:
Preparation of Ephedrine Hydrochloride Impurity:
Weigh and transfer accurately about 3.0 mg of Ephedrine Hydrochloride to a 100 mL of volumetric flask add 70
mL diluent and sonicate for 15 minutes. Dilute upto mark with diluent and mix. Further dilute 2 mL of this solution
to 50 mL with diluent and mix.
Preparation of Nor-ephedrine Hydrochloride Impurity:
Weigh and transfer accurately about 3.0 mg of Nor ephedrine Hydrochloride to a 100 mL of volumetric flask add

70 mL diluent and sonicate for 15 minutes. Dilute upto mark with diluent and mix. Further dilute 2 mL of this
solution to 50 mL with diluent and mix.
Preparation of Nor-Pseudoephedrine Hydrochloride Impurity:
Weigh and transfer accurately about 3.0 mg of Nor-Pseudoephedrine Hydrochloride to a 100 mL of volumetric
flask add 70 mL diluent and sonicate for 15 minutes. Dilute upto mark with diluent and mix. Further dilute 2 mL of
this solution to 50 mL with diluent and mix.
Preparation of Test solution spiked with known Impurities:
Weigh powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL of volumetric
flask, add 70 mL of diluent to flask. Sonicate for 15 minutes. Make up to volume with diluent and mix. Centrifuge
this solution at 8000 rpm for 10 minutes. Decant the supernatant solution into another test tube and transfer 10 mL
of supernatant solution and 2 mL each of impurity stock solution into another 50 mL volumetric flask and make up
the volume with diluent. Filter the solution through 0.45 µm nylon membrane filter.
Perform the analysis on HPLC equipped with Photo Diode Array detector.
The sequence of injections for selectivity is given in table 2.
Table 2: Sample Sequence for Selectivity Study
Sr. No.

Sample Name

No. of Injections


1
2
3
4
5
6
7
8

9
10

Diluent blank
System suitability solution
Diluent blank
Excipient blend solution
Ephedrine Hydrochloride Impurity
Nor ephedrine Hydrochloride Impurity
Nor-Pseudoephedrine Hydrochloride Impurity
Test solution
Test solution spiked with Known impurities
Bracketing standard

1
5
1
1
1
1
1
1
1
2

Data evaluation:
Record each chromatogram. All the injections will be processed at the wavelength provided in the method and
selectivity will be demonstrated with regards to non-interference from diluent blank, excipient blend solution and
known impurities with Pseudoephedrine Hydrochloride peak.
1.1.2

Forced Degradation – Experiment:
Degrading agents will be added separately to Pseudoephedrine Hydrochloride API, Excipient blend and Cetirizine
Hydrochloride 5 mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets. Degrading agents will
be 10N HCl (Acid degradation), 10N NaOH (Base degradation), 50% Hydrogen peroxide (Oxidative degradation),
Heat (60°C) Humidity (75% R.H.) and Photolysis.
Forced degradation samples will be prepared as follows as given in table 3, 4 & 5.

Sr.
No.
1

2

3

4

Table 3: Preparation of Solutions for Forced Degradation Studies (API)
Degrading
Condition
Sample Preparations
Agents
Untreated
---Weigh accurately about 60 mg of API and transfer into 100 mL volumetric
sample
flask, add 70 mL of diluent, sonicate to dissolve and dilute to volume with
diluent and mix. Dilute 10 mL of this solution to 50 mL with diluent and
mix.
Acid
10N HCl

Weigh accurately about 60 mg of API and transfer into 100 mL volumetric
degradation
flask, add 70 mL diluent, sonicate to dissolve. Add 10 mL of 10N HCl
(aqueous) to the flask. Keep the flask to room temperature for 24 Hrs.
Neutralize this solution with 10N NaOH (aqueous) and dilute to volume with
diluent and mix. Dilute 10 mL of this solution to 50 mL with diluent and
mix.
A blank solution should be prepared in similar way.
Base
10N NaOH Weigh accurately about 60 mg of API and transfer into 100 mL volumetric
degradation
flask, add 70 mL diluent, sonicate to dissolve. Add 10 mL of 10N NaOH
(aqueous) to the flask. Keep the flask to room temperature for 24 Hrs.
Neutralize this solution with 10N HCl (aqueous) and dilute to volume with
diluent and mix. Dilute 10 mL of this solution to 50 mL with diluent and
mix.
A blank solution should be prepared in similar way.
Peroxide
50% H2O2 Weigh accurately about 60 mg of API and transfer into 100 mL volumetric
degradation
flask, add 70 mL, diluent sonicate to dissolve. Add 10 mL of 50% H2O2


Sr.
No.

Condition

Degrading
Agents

5

Heat
degradation
(Solid state)

60°C

6

Heat
degradation
(solution
state)
Humidity
degradation

60°C

7

8

Photolytic
degradation

75% R.H.

1.2 million

lux hours

Sample Preparations
(aqueous) to the flask. Heat the flask at 80°C for 6 Hours Cool the flask to
room temperature and dilute to volume with diluent and mix. Dilute 10 mL of
this solution to 50 mL with diluent and mix.
A blank solution should be prepared in similar way.
Weigh about 1.0 g of API and transfer in a Petri dish. Keep it at 60°C in hot
air oven. Withdraw after 24 hours. Weigh accurately about 60 mg of treated
API and transfer into 100 mL volumetric flask, Add 70 mL diluent, sonicate
to dissolve. Dilute to volume with diluent and mix. Dilute 10 mL of this
solution to 50 mL with diluent and mix.
Weigh accurately about 60 mg of API and transfer into 100 mL volumetric
flask, add 70 mL of diluent, sonicate to dissolve. Keep the flask at 60°C for 8
hours. Cool the flask to room temperature and dilute to volume with diluent
and mix. Dilute 10 mL of this solution to 50 mL with diluent and mix.
Weigh about 1.0 g of API and transfer in a Petri dish. Keep it at 75% R.H.
Withdraw the sample after 24 hours. Weigh accurately about 60 mg of the
treated API and transfer into 100 mL volumetric flask, add 70 mL diluent
sonicate to dissolve and dilute to volume with diluent and mix. Dilute 10 mL
of this solution to 50 mL with diluent and mix.
Weigh about 1.0 g of API and transfer in a Petri dish. Keep it in photolytic
chamber for a sufficient period to give total exposure of about 1.2 million lux
hours. After the desired exposure withdraw the sample. Weigh accurately about
60 mg of treated API and transfer into 100 mL volumetric flask, add 70 mL of
diluent sonicate to dissolve. Dilute to volume with diluent and mix. Dilute 10
mL of this solution to 50 mL with diluent and mix.

Table 4: Preparation of Solutions for Forced Degradation Studies (Excipient Blend)

Sr.
No.
1

Condition
Untreated
Excipient
blend

Degrading
Agents
------

2

Acid
degradation

10N HCl

3

Base
degradation

10N NaOH

4

Peroxide
degradation

50% H2O2

Sample Preparations
Weigh accurately about 187.2 mg of excipient blend and transfer into 100 mL
volumetric flask, Add 70 mL of diluent sonicate for 15 minutes and make up
to volume with diluent and mix. Centrifuge this solution at 8000 rpm for 10
minutes. Decant the supernatant solution into another test tube and transfer
10 mL of supernatant solution into another 50 mL volumetric flask and make
up the volume with diluent. Filter the solution through 0.45 µm nylon
membrane filter.
Weigh accurately about 187.2 mg of excipient blend and transfer into 100 mL
volumetric flask, Add 70 mL diluent sonicate for 15 minutes. Add 10 mL of
10N HCl (aqueous) to the flask. Keep the flask to room temperature for 24
Hrs. Neutralize this solution with 10N NaOH (aqueous) and make up to
volume with diluent and mix. Centrifuge this solution at 8000 rpm for 10
minutes. Decant the supernatant solution into another test tube and transfer
10 mL of supernatant solution into another 50 mL volumetric flask and make
up the volume with diluent. Filter the solution through 0.45 µm nylon
membrane filter.
Weigh accurately about 187.2 mg of excipient blend and transfer into 100 mL
volumetric flask, Add 70 mL diluent sonicate for 15 minutes. Add 10 mL of
10N NaOH (aqueous) to the flask. Keep the flask to room temperature for 24
Hrs. Neutralize this solution with 10N HCl (aqueous) and make up to volume
with diluent and mix. Centrifuge this solution at 8000 rpm for 10 minutes.
Decant the supernatant solution into another test tube and transfer 10 mL of
supernatant solution into another 50 mL volumetric flask and make up the
volume with diluent. Filter the solution through 0.45 µm nylon membrane

filter.
Weigh accurately about 187.2 mg of excipient blend and transfer into 100 mL
volumetric flask, Add 70 mL diluent sonicate for 15 minutes. Add 10 mL of
50% H2O2 (aqueous) to the flask. Heat the solution at 80°C for 6 hours. Cool

5

Heat
degradation
(Solid state)

60°C

6

Heat
degradation

60°C

the flask to room temperature and dilute to volume with diluent and mix.
Centrifuge this solution at 8000 rpm for 10 minutes. Decant the supernatant
solution into another test tube and transfer 10 mL of supernatant solution into
another 50 mL volumetric flask and make up the volume with diluent. Filter
the solution through 0.45 µm nylon membrane filter.
Keep about 2.0 g of excipient blend and transfer in a Petri dish. Keep it at
60°C in hot air oven. Withdraw after 24 hours. Weigh accurately about 187.2
mg of the treated sample and transfer into 100 mL volumetric flask, add 70
mL of diluent, sonicate 15 minutes and make up to volume with diluent and
mix. Centrifuge this solution at 8000 rpm for 10 minutes. Decant the

supernatant solution into another test tube and transfer 10 mL of supernatant
solution into another 50 mL volumetric flask and make up the volume with
diluent. Filter the solution through 0.45 µm nylon membrane filter.
Weigh about 187.2 mg of excipient blend and transfer into 100 mL
volumetric flask, add 70 mL of diluent and sonicate for 15 minutes. Keep the


Weigh and crush 20 tablets to a fine powder. Use this sample powder for the preparation of Forced Degradation
study.
Table 5: Preparation of Solutions for Forced Degradation Studies (Cetirizine Hydrochloride 5 mg and
Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets)


Sr.
No.
1

Condition
Untreated
Sample

Degrading
Agents
----

2

Acid
degradation

10N HCl

3

Base
degradation

10N NaOH

4

Peroxide
degradation

50% H2O2

5

Heat
degradation
(solid state)

60°C

6

Heat
degradation
(solution

60°C

Sample Preparations
Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine
Hydrochloride and transfer to 100 mL of volumetric flask, add 70 mL of
diluent to flask and sonicate for 15 minutes. Make up to volume with diluent
and mix. Centrifuge this solution at 8000 rpm for 10 minutes. Decant the
supernatant solution into another test tube and transfer 10 mL of supernatant
solution into another 50 mL volumetric flask and make up the volume with
diluent. Filter the solution through 0.45 µm nylon membrane filter.
Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride
and transfer to 100 mL of volumetric flask. Add 70ml of diluent, sonicate for 15
minutes add 10 mL of 10N HCl (aqueous) to the flask.. Keep the flask to room
temperature.for 24 Hrs. Neutralize this solution with 10N NaOH (aqueous) and
make up to volume with diluent and mix. Centrifuge this solution at 8000 rpm
for 10 minutes. Decant the supernatant solution into another test tube and
transfer 10 mL of supernatant solution into another 50 mL volumetric flask and
make up the volume with diluent. Filter the solution through 0.45 µm nylon
membrane filter.
Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine Hydrochloride
and transfer to 100 mL of volumetric flask. Add 70ml of diluent, sonicate for 15
minutes add 10 mL of 10N NaOH (aqueous) to the flask.. Keep the flask to room
temperature for 24 Hrs. Neutralize this solution with 10N HCl (aqueous) and
make up to volume with diluent and mix. Centrifuge this solution at 8000 rpm
for 10 minutes. Decant the supernatant solution into another test tube and
transfer 10 mL of supernatant solution into another 50 mL volumetric flask and
make up the volume with diluent. Filter the solution through 0.45 µm nylon
membrane filter..
Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine
Hydrochloride and transfer to 100 mL of volumetric flask. Add 70ml of

diluent sonicate for 15 minutes, add 10 mL of 50% H2O2 (aqueous) to the
flask. Heat the solution at 80°C for 6 hours. Cool the flask to room
temperature and make up to volume with diluent and mix. Centrifuge this
solution at 8000 rpm for 10 minutes. Decant the supernatant solution into
another test tube and transfer 10 mL of supernatant solution into another 50
mL volumetric flask and make up the volume with diluent. Filter the solution
through 0.45 µm nylon membrane filter.
Weigh about 2.0 g of tablet powder and transfer in a Petri dish. Keep it at
60°C in hot air oven. Withdraw after 24 hours. Weigh treated tablet powder
equivalent to 60.0 mg of Pseudoephedrine Hydrochloride and transfer to 100
mL of volumetric flask. Add 70ml of diluent, sonicate for 15 minutes make
up to volume with diluent and mix. Centrifuge this solution at 8000 rpm for
10 minutes. Decant the supernatant solution into another test tube and
transfer 10 mL of supernatant solution into another 50 mL volumetric flask
and make up the volume with diluent. Filter the solution through 0.45 µm
nylon membrane filter.
Weigh tablet powder equivalent to 60.0 mg of Pseudoephedrine
Hydrochloride and transfer to 100 mL of volumetric flask, add 70 mL of
diluent and sonicate for 15 minutes. Keep the flask at 60°C for 8 hours. Cool


Note : 494.4 mg of tablet blend is equivalent to 120 mg of Pseudoephedrine Hydrochloride
Perform the above analysis on HPLC equipped with Photo Diode Array detector. The sequence of injections for forced
degradation is given in table 6.
Table 6: Sample Sequence for Forced Degradation Studies
Sample Name
Diluent blank
System suitability solution
Blank – HCl
Blank – NaOH

Blank – H2O2
Excipient blend – Untreated
Excipient blend – Heat (Solid state)
Excipient blend Heat (Solution state)
Excipient blend – Humidity
Excipient blend – Photolytic degradation
Excipient blend – HCl
Excipient blend – NaOH
Bracketing standard – 1
Excipient blend – H2O2
Bracketing standard – 2

No. of Injections
1
5
1
1
1
1
1
1
1
1
1
1
2
1
2

Note :

Sequence for Forced Degradation of Pseudoephedrine Hydrochloride API and Tablets is carried out as per given
sequence replacing Excipient blend with API & Tablets respectively.
1.2.1 Data evaluation:
Record each chromatogram. The peak purity will be determined for the Pseudoephedrine Hydrochloride peak.
The degradation if any will be compared with untreated sample. The peaks due to degradation and of excipient
blend should be well separated from the Pseudoephedrine Hydrochloride peak.
2.
Linearity and Range:
2.1
Experiment:
Prepare a series of standard preparations (five preparations) of Pseudoephedrine Hydrochloride, over a range
starting from 50% to at least 150% of the specified limits of assay.
2.1.1

Preparation of linearity stock solutions:
Stock solution preparation of Pseudoephedrine Hydrochloride:
Weigh accurately about 60 mg of Pseudoephedrine Hydrochloride working standard to a 100 mL volumetric
flask. Add 30 mL of diluent & sonicate for 15 minutes to dissolve. Dilute to volume with diluent and mix.
The preparation of linearity solutions is given in table 7.
Inject each of the linearity preparation in triplicate and then take average area count for calculations.
Table 7: Dilutions for Linearity


The
Linearity
level

Sample
concentration

Amount of stock solution
preparation of
Pseudoephedrine
Hydrochloride to be
transferred (mL)

Volume made
up to (mL) with
Diluent

Concentration of
Pseudoephedrine
Hydrochloride
(ppm)

50
50
50
50
50

60
90
120
150
180

Level – 1
50%
5.0 mL

Level – 2
75%
7.5 mL
Level – 3
100%
10.0 mL
Level – 4
125%
12.5 mL
Level – 5
150%
15.0 mL
sequence of injections for linearity is given in table 8.
Table 8: Sequence for Linearity
Sample Name
Diluent blank
System suitability solution
Linearity Level 1
Linearity Level 2
Linearity Level 3
Bracketing standard
Linearity Level 4
Linearity Level 5
Bracketing standard
2.2

No. of Injections
1
5
3

3
3
2
3
3
2

Data evaluation:
Plot a linearity graph of average area at each level against the concentration (%) and determine the correlation
coefficient.
The range of the analytical method in concentration (%) will be reported.

3.
3.1
3.1.1

Precision:
System Precision:
Experiment:
Make ten replicate injections of system suitability solution into the HPLC using the method of analysis.
The sequence of injections for system precision is given in table 9.
Table 9: Sequence for System Precision
Sample Name
Diluent blank
System suitability solution

3.1.2

No. of Injections
1

10

Data evaluation:
Calculate and report standard deviation and % RSD of peak areas of 10 replicate injections and also report
theoretical plates and tailing factor for the last injection of system suitability solution.


3.2

Method Precision:

3.2.1

Experiment:
Prepare six test solutions of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride 5 mg and
Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets and inject into the HPLC as per the
analytical method.
The sequence of injections for method precision is given in table 10.
Table 10: Sequence for Method Precision
Sample Name
Diluent blank
System suitability solution
Test solution - 1
Test solution - 2
Test solution - 3
Test solution - 4
Test solution - 5
Bracketing standard-1
Test solution - 6
Bracketing standard-2

3.2.2

No. of Injections
1
5
2
2
2
2
2
2
2
2

Data evaluation:
The % assay of Pseudoephedrine Hydrochloride will be calculated and reported along with the standard
deviation and % RSD of the six test solutions.

3.3

Intermediate Precision:

3.3.1

Experiment:

Prepare six test solutions of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride 5 mg and
Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets of the same lot (as used in 3.2) using a
different analyst on a different column and inject into a different HPLC system (other than that used in 3.2) on

different day as per the analytical method.
The sequence of injections for intermediate precision is given in table 11.
Table 11: Sequence for Intermediate Precision
Sample Name
Diluent blank
System suitability solution
Test solution - 1
Test solution - 2
Test solution - 3
Test solution - 4
Test solution -5
Bracketing standard - 1
Test solution - 6
Bracketing standard - 2
3.3.2

Data evaluation:

No. of Injections
1
5
2
2
2
2
2
2
2
2

The % assay of Pseudoephedrine Hydrochloride will be calculated and reported along with the % RSD of the
twelve test solutions (six of method precision & six of intermediate precision).
4. Accuracy (% Recovery):
4.1 Experiment:
Weigh and transfer accurately, in triplicate at each level, about 187.2 mg of excipient blend and add to it
Pseudoephedrine Hydrochloride API at 50%, 100% and 150% of the working concentration. Working (assay)
concentration of Pseudoephedrine Hydrochloride is 120 ppm. Prepare the solutions as per the analytical method.
The preparation of accuracy solutions is given in table 12.
Inject each of the samples in triplicate and then take average area count for calculations.
Table 12: Dilutions for Accuracy

Level

First level

Second level

Third level

Sample Name
Rec-50% / 1
Rec-50% / 2
Rec-50% / 3
Rec-100% / 1
Rec-100% / 2
Rec-100% / 3
Rec-150% / 1
Rec-150% / 2
Rec-150% / 3

Amount of
Pseudoephedrine
Hydrochloride API to
be weighed (mg)
30
30
30
60
60
60
90
90
90

Make up
volume in
mL
100
100
100
100
100
100
100
100
100

Further transfer 10 mL of each of the above solution to 50 mL and dilute with diluent and mix. The sequence of
injections for accuracy is given in table 13.

Table 13: Sequence for Accuracy
Sample Name
Diluent blank
System suitability solution
Rec-50% / 1
Rec-50% / 2
Rec-50% / 3
Bracketing standard - 1
Rec-100% / 1
Rec-100% / 2
Rec-100% / 3
Bracketing standard - 2
Rec-150% / 1
Rec-150% / 2
Rec-150% / 3
Bracketing standard - 3

No. of Injections
1
5
3
3
3
2
3
3
3
2
3
3

3
2


4.2

Data evaluation:

The percent actual concentration and theoretical concentration will be calculated and percent recovery at each level
will be calculated and reported along with percent mean recovery at each level.
5. Filter Validation:
5.1 Experiment:
Weigh accurately tablet powder equivalent to 60 mg of Pseudoephedrine Hydrochloride and transfer to 100 mL
volumetric flask. Add about 70 mL of diluent and sonicate for 15 minutes. Dilute to volume with the diluent and
mix. Centrifuge a portion of the resulting solution at about 8000 rpm for about 10 minutes. Decant the supernatant
solution into another test tube and transfer 10 mL of supernatant solution into another 50 mL volumetric flask and
make up the volume with diluent. Use this solution as unfiltered test solution. Dilute 10 mL of remaining portion of
the supernatant solution to 50 mL with diluent and filter it through 0.45 µm nylon membrane filter paper. Fill 5
vials of this solution. Use this as filtered test solution. Analyze all filtered and unfiltered solutions in single
sequence. The sequence of injections for filter validation is given in table 14.
Table 14: Sample Sequence for Filter Validation
Sample Name
Diluent blank
System suitability solution
Unfiltered test solution
Filtered test solution – 1
Filtered test solution – 2
Filtered test solution – 3
Filtered test solution – 4
Filtered test solution – 5

Bracketing standard

No. of Injections
1
5
1
1
1
1
1
1
2

5.2 Data evaluation:
The % assay of Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride 5 mg and Pseudoephedrine
Hydrochloride 120 mg Extended Release Tablets with filtered and unfiltered test solution will be calculated and
reported along with the absolute difference between unfiltered and filtered test solution.
6. Robustness:
6.1
Experiment:
Prepare two test solution of the same lot (as used in 3.2 and 3.3) of Pseudoephedrine Hydrochloride in Cetirizine
Hydrochloride 5 mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets as per analytical method.
Inject this solution along with the blank and system suitability solution along different chromatographic conditions as
shown below:
6.1.1
Change in column lot (same make, different serial no.)
6.1.2

Change in flow rate (± 0.2 mL/minutes)

6.1.3
Change in wavelength (± 2 nm)
6.1.4
Change in column oven temperature (± 5°C)
6.1.5
Change in pH of buffer (± 0.2 units)
The sequence of injections for robustness study is given in table 15 to table 23.


6.1.1

Change in Column Lot:
[Normal Experimental Condition:
C18, 150 mm x 4.6 mm, 5 µm (Zorbax SB or Waters symmetry) Sr. No.:
Table 15: Sample sequence for change in column lot

6.1.2

Sample Name
No. of Injections
Diluent blank
1
System suitability solution
5
Test solution-1
1
Test solution -2
1
Bracketing standard
2

This experiment can be performed as a part of intermediate precision study.
Change in Flow Rate:
(Normal Experimental Condition: 1.5 mL/minute)
Table 16: Sample sequence for change in flow rate (1.3 mL/minute)
Sample Name
Diluent blank
System suitability solution
Test solution -1
Test solution -2
Bracketing standard

No. of Injections
1
5
1
1
2

Table 17: Sample sequence for change in flow rate (1.7 mL/minute)
Sample Name
Diluent blank
System suitability solution
Test solution -1
Test solution -2
Bracketing standard
6.1.3

No. of Injections
1
5

1
1
2

Change in Wavelength:
(Normal Experimental Condition; 215 nm)
Table 18: Sample sequence for change in wavelength (213 nm)
Sample Name
Diluent blank
System suitability solution
Test solution -1
Test solution -2
Bracketing standard

No. of Injections
1
5
1
1
2

Table 19: Sample sequence for change in wavelength (217 nm)
Sample Name
Diluent blank
System suitability solution
Test solution -1
Test solution -2
Bracketing standard

No. of Injections

1
5
1
1
2

]


6.1.4

Change in pH of Buffer:
(Normal Experimental Condition; pH 4.0)
Table 20: Sample sequence for change in pH of buffer (3.8)
Sample Name
Diluent blank
System suitability solution
Test solution -1
Test solution -2
Bracketing standard

No. of Injections
1
5
1
1
2

Table 21: Sample sequence for change in pH of buffer (4.2)
Sample Name

Diluent blank
System suitability solution
Test solution -1
Test solution -2
Bracketing standard
6.1.5

No. of Injections
1
5
1
1
2

Change in Temperature:
(Normal Experimental Condition; 40°C)
Table 22: Sample sequence for change in column oven temperature (35°C)
Sample Name
Diluent blank
System suitability solution
Test solution -1
Test solution -2
Bracketing standard

No. of Injections
1
5
1
1
2

Table 23: Sample sequence for change in column oven temperature (45°C)
Sample Name
Diluent blank
System suitability solution
Test solution -1
Test solution -2
Bracketing standard
6.2

No. of Injections
1
5
1
1
2

Data evaluation:
System suitability is to be reported for each experiment.
% RSD between mean of method precision results and two results obtained with changed condition is to be
reported for each experiment.

7. Stability of Analytical Solution:
7.1 Experiment:
Prepare the Pseudoephedrine Hydrochloride standard solution, and test solution of Pseudoephedrine Hydrochloride
in Cetirizine Hydrochloride 5 mg and Pseudoephedrine Hydrochloride 120 mg Extended Release Tablets on 1 st, 3rd,
5th, 6th and 7th day of experiment. Store these test solutions at room temperature for every time interval upto 8 th day.


Analyze these test solutions on 8 th day with freshly prepared test solution. Prepare the Pseudoephedrine

Hydrochloride standard solution freshly at the time of analysis and calculate the assay of Pseudoephedrine
Hydrochloride in the sample.
The sequence of injections for solution stability for the initial analysis is given in table 24.
Table 24: Sequence for solution stability
Sample Name
Diluent blank
Freshly prepared standard solution
7th day prepared standard
6th day prepared standard
5th day prepared standard
3rd day prepared standard
1st day prepared standard
Freshly prepared test solution
7th day prepared test solution
6th day prepared solution
5th day prepared solution
3rd day prepared solution
Bracketing standard -1
1st day prepared solution
Bracketing standard -2

No. of Injections
1
5
1
1
1
1
1
1

1
1
1
1
2
1
2

7.2 Data evaluation:
The test solution for Pseudoephedrine Hydrochloride in Cetirizine Hydrochloride 5 mg and Pseudoephedrine
Hydrochloride 120 mg Extended Release Tablets is said to be stable up to the time point till which there is no
significant change in % assay.
VI. VALIDATION REPORT:
Prepare the validation report using the following headers:
• Introduction
• The instruments and samples used including standards.
• Validation results and Discussion with following headings:


Specificity



Selectivity



Forced Degradation



Linearity and Range



Precision



System Precision



Method Precision



Intermediate Precision



Accuracy (% Recovery)



Filter validation



Robustness



Stability of Analytical Solution


System suitability data – that obtained during different days of analytical method validation


•

(including robustness)
Summary

•

Conclusion

Figures should include chromatograms of diluent blank, standard preparation, excipient blend, test solution,
chromatograms of selectivity, forced degradation study and linearity graph of Pseudoephedrine Hydrochloride.

ANNEXURE – 1
VII.
ANALYTICAL METHOD: (Method No.: CPH/ASY-02/0)
Chromatographic conditions:
Column
: C18, 150 mm X 4.6 mm, 5 µm
(Zorbax SB, Waters symmetry or equivalent)
Buffer
: Weigh accurately and transfer 11.5 g of Ammonium dihydrogen

orthophosphate to a 1000 ml volumetric flask. Add about 980 ml of water,
dissolve and dilute to volume with water. Add 1.0 ml of Triethylamine to the
1000 ml of buffer solution prepared. Adjust the pH to 4.0 ± 0.05 with
Mobile Phase

Wavelength
Flow Rate
Column temperature
Injection volume
Run time
Blank solution
Diluent

Orthophosphoric acid. Filter and degas the Buffer
: For isocratic system, prepare a mixture of Methanol and buffer in the
proportion 10: 90 respectively. Mix well. Filter through 0.45 µ Nylon
membrane filter paper and degas prior to use.
: 215 nm
: 1.5 ml / minute
: 40°C ± 2°C
: 10 μl
: 10 minutes
: Use 0.1N Hydrochloric acid as blank
: Use 0.1N Hydrochloric acid as diluent


Preparation of Pseudoephedrine HCl Standard Stock Solution:
Weigh accurately about 30 mg of Pseudoephedrine HCl working standard and transfer to a 50 ml volumetric flask.
Add 30 ml of diluent and sonicate to dissolve. Dilute to volume with diluent and mix. (Dilution scheme: 30 mg →
50 ml)

Preparation of Pseudoephedrine HCl Standard working Solution:
Transfer 10.0 ml of Pseudoephedrine HCl standard stock solution into a 50 ml of volumetric flask and dilute to
volume with the diluent. Mix well.
(Dilution scheme: 10.0 ml → 50 ml)

Preparation of Test Solution:
Weigh and crush 20 tablets to a very fine powder. Transfer an accurately weighed 240 mg of compressed tablet
powder and 247.2 mg of coated tablet powder i.e. equivalent to 60 mg of Pseudoephedrine into a 100 ml
volumetric flask. Add about 70 ml of diluent and sonicate for 15 minutes. Dilute up to mark with diluent.
Centrifuge this solution at 8000 rpm for 10 minutes. Decant the supernatant solution into another test tube and
transfer 10.0 ml of supernatant solution into another 50 ml volumetric flask and make up the volume with diluent.
Filter the solution through 0.45 µm nylon membrane filter.
(Compressed tablets: Dilution scheme: 240 → 100 ml/ 10.0 ml → 50 ml)
(Coated tablets: Dilution scheme: 247.2 → 100 ml/ 10.0 ml → 50 ml)
System Suitability Solution:
Use Pseudoephedrine HCl standard working solution as system suitability solution.
Procedure:
Separately inject equal volumes of blank, five replicate injections of system suitability solution (Pseudoephedrine
HCl standard working solution). Then inject two injections of test solution and record the chromatograms.
Disregard any peak due to blank in the test solution. Calculate % RSD of five replicate injections of system
suitability solution (Pseudoephedrine HCl standard working solution). Check tailing factor and theoretical plates of
the peak in the chromatogram obtained with 5th injection of system suitability solution (Pseudoephedrine HCl
standard working solution).
The limits are as below,
1) Theoretical plates should be not less than 3000.
2) Tailing factor should be less than 2.0.
3) % RSD should be not more than 2.0%.
Note: Inject system suitability solution (Bracketing) in two replicates after completion of sequence and after
injecting 10 injections of Test Solution, whichever is earlier. Check the RSD of bracketing standards with earlier
injected standards.

Injection scheme:
Sr. No.

Solutions to be injected

No. of injections

1

Diluent Blank solution

1

2

System suitability solution

5


(Pseudoephedrine HCl standard working solution)
3

Test Solution

2

4

Bracketing standard (system suitability solution) after

completion of sequence and / or after injecting 10 injections
of test solution whichever is earlier.

2

Calculations:
% Assay =
Where,
AT
AS
WS
WT
AW
P

AT
AS

X

WS
50

X

10
50

X

100
X
WT

50
X
10

P
120

X AW

Average peak area of Pseudoephedrine in test solution.
Mean peak area of Pseudoephedrine in system suitability solution.
Weight of Pseudoephedrine HCl working standard in mg.
Weight of sample taken.
Average weight of Tablets.
Assay of Pseudoephedrine HCl working standard in % on as is basis.

Express the results up to two decimals.