ANALYTICAT METHOD VAI-IDATION PROTOCOL

Material Name Montelukast Sodium INN Page No. Lof12

ffiriems
Protocol No.: Method No. rP/u.13010/01 Test parameter Assay by HPLC
vPL/MV-01s Revision No. 00 Date Revised New

TABLE OF CONTENTS
Serial No. Contents Page No.

1 Purpose 02

2 Objective 02

3 Scope 02

4 Experimental Plan 02

5 Materials, Reagent and Column 03

5.1 Primary material 03

5.2 Regents 03

5.3 Column 03

6 nstrument Qualif ication

I
03

7 Analytical Method 04-05

8 Methods Validation Analytical Performance Characteristics To Be Evaluated 06

8.1 Accuracy 06

8.2 Precision 07

8.2.1 Repeatabi lity ( Method Precision) 07

8.2.2 lntermediate Precision 08

8.3 Linearity and range 09

8.4 Speclficity L0

8.4.1- Blank effect 10

8.5 System Suitability 10-11

9 Maintenance of raw data 1,1

10 Deviation 11

1.7 Report LL-L2

L2 Revision History 12

Prepared bv: Ghecked bv: Approved by:

Name Farhana Sultana Md. Tarikul lslam Bassunia Md Moniruzzaman

Desionation PD Officer Asst. Manaqer, QC. QA Manaqer

Signature
f crn L*-a &,*ht-\ $odr
Date L{t t)tt4 '6/Ai/ r\ +1-CI(b.l v
 ANALYTICAL M ETHOD VALIDATION PROTOCOL
Montelukast Sodium INN Page No. 2ofL2
ve ritas Material Name

Method No. TPlL7130tOl01 Test parameter Assay by HPI-C

ProtocolNo.:
VPL/MV-01s Revision No. 00 Date Revised New

1" purpose: To detail the standard procedures for validation of analytical method of Montelukast Sodium"

for analysis of
objective: The objective of validation of the analytical method is to demonstrate that the method is suitable
2.
Montelukast Sodium with accuracy, precision and reliability within defined range.

3. Scope: To evaluate the acceptability of analytical method for its intended purpose. lt will define the
procedure,
Montelukast Sodium'
documentation, references and acceptance criteria to be used in the validation of assay method of

4. Experimental Plan:

Parameters Experiment Acceptance Criteria

Accuracy Prepare samples at three different levels 80%, 100% Assay value - 98% lo 102% and RSD
and !20% in triplicate (total nine determinations) value of nine determinations must be
and determine the active content. than 2%.
less

Repeatability Prepare six samples atLOO% concentration label and RSD of the active contents of six

(Method Precision) determine active content of each sample and also samples must be less than 2%.
calculate their RSD value.
lntermediate Prepare six samples at L00% concentration label RSD of the active contents of six
Precision from the same batch at next day by another analyst samples must be less than 2Yo.as per
and determine active content of each sample and Repeatability day 1.
also calculate their RSD value.
Linearity & Range Linearity to be performed in the range of about 60- The calibration curve should be linear (

140% with LOO% as working level concentration visually) and the value of R2 must be >

0.99

Specificity lnject standard, sample and blank solution according will be no interference at the
There-legion
to method. peak of Montelukast due to
blank.
System Suitability Prepare a standard solution al !00% concentration RSD of the area, retention time &
label and run six injections. height of peaks is within 2%, Avg' tailing
factor not more than 2.

Prepared by: Checked bv: Aooroved bv:

Name Farhana Sultana Md. Tarikul lslam Bassunia Md. Moniruzzaman

QA Manaqer
Desiqnation
Signature
PD Officer
ta.v haro-
Asst. Manaoer, QC
'trtJ"-(
1At 0<'/
&;{
9t.,efb'lV
Date 1-<lh, I 4 11
 ANALYTICAL METHOD VALIDATION PROTOCOL
Material Name Montelukast Sodium INN

TPl1L13O1.O/01 Test parameter Assay by HPLC

5. MATERIALS, REAGENT AND EqUIPMENTS

protocol:
The following materials will be used during the execution of this
5.1 PRIMARY MATERIAL
Sou rce Descri nti on
Sr. No. Name
TVpe Raw Material
01 Montelukast Sodium INN
Batch No.
Shelf life
TVpe Working Standard
02 Montelukast Sodium INN
Batch No.
Shelf life

5,2 REAGENT
Manufacture Batch No. Shelf Life
Sr. No. Reagent Name
01 Ammonium Acetate
02 Glacial Acetic Acid
03 Methanol
5.3 Column
ID
Sr. No. Description
01 Kromacil l-00-Crs HD, 1-50 x4.6 mm, 5pr or Equivalent

5. INSTRUM ENT qUALIFICATION

The below instruments should be used for this validation:
QAD No. Calibration Recqrd Doc. No.
Sr. No. lnstrument Name Brief Description
01 HPLC PerkinElmer Flexar HPLC QC-LC-o1 Date of Calibration
with Next due date \
integrated PDA
Calibration done bY
detector
Analytical Balance BOECO Electronic Balance QC-AB-18 Date of Calibration
02
Next due date
Callbration done bY

Prepared by: Checked bv: Aooroved bY:

Name Farhana Sultana Md. Tarikul lslam Bassunia Md. Moniruzzaman

Asst. Manaqer, QC QA Manaqer

Desionation
Signature
PD Officer

f-ar )-a^r q'*"J

,14/o\
^

Date /t-< l 0t ll4 /11 'LEr.t5 'b.lY

 ANALYTrcAT METHOD VALIDATION PROTOCOL
Material Name Montelukast Sodium INN

Test parameter Assay bY HPLC

TPll-11301o/01

7. Proposed AnalYtical Method

(HPLC)
Method: Hish Pressure Liquid Chromatosraphv

Principle:
of High
are injected consecutively into a suitable column
A prepared standard solution and sample solution
sodium present in sample is calculated
I
I
performance Liquid chromatography (Hplc). The content of Monterukast
I
I

bycomparingthepeakareaofactiveMontelukastSodiumpresentinsamplepreparationwiththatofstandard
preparation.

Procedure:
detector
Apparatus PerkinElmer Flexar HPLC integrated with PDA
I

Column Kromacil 100-C1s HD, 150 x4'6 mm, 5pt or Equivalent

ml of water and
Dissolve 1.9 g of Ammonium Acetate in 500
Buffer
adjust pH 3.5 t 0.05 by glacial acetic acid'

Mobile Phase Buffer : Methanol = L5 : 85

Diluting solution Water: Methanol= 30:70

Temperature 30'c
1.5 ml/rnin.
Flow rate

Monitoring wavelength 254 nm

Load
L0 pl

Retention Time Montelukast : 5.0 minutes (Approx')

Preparation of standard solution:

a clean and dry
workrng standard of Monterukast sodium in to
weigh accuratery and transfer about 10.0 mg of S0nicate for 5
100 mr vorumetric frask. Add about 60 mr of
diruting sorution in to it, shake for 5 rninutes and
and shake to mix well.. Filter this through 0'2
micron disk
minutes. volume up to the mark with same solvent
filter, Call this solution A.

Farhana Sultana trltO. fariXut lslam Bassunia

 Af.tnl-Wrcnt METHOD VALIDATION PROTOCOL
MaterialName Montelukast Sodium INN
veritas
TP/LL73O1O(0t Test parameter

Sample PreParation:
about
in to a crean and dry 100 mr vorumetric flask' Add
weigh accuratery and transfer about r.0.0 mg of sampre mark with
and sonicate for 5 minutes. Vorume up to the
60 mr of diruting sorution in to it, shake for 5 minutes
0,2 micron disk firter. cau this sorution B.
same sorvent and shake to mix weil.. Firter this through

Procedure:

A and sample preparation B into the tray of the auto sampler of the
Place vials containing standard preparation
chromatogram. Calculate the quantity of Montelukast in sample using
HPLC. Run the instrument and record the
the following equation.

Calculation:

1) Montelukast content (%)

Peak area of sample, W.ighl "

f
'ltt9l-g)
P*k x Weight of sample (mg) x 1'037
"t., "f.iandard
Where,
as it is basis'
Y= Strengthof Montelukast sodium (working standard) on
1.037= Conversion ratio of Montelukast sodium
to Montelukast'

Farhana Sultana Md. Tarikul lslam Bassunta

 NNTNI-WICAI M ETHOD VALIDATION PROTOCOL
MaterialName Montelukast Sodium INN

Test parameter Assay bY HPLC

TP/11.13010/01

S.METHoDSVAIIDATIoNANALYTICALPERFoRMANCECHARACTERISTICSToBEEVALUATED

8.1 AccuracY
accepted as either
between the varue found and the varue that is
a
Accuracy expresses the croseness of agreement the assay of known'
value. lt may often be expressed as the recovery by
conventional true value or an accepted reference g over a minimum of
using preferably a minimum of determinations
added amounts of anaryte. Accuracy shourd be assessed procedure')
(e.g. 3 concentrations /3 repricates each of totar analytical
3 concentration revers covering the specified range
Experimental Determination of Accuracy

* Prepare a standard solution as per method'

conc'
* Prepare three samples at about 80% of nominal
at about tOl% of nominal conc'
'f' Prepare three samples
of nominal conc'
* Prepare three samples at about t20%
{. lnject each preparation according to method'
preparation
at each concentration level comparing with standard
n calculate the active content of the three samples

Sample Weight (mg)

less than 2%
Average recovery at each level within 98% to 102% & RSD of nine determinations must be
Acceptance criteria:
Result: n Complies tr Not ComPlies
Data Attached

Farhana Sultana Md. Tarikul lslam Bassunia

 Arualwtcnl METHoD vALIDATIoN PRorocoL
MaterialName Montelukast Sodium INN

Test parameter Assay by HPLC

ProtocolNo.; TPlLL73O1.0l07
vPL/MV-01s

8.2 Precision
The precision of an analytical procedure expresses the closeness
of agreement (degree of scatter) between a series of
measurements obtained from multiple sampling of the
same homogeneous sample under the prescribed conditions'
precision may be considered at three levels: repeatability, intermediate precision and reproducibiiity'

it is not possible to obtain

precision should be investigated using homogeneous, authentic samples' However' if
a

prepared samples or a sample solution

homogeneous sample it may be investigated using artificially

8.2.1 Repeatability (Method Precision)

is
conditions over a short interval of time' Repeatability
Repeatability expresses the precision under the same operating the intended
also termed intra-assay precision. A minimum of three
determinations at each of three concentrations across
test concentration is to used'
range, or a minimum of six determinations at the
Experimental Determination of Repeatability

* Prepare a standard solution as per method'

.i. Prepare six samples at test (100%) concentration label'
* Run each samples using the proposed analytical
method
*Calculatethepercentofactivetoeachsamplecomparingwithstandardsolution

Result

Av. AssaY (o/o)

AcCeptancg critgria: RSD of six samples should not be more than 2'0%

Result: tr Complies tJ Not ComPlies

Data Attached

Prepared bv: Checked by: Aoproved bY:

Md. Tarikul lslam Bassunia Md. Moniruzzaman
Name Farhana Sultana

PD Officer Asst. Manaqer, QC QA Manager

Desiqnation
Signature
-FonL-a..t-
'ffuk,-\
Date .\,cl09llh ------87 0",/ 14 '1-{-r-\'1t,5'l Y
 NNNIWICNI METHOD VALIDATION PROTOCOL
Montelukast Sodium INN
veritas
Assay bY HPLC
ProtocolNo.: Method No. TP/L1.13010101
vPL/MV-01s

8.2.2. lntermediate Precision

instruments' by
lntermediate precision refers to variations within a laboratory as with different days, with different
different analYsts etc.

sample but using different equipment, different column

on different day, another analyst will carry out the test from same
(but same dimension and packing material) etc'

Experimental Determination of lntermediate precision

* Using Experimental Determination of Repeatability arrange for analysis with second daY
Result:

Analyst 1
Analyst 2

Done By

Location

lnstrument
Used

Date of

Analysis
Mean
Mean Label spl
Label spl Assay% Assay STDEV RSD%
Assay% STDEV RSD%
Assay% Claim% No.
Claim% No. %

L
7

2
2

100 3
100 3

,4 4

5
5

6
6

Acceptance criteria: RSD of six determinations must be less than 2/o as per repeatability day1"

Remarks: ! complies ! Not Complies

Data Attached

Md. Tarikul lslam Bassunia Md. Moniruzzaman

Farhana Sultana
 ar.tAlWtCal M ETH OD VA!-iDATION PROTOCOL
Material Name Montelukast Sodium INN

Test parameter Assay bY HPLC

TPl1113010101

8.3 LinearitY and range - -^-^-&:^

proportional
(within a given range) to obtain a response that is directly
Linearity evaruates the analyticar procedure,s abirity defined
is linear, the test results are directly, or by well
to the concentration (amount) of analyte standard. rf the method a given range' Range is the
of analyte in samples within
rnathematical transformation, proportional to the concentration
(amounts) of analyte in the sample (including these concentrations)
interval between the upper and lower concentrations
procedure has a suitable level of precision, accuracy, and linearity'
for which it has been demonstrated that the analytical
Experimental Determination of Linearity & range

* Prepare a stock standard solution'

and 1'40%
* Prepare standards at about 60%,80%,1'00%'120%
method
* Run each samples using the proposed analytical
coefficient and y-intercept'
* Using linear regressionlnalysis, calculate the correlation

Result:

Peak area of
Montelukast

Montelukast
of must be > 0'99
The calibration curve should be linear (visually) and the value
R2
Acceptance criteria:
Remarks: I Complies E Not Complies

Data Attached

Checked by: Approved bY:

Prepared bY:
Md. Tarikul lslam Bassunia Md. Moniruzzaman
Name Farhana Sultana

Asst. Manaqer, QC QA_lVl_anager

Desionation
Signature
PD Officer
[--c.r ha.r.a.
tilrlt"' *Wze"^
- Rq's9'tv
Date "t{t0)ll4 Y5 ,' 6 i,t t1
 ANALYTICAL METHOD VALIDATION PROTOCOL
Montelukast Sodium INN
feritas Material Name

rP/fi]-,3]t1lot Test paranneter Assay bY HPLC

8.4 SpecificitY
8.4.1 Blank effect
presence of components that may be expected to be
specificity is the ability to assess unequivocally the analyte in the
must be inarguable data for a method to be specific'
present such as impurities, degradation products, and excipients. There
from other species which might be present;
specificity measures only the desired component without interference
separation is not necessarily required'
Experimental Determination of Specificity:
*Preparestandardsolutionaccordingtomethod&measurePeakArea.
* Prepare Blank & run according to propose method
i. Determine percentage of interference due to blank"

%lnterference bY Blank

due to blank solution'

Acceptance criteria: There will be no interference at the peak reglon of Montelukast

Remarks: u complies n Not ComPlies

Data Attached

8.6 System SuitabilitY

system suitability is the evaluation of the components of an

analyticai system to show that the performance of a systern
evaluation usually contains its own set of parameters;
for
meets the standards required by a method. A system suitability
chromatographic assays, these may include tailing factors, resolution,
and precision of standard. peak areas' and
tinnes, theoretical plates, and calibration curve iinearity"
comparison to a confirmation standard, capacity factors, retention

Experimental Determination of System Suitability

* Prepare a stock standard solution'

* PrePare standards at 100% label
.|. Run the standard for six times according to proposed method
parameters and also ealculate
* Determine the peak area, tailing factoi, retention time & other system suitability
their RSD values where applicable'

Md. Tarikul lslam Bassunia

 NruNIWICNI METHOD VALIDATION PROTOCOL
Montelukast Sodium INN L! of LZ
Material Name
veritas
T?/LL13O7Ol0t Test parameter
Protocol No.:
VPL/MV-01s

Result:

lnjection Tailing Factor Theoretical Plate Count

Area Retention Time (min.)
No.

Avg. (n=6)
%RSD

RSD%: NMT 2.0% NMT 2 NLT 15OO

Limit: RSD%: NMT 2.0%

Remarks: ! Complies tr Not ComPlies

Data Attached
r

9. MAINTENANCE OF RAW DATA \

auditing the study during its progress, and before the issuance
original data, or copies, will be available at ec lab to facilitate
paper data, all magnetically encoded records' and a copy of
of a final report. when the final report is completed, all original
Assurance Department'
the final report will be retained in the archives of Quality

10" Deviation

justified.
lf any deviation observed it must be mentioned and

11. REPORT
blank spaces in calculation and result will filled and will
be
Upon completion of the method validation experiments, the
piesented to QAM for, evaluation, review, and comment, after which a final
report will be issued'
& Recommendation as a result of Method Validation study shall be prepared and
summary & Discussion and conclusion
documented in the following format:

Md. Tarikul Islam Bassunia

 ANATYTICAL M ETH OD VALI DATION PROTOCOL
Material Name Montelukast Sodium INN Page No. 72of 12

$ritas
ProtocolNo.: Method No. TP11.1.73010/01 Test parameter Assay by HPLC
vPr/MV-01s Revision No. 00 Date Revised New

Seritas
Department : Product DevetL
Material Name:
Summary & Discussion :

The data collected so far suggests that method is suitable and there is no significant change with respect to the established
limits.
Conclusion & Recommendation :
Based on this it is concluded that this method can be assigned as Analytical method for the assty of
C"rt''f'*t" .f Vrl'td.tl"*

t--_l Method is considered as validate.

l--J Method is not suitable.

Prepared by:
Signature:
Date :

Checked by:
Signature
Date

Approved by:
Signature:
Date :

12. Revision History

Sl. No. Versi6n & lssue details Revision/Change in brief Reason for change

1 Version : 00, Date of issue: Newly created. lnitial document

March-2014

Preoared bv: Checked bv: Aooroved bv:

Name Farhana Sultana Md. Tarikul lslam Bassunia Md. Moniruzzaman

Desiqnation PD Officer Asst. Manaqer, QC QA Manaqer

Signature
Fc,v L^r-..c' ffilk,.,l &*-*
Date 1l.-l() \l14 "L\ / O',+,,' t4 ryq* $(5''V