PHARMA DEVILS

QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

ANALYTICAL METHOD VALIDATION

FOR
GLIMEPIRIDE (CLEANING VALIDATION)
 PHARMA DEVILS
QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

Table of Contents

S.No. Title Page No.

1. Protocol Approval Sheet 4
2. Objective 5
3. Scope 5
4. Introduction and Overview 5
5. Validation Team 5
6. Methodology 5
7. Equipment and Materials 7
8. Validation Parameters of Assay Method 7
9. System Suitability 8
10. Specificity 9
11. Accuracy 9
12. Linearity and Range 10
13. LOD 10
14. LOQ 11
15. Overall Conclusion 12
16. Abbreviation 14
17. Post Approval Sheet 14
 PHARMA DEVILS
QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

1.0 Protocol Approval Sheet:

Prepared By Signature Date

(Name & Designation)
Quality Control

Checked By Signature Date

(Name & Designation)
Quality Control

Quality Assurance

Approved By Signature Date

(Name & Designation)
Quality Assurance
 PHARMA DEVILS
QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

2.0 Objective: To perform the validation of analytical method for determination of traces of API content
of glimepiride in swab by HPLC, And documented evidence and provide the procedure for the same.

3.0 Scope: The scope should evaluate the acceptability of an analytical method for determination of trace
of API content in swab by HPLC. This protocol should define the procedure, documentation,
reference acceptance criteria and result evaluated for determination of trace of API content in Swab by
HPLC.

4.0 Introduction and Overview:

4.1 This protocol provides detailed information on all the aspects of validation of the method of
analysis for trace of API Content.
4.2 All instrument/equipment shall be qualified and validated.
4.3 Acceptance criteria and rationale for assessing validation are as per ICH guideline Q2(R1)

5.0 Validation Team:

S.No. Responsibility Department
1. Development of Validation Protocol QC
2. Execution of Validation Protocol QC
3. Review and assembling of data into final Protocol QC
4. Approval Protocol QA

6.0 Methodology:
Chromatographic Conditions:
Column : : Stainless steel column 125 x 4.6 mm, packed with octadecylsilane bonded to
porous silica 5 µm or equivalent
Flow Rate : 1.0 ml/min
Detector : 228 nm
Temperature : 25ºC
Injection : 20 µL
Volume
Run Time : 12 min
Diluent: Prepaid a mixture of 80 volume acetonitrile and 20 volume of water.
 PHARMA DEVILS
QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

Preparation of Mobile Phase:

Prepare a mixture of 50 volume of a solution containing 0.5 g of monobasic sodium phosphate in 500
ml water, adjusted to Ph 2.1 with orthophophoric acid and 50 volume of acetonitrile.
Preparation of Standard Solution:
Weigh accurately about 20 mg of Glimepiride working standard into a 100 mL volumetric flask add
10 ml of diluent and sonicate to dissolve. Dilute to volume with same solvent. Mix well Filter the
solution through 0.45 µ nylon membrane filter. Take 1 ml of solution into 100 ml volumetric flask
and dilute to volume with Diluent and mix well. Diluted 1.0 ml of this solution to 10 ml with diluent.
Procedure:
Inject 20 µl of blank, standard solution (five replicate injections), sample solution (in duplicate).
Record the chromatograms and measure the peak areas.
Evaluation of System Suitability:
1. % RSD of Glimepiride and peak for 5 replicate injection of standard solution is not more than 2.0.
2. The column efficiency as determined from the Glimepiride peak is not less than 1500.
3. The tailing factor for Glimepiride peak is not more than 2.0.
Calculations:
Glimepiride (%) labeled amount
AT WS 1 1 P
-------X ---------X--------X--------X--------- X 1000 =----------ppm
AS 100 100 10 100

AT = Area count of Glimepiride peak in sample solution

AS = Average area count of Glimepiride peak in standard solution

WS = Weight of Glimepiride working standard taken in mg

P = Purity of Glimepiride working standard used (On as is basis)

 PHARMA DEVILS
QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

Equipment’s and Materials:

Equipment’s
Instrument Name Instrument ID Make
HPLC --- Shimadzu
Analytical Balance --- Sartorius
Analytical Balance --- Sartorius
pH Meter --- V-Tech

Table-1
Standards, Chemicals, Column and Samples
1. Glimepiride working standard No. ---
2. Potency of Glimepiride Working Standard 98.96%
3. Acetonitrile (HPLC Grade) ---
4. Orthophophoric acid ---
5. Sodium Dihydrogen orthophosphate ---

7.0 Method Validation parameters:

Analytical method has to be validated as per protocol on validation of API Swab method of
Glimepiride in Glimepiride Swab for the analytical parameters of System Suitability, Specificity,
System Precision, Precision, Linearity and Range, Accuracy.

8.0 System Suitability:

8.1 Objective: To demonstrate and verify that the system suitability parameters of the
chromatographic system are adequate for the subjected analysis.
8.2 Procedure: Inject standard solution (Six replicates) as described under Standard Test
Procedure
The results shall be tabulated in table-2
 PHARMA DEVILS
QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

Table-2
System Suitability
Replicates Glimepiride
1 `
2 54496
3 54687
4 54883
5 54959
6 55402
Avg. 54837
Std. Dev. 326.273
% RSD 0.59
Theoretical Plates 4083
Tailing Factor 0.93

Acceptance Criteria: Relative standard deviation for peak areas of Glimepiride should not be more
than 2.0%, tailing factor should be more than 2.0 and theoretical plates should not be less than 1500.

9.0 Specificity:
9.1 Objective: To demonstrate the ability of the analytical method to separate the analyze and
there is no interference in the peaks of analyze due to other component that may be present in
the sample matrix.
9.2 Procedure: Peak purity analysis shall be done by injecting one injection of blank, one
injection of placebo preparation (blank with swab stick), and one injection of standard
preparation in to the HPLC using chromatographic condition as per methodology.
Table-3
S.No. RT Peak Purity Purity Threshold
Blank NA NA NA
Placebo NA NA NA
Standard of Glimepiride 6.41 NA NA

Acceptance Criteria: No any peak inference shall be observed at retention time Glimepiride and
peak purity analysis for sample shall pass.
 PHARMA DEVILS
QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

10.0 Accuracy:
10.1 Objective: To study of the reliability, suability, and Accuracy of the method recovery
experiment were carried out for cleaning method validation for residual determination of
glimepiride. Accuracy is the analytical procedure is the closeness of the test result obtained by
that procedure to the true value.
Stock Solution: Weigh accurately about 20 mg of Glimepiride working standard into a 100 mL
volumetric flask add 10 ml of diluent and sonicate to dissolve. Dilute to volume with same
solvent. Mix well Filter the solution through 0.45 µ nylon membrane filter.
Standard solution: Take 1.5 ml of stock solution into 100 ml volumetric flask and dilute to
volume with Diluent and mix well.
 Accuracy Solution for 50% I: Select clean and dried 10 cm x 10 cm surface area stain less steel
plates spread 0.75 ml spiking solution on dried 10 cm x10 cm surface stain less steel plate, taking
utmost care to avoid any spillage. Dry the plate at room temperature using 10 ml of accurately
measured diluent recovered the test sample from 10 cm x10 cm surface area stain less steel plate,
by gently swirling filter and inject into HPLC. Finally recorded the area of test sample in swab
recovery on stainless steel plate calculate the % swab recovery.
Accuracy Solution for 50% II: Select clean and dried 10 cm x 10 cm surface area stain less steel
plates spread 0.75 ml spiking solution on dried 10 cm x10 cm surface stain less steel plate, taking
utmost care to avoid any spillage. Dry the plate at room temperature using 10 ml of accurately
measured diluent recovered the test sample from 10 cm x10 cm surface area stain less steel plate,
by gently swirling filter and inject into HPLC. Finally recorded the area of test sample in swab
recovery on stainless steel plate calculate the % swab recovery.
 Accuracy Solution for 50% III: Select clean and dried 10 cm x 10 cm surface area stain less
steel plates spread 0.75 ml spiking solution on dried 10 cm x10 cm surface stain less steel plate,
taking utmost care to avoid any spillage. Dry the plate at room temperature using 10 ml of
accurately measured diluent recovered the test sample from 10 cm x10 cm surface area stain less
steel plate, by gently swirling filter and inject into HPLC. Finally recorded the area of test sample
in swab recovery on stainless steel plate calculate the % swab recovery.
 Accuracy Solution for 100% I: Select clean and dried 10 cm x 10 cm surface area stain less
steel plates spread 0.75 ml spiking solution on dried 10 cm x10 cm surface stain less steel plate,
taking utmost care to avoid any spillage. Dry the plate at room temperature using 10 ml of
accurately measured diluent recovered the test sample from 10 cm x10 cm surface area stain less
 PHARMA DEVILS
QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

steel plate, by gently swirling filter and inject into HPLC. Finally recorded the area of test sample
in swab recovery on stainless steel plate calculate the % swab recovery.
 Accuracy Solution for 100% II: Select clean and dried 10 cm x 10 cm surface area stain less
steel plates spread 1.5 ml spiking solution on dried 10 cm x10 cm surface stain less steel plate,
taking utmost care to avoid any spillage. Dry the plate at room temperature using 10 ml of
accurately measured diluent recovered the test sample from 10 cm x10 cm surface area stain less
steel plate, by gently swirling filter and inject into HPLC. Finally recorded the area of test sample
in swab recovery on stainless steel plate calculate the % swab recovery.
 Accuracy Solution for 100% III: Select clean and dried 10 cm x 10 cm surface area stain less
steel plates spread 1.5 ml spiking solution on dried 10 cm x10 cm surface stain less steel plate,
taking utmost care to avoid any spillage. Dry the plate at room temperature using 10 ml of
accurately measured diluent recovered the test sample from 10 cm x10 cm surface area stain less
steel plate, by gently swirling filter and inject into HPLC. Finally recorded the area of test sample
in swab recovery on stainless steel plate calculate the % swab recovery.
 Accuracy Solution for 150% I: Select clean and dried 10 cm x 10 cm surface area stain less
steel plates spread 2.25 ml spiking solution on dried 10 cm x10 cm surface stain less steel plate,
taking utmost care to avoid any spillage. Dry the plate at room temperature using 10 ml of
accurately measured diluent recovered the test sample from 10 cm x10 cm surface area stain less
steel plate, by gently swirling filter and inject into HPLC. Finally recorded the area of test sample
in swab recovery on stainless steel plate calculate the % swab recovery.
 Accuracy Solution for 150% II: Select clean and dried 10 cm x 10 cm surface area stain less
steel plates spread 2.25 ml spiking solution on dried 10 cm x10 cm surface stain less steel plate,
taking utmost care to avoid any spillage. Dry the plate at room temperature using 10 ml of
accurately measured diluent recovered the test sample from 10 cm x10 cm surface area stain less
steel plate, by gently swirling filter and inject into HPLC. Finally recorded the area of test sample
in swab recovery on stainless steel plate calculate the % swab recovery.
.
 Accuracy Solution for 150% III: Select clean and dried 10 cm x 10 cm surface area stain less
steel plates spread 2.25 ml spiking solution on dried 10 cm x10 cm surface stain less steel plate,
taking utmost care to avoid any spillage. Dry the plate at room temperature using 10 ml of
accurately measured diluent recovered the test sample from 10 cm x10 cm surface area stain less
 PHARMA DEVILS
QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

steel plate, by gently swirling filter and inject into HPLC. Finally recorded the area of test sample
in swab recovery on stainless steel plate calculate the % swab recovery.
Table-8a
Recovery
Level No./Level in % Actual Amount of Amount of Glimepiride % Recovery
Glimepiride added in found in mg
mg
37.76 36.86 98.30
Level-1
37.77 37.25 98.63
(50%)
37.68 37.66 99.94
50.38 50.36 99.67
Level-2
50.85 50.54 99.70
(100%)
50.32 50.05 99.46
75.64 74.47 98.45
Level-3
75.8 74.98 98.92
(150%)
75.88 74.84 98.63
Acceptance Criteria: The recovery obtained should be within range 80-100%.

11.0 Linearity and Range:

11.1 Linearity: To demonstrate that the analytical method is capable to obtain test results, which
are directly proportional to the concentration of analyze.
11.2 Range: Provide acceptable degree of linearity, accuracy and precision when applied to
samples containing amounts of analyze within or at the extreme of the specified range of the
analytical procedure.
11.3 Procedure:
Stock Solution: Weigh accurately about 25 mg of Glimepiride working standard into a 100
mL volumetric flask add 10 ml of diluent and sonicate to dissolve. Dilute to volume with
same solvent. Mix well Filter the solution through 0.45 µ nylon membrane filter.
A. 25 %: Dilute 1 ml of stock solution in 100 ml volumetric flask and makeup with mobile
phase.
B. 50 %: Dilute 2 ml of stock solution in 100 ml volumetric flask and makeup with mobile
phase.
C. 75 %: Linearity Solution: Dilute 3.0 ml stock solution with 100 ml mobile phase
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QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

D. 100%: Dilute 4 ml of stock solution in 100 ml volumetric flask and makeup with mobile
phase
E. 125%: Dilute 5 ml of stock solution in 100 ml volumetric flask and makeup with mobile
phase.
F. The results of linearity shall be tabulated in table-9

Table-9
Linearity

S.No. Target Concentration in % Area of Glimepiride

1. 25 159814
2. 50 327259
3. 75 477501
4. 100 645380
5. 125 803938
Correlation Coefficient 0.9998
Acceptance Criteria: Correlation Coefficient should not be less than 0.99.

12.0 LOD and LOQ

LOD is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated,
under the stated experimental conditions.
Limit of detection (LOD) =3.3 x (residual standard deviation) /slope
LOQ is the lowest amount of analyte in a sample that can be quantitated with acceptable precision, under
the stated experimental conditions.
Limit of quantification (LOQ) = (10 x residual standard deviation)/slope
Preparation of LOD solution = about 0.2 ml of standard stock solution in 100 ml volumetric flask and
diluted up to mark with diluent. Injected in triplicate. The LOD experimental result are recorded.

Table-10
LOD STD

Trial Area of Glimepiride

1 15959
2 16072
3 15824
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ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

Preparation of LOQ solution = about 0.2 ml of standard stock solution in 100 ml volumetric flask and
diluted up to mark with diluent. Injected in triplicate. The LOD experimental result are recorded.

Table-11
LOQ STD
Replicates Glimepiride
1 54596
2 54496
3 54687
4 54883
5 54959
6 55402
Avg. 54837
Std. Dev. 326.273
% RSD 0.59
Theoretical Plates 4083
Tailing Factor 0.93

12.0 Overall Conclusion:

Cleaning method has been validated for clean for Glimepiride. To validate the method used, System
suitability, Accuracy, and Linearity and range has been performed and results obtained found within
the specified limits of each parameters.
Hence based on above it is concluded that the cleaning method has been validated successfully and
used for clean for Glimepiride.
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QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

13.0 Abbreviation:
SD Standard Deviation
RSD Relative Standard Deviation
S. No Serial Number
QC Quality Control
QA Quality Assurance
Mg Milligram
ICH International conference on harmonization
HPLC High Performance Liquid Chromatography
µl Microliter
ml/min Millimeter per minute
% Percentage
AR Analytical Reagent
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QUALITY CONTROL DEPARTMENT

ANALYTICAL METHOD VALIDATION FOR CLEANING VALIDATION

14.0 Post Approval Sheet:

Executed By Signature Date
(Name & Designation)
Quality Control

Reviewed By Signature Date

(Name & Designation)
Quality Assurance

Approved By Signature Date

(Name & Designation)
Quality Assurance