ANALYTICAL METHOD VERIFICATION PROTOCOL FOR IDENTIFICATION BY TLC OF ERYTHROMYCIN Ph. Eur.

ANALYTICAL METHOD VERIFICATION PROTOCOL FOR IDENTIFICATION BY TLC OF ERYTHROMYCIN Ph. Eur.

Superseded Protocol No.	Nil
Effective Date

Table of contents :

Sr. No.	Subject	Page No.
	Protocol Approval
	Objective
	Scope
	Responsibility
	Product profile
	Methodology
	Verification parameters
	Incident/Deviation
	Summary/Final conclusion/Recommendation
	Abbreviation
	Revision History

1. Protocol Approval:

Prepared By:

Functional Area	Name	Designation	Signature / Date
Quality Control

Reviewed By:

Functional Area	Name	Designation	Signature / Date
Quality Assurance
Head Quality Control

Approved By:

Functional Area	Name	Designation	Signature / Date
Head QA

• Objective:

The objective of this protocol is to verify the suitability of Identification by TLC (Thin Layer Chromatography) of Erythromycin Ph. Eur. by considering thefollowing parameters:

Parameters	Erythromycin
Specificity	yes
Precision
Method Precision	yes
Intermediate Precision	yes
System Suitability	yes

• Scope :

This protocol is applicable for the verification of Identification of Erythromycin Ph. Eur. by TLC.

• Responsibility of Validation Team:

Departments	Responsibilities
QC	Preparation & Review of Protocol.
Analysis of samples and recording of data.
Compilation and checking of data
Preparation of Summary Report.
To impart training of protocol to concerned department/persons.
QA	Review of protocol.
Co-ordination with QC to carryout Verification.
Review of data and summary report.
Head QA	Approval of Protocol

• Product Profile:

Category	Macrolide antibiotic
Reason for verification	First Verification
Active Ingredient	Erythromycin Ph. Eur.
Method Reference	European Pharmacopoeia
Specification Limits	Principal spot due to test solution is similar in position, color and size to the principal peak obtained with reference solution.

• Methodology:

Chemical, reagents and filters:

Table 1.0: Chemical, reagents and filters

Sr. No	Material /Chemicals/Filters	Grade
	Glacial Acetic acid	AR/HPLC grade
	Methanol	AR/HPLC grade
	Sulphuric acid	AR/HPLC grade
	2-Propanol	AR/HPLC grade
	Ammonium acetate	AR/HPLC grade
	Ammonia	AR/HPLC grade
	Ethyl Acetate	AR/HPLC grade
	Anisaldehyde	AR/HPLC grade
	Silica Gel TLC plate	Merck or equivalent

Reagent preparations:

Anisaldehyde solution:

85 ml of ice-cooled methanol mixed with 10 ml of glacial acetic acid, 5 ml of sulphuric acid, 5 ml of sulphuric acid and 0.5 ml of p-anisaldehyde.

Ammonium Acetate solution:

• Weigh 3.75 g of ammonium acetate in 25 ml volumetric flask.
• Dissolve and dilute to volume with water.
• Adjust the pH to 9.6 with ammonia solution.

Test solution

• Weigh accurately 10 mg of sample in 10 ml volumetric flask.
• Dissolve and dilute to volume with methanol.

Reference solution (a):

• Weigh accurately 10 mg Erythromycin A CRS in 10 ml volumetric flask.
•  Dissolve and dilute to volume with methanol.

Reference solution (b):

• Weigh accurately 20 mg of Spiramycin CRS in 10 ml volumetric flask.
• Dissolve and dilute to volume with methanol.

Mobile phase

Mix 20 ml of 2-Propanol, 40 ml of ammonium acetate solution and 45 ml of ethyl acetate, allow for settle and use the upper layer as mobile phase.

Procedure:

1. Obtain a silica gel TLC plate of dimension 10 x 16 cm.
2. Pencil mark the TLC plate as Test solution as T, reference solution (A) as ‘a’ and reference solution (b) as ‘b’.
3. Spot accurately 10 µl of Test solution, reference solution (a) and reference solution (b).
4. Develop the TLC plate over 2/3 of the plate using above mobile phase.
5. Remove the TLC plate and air dry it.

Detection:

• Spray the plate with anisaldehyde solution and heat at 110°C for 5 minutes.
• Examine the late in day light.

Result:

The principle spot due to test solution is similar in position, colour and size to the principal spot obtained with reference solution (a) and its position and colour are different from those of the spots obtained with reference solution (b).

• Verification parameters:

The following parameters to be perform for the verification activity.

      Specificity

      Precision

• Specificity:

Specificity of analytical method is its ability to assess unequivocally the analyte in presence of components that may be expected to be present in the blank and placebo.

Specificity of test method shall be established by separate injecting of blank solution (solvent mixture), reference solution (b) and test solution and on TLC plate. Sample solution shall be prepared as per method of analysis given in section 6.0.

Blank Solution:

Apply separately to the plate10 µl of Methanol.

Reagent preparations:

Anisaldehyde solution:

85 ml of ice-cooled methanol mixed with 10 ml of glacial acetic acid, 5 ml of sulphuric acid, 5 ml of sulphuric acid and 0.5 ml of p-anisaldehyde.

Ammonium Acetate solution:

• Weigh 3.75 g of ammonium acetate in 25 ml volumetric flask.
• Dissolve and dilute to volume with water.
• Adjust the pH to 9.6 with ammonia solution.

Test solution

• Weigh accurately 10 mg of sample in 10 ml volumetric flask.
• Dissolve and dilute to volume with methanol.

Reference solution (a):

• Weigh accurately 10 mg Erythromycin A CRS in 10 ml volumetric flask.
•  Dissolve and dilute to volume with methanol.

Reference solution (b):

• Weigh accurately 20 mg of Spiramycin CRS in 10 ml volumetric flask.
• Dissolve and dilute to volume with methanol.

Mobile phase

Mix 20 ml of 2-Propanol, 40 ml of ammonium acetate solution and 45 ml of ethyl acetate, allow for settle and use the upper layer as mobile phase.

Procedure:

• Obtain a silica gel TLC plate of dimension 10 x 16 cm.
• Pencil mark the TLC plate as Test solution as T, reference solution (A) as ‘a’ and reference solution (b) as ‘b’.
• Spot accurately 10 µl of Test solution, reference solution (a) and reference solution (b).
• Develop the TLC plate over 2/3 of the plate using above mobile phase.
• Remove the TLC plate and air dry it.

Detection:

1. Spray the plate with anisaldehyde solution and heat at 110°C for 5 minutes.
2. Examine the late in day light.

Acceptance Criteria:

1. There shall be no interference of the Methanol at zone of Erythromycin A and Spiramycin region.
2. The principle spot due to test solution is similar in position, colour and size to the principal spot obtained with reference solution (a) and its position and colour are different from those of the spots obtained with reference solution (b).
   1. Precision:
      1. Method Precision:

The precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatability to multiple samplings of homogenous sample. It is usually expressed by visual inspection in UV cabinet.

Test Procedure:

Prepare the six sample of same batch and analyze as per method of analysis, record the observation as given below in table.

Table 2.0: Method precision results for TLC

Sample no.	Sample wt. (mg)	Observation
1
2
3
4
5
6

Acceptance Criteria:

The principle spot due to test solution is similar in position, colour and size to the principal spot obtained with reference solution (a) and its position and colour are different from those of the spots obtained with reference solution (b).

• Intermediate Precision (Ruggedness):

Intermediate precision expresses within laboratory variation with different analysts or different/same equipment or different days using same batch of drug substance as per method of analysis.

Test Procedure:

Prepare the six sample of same batch and analyze as per method of analysis, record the record the observation as given below in table.

Table 3.0: Intermediate precision results for TLC

Sample no.	Sample wt. (mg)	Observation
1
2
3
4
5
6

Acceptance Criteria:

The principle spot due to test solution is similar in position, colour and size to the principal spot obtained with reference solution (a) and its position and colour are different from those of the spots obtained with reference solution (b).

Table 4.0: Cumulative Observation of Method Precision and Intermediate Precision

Sample No.	Sample wt. (mg)	Identification
Observation
ANALYST I
1
2
3
4
5
6
ANALYST II
1
2
3
4
5
6

Acceptance Criteria:

The principle spot due to test solution is similar in position, colour and size to the principal spot obtained with reference solution (a) and its position and colour are different from those of the spots obtained with reference solution (b) by two different analyst observation.

• Incident /Deviation:

Any incident or deviation observed during analytical method verification shall be recorded and reported as per SOP.

• Summary/ Conclusion / Recommendation:

Final conclusion should be drawn from analytical method verification for its use to analyze the Identification by TLC method.

Summary of verification report shall be prepared and accordingly standard testing procedure to be updated if required.

Abbreviation

REL                 :           Related substance

            VER                :           Verification

            P                      :           Protocol

            TLC                 :           Thin Layer Chromatography

            ml                    :           Milliliter

            mg                   :           Milligram

            min.                 :           Minutes

            QA                   :           Quality Assurance

            QC                  :           Quality Control

            %                     :           Percentage

            hrs                   :           Hours

            µm                   :           Micrometer

            µl                     :           Microlitre

            Ph.Eur.            :           European Pharmacopoeia

            NLT                 :           Not less than

NMT                :           Not more than

Vol                    :           Volume Revision History:

Revision No.	Details of changes	Reason
00	Nil	New Document