ANALYTICAL METHOD VERIFICATION PROTOCOL FOR CONTENT UNIFORMITY OF ERYTHROMYCIN 250 MG GASTRO-RESISTANT TABLETS

ANALYTICAL METHOD VERIFICATION PROTOCOL FOR CONTENT UNIFORMITY OF ERYTHROMYCIN 250 MG GASTRO-RESISTANT TABLETS

Superseded Protocol No.	Nil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 method of analysis for the content uniformity of Erythromycin 250 mg gastro-resistant tablets by considering thefollowing parameters:

Parameters	Erythromycin 250 mg gastro-resistant tablets
Specificity	yes
Precision
System Precision	yes
Method Precision	yes
Intermediate Precision	yes
System Suitability	yes

• Scope :

This protocol is applicable for the verification of content uniformity of Erythromycin 250 mg gastro-resistant tablets.

• 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	Erythromycin 250 mg Gastro-Resistant Tablets Method of Analysis
Specification Limits	Content uniformity Acceptance Value : NMT-15

• Methodology:

Content uniformity

Chemical, reagents and filters:

Table 1.0: Chemical, reagents and filters

Sr. No	Material /Chemicals/Filters	Grade
1.	Dipotassium hydrogen phosphate Anhydrous (K2HPO4)	AR Grade
2.	Disodium hydrogen phosphate Dodecahydrate (Na2HPO4.12H2O)	AR Grade
3.	Citric Acid Monohydrate	AR Grade
4.	Acetonitrile	HPLC Grade
5.	Methanol	HPLC Grade
6.	Orthophosphoric acid	HPLC Grade
7.	Water	Milli Q Water
8.	Tert-Butanol	HPLC Grade

Chromatographic conditions:

Column                :    Stainless steel column (25 cm x 4.6 mm) packed with styrene-demethylbenzene copolymer (8 µm) with a pore size of 100 nm (PLRP-S is suitable).

Flow rate              :    2.0 ml/min

Wavelength         :    215 nm

Temperature            :        Maintained at 70°C

Injection volume  :    80 µl (0.08 ml)

Note: Column and at least one third of the tubing preceding the column shall maintain at 70°C.

Preparation of solvent A:

Mix 1 volume of methanol and 3 volumes of citro-phosphate buffer pH 7.0.

Preparation of citro-phosphate buffer pH 7.0:

Mix 82.4 ml of a 7.15% w/v solution of disodium hydrogen orthophosphate dodecahydrate with 17.6 ml of a 2.1 % w/v solution of citric acid monohydrate.

Preparation on mobile phase:

To 50 ml of a 3.5% w/v solution of Dipotassium hydrogen orthophosphate adjusted to pH 9.0 with 1M orthophosphoric acid add 400 ml of water, 180 ml of 2-methylpropan-2-ol and 30 ml of Acetonitrile and dilute to 1000 ml with water.

Sample preparation:

Weigh and transfer one tablet (if necessary, remove the coating from the tablet using a sharp blade, taking care not to damage the core tablet) in to 50 ml volumetric flask, Add about 25 ml of a mixture of 1 volume of methanol and 3 volumes of citro-phosphate buffer pH 7.0, dissolve, dilute with same solvent. Mix well and filter.

Standard preparation:

Transfer about 50 mg of Erythromycin RS/WS accurately weighed to a 10 ml volumetric flask, add 5.0 ml of mixture of 1 volume of methanol and 3 volumes of citro-phosphate buffer pH 7.0, dilute to volume with same solvent and mix.

Procedure:

Separately inject equal volumes (80µl) of standard and sample preparations into the chromatograph, record the chromatographs, and measure the responses for the major peaks. Calculate the active substance content expressed as percentage of label claim of each tablet using the formula.

Calculation:

Sample peak area    Std. Weight        50        Purity of Std.          100

—————————x——————-x———-x———————–x—————– = content in %

Standard peak area         10                1             100                  Label claim

Calculate the acceptance value.

Acceptance Criteria

1. Acceptance value (AV) =   M – X  + ks

Where,

k = acceptability constant

If the number of tablets is 10, then k = 2.4

If the number of tablets is 30, then k = 2.0

s = Sample standard deviation

• Calculate the mean of individual contents (expressed as % of label claim) and note down as X.
• Determine the value of M as follows
• If 98.5 % < x < 101.5 % then M = X
• If X < 98.5% then M = 98.5 %
• If X > 101.5 % then M = 101.5 %

Calculate the acceptance value (AV) by substituting the values of M, X and s respectively.

The requirements for dosage uniformity are met if the acceptance value of the first 10 dosage units is less than or equal to L1 (L1 = 15.0)

• Verification parameters:

The following parameters to be perform for the verification activity.

       Specificity

       Precision

         System Suitability

• 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 solution and placebo.

Specificity of test method should be established by separately injecting blank, placebo solution, standard solution, test solution and spike test solution. Test solution to be prepared as per method of analysis.

Blank: Solvent A

Note: Quantity of standard can be reduce to achieve the final concentration same.

Sample preparation:

Weigh and transfer one tablet (if necessary, remove the coating from the tablet using a sharp blade, taking care not to damage the core tablet) in to 50 ml volumetric flask, Add about 25 ml of a mixture of 1 volume of methanol and 3 volumes of citro-phosphate buffer pH 7.0, dissolve, dilute with same solvent. Mix well and filter.

Standard preparation:

Transfer about 50 mg of Erythromycin RS/WS accurately weighed to a 10 ml volumetric flask, add 5.0 ml of mixture of 1 volume of methanol and 3 volumes of citro-phosphate buffer pH 7.0, dilute to volume with same solvent and mix.

Preparation of solvent A: (Diluent)

Mix 1 volume of methanol and 3 volumes of citro-phosphate buffer pH 7.0.

Placebo solution: Weight and powder quantity of 82 mg of placebo powder into 50 ml volumetric flask, Add about 25 ml of a mixture of 1 volume of methanol and 3 volumes of citro-phosphate buffer pH 7.0, dissolve, dilute with same solvent. Mix well and filter.

Spiked test solution:

Weight and powder quantity of 82 mg of placebo powder + 250 mg of erythromycin API into 50 ml volumetric flask, Add about 25 ml of a mixture of 1 volume of methanol and 3 volumes of citro-phosphate buffer pH 7.0, dissolve, dilute with same solvent. Mix well and filter.

Procedure: Inject the preparation of blank solution, placebo solution, Standard solution (six replicate), test solution and spike test solution on a HPLC system with a Diode array detector (DAD) as follows in Table 2.0. Determine the purity of the individual peak of interest. Record the retention times of all peaks obtained from the respective solution.

Table 2.0: Sequence of Injection (Specificity)

Solution	No of Injection to be injected in Sequence
Blank solution	1
Placebo solution	1
Standard solution	6
Sample solution	1
Spike test solution	1
Standard solution + Bracketing	1

Table 3.0: Specificity data

Sr. No	Sample	RT (min.)	Peak purity
	Blank solution		NA
	Placebo solution		NA
	Standard solution	Erythromycin
	Sample solution	Blank		NA
Placebo		NA
Erythromycin
	Spike solution	Blank		NA
Placebo		NA
Erythromycin

Acceptance Criteria:

1. There should be no interference of the solution A and placebo at the retention time of analyte peak.
2. Analyte peak in standard solution, test solution and spike test solution should be spectrally pure.
   1. Precision:
      1. System Precision:

The system precision is the closeness of agreement between the responses of detector. It is usually expressed as the relative standard deviation (RSD). Standard solution shall be prepared as per method of analysis and six replicate injections to be injected in sequence and recorded the area response and retention time of main analyte peak. Calculate the % RSD of area and Retention time of main analyte peak.

Table 4.0: System Precision- Repeatability of Standard Injections

Sr. No.	Erythromycin
Peak Area	Retention time (min.)
1
2
3
4
5
6
Mean
% RSD

Acceptance criteria:

% RSD for peak area and retention time of replicate standard solution injections should be NMT 2.0% and 1.0% respectively.

• 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 as the standard deviation and the relative standard deviation. Check for system suitability criteria.

Test Procedure for Content uniformity:

Prepare the Ten sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % content uniformity, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.

Table 6.0: Method precision for content uniformity of Erythromycin 250 mg tablet

Tablet No.	Erythromycin
Peak Area	% Release Content
1
2
3
4
5
6
7
8
9
10
Mean
Minimum
Maximum
SD
% RSD
≤ L1% , L1 = 15

Acceptance Criteria:

1. L1 (VA) ≤ 15.0 (For 10 Dosage Units)
2. L2 ≤ 25.0 (For 30 Dosage Units)
   1. Intermediate Precision ( Ruggedness ):

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

Standard solution shall be prepared as per method of analysis and six replicate injections to be injected in sequence and recorded the area response and retention time of main analyte peak. Calculate the % RSD of area and Retention time of main analyte peak.

Table 6.0: Repeatability of standard injections

Sr. No.	Erythromycin
Peak Area	Retention time (min.)
1
2
3
4
5
6
Mean
% RSD

Acceptance criteria:

% RSD for peak area and retention time of replicate standard solution injections should be NMT 2.0% and 1.0% respectively.

Test Procedure:

Prepare the ten sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % content uniformity, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.

          Table 7.0: Intermediate precision results for content uniformity

Tablet No.	Erythromycin
Peak Area	% Release Content
1
2
3
4
5
6
7
8
9
10
Mean
Minimum
Maximum
SD
% RSD
≤ L1% , L1 = 15

Acceptance Criteria:

1. L1 (VA) ≤ 15.0 (For 10 Dosage Units)
2. L2 ≤ 25.0 (For 30 Dosage Units)
   1. System Suitability

To ensure that during each analysis, the analytical procedure is giving accurate and precise results, system suitability parameters have been set. The set limits are given below. The data obtained will be summarized in Table.

Table 9.0: System Suitability Criteria

Sr. No.	System Suitability Parameter	Set Limits
1.	The % RSD value of erythromycin in standard solution.	NMT – 2.0%

• Incident /Deviation:

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

• Summary/ Conclusion / Recommendation:

Final conclusion should be drawn from analytical method verification for its use to analyze the content uniformity test of Erythromycin 250 mg Gastro-Resistant tablets by HPLC method.

Summary of verification report shall be prepare and accordingly conclusion and recommendation to be given.

Abbreviation

UOC                :           Uniformity of Content

            VERP              :           Verification Protocol

            SD                   :           Standard deviation

            HPLC              :           High performance liquid chromatography

DAD                :           diode-array detector

            RT                   :           Retention Time

            Temp.              :           Temperature

            ml                    :           Milliliter

            mg                   :           Milligram

            min.                 :           Minutes

            QA                   :           Quality Assurance

            QC                  :           Quality Control

            %                     :           Percentage

            ºC                    :            Degree centigrade

            µl                     :           Microlitre

            EP                   :           Europeian Pharmacopoeia

            CRS                :           Current reference standard

            RSD                :           Relative standard deviation

            NLT                 :           Not less than

NMT                :           Not more than

Ws                   :           Working standard

Wt                    :           Weight

Vol                   :           Volume

Revision History:

Revision No.	Details of changes	Reason
00	Nil	New Document