by ANALYTICAL METHOD VERIFICATION PROTOCOL FOR RELATED SUBSTANCE OF ERYTHROMIN 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| Superseded Protocol No. | Nil |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 |
- 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 related substance 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 related substance 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 | Related substance Erythromycin B : Not more than 5% m/m Erythromycin C : Not more than 5% m/m Impurity A : Not more than 3% m/m Impurity B : Not more than 3% m/m Impurity C : Not more than 3% m/m Impurity D : Not more than 3% m/m Impurity E : Not more than 3% m/m Impurity F : Not more than 3% m/m Any other impurity : Not more than 3% m/m Total impurities (Excluding Erythromycin B & Erythromycin C) : Not more than 7.0% m/m |
- Methodology:
Related substance
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 |
Preparation of solution 1:
Weigh and powder 20 tablets (if necessary, remove the coating from 20 tablets using a sharp blade, taking care not to damage the cores, weigh the cores and powder). Dissolve a quantity of the powdered tablets containing 40 mg ofErythromycin in 10 ml of a solvent A, filter and use the filtrate.
Preparation of solution 2:
Weigh accurately 40 mg of Erythromycin A CRS/WS and transfer into 10 ml volumetric flask, dissolve and dilute to volume with solvent A.
Preparation of solution 3:
Weigh accurately 20 mg of each Erythromycin B CRS/WS and Erythromycin C CRS transfer into 100 ml volumetric flask, dissolve and dilute to volume with solvent A.
Preparation of solution 4:
Weigh accurately 12 mg of Erythromycin A CRS/WS and transfer into 100 ml volumetric flask, dissolve and dilute to volume with solvent A.
Preparation of solution 5:
Weigh accurately 5 mg of N-Demethylerythromycin A EPCRS and transfer into 25 ml volumetric flask, dissolve in solution 3, add 1 ml of solution 2 and make up to volume with solution 3.
Preparation of solution 6:
Transfer 40 mg of Erythromycin A CRS to a glass vial and spread evenly such that it forms a layer not more than about 1 mm thick. Heat at 130°C for 4 hours, allow to cool and dissolve in sufficient of solvent A to produce 10 ml (generation of impurities E and F). The solutions can be used within one day if stored at 5°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.
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 : 100 µl (0.1 ml)
Note: Column and at least one third of the tubing preceding the column shall maintain at 70°C.
Procedure 1: benzene
Inject 100 µl (0.1 ml) of each solution. For solution (1) allow the chromatography to proceed for 5 times the retention time of the peak corresponding to Erythromycin A.
When the chromatograms are recorded using the prescribed conditions the retention time of Erythromycin A is about 15 minutes. The retention times relatives to Erythromycin A are:
Impurity RRT
Impurity A about 0.3
Impurity B about 0.45
Erythromycin C about 0.5
Impurity C about 0.9
Impurity D about 1.4
Impurity F about 1.5
Erythromycin B about 1.8
Impurity E about 4.3
Identify any peaks in the chromatogram obtained with solution (1) corresponding to impurities E and F using solution (6) and multiply the areas of these peaks by the corresponding correction factors: Impurity E, 0.09; Impurity F, 0.15.
Limits:
In the chromatogram obtained with solution (1) the area of any peak other than those peaks corresponding to Erythromycin A, Erythromycin B and Erythromycin C, identified from the peaks in the chromatograms obtained with solution (2) and (3), is not greater than the area of the principal peak in the chromatogram obtained with solution (4) (3%) and the sum of the areas of any such peaks is not greater than 2.3 times the area of the principal peak in the chromatogram obtained with solution (4) (7%). Disregard any peak with an area less than 0.02 times the area of the principal peak in the chromatogram obtained with solution (4) (0.06%).
The content of each of Erythromycin B and Erythromycin C, as determined under assay is not more than 5%.
Related substance Erythromycin content = Sum of Erythromycin A + B + C
- Verification parameters:
The following parameters to be perform for the verification activity.
Specificity
Precision
System Suitability
Note: Reduce quantity of standards/Impurity can be use by keeping final concentration same.
- 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.
The Chromatographic condition and solution preparations are same as described in assay test, therefore the specificity shall be considered from assay test.
- Precision:
- System Precision:
The system precision is the closeness of agreement between the responses of detector. It is usually expressed as the standard deviation (SD) or the relative standard deviation (RSD). Solution 4 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 2.0: System Precision- Repeatability of Standard Injections
| Sr. No. | Erythromycin A | |
| 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 5.0% 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 Related substance:
Prepare the six sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % impurity, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 3.0: Method precision results for related substance
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total impurity ( ≥0.06%) | ||||||||
| Impurity A | Impurity B | Impurity C | Impurity D | Impurity E | Impurity F | Erythromycin B | Erythromycin C | 1 | 2 | |||
| RRT | ||||||||||||
| 1 | ||||||||||||
| 2 | ||||||||||||
| 3 | ||||||||||||
| 4 | ||||||||||||
| 5 | ||||||||||||
| 6 | ||||||||||||
| Mean | ||||||||||||
| % RSD |
Acceptance Criteria:
- % Impurity should be within acceptance limit as per specification.
- % RSD of known, unknown and total impurity content should be not more than the limits specified below.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for % RSD.
- 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.
Solution 4 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: Repeatability of standard injections
| Sr. No. | Erythromycin A | |
| 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 six sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % related substance, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 5.0: Intermediate precision results for related substance
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total impurity ( ≥0.06%) | ||||||||
| Impurity A | Impurity B | Impurity C | Impurity D | Impurity E | Impurity F | Erythromycin B | Erythromycin C | 1 | 2 | |||
| RRT | ||||||||||||
| 1 | ||||||||||||
| 2 | ||||||||||||
| 3 | ||||||||||||
| 4 | ||||||||||||
| 5 | ||||||||||||
| 6 | ||||||||||||
| Mean | ||||||||||||
| % RSD |
Acceptance Criteria:
- % Impurity should be within acceptance limit as per specification.
- % RSD of known, unknown and total impurity content should be not more than the limits specified below.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for % RSD.
Table 6.0 Cumulative results of Method Precision and Intermediate Precision
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total impurity (≥0.06%) | ||||||||
| Impurity A | Impurity B | Impurity C | Impurity D | Impurity E | Impurity F | Erythromycin B | Erythromycin C | 1 | 2 | |||
| Method Precision | ||||||||||||
| RRT | ||||||||||||
| 1 | ||||||||||||
| 2 | ||||||||||||
| 3 | ||||||||||||
| 4 | ||||||||||||
| 5 | ||||||||||||
| 6 | ||||||||||||
| Intermediate Precision | ||||||||||||
| RRT | ||||||||||||
| 1 | ||||||||||||
| 2 | ||||||||||||
| 3 | ||||||||||||
| 4 | ||||||||||||
| 5 | ||||||||||||
| 6 | ||||||||||||
| Mean | ||||||||||||
| SD | ||||||||||||
| % RSD |
Acceptance Criteria:
- % Impurity should be within acceptance limit as per specification.
- % RSD of known, unknown and total impurity content should be not more than the limits specified below.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for % RSD.
- 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 7.0: System Suitability Criteria
| Sr. No. | System Suitability Parameter | Set Limits |
| 1. | Resolution factor between the peaks corresponding to N-demethyleryhromycin A and Erythromycin C | At least 0.8 |
| 2. | resolution factor between the peaks corresponding to N-demethylerythromycin A and Erythromycin A | At least 5.5 |
If necessary, adjust the concentration of 2-methylpropan-2-ol in the mobile phase (180 ml has been found to be suitable) or reduce the flow rate to 1.5 or 1.0 ml per minutes.
- 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 related substance test of Erythromycin 250 mg Gastro-Resistant tablets by HPLC.
The analytical methodology is same for Related substance test and related substance test, therefore the specificity test shall be performed once and same shall be considered for both tests.
Summary of verification report shall be prepare and accordingly conclusion and recommendation to be given.
Abbreviation:
ASS : Related substance
VERP : Verification Protocol
REL : Related Substances
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 |
