by ANALYTICAL METHOD VERIFICATION PROTOCOL FOR ASSAY 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| Superseded Protocol No. | Nil |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 |
- 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 assay 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 assay 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 |
| Specification Limits | Assay Calculated as the sum of Erythromycin A, Erythromycin B, Erythromycin C. 237.5 to 262.5 mg (95.0 to 105.0 %) |
- Methodology:
Assay
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/ HPLC water |
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 of Erythromycin 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-demethylbenze 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.
System suitability:
Inject 0.1 ml of solution (5). Adjust the sensitivity of the detector so that the height of the peaks is at least 25 % of the full scale of the recorder. The substances are eluted in the following order: N-Demethylerythromycin-A, Erythromycin C, Erythromycin A and Erythromycin B.
The test is not valid unless the resolution factor between the peaks corresponding to N-demethyleryhromycin A and Erythromycin C at least 0.8 and the resolution factor between the peaks corresponding to N-demethylerythromycin A and Erythromycin A is 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.
Procedure:
Inject alternately 0.1 ml of solution (1), (2) and (3). Calculate the content of Erythromycin A using the chromatograms obtained with solution (1) and (2) and from the declared content of C37H67NO13 in Erythromycin A CRS. Calculate the contents of Erythromycin B and Erythromycin C using the chromatograms obtained with solution (1) and (3) and from the declared contents of C37H67NO12 and C36H65NO13 in Erythromycin B CRS respectively.
Calculation:
- Content of Erythromycin A
Erythromycin A peak area in sol-1 x WS1 x 10 x PUS1 x Average weight
—————————————————————————————————
Erythromycin A peak are in sol-2 x 10 x WS2 x 100
- Content of Erythromycin B
Erythromycin B peak area in sol-1 x WS3 x 10 x PUS2 x Average weight
—————————————————————————————————
Erythromycin B peak are in sol-3 x 100 x WS2 x 100
- Content of Erythromycin C
Erythromycin C peak area in sol-1 x WS4 x 10 x PUS3 x Average weight
—————————————————————————————————
Erythromycin C peak are in sol-3 x 100 x WS2 x 100
Where,
WS 1 : Weight of Working /Reference standard of Erythromycin A in mg
WS 3 : Weight of Working /Reference standard of Erythromycin B in mg
WS 4 : Weight of Working /Reference standard of Erythromycin C in mg
WS2 : Weight of sample in mg
PUS1 : % Purity of Working/Reference standard of Erythromycin A
PUS2 : % Purity of Working/Reference standard of Erythromycin B
PUS3 : % Purity of Working/Reference standard of Erythromycin C
Assay Erythromycin content = Sum of Erythromycin A + B + C
- 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, reference solution, test solution and spike test solution. Test solution to be prepared as per method of analysis.
Blank: Solvent A
Note: Reduce quantity of standards/Impurity can be use by keeping final concentration same.
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 of Erythromycin in 10 ml of a solvent A, filter and use the filtrate.
Preparation of solution 2:
Weigh accurately 40 mg of Erythromycin A CRS 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 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 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: (Diluent)
Mix 1 volume of methanol and 3 volumes of citro-phosphate buffer pH 7.0.
Preparation of Identification solutions:
Note: Due to unavailability of Impurity C, it can not to be injected so it shall be considered on RRT basis in system suitability solution.
Stock solution of Erythromycin B:
Weigh accurately about 5.0 mg of Erythromycin B and transferred to 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Erythromycin B:
Dilute 4.0 ml of stock solution of Erythromycin B to 10.0 ml with diluent, mix.
Stock solution of Erythromycin C:
Weigh accurately about 5.0 mg of Erythromycin C and transferred to 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Erythromycin C:
Dilute 4.0 ml of stock solution of Erythromycin C to 10.0 ml with diluent, mix.
Stock solution of Impurity A:
Weigh accurately about 3.0 mg of Impurity A and transferred to 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity A:
Dilute 4.0 ml of stock solution of impurity A to 10.0 ml with diluent, mix.
Stock solution of Impurity B:
Weigh accurately about 3.0 mg of Impurity B and transferred to 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity B:
Dilute 4.0 ml of stock solution of impurity B to 10.0 ml with diluent, mix.
- Stock solution of Impurity D:
Weigh accurately about 3.0 mg of Impurity D and transferred to 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity D:
Dilute 4.0 ml of stock solution of impurity D to 10.0 ml with diluent, mix.
- Stock solution of Impurity E:
Weigh accurately about 3.0 mg of Impurity E and transferred to 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity E:
Dilute 4.0 ml of stock solution of impurity E to 10.0 ml with diluent, mix.
- Stock solution of Impurity F:
Weigh accurately about 3.0 mg of Impurity F and transferred to 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity F:
Dilute 4.0 ml of stock solution of impurity F to 10.0 ml with diluent, mix.
Impurity Stock Solution : Weigh accurately about 5.0 mg of Erythromycin B & Erythromycin C and 3.0 mg of Impurity A, Impurity B, Impurity D, Impurity E and Impurity F into 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Spiked test solution:
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 and also add 4.0 ml of Impurity Stock solution filter and use the filtrate.
Procedure: Inject the preparation of blank solution, placebo solution, solution 5, solution 2 (six replicate), solution 3 (two replicate), Solution 6 (to identify impurity E and Impurity F), ID solutions (Impurity A, Impurity B, Impurity D, Impurity E, Impurity F, 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 |
| Solution 5 (System Suitability) | 1 |
| Blank solution | 1 |
| Placebo solution | 1 |
| Solution 2 | 6 |
| Solution 3 | 2 |
| Solution 6 | 1 |
| Identification solution of Impurity A | 1 |
| Identification solution of Impurity B | 1 |
| Identification solution of Impurity D | 1 |
| Identification solution of Impurity E | 1 |
| Identification solution of Impurity F | 1 |
| Solution 1 (Test solution) | 1 |
| Spike test solution | 1 |
| Solution 2 + Bracketing | 1 |
Table 3.0: Specificity data
| Sr. No | Sample | RT (min.) | Peak purity | |
| Blank solution | NA | |||
| Placebo solution | NA | |||
| Solution 5 | N-Demethylerythromycin | |||
| Erythromycin A | ||||
| Erythromycin B | ||||
| Erythromycin C | ||||
| Solution 4 | Erythromycin A | |||
| Solution 2 | Erythromycin A | |||
| Solution 3 | Erythromycin B | |||
| Erythromycin C | ||||
| Solution 6 | Erythromycin A | |||
| Impurity F | ||||
| Impurity E | ||||
| Identification solution of Impurity A | ||||
| Identification solution of Impurity B | ||||
| Identification solution of Impurity D | ||||
| Identification solution of Impurity F | ||||
| Identification solution of Erythromycin B | ||||
| Identification solution of Erythromycin A | ||||
| Solution 1 (Test solution) | Blank | |||
| Placebo | ||||
| Known Impurity | ||||
| Unknown Impurity | ||||
| Spike Solution 1 (Test solution) | Blank | |||
| Placebo | ||||
| Known Impurity | ||||
| Unknown Impurity |
Acceptance Criteria:
- There should be no interference of the Erythromycin A and placebo at the retention time of Analyte peak.
- Blank peak, placebo peak, and known impurity should be well resolved from active peak and each other.
- Analyte peak in Identification
solutions, test solution and spike test solution should be spectrally pure.
- Precision:
- System Precision:
- 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 2 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 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.
- 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 Assay:
Prepare the six sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % assay, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 5.0: Method precision results for Assay
| Sr. No | Sample wt. (mg) | Erythromycin A | Erythromycin B | Erythromycin C | Sum of Assay of Erythromycin A,B & C | |||
| Area | % Assay | Area | % Assay | Area | % Assay | |||
| 1 | ||||||||
| 2 | ||||||||
| 3 | ||||||||
| 4 | ||||||||
| 5 | ||||||||
| 6 | ||||||||
| Average | ||||||||
| Minimum | ||||||||
| Maximum | ||||||||
| % RSD |
Acceptance Criteria:
- Calculated as the sum of Erythromycin A (C37H67NO13), Erythromycin B (C37H67NO12) and Erythromycin C (C36H65NO13) in mg should be between 95.0-105.0 %.
- % RSD for six replicate preparations
of assay should be NMT 2.0%.
- 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 2 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 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 % assay, 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 assay
| Sr. No | Sample wt. (mg) | Erythromycin A | Erythromycin B | Erythromycin C | Sum of Assay of Erythromycin A,B & C | |||
| Area | % Assay | Area | % Assay | Area | % Assay | |||
| 1 | ||||||||
| 2 | ||||||||
| 3 | ||||||||
| 4 | ||||||||
| 5 | ||||||||
| 6 | ||||||||
| Average | ||||||||
| Minimum | ||||||||
| Maximum | ||||||||
| % RSD |
Acceptance Criteria:
- Calculated as the sum of Erythromycin A (C37H67NO13), Erythromycin B (C37H67NO12) and Erythromycin C (C36H65NO13) in mg should be between 95.0-105.0 %.
- % RSD for six replicate preparations of assay should be NMT 2.0%.
Table 8.0: Cumulative results of assay of Method Precision & Intermediate Precision
| Parameter | Sr. No. | Sample wt. (mg) | Sum of Assay of Erythromycin A,B & C |
| Method Precision | 1 | ||
| 2 | |||
| 3 | |||
| 4 | |||
| 5 | |||
| 6 | |||
| Intermediate Precision | 1 | ||
| 2 | |||
| 3 | |||
| 4 | |||
| 5 | |||
| 6 | |||
| Mean | |||
| Cumulative % RSD |
Acceptance Criteria:
- Calculated as the sum of Erythromycin A (C37H67NO13), Erythromycin B (C37H67NO12) and Erythromycin C (C36H65NO13) in mg should be between 95.0-105.0 %.
- % RSD for six replicate preparations
of assay should be NMT 2.0%.
- 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. | 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 assay test of Erythromycin 250 mg Gastro-Resistant tablets by HPLC.
The analytical methodology is same for Assay 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 : Assay
VERP : Verification Protocol
T : Tablets
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 |
