by ANALYTICAL METHOD VERIFICATION PROTOCOL FOR DISSOLUTION 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| Superseded Protocol No. | Nil |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 | ||
| Validation 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 validate the suitability of dissolution 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 dissolution of Erythromycin 250 mg gastro-resistant.
- 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 and approval of protocol. |
| Co-ordination with QC to carryout Validation. | |
| Review of data and summary report. | |
| Head Quality | Authorization 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 | Dissolution Acid stage : Not more than 10% of the labeled amount of Erythromycin is dissolved in 120 minutes. Buffer stage : Not less than 75 % (Q) of the labeled amount of Erythromycin is dissolved in 60 minutes. |
- Methodology:
Chemical and reagents:
Table 1.0: Chemical and reagents
| 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 |
| 9. | Potassium dihydrogen orthophosphate | AR Grade |
| 10. | Hydrochloric acid | AR Grade |
| 11. | Sodium Hydroxide | AR 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 : 100 µl (0.1 ml)
Note: Column and at least one third of the tubing preceding the column shall maintain at 70°C.
Dissolution parameter:
Method : Apparatus I (Basket)
Medium : Stage I – Acid stage 900 ml of simulated gastric fluid without pepsin (Use 0.1 M
Hydrochloric acid).
Stage II – Buffer stage 900 ml of pH 6.8 Phosphate buffer
RPM : 100
Time : Stage I – 120 minutes
Stage II – 60 minutes
Preparation of Phosphate buffer pH 6.8 (0.2M Mixed)
Mix 51 ml of a 2.72% w/v solution of potassium dihydrogen orthophosphate with 49 ml of 7.16% w/v of disodium hydrogen orthophosphate and adjust the pH if necessary.
Preparation of 0.1M hydrochloric acid:
Dilute 8.5 ml of hydrochloric acid with sufficient water to produce 1000 ml. adjust the volume as per required volume.
Standard preparation:
Transfer an accurately weighed quantity of about 28 mg of Erythromycin working standard into a 100 ml volumetric flask. Dissolve and dilute up to mark with dissolution medium.
Sample preparation:
Use 5 ml of a filtered sample for injection.
Calculate the content of Erythromycin dissolved from the following calculation
Calculation:
% Release =
Sample peak area wt. of Std. (mg) 900 standard purity 100
—————————x———————x———-x———————–x———- = % Erythromycin
Standard peak area 100 1 250 100
- 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 and placebo.
Specificity of test method should be established by separately injecting blank solution (dissolution medium), Placebo, standard solution, sample solution and spiked sample solution. Sample solution to be preparing as per method of analysis.
Dissolution in acid medium:
Standard preparation:
Transfer an accurately weighed quantity of about 28 mg of Erythromycin working standard into a 100 ml volumetric flask. Dissolve and dilute up to mark with 0.1N Hydrochloric acid.
Preparation of specificity solutions for dissolution test in acid medium:
In the set of 4 dissolution vessels add 900 ml of dissolution medium.
- Blank solution : Dissolution medium of 0.1N hydrochloric acid
- Placebo solution : Weight and transfer 82 mg of placebo into 0.1N hydrochloric acid.
- Test solution : Weight one tablet and transfer into 0.1 hydrochloric acid.
- Spike test solution : Weight 82 mg placebo + 250 mg Erythromycin API into 0.1N hydrochloric acid.
Run the apparatus as per given condition. After specified time, withdraw specified volume of aliquot from each vessel and filter, discard first 2-3 ml of the filtrate. Sequence shall be prepare as per below table.
Table 2.0: Sequence for dissolution test in acid medium
| Solution | No of Injection to be injected in Sequence |
| Blank solution | 1 |
| Placebo Solution | 1 |
| Standard solution | 6 |
| Test Solution | 1 |
| Spiked Test Solution | 1 |
| Standard Solution + Bracketing | 1 |
Dissolution in Buffer medium:
Standard preparation:
Transfer an accurately weighed quantity of about 28 mg of Erythromycin working standard into a 100 ml volumetric flask. Dissolve and dilute up to mark with pH 6.8 Phosphate buffer.
Preparation of specificity solutions for dissolution test:
In the set of 4 dissolution vessels add 900 ml of dissolution medium.
- Blank solution : Dissolution medium of 6.8 phosphate buffer
- Placebo solution : Weight and transfer 82 mg of placebo into pH 6.8 phosphate buffer.
- Test solution : Weight one tablet and transfer into pH 6.8 phosphate buffer.
- Spike test solution : Weight 82 mg placebo + 25 mg Erythromycin API into pH 6.8 Phosphate buffer.
Run the apparatus as per given condition. After specified time, withdraw specified volume of aliquot from each vessel and filter, discard first 2-3 ml of the filtrate.
Table 3.0: Sequence for dissolution test in Buffer medium
| Solution | No of Injection to be injected in Sequence |
| Blank solution | 1 |
| Placebo Solution | 1 |
| Standard solution | 6 |
| Test Solution | 1 |
| Spiked Test Solution | 1 |
| Standard Solution + Bracketing | 1 |
Table 4.0: Specificity data of dissolution in acid medium
| Sr. No | Sample | RT (min.) | Peak purity |
| Blank solution | NA | ||
| Placebo solution | NA | ||
| Standard solution | |||
| Sample solution | |||
| Spike solution |
Table 5.0: Specificity data of dissolution in Buffer medium
| Sr. No | Sample | RT (min.) | Peak purity |
| Blank solution | NA | ||
| Placebo solution | NA | ||
| Standard solution | |||
| Sample solution | |||
| Spike solution |
Acceptance Criteria:
- There should be no interference of the diluent, placebo at the retention time of analyte peak,
- Blank peak and Placebo peak should be well resolved from active peak and each other,
- Analyte peak in standard solution, sample
solution and spiked sample 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 the relative standard deviation (RSD).
Standard solution will be prepared in buffer medium 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 % area RSD and % RT RSD of main analyte peak.
Table 6.0: 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 relative standard deviation.
Test Procedure for Dissolution:
Prepare the six sample of same batch and analyze as per method of analysis in both acid medium and buffer medium, record the area on testing data sheet and calculate the % release, mean and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 7.0: Method precision results for Dissolution
| Tablet No. | Erythromycin 250 mg | |||
| Acid Medium | Buffer Medium | |||
| Peak Area | % Release | Peak Area | % Release | |
| 1 | ||||
| 2 | ||||
| 3 | ||||
| 4 | ||||
| 5 | ||||
| 6 | ||||
| Mean | ||||
| % RSD |
Acceptance Criteria:
Calculate the % release content of erythromycin for each tablet and calculate the mean and % RSD.
- In acid medium, % Release of Erythromycin content should not be more than 10 %.
- In buffer medium % Release of Erythromycin content should not less than 75 % (Q).
- %
RSD of the six analyses shall be not more than 5 for the release above 85 % and
not more than 10 for the release below 85% in buffer medium.
- 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 will be prepared as per method of analysis and injected six replicate injections to be injected in sequence and recorded the area response and retention time of main analyte peak. Calculate the % area RSD and % RT RSD of main analyte peak.
Table 8.0: 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 six sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % released, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 9.0: Intermediate precision results for Dissolution
| Tablet No. | Erythromycin 250 mg | |||
| Acid Medium | Buffer Medium | |||
| Peak Area | % Release | Peak Area | % Release | |
| 1 | ||||
| 2 | ||||
| 3 | ||||
| 4 | ||||
| 5 | ||||
| 6 | ||||
| Mean | ||||
| % RSD |
Acceptance limit:
Calculate the % release content of erythromycin for each tablet and calculate the mean and % RSD.
- In acid medium, % Release of Erythromycin content should not be more than 10 %.
- In buffer medium % Release of Erythromycin content should not less than 75 % (Q).
- % RSD of the six analyses shall be not more than 5 for the release above 85 % and not more than 10 for the release below 85% in buffer medium.
Table 10.0: Cumulative Results of % Release of Method Precision and Intermediate Precision
| Parameter | Tablet No. | Erythromycin content | |
| % Release in Acid Medium | % Release in Buffer Medium | ||
| Method Precision | 1 | ||
| 2 | |||
| 3 | |||
| 4 | |||
| 5 | |||
| 6 | |||
| Intermediate Precision | 1 | ||
| 2 | |||
| 3 | |||
| 4 | |||
| 5 | |||
| 6 | |||
| Mean | |||
| % RSD |
Acceptance Criteria:
Calculate the % release content of erythromycin for each tablet and calculate the mean and % RSD.
- In acid medium, % Release of Erythromycin content should not be more than 10 %.
- In buffer medium % Release of Erythromycin content should not less than 75 % (Q).
- %
RSD of the six analyses shall be not more than 5 for the release above 85 % and
not more than 10 for the release below 85% in buffer medium.
- 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 11.0: System suitability parameters
| Sr. No. | Parameter | Set limits |
| RSD for the peak area of Erythromycin in the five replicate injections of standard solution in buffer medium. | NMT – 2.0% |
- Incident /Deviation:
Any incident or deviation observed during analytical method verification should be recorded and investigate as per SOP.
- Summary/ Conclusion / Recommendation:
Final conclusion should be drawn from analytical method verification for its use to analyze the % release of Erythromycin content in dissolution test of Erythromycin 250 mg in both acid medium and buffer medium by HPLC.
Summary of verification report shall be prepared and accordingly recommendation shall be updated if any.
Abbreviation:
DIS : Dissolution
VERP : Verification Protocol
SD : Standard deviation
HPLC : High performance liquid chromatography
DAD : diode-array detector
RT : Retention Time
mL : Milliliter
mg : Milligram
min. : Minutes
QA : Quality Assurance
QC : Quality Control
SOP : Standard operating procedure
% : Percentage
ºC : Degree centigrade
hrs : Hours
µm : Micrometer
µl : Microlitre
RSD : Relative standard deviation
NLT : Not less than
NMT : Not more than
WS : Working standard
Vol : Volume
Revision History:
| Revision No. | Details of changes | Reason |
| 00 | Nil | New Document |
