by ANALYTICAL METHOD VERIFICATION PROTOCOL FOR ASSAY 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| Superseded Protocol No. | Nil |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 |
- 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 Erythromycin Ph. Eur. by considering the following parameters:
| Parameters | Erythromycin Ph. Eur. |
| 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 Ph. Eur.
- Responsibility of Validation Team:
| Departments | Responsibilities |
| QC | Preparation & Review of Protocol. |
| Analysis of samples and recording of data. | |
| Compilation and Review 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 | Assay (On anhydrous basis) Sum of Erythromycin A,B and C : 93.0 to 102.0% Erythromycin B : Not more than 5.0% Erythromycin C : Not more than 5.0% |
- Methodology:
- Assay
Chemical, reagents and filters:
Table 1.0: Chemical, reagents and filters
| Sr. No | Material /Chemicals/Filters | Grade |
| 1. | Dipotassium hydrogen phosphate (K2HPO4) | AR Grade |
| 2. | Orthophosphoric acid | HPLC Grade |
| 3. | Methanol | HPLC Grade |
| 4. | Acetonitrile | HPLC Grade |
| 5. | Water | Milli Q Water/ HPLC water |
Mobile phase and diluent
Buffer pH 7.0
K2HPO4 solution:
Accurately weigh 35 g of K2HPO4 into 900 ml volumetric flask, dissolve and dilute to volume with water, adjust the pH to 7.0 ± 0.05 with dilute phosphoric acid, dilute to 1000 ml with water.
Mobile phase A:
Phosphate buffer solution pH 7.0: Acetonitrile: Water (5:35:60 v/v/v)
Mobile phase B:
Phosphate buffer solution pH 7.0: Water: Acetonitrile (5:45:50 v/v/v)
Solution A:
K2HPO4 solution: Accurately weigh 11.5 g of K2HPO4 into 900 ml water, adjust the pH to 8.0 ± 0.05 with dilute phosphoric acid and dilute to 1000 ml with water.
Solvent mixture:
Mix Methanol and Solution A in the ratio (40: 60 v/v)
Chromatographic conditions:
Column : X TERRA RP18, 4.6 x 250 mm, 3.5 µm
Flow rate : 1.0 ml/min
Wavelength : 210 nm
Column Temp. : 65°C (preheating of the mobile phase is required by extending the inlet in the oven to 30 cm)
Sample Temp : 4°C
Injection Volume : 100 µl
Run time : For blank and Test preparation run full gradient For Reference solution (a) and Reference solution (b) – tR + 5
Program : Gradient
Note: Modify gradient program as per Retention time of Erythromycin B peak (tR) by injecting 10µl of reference solution (b) and eluting with mobile phase A.
(For Assay also)
| Time (min) | Mobile Phase-A | Mobile Phase-B |
| 0 – tR | 100 | 0 |
| tR – (tR + 2) | 100 to 0 | 0 to 100 |
| (tR + 2) – (tR + 15) | 0 | 100 |
Reference solutions and Test solutions
Reference solution (a):
Weigh accurately 40.0 mg of Erythromycin A CRS into 10 ml volumetric flask, dissolve and dilute to volume with solvent mixture.
Reference solution (b):
Weigh accurately 10.0 mg each of Erythromycin B CRS and Erythromycin C CRS into 50 ml volumetric flask, dissolve and dilute to volume with solvent mixture.
Test solution:
Weigh accurately 40.0 mg of sample into 10 ml volumetric flask, dissolve and dilute to volume with solvent mixture.
Note: If possible use same reference solution (a), reference solution (b) and test solution preparations for related substance testing.
Procedure:
- Inject blank (Solvent mixture) and check for baseline drifting, noise or any other sign of system instability. Repeat this procedure until the system becomes stable.
- Inject reference solution (a) once to check for system suitability requirements.
- Compare the retention time with the expected one and calculate symmetry factor, Symmetry factor should be NMT 2.0 for Erythromycin peak.
- Inject blank (Diluent) and check for carryover.
- Inject Reference solution (a) repeatedly six times and calculate the % RSD, should be NMT 1.0 %.
- Inject reference solution (b) repeatedly six times and calculate the %RSD, should be NMT 5.0%.
- If all system suitability requirements meets continue with sample analysis
- Inject sample preparation once.
- Inject once reference solution (a) as bracketing standard at the end of the sequence.
- Retention time and relative retention times of individual peaks
| Impurity | ≈Relative retention time (RRT) |
| Erythromycin C | 0.5 |
| Erythromycin A | 1.0 |
| Erythromycin B | 1.6 |
Note:
- RRT are with reference to Erythromycin A retention time about 23 min.
- If system suitability criterion not meeting, adjust Acetonitrile composition in mobile phase or gradient.
Calculations:
Calculate the contents of Erythromycin B and Erythromycin C by following expressions
Au x Ws x 10 x f 100
% Impurity content = ———————————— x —————– x 100
As x 50 x Wu x 100 (100 – W)
Where;
Au : Area of respective impurity in sample chromatogram
As : Mean area of respective impurity in reference solution (b).
Wu : Weight of sample taken for analysis.
Ws : Weight of respective impurity in reference solution (b).
f : Purity of respective impurity in %.
W: Water content of sample.
Calculate the Assay of Erythromycin A by following expression
Au x Ws x 10 x f 100
%Assay = ———————————— x ——————- x 100
As x 10 x Wu x 100 (100 – W)
Where;
Au : Area of Erythromycin A in sample chromatogram
As : Mean area of Erythromycin A in reference solution (a).
Wu : Weight of sample taken for analysis.
Ws : Weight of Erythromycin A CRS/WS in reference solution (a) preparation.
f : Purity of Erythromycin A CRS/ WS.
W : Water content of sample.
- Water content:
Equipment: Karl Fischer Apparatus
Reagents
100 g/L solution of Imidazole in anhydrous Methanol
Composite 2 KF reagents
Procedure: Perform in duplicate
- Turn on the titrator.
- Turn on the printer; check the connection between Titrator and printer.
- Press method on titrator and enter the same name details and Press RUN.
- After pre-titration, accurately weigh 0.2 g of the sample.
- Transfer the weighed sample into the designated beaker containing 40 ml of 100 g/L solution of Imidazole in anhydrous Methanol.
- Press sample on the titrator and enter the transferred weight and press RUN.
- After the specified time in the method, titration starts and report will be printed.
Limit: Not more than 6.5 %
- 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.
Specificity of test method should be established by separately injecting blank solution (solvent mixture), reference solution (a) and test solution (b). Test solution to be prepared as per method of analysis.
Preparation of Identification solutions:
Identification solution of Erythromycin B:
Weigh accurately about 2 mg of Erythromycin B and transferred to 10 ml volumetric flask, add 5.0 ml of diluent and dissolve. Make up the volume to 10.0 ml with diluent and mix.
Identification solution of Erythromycin C:
Weigh accurately about 2 mg of Erythromycin C and transferred to 10 ml volumetric flask, add 5.0 ml of diluent and dissolve. Make up the volume to 10.0 ml with diluent and mix.
(Note: Depending on purity of Impurity, adjust the weight of Impurity and the weight of impurity can be reduce to achieve the final concentration.)
Reference solutions and Test solutions
Blank: Solvent mixture
Reference solution (a):
Weigh accurately 40.0 mg of Erythromycin A CRS into 10 ml volumetric flask, dissolve and dilute to volume with solvent mixture.
Reference solution (b):
Weigh accurately 10.0 mg each of Erythromycin B CRS and Erythromycin C CRS into 50 ml volumetric flask, dissolve and dilute to volume with solvent mixture.
Test solution:
Weigh accurately 40.0 mg of sample into 10 ml volumetric flask, dissolve and dilute to volume with solvent mixture.
Procedure: Inject the preparation of blank solution (solvent mixture), Reference solution (a), Identification solution of Erythromycin B & C, Reference solution (b), and test solution and on a HPLC system with a Diode array detector (DAD) as follows in Table 4.0. Determine the purity of the individual peak of interest. Record the retention times of all peaks obtained from the respective solution and check for system suitability parameters for Erythromycin peak obtained.
Table 3.0: Sequence of Injection (Specificity)
| Solution | No of Injection to be injected in Sequence |
| Blank | 1 |
| Reference solution (a) | 6 |
| ID solution of Erythromycin B | 1 |
| ID solution of Erythromycin C | 1 |
| Reference solution (b) | 6 |
| Test solution | 1 |
| Reference solution (a) + Bracketing | 1 |
Table 4.0: Specificity data
| Sr. No | Sample | RT (min.) | Peak purity | |
| Blank | NA | |||
| Reference solution (a) | Erythromycin A | |||
| ID solution of Erythromycin B | ||||
| ID solution of Erythromycin C | ||||
| Reference solution (b) | Erythromycin B | |||
| Erythromycin C | ||||
| Test solution |
Acceptance Criteria:
- There should be no interference of the solvent mixture at the retention time of analyte peak.
- Blank peak should be well resolved from active peak and each other,
- Analyte peak in Reference solution (a) & (b), ID solutions and test solution should be spectrally pure.
- 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). Reference solution (a) and Reference solution (b) is prepared as per method of analysis and six replicate injections of each solution to be injected in sequence and recorded the area response and retention time of Erythromycin A, Erythromycin B & Erythromycin C. Calculate the % RSD for area and Retention time.
Table 5.0: System Precision- Repeatability of Standard Injections
| Sr. No. | Erythromycin A | Erythromycin B | Erythromycin C | |||
| Peak Area | Retention time (min.) | Peak Area | Retention time (min.) | Peak Area | Retention time (min.) | |
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
| Mean | ||||||
| SD | ||||||
| % RSD |
Acceptance criteria:
In reference solution (a), % RSD for peak area and retention time of Erythromycin A in replicate injections should be NMT 2.0% and 1.0% respectively.
In reference solution (b), % RSD for peak area and retention time of Erythromycin B and Erythromycin C in replicate 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 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 of sum of Erythromycin A,B and C (on anhydrous basis), mean, standard deviation and % relative standard deviation. Obtained results shall be report in tabulated manner as given below.
Table 6.0: 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 | ||||||||
| SD | ||||||||
| % RSD |
Acceptance Criteria:
Calculate the % assay of sum of Erythromycin A, B & C for each analysis on anhydrous basis and calculate the mean, SD and % RSD. % RSD for assay values 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.
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 7.0: Repeatability of Standard Injections
| Erythromycin A | Erythromycin B | Erythromycin C | ||||
| Peak Area | Retention time (min.) | Peak Area | Retention time (min.) | Peak Area | Retention time (min.) | |
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
| Mean | ||||||
| SD | ||||||
| % RSD |
Acceptance criteria:
In reference solution (a), % RSD for peak area and retention time of Erythromycin A in replicate injections should be NMT 2.0% and 1.0% respectively.
In reference solution (b), % RSD for peak area and retention time of Erythromycin B and Erythromycin C in replicate injections should be NMT 5.0% 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 (on anhydrous basis), mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 8.0: 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 | ||||||||
| SD | ||||||||
| % RSD |
Acceptance Criteria:
Calculate the % assay of sum of Erythromycin A, B & C for each analysis on anhydrous basis and calculate the mean, SD and % RSD. % RSD for assay values should be NMT 2.0%.
Table 9.0: Cumulative results of Assay of method precision and 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:
Calculate the cumulative % assay of sum of Erythromycin A, B & C for each analysis on anhydrous basis and calculate the mean, SD and % RSD. % RSD for assay values 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 10.0: 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 investigation 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 Ph. Eur. by HPLC.
Summary of verification report shall be prepare and accordingly conclusion and recommendation to be given.
Abbreviation:
ASS : Assay
VER : Verification
P : Protocol
T : Tablets
SD : Standard deviation
HPLC : High performance liquid chromatography
DAD : diode-array detector
RT : Retention Time
mL : Milliliter
ID : Identification solution
mg : Milligram
min. : Minutes
QA : Quality Assurance
QC : Quality Control
% : Percentage
ºC : Degree centigrade
µl : Microlitre
EP/Ph.Eur. : Europeian Pharmacopoeia
RSD : Relative standard deviation
NLT : Not less than
NMT : Not more than
Ws : Working standard
Wt : Weight
Vol : Volume
As : Standard Area
At : Test Area
- Revision History:
| Revision No. | Details of changes | Reason |
| 00 | Nil | New Document |
