by ANALYTICAL METHOD VERIFICATION PROTOCOL OF RELATED SUBSTANCES OF ERYTHROMYCIN Ph. Eur.
| Superseded Protocol No. | Nil |
| Effective Date |
TABLE OF CONTENTS:
| Sr. No. | Subject | Page No. |
| Protocol Approval | ||
| Objective | ||
| Scope | ||
| Responsibility of validation team | ||
| 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 verification is to provide documentary evidence that analytical methodology used for related substances of Erythromycin Ph. Eur. by pharmacopeia method is consistent and reliable results within the predetermined acceptance criteria.
Analytical method verification will be performed by considering thefollowing parameters:
| Parameters | Erythromycin Ph. Eur. |
| Specificity | |
| Precision | |
| System Precision | |
| Method Precision | |
| Intermediate Precision (Ruggedness) | |
| System Suitability |
- Scope :
The scope of this protocol is applicable for the verification of method of analysis of related substances of Erythromycin Ph. Eur.
- 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 | 1st verification |
| Active Ingredient | Erythromycin Ph. Eur. |
| Method Reference | European Pharmacopoeia |
| Specification Limits | Specified impurities 1. Impurity A : NMT-2.0 % 2. Impurity B : NMT-2.0 % 3. Impurity C : NMT-3.0 % 4. Impurity D : NMT-1.0 % 5. Impurity E : NMT-1.0 % 6. Impurity F : NMT-1.0 % 7. Impurity H : NMT-1.0 % 8. Impurity L : NMT-0.4 % 9. Impurity M : NMT-1.0 % Unspecified Impurities Any other Impurity : NMT-0.4% Total Impurity : NMT-7.0% Reporting Threshold : 0.2 % Disregard peaks due to Erythromycin B and Erythromycin C |
- Methodology:
Reagent:
Table 1.0: Chemicals/ Reagents
| Sr. No. | Reagent | Grade |
| 1. | Dipotassium hydrogen phosphate | AR Grade |
| 2. | Orthophosphoric acid | AR Grade |
| 3. | Acetonitrile | HPLC Grade |
| 4. | Methanol | HPLC Grade |
| 5. | Water | HPLC grade or Milli Q Water |
Mobile phase and diluent:
Buffer pH 7.0
K2HPO4 solution:
Accurately weigh 35 g of K2HPO4 into 900 ml volumetric flask, dissolve, 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, dissolve and 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 P/N 186001472
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 Vol. : 100 µl
Run time : For blank, Reference solution (d), Reference solution (e) and Test preparation run full gradient. For Reference solution (b) and Reference solution (c) –tR + 5 minutes
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.
(Use same procedure for Assay)
| 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 / working standard (WS) 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.
Reference solution (c):
Pipette out 1.0 ml of reference solution (a) into 100 ml volumetric flask, dissolve and dilute to volume with Solvent mixture.
Reference solution (d):
Weigh accurately 4.0 mg of Erythromycin for system suitability CRS (containing impurities A, B, C, D, E, F, H and L) into 1 ml volumetric flask, dissolve and dilute to volume with Solvent mixture.
Reference solution (e):
Weigh accurately 4.0 mg of Erythromycin for impurity M CRS into 1 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 diluent.
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 (d) once to check system suitability and identify retention times.
- Calculate the resolution, resolution between Impurity B and Erythromycin C, should be NLT 1.2.
- Calculate the Peak to valley ratio (Hp/Hv) between impurity F and Erythromycin B, should be NLT 1.5.
Where,
Hp = Height above the baseline of the peak due to Impurity F.
Hv = Height above the baseline of the lowest point of the curve separating Impurity F and Erythromycin B.
- Calculate the Peak to valley ratio between Impurity C and Erythromycin A, should be NLT 2.0.
Where,
Hp = Height above the baseline of the peak due to Impurity C.
Hv = Height above the baseline of the lowest point of the curve separating impurity C ad Erythromycin A.
Note: If system suitability criterion not meeting, adjust Acetonitrile composition in mobile phase or gradient.
Retention time and relative retention time of individual peak.
| Impurity | Relative retention time (≈RRT) | Correction Factor (F) |
| Impurity H | 0.3 | 1 |
| Impurity A | 0.4 | 1 |
| Impurity B | 0.5 | 1 |
| Erythromycin C | 0.55 | 1 |
| Impurity M | 0.58 | 1 |
| Impurity L | 0.63 | 0.11 |
| Impurity C | 0.9 | 1 |
| Erythromycin A | 1.0 | 1 |
| Impurity D | 1.61 | 2 |
| Erythromycin B | 1.75 | 1 |
| Impurity F | 1.81 | 0.08 |
| Impurity E | 2.3 | 0.08 |
Note: RRT are with reference to Erythromycin A retention time about 23 minutes.
- Inject Reference solution (e) once to identify peak due to Impurity M.
- Inject Reference solution (b) once to identify peak due to Erythromycin B and C.
- If all system suitability criterions meet, inject Reference solution (c) repeatedly six times and % RSD of six replicate injections is NMT 5.0 %.
Once all system suitability criterion meets proceed with sample analysis.
- Inject sample solution once.
- Inject Reference solution (d) once at the end of the sequence to identify retention times only.
Calculation
- Calculate all specified impurities using the below expression
Au x Ws x 1 x 10 x F x f
% Impurity content = ————————————— x 100
As x 10 x 100 x Wu x 100
Where,
Au : Area of respective specified and unspecified Impurity in test preparation.
As : Mean Area of Erythromycin A CRS/WS in Reference solution (c) injections
Ws : Weight of Erythromycin A CRS/WS in Reference solution (a) preparation
Wu : Weight of sample in test preparation
F : Correction factor for specified impurities
f : Purity for Erythromycin A CRS / WS
- Calculate all unspecified impurities using the below expression
Au x Ws x 1 x 10 x f
% Impurity content = ————————————— x 100
As x 10 x 100 x Wu x 100
Where,
Au : Area of respective specified and unspecified Impurity in test preparation.
As : Mean Area of Erythromycin A CRS/WS in Reference solution (c) injections
Ws : Weight of Erythromycin A CRS/WS in Reference solution (a) preparation
Wu : Weight of sample in test preparation
f : Purity for Erythromycin A CRS / WS
Reporting Threshold: 0.2 %
Disregard peaks due to Erythromycin B and Erythromycin 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 solvent mixture.
Specificity of test method should be established by separately injecting blank solution, Reference solution (d),Reference solution (e), Reference solution (b), Reference solution (c) (six times), identification solution of Impurity A, Impurity B, Impurity D, Impurity E, Impurity F, Impurity H, test solution and spiked test solution. Impurity L, Impurity C & impurity M is not available so it shall be considered on RRT basis. Identification solution and impurities spiked test solution shall be prepared as per specification limit level. Test solution to be prepared as per method of analysis.
Blank: Solvent Mixture
Preparation of Identification solutions:
Stock solution of Impurity A:
Weigh accurately about 2.0 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 2.0 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 2.0 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 2.0 ml of stock solution of impurity D to 10.0 ml with diluent, mix.
Stock solution of Impurity E:
Weigh accurately about 2.0 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 2.0 ml of stock solution of impurity E to 10.0 ml with diluent, mix.
- Stock solution of Impurity F:
Weigh accurately about 2.0 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 2.0 ml of stock solution of impurity F to 10.0 ml with diluent, mix.
- Stock solution of Impurity H:
Weigh accurately about 2.0 mg of Impurity H and transferred to 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Identification solution of Impurity H:
Dilute 2.0 ml of stock solution of impurity H to 10.0 ml with diluent, mix.
Reference solutions and Test solutions
Reference solution (a):
Weigh accurately 40.0 mg of Erythromycin A CRS / working standard (WS) 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.
Reference solution (c):
Pipette out 1.0 ml of reference solution (a) into 100 ml volumetric flask, dissolve and dilute to volume with Solvent mixture.
Reference solution (d):
Weigh accurately 4.0 mg of Erythromycin for system suitability CRS (containing impurities A, B, C, D, E, F, H and L) into 1 ml volumetric flask, dissolve and dilute to volume with Solvent mixture.
Reference solution (e):
Weigh accurately 4.0 mg of Erythromycin for impurity M CRS into 1 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 diluent.
Impurity Stock Solution I: Weigh accurately about 2.0 mg of Impurity A and Impurity B into 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Impurity Stock Solution II: Weigh accurately about 2.0 mg of Impurity D, Impurity E, Impurity F and Impurity H into 10 ml volumetric flask. Dissolve and dilute to volume with diluent, mix.
Spike Test Solution:
Weigh accurately 40.0 mg of sample into 10 ml volumetric flask, Transfer 4.0 ml of Impurity stock solution I, 2.0 ml of impurity stock solution II. Dissolve and dilute to volume with diluent.
Procedure: Inject the preparation of blank solution, reference solution (d) or system suitability solution, reference solution (e), reference solution (b), reference solution (c) (six times) and identification solution of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity H, test solution and spiked test solution on a HPLC system with a Diode array detector (DAD) as follows in table 2.0. Determine the purity of the individual peaks of interest. Record the retention times and check for system suitability parameters. Obtained specificity data shall be reported in tabular manner with reference of table 3.0 and 4.0.
Table 2.0: Sequence
| Solutions | No of Injection to be injected in Sequence |
| Blank solution | 1 |
| Reference solution (d) | 1 |
| Reference solution (e) | 1 |
| Reference solution (b) | 1 |
| Reference solution (c) | 6 |
| 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 |
| Identification solution of Impurity H | 1 |
| Test solution | 1 |
| Spike test solution | 1 |
| Blank (to avoid carry over) | 1 |
| Reference solution (c) bracketing | 1 |
Table 3.0 Specificity data
| Sr. No | Sample | RT (min.) | RRT | Peak purity | |
| Blank | NA | ||||
| Reference solution (d) (Erythromycin for system suitability CRS) | Impurity A | ||||
| Impurity B | |||||
| Impurity C | |||||
| Impurity D | |||||
| Impurity E | |||||
| Impurity F | |||||
| Impurity H | |||||
| Impurity L | |||||
| Reference solution (e) (Erythromycin for Impurity M CRS) | Erythromycin A | ||||
| Impurity C | |||||
| Impurity M | |||||
| Impurity L | |||||
| Impurity A | |||||
| Reference solution (b) | Erythromycin B | ||||
| Erythromycin C | |||||
| Reference solution (c) | Erythromycin A | ||||
| Identification solution of Impurity A | |||||
| Identification solution of Impurity B | |||||
| Identification solution of Impurity C | |||||
| Identification solution of Impurity D | |||||
| Identification solution of Impurity E | |||||
| Identification solution of Impurity F | |||||
| Identification solution of Impurity H | |||||
| Test Solution (a) | Erythromycin A | ||||
| Unknown Impurity | NA |
Table 6.0 Spiked test solution
| Sr. No | Sample | RT (min.) | RRT | Peak purity |
| Blank | NA | |||
| Erythromycin A | ||||
| Impurity A | ||||
| Impurity B | ||||
| Impurity C | ||||
| Impurity D | ||||
| Impurity E | ||||
| Impurity F | ||||
| Impurity H | ||||
| Unknown Impurity | NA |
Acceptance Criteria:
- There should be no interference of the diluent, impurities at the retention time of Analyte peak.
- Impurity peaks should be well resolved from active peak and each other.
- Analyte peak in Reference solution and known Impurity peaks in Identification solution, spiked sample 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 (c) shall be prepared as per method of analysis and six replicate injections to be injected in sequence and recorded the area response of main analyte peak. Calculate the area % RSD and Retention time % RSD of main analyte peak.
Table 7.0 System Precision- Repeatability of Standard Injections
| Sr. No. | Erythromycin A | |
| Peak Area | Retention time (min.) | |
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| % RSD |
Acceptance criteria:
% RSD for peak area and retention time of Erythromycin A in replicate injections of Reference (c) 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.
Test Procedure:
Prepare the six samples of same batch as per standard analytical procedure. Record the area response of impurity in test solution and calculate the % impurity, mean, standard deviation and % relative standard deviation of six samples in tabular manner as given below.
Table 6.0 Method precision results by 1st analyst
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total impurity ( ≥0.2%) | |||||||||
| Impurity A | Impurity B | Impurity C | Impurity D | Impurity E | Impurity F | Impurity H | Impurity L | Impurity M | 1 | 2 | |||
| RRT | |||||||||||||
| 1 | |||||||||||||
| 2 | |||||||||||||
| 3 | |||||||||||||
| 4 | |||||||||||||
| 5 | |||||||||||||
| 6 | |||||||||||||
| Mean | |||||||||||||
| SD | |||||||||||||
| % RSD |
Acceptance criteria:
% RSD of known, unknown and total impurity content should be not more than the limits specified below. Reporting threshold value is 0.2%.
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.
Reference (c) will be prepared as per method of analysis and six replicate injections to be injected in sequence and recorded the area response of main analyte peak. And calculate the % area RSD and % RT RSD of main analyte peak.
Table 7.0 System Precision- Repeatability of Standard Injections
| Sr. No. | Erythromycin A | |
| Peak Area | Retention time (min.) | |
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| % RSD |
Acceptance criteria:
% RSD for peak area and retention time of Erythromycin A in replicate injections of Reference (c) should be NMT 5.0% and 1.0% respectively.
Test Procedure:
Prepare the six samples of same batch as per standard analytical procedure and analyzed. Record the area response of impurity obtained in test solution. Calculate the % impurity, mean, standard deviation and % relative standard deviation of six samples in tabular manner as given below.
Table 8.0 Intermediate (ruggedness) precision results by 2nd analyst
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total impurity ( ≥0.2%) | |||||||||
| Impurity A | Impurity B | Impurity C | Impurity D | Impurity E | Impurity F | Impurity H | Impurity L | Impurity M | 1 | 2 | |||
| RRT | |||||||||||||
| 1 | |||||||||||||
| 2 | |||||||||||||
| 3 | |||||||||||||
| 4 | |||||||||||||
| 5 | |||||||||||||
| 6 | |||||||||||||
| Mean | |||||||||||||
| SD | |||||||||||||
| % RSD |
Acceptance criteria:
% RSD of known, unknown and total impurity content should be not more than the limits specified below. Reporting threshold value is 0.2%.
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 9.0 Pooled results of analyst – I & analyst – II
| Sr. No. | Sample Weight (mg) | Known Impurities (%) | Unknown Impurities (%) | %Total impurity ( ≥0.2%) | ||||||||||
| Impurity A | Impurity B | Impurity C | Impurity D | Impurity E | Impurity F | Impurity H | Impurity L | Impurity M | 1 | 2 | ||||
| ANALYST I | ||||||||||||||
| RRT | ||||||||||||||
| 1 | ||||||||||||||
| 2 | ||||||||||||||
| 3 | ||||||||||||||
| 4 | ||||||||||||||
| 5 | ||||||||||||||
| 6 | ||||||||||||||
| ANALYST II | ||||||||||||||
| RRT | ||||||||||||||
| 1 | ||||||||||||||
| 2 | ||||||||||||||
| 3 | ||||||||||||||
| 4 | ||||||||||||||
| 5 | ||||||||||||||
| 6 | ||||||||||||||
| Mean | ||||||||||||||
| SD | ||||||||||||||
| % RSD | ||||||||||||||
Acceptance criteria:
Pooled % 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:
System suitability tests are based on concept that the equipment, electronics, analytical operations and sample to be analyzed, system suitability test provide the added assurance that on specific occasion the method is given accurate and precise results.
The system suitability should be as per below mention criteria in Table 10.0.
Table 10.0: System Suitability Criteria
| Sr. No. | Parameters | System Suitability Criteria | Limit |
| 1. | Resolution | Resolution between Impurity B and Erythromycin C | NLT-1.2 |
| 2. | Peak to Valley Ratio | Peak to valley ratio (Hp/Hv) between impurity F and Erythromycin B. | NLT 1.5 |
| Peak to valley ratio between Impurity C and Erythromycin A. | NLT 2.0 | ||
| 3. | % RSD | Reference solution (c) repeatedly six times and % RSD of six replicate injections. | NMT 5.0 %. |
- 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 related substances of Erythromycin Ph. Eur. by pharmacopeia method.
Summary of verification report shall be prepare and accordingly conclusion and recommendation to be given.
Abbreviations:
REL : Related substance
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
% : 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
Revision History :
| Revision No. | Details of changes | Reason for change |
| 00 | Nil | New Document |
