ANALYTICAL METHOD VERIFICATION PROTOCOL FOR ASSAY OF ERYTHROMYCIN Ph. Eur.

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Superseded Protocol No.	Nil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

1. 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) – t_R + 5

Program                :  Gradient

Note: Modify gradient program as per Retention time of Erythromycin B peak (t_R) 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:

1. 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.
2. Inject reference solution (a) once to check for system suitability requirements.
3. Compare the retention time with the expected one and calculate symmetry factor, Symmetry factor should be NMT 2.0 for Erythromycin peak.
4. Inject blank (Diluent) and check for carryover.
5. Inject Reference solution (a) repeatedly six times and calculate the % RSD, should be NMT 1.0 %.
6. Inject reference solution (b) repeatedly six times and calculate the %RSD, should be NMT 5.0%.
7. If all system suitability requirements meets continue with sample analysis
8. Inject sample preparation once.
9. Inject once reference solution (a) as bracketing standard at the end of the sequence.
10. 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:

1. RRT are with reference to Erythromycin A retention time about 23 min.
2. 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

1. Turn on the titrator.
2. Turn on the printer; check the connection between Titrator and printer.
3. Press method on titrator and enter the same name details and Press RUN.
4. After pre-titration, accurately weigh 0.2 g of the sample.
5. Transfer the weighed sample into the designated beaker containing 40 ml of 100 g/L solution of Imidazole in anhydrous Methanol.
6. Press sample on the titrator and enter the transferred weight and press RUN.
7. 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:

1. There should be no interference of the solvent mixture at the retention time of analyte peak.
2. Blank peak should be well resolved from active peak and each other,
3. 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