ANALYTICAL METHOD VERIFICATION PROTOCOL FOR RESIDUAL SOLVENT OF ERYTHROMYCIN Ph. Eur.

ANALYTICAL METHOD VERIFICATION PROTOCOL FOR RESIDUAL SOLVENT 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Superseded Protocol No.	Nil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 verification is to provide documentary evidence that analytical methodology used for residual solvent of Erythromycin Ph. Eur. by GC method is consistent and reliable results within the predetermined acceptance criteria. This verification study is intended to show that the method is suitable for release of residual solvent of manufacturing batches of API.

Analytical method verification will be performed by considering thefollowing parameters:

Parameters	Erythromycin Ph. Eur.
Specificity	yes
Precision
System Precision	yes
Method Precision	yes
Intermediate Precision (Ruggedness)	yes
System Suitability	yes

• Scope :

The scope of this protocol is applicable for the verification of method of analysis of residual solvent of Erythromycin Ph. Eur. By GC method.

• 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	Residual solvent Dichloromethane :  NMT 600 ppm

• Methodology:

Solvents:

Table 1.0: Chemical

Sr. No.	Solvents	Grade
	Dichloromethane	GC Grade
	N,N-Dimethylformamide	GC Grade

Instrumentation

A Gas chromatograph equipped with head space and auto injector, Flame Ionization as a detector and an auto sampler.

Data handling system: Empower or equivalent.

Chromatographic conditions:

Column                                         :           DB-624, 60 m x 0.530 mm, 3.0 µm (Part No: 125-1364)

Oven Programming                      :          

Initial temperature (°C)           –           100

                        Initial time (min)                      –           2.0

                        Rate 1 (°C/min)                       –           5

                        Temperature 1 (°C)                –           120

                        Hold time (min)                       –           2.0

                        Rate 2 (°C/min)                       –           25

                        Temperature 2(°C)                 –           240

                        Hold time (min)                       –           5

                        Run time (in min)                    –           17.8

Injection Mode                              :           Split

Split ratio                                       :           3: 1

Carrier gas flow rate (ml/min)       :           4

Injection Port temperature (°C)    :           140

Detector                                        :           FID

Detector temperature (°C)            :           250

Carrier gas                                    :           Nitrogen

Make up gas                                 :           Nitrogen

Make-Up Flow (ml/min)                :           45

H2 Flow (ml/min)                          :           40

Air Flow (ml/min)                          :           450

Sampling rate                                :           5.0

Data sensitivity                              :           High

Head space condition                   :           Oven temperature                  –           60°C

Loop temperature                   –           110°C

                        Transfer line temperature       –           150°C

                        GC cycle time                         –           21 minutes

                        Vial equilibration time              –           15 min.

                        Vial pressure time                   –           0.5 min

                        Loop filling time                       –           0.10 min

                        Loop equilibration time            –           0.20 min.

                        Injection time                           –           0.2 min.

                        Vial pressure                           –           15 psi

                        Vial shake mode                     –           High

                        Injection Volume                     –           1 ml

Diluent                                          :           N,N-Dimethylformamide

Blank preparation:

1. Pipette out 5.0 ml of diluent into 20 ml headspace vial.
2. Close tightly with PTFE / Silicone septum and aluminum crimp cap.
3. Label and insert into the auto sampler compartment.

Standard preparation:

1. Accurately weigh and transfer about 60 mg of Dichloromethane standard into a 100 ml volumetric flask containing 10 ml of diluent.
2. Dissolve and dilute to volume with diluent.
3. Pipette out 10 ml of stock standard solution into a 100 ml volumetric flask. Dilute to volume with diluent.

Head space standard preparation:

1. Pipette out 5.0 ml of working standard solution into 20 ml headspace vial.
2. Close tightly with PTFE / Silicone septum and aluminum crimp cap.
3. Label and insert into the auto sampler compartment.

Sample preparation:

1. Accurately weigh and transfer about 1000 mg of sample into 10 ml volumetric flask, dissolve and dilute to volume with diluent.
2. Pipette out 5.0 ml of above solution into 20 ml headspace vial.
3. Close tightly with PTFE / Silicone septum and aluminum crimp cap.
4. Label and insert into the auto sampler compartment.

Chromatographic procedure:

1. Inject blank, check baseline noise and drift or any other sign of system instability. Repeat injections until the system is stable.
2. Inject standard preparation repeatedly six times, calculate the % RSD. % RSD should be NMT 15%.
3. Inject blank preparation once and check for any carryover.

Once system suitability conditions met continue with sample analysis.

• Inject sample preparation once.
• Inject standard preparation once; calculate the %RSD for calibration standards and the bracketing standard. %RSD should be NMT 15 %.

The retention times (RT) for the solvent peak is given below.

Name of the Solvent	~RT (min)
Dichloromethane	4.7

Calculation:

Calculate the content of all residual solvents by the following expression:

                              Au  x Ws  x 10 x 10 x purity of standard            

ppm of solvent = —————————————————————— x 1000000

                              As  x 100  x 100  x Wu X 100

Where;

Au  :           Area of respective solvent peak in sample chromatogram

As  :           Average area of respective solvent peak in standard chromatogram

Wu :           Weight of sample taken for analysis

Ws :           Weight of respective solvent peak in standard preparation.

Quantification Limit (QL)

Dichloromethane  : 31 ppm

Note: Report results as Below Quantification Limit (BQL) if the, Dichloromethane content is below specified Quantification Limits (QL).     

• Verification parameters:

The following parameters to be perform for the verification activity.

1. Specificity
   1. Precision
   1. System Suitability
   1. Specificity:

Specificity is the ability to assess unequivocally the Analyte in the presence of components which may be expected to be present.

Specificity of test method should be established by separately injecting blank solution (N,N-Dimethyl formamide), spiked solution and test solution. Spike sample shall be prepared by adding Dichloromethane standard in test solution at specification limit level and test solution to be prepared as per method of analysis. Sequence shall be prepare as per the sequence mentioned in table no 2.0 and data shall be represent in report as per the table no 3.0.

Diluent: N, N-Dimethyl formamide

Blank preparation:

1. Pipette out 5.0 ml of diluent into 20 ml headspace vial.
2. Close tightly with PTFE / Silicone septum and aluminum crimp cap.
3. Label and insert into the auto sampler compartment.

Standard preparation:

1. Standard Stock Solution: Accurately weigh and transfer about 60 mg of Dichloromethane standards into a 100 ml volumetric flask containing 10 ml of diluent.
2. Dissolve and dilute to volume with diluent.
3. Final standard dilution: Pipette out 10 ml of stock standard solution into a 100 ml volumetric flask. Dilute to volume with diluent.

Head space standard preparation:

1. Pipette out 5.0 ml of working standard solution (Final) into 20 ml headspace vial.
2. Close tightly with PTFE / Silicone septum and aluminum crimp cap.
3. Label and insert into the auto sampler compartment.

Sample preparation:

1. Accurately weigh and transfer about 1000 mg of sample into 10 ml volumetric flask, dissolve and dilute to volume with diluent.
2. Pipette out 5.0 ml of above solution into 20 ml headspace vial.
3. Close tightly with PTFE / Silicone septum and aluminum crimp cap.
4. Label and insert into the auto sampler compartment.

Spike Sample preparation:

1. Accurately weigh and transfer about 1000 mg of sample into 10 ml volumetric flask, dissolve and dilute to volume with final standard dilution.
2. Pipette out 5.0 ml of above solution into 20 ml headspace vial.
3. Close tightly with PTFE / Silicone septum and aluminum crimp cap.
4. Label and insert into the auto sampler compartment.

Tablet 2.0: Sequence

Sr. No	Solution	No of Injection to be injected in Sequence
1	Blank	1
2	Standard Solution (Six vials)	6
9	Blank (to avoid carry over)	1
10	Test Solution	1
11	Spiked Test Solution	1
12	Standard solution + Bracketing	1

Table 3.0 Specificity data

Sr. No	Sample	RT (min.)
1	Blank	N,N-Dimethyl formamide
2	Standard solution	N,N-Dimethyl formamide
Dichloromethane
3	Test solution	N,N-Dimethyl formamide
Dichloromethane
4	Spike Test solution	N,N-Dimethyl formamide
Dichloromethane

Acceptance Criteria:

1. There should be no interference of the blank and solvent content at the same retention time.
2. Solvent peaks should be well resolved from diluent peak and observe the elution of pattern.
   1. Precision:
      1. 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).

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 of main analyte peak. And calculate the % area RSD and % RT RSD of main analyte peak.

Table 4.0 System Precision- Repeatability of Standard Injections

Sr. No.	Dichloromethane
Peak Area	Retention time (min.)
1
2
3
4
5
6
Mean
SD
% RSD

Acceptance Criteria:

% RSD for peak area and retention time of replicate standard solution injections should be NMT 15.0% 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 and analyzed by spiking the known solvent content of Dichloromethane in test solution at specification limit level. As such sample will be also analyzed to identify the known solvent content in sample. Obtained known solvent in as such sample will be subtracted in spiked sample to calculate the actually known spiked amount of solvent content.  Record the area on data sheet and calculate the ppm of solvent content. Mean, standard deviation and % relative standard deviation shall be reported.

Table 5.0 Method precision results spiked sample at limit level

Sr. No	Sample wt. (mg)	Dichloromethane
Peak Area	Results (ppm)
1
2
3
4
5
6
Mean
SD
%RSD

Acceptance criteria:

Calculate the solvent content in ppm for each analysis and report the mean, SD and % RSD.

% RSD of each of solvent content should be NMT 15.0%.

• 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 substance 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 of main analyte peak. And calculate the % area RSD and % RT RSD of main analyte peak.

Table 6.0 System Precision- Repeatability of Standard Injections

Sr. No.	Dichloromethane
Peak Area	Retention time (min.)
1
2
3
4
5
6
Mean
SD
% RSD

Acceptance Criteria:

% RSD for peak area and retention time of replicate standard solution injections should be NMT 15.0% and 1.0% respectively.

Test Procedure:

Prepare the six samples of same batch as per standard analytical procedure and analyzed by spiking the known solvent of each content at limit level. As such sample will be also analyzed to identify the known solvent content in sample. Obtained known solvent in as such sample will be subtracted in spiked sample to calculate the actually known spiked amount of solvent content.  Record the area on data sheet and calculate the ppm of solvent content. Mean, standard deviation and % relative standard deviation shall be reported.

Table 7.0 Intermediate precision results spiked sample at limit level

Sr. No	Sample wt. (mg)	Dichloromethane
Peak Area	Results (ppm)
1
2
3
4
5
6
Mean
SD
%RSD

Acceptance criteria:

Calculate the solvent content in ppm for each analysis and report the mean, SD and % RSD. % RSD of each of solvent content should be NMT 15.0%.

Table 8.0 Cumulative results of Method Precision and Intermediate Precision

Sr. No	Sample wt. (mg)	Dichloromethane
Peak Area	Results (ppm)
Method Precision
1
2
3
4
5
6
Intermediate Precision
1
2
3
4
5
6
Cumulative Mean
Cumulative SD
Cumulative %RSD

Acceptance criteria:

The Cumulative % RSD of each of solvent content of Method precision and Intermediate precision should be NMT 15.0%.

• 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 9.0.

Table 9.0

Parameters	System Suitability Criteria	Limit
% RSD	The area %RSD of six replicate injection of each of solvent content in standard solution	NMT – 15.0%

• Incident/Deviation:

Any Incident or Deviation observed during Analytical Method verification shall be recorded and investigate as per the current SOP.

• Summary/Conclusion/recommendation:

Final conclusion should be drawn from analytical method verification for its use to analyze the residual solvent test of Erythromycin Ph. Eur. by GC.

Summary of verification report shall be prepare and accordingly conclusion and recommendation to be given.

Abbreviations

RES                :           Residual solvent

            VERP              :           Verification Protocol

            RSD                :           Related standard deviation

            GC                  :           Gas chromatography

            mL                   :           Milliliter

            mg                   :           Milligram

            PPM                :           Parts per million

            min.                 :           Minutes

            QA                   :           Quality Assurance

            QC                  :           Quality Control

            %                     :           Percentage

            ºC                    :           Degree centigrade

            hrs                   :           Hours

            µm                   :           Micrometer

            Ph.Eur.            :           Europeon Pharmacopoeia

            µl                     :           Microlitre

            RT                   :           Retention time

            NLT                 :           Not less than

NMT                :           Not more than

SOP                :           Standard Operating Procedure

Vol                   :           Volume

API                  :           Active product ingredient

Revision History :

Revision No.	Details of changes	Reason for change
00	Nil	New Document