by ANALYTICAL METHOD VALIDATION PROTOCOL FOR DETERMINATION OF N-METHYL-3-PYRROLIDINOL IN GLYCOPYRRONIUM BROMIDE BY GAS CHROMETOGRAPHY
| PROTOCOL NUMBER: |
TABLE OF CONTENT
| S.NO | CONTENTS | PAGE NO |
| Protocol approval | ||
| Objective | ||
| Scope | ||
| Responsibility | ||
| Procedure | ||
| Standards | ||
| Samples | ||
| Equipment | ||
| Test Methodology | ||
| Validation Parameters to be validated | ||
| Deviation | ||
| Summary | ||
| Conclusion | ||
| Abbreviation | ||
| Annexure |
PROTOCOL APPROVAL
Prepared by:
| S.# | Name | Designation | Signature | Date |
Reviewed by:
| S.# | Name | Designation | Signature | Date |
Validation Team:
| S.# | Name | Designation | Signature | Date |
Approved by:
| S.# | Name | Designation | Signature | Date |
1. Objective :
Objective of this method validation is to provide documentary evidence that analytical methodology used in the determination of the N-methyl-3-pyrrolidinol in Glycopyrronium bromide will yield consistent and reliable results within the predetermined acceptance criteria.
The method validation for N-methyl-3-pyrrolidinolwill be performed as per Protocol. This protocol describes the methodology which will be used in the validation study for the following parameters;
1. Specificity
2. Precision
3 Limit of Detection and Limit of Quantitation (LOD & LOQ)
4. Linearity and Range
5. Intermediate Precision (Ruggedness)
6. Accuracy (Recovery)
7. Robustness
8. Stability of Analytical solutions
2. Scope:
The scope of this method validation protocol is limited to analytical method for determination of N-methyl-3-pyrrolidinol(NMP) in Glycopyronium bromide drug substance.
3. Responsibility:
| Jr Scientist-Analytics: |
| Responsible to prepare, execute the validation as per approved validation protocol and prepare the validation report. |
| Sr scientist –Analytics: Responsible to check the validation protocol, raw data, validation report and to review the validation document. |
| Head –QA: Responsible to review and approve the validation protocol and validation report . |
4. Procedure:
Method validation of N-methly-3-pyrrolidinol in Glycopyrronium bromide is to be done as per Protocol and after actual experiment report is generated along with chromatograms.
4.1 GENERAL INFORMATION:
| Category | : | Related substance method | |||
| Methodology | : | Non-compendia | |||
| Reference | : | ICH Q2 (R1) | |||
| Method reference | : | In-house | |||
| Reason for validation | : | Regulatory requirement | |||
| Type of validation | : | Complete | |||
| ICH | In-house | ||||
| Specification (%) | : | N-methyl-3-pyrrolidinol | : | ————– | NMT 0.10% |
| * Specifications are as per Inhouse.. |
5. STANDARDS:
| S. # | Name | Batch / Lot# | Manufacturer | % Purity |
| 1 | Methanol | |||
| 2 | N-methyl-3-pyrrolidinol |
| 6. | SAMPLES: | |||
| S. # | Name | Batch No. | Manufacturer | |
| 1 | Glycopyrronium bromide |
| 7. | EQUIPMENTS: | |||
| S. # | Name | Equipment details | ID # | |
| 1 | GC-autosampler | |||
| 2 | Analytical Balance |
8. TEST METHODOLOGY
GC Chromatographic conditions: (For Agilent GC)
| Column | DB-1 (30 m x 0.53 mm x 1.5µm) or equivalent |
| Initial Oven temp | 80oC |
| Initial Hold time | 5.0 minute |
| Ramp(1) | 20oC/minute |
| Oven temperature(1) | 270oC |
| Hold time(1) | 10 minute |
| Injection Port | |
| Injection mode | Split as per STP split less |
| Injector temperature | 225oC |
| Carrier gas | Nitrogen |
| Column pressure | 4.00 PSI(27.6 kPa) |
| Purge flow | 1.0 ml/min |
| Column mode | Constant flow |
| Detector Port | |
| Average velocity | 40.0 cm /sec |
| Detector temperature | 300oC |
| Total flow | 8.8 ml/min |
| Flow rate | 5.0 ml/min |
| Hydrogen flow | 45 mL/minute |
| Air flow | 450 mL/minute |
| Run time | 24.5 min |
| Auto injector parameter | |
| Rinse solvent | Methanol |
| Injection speed | Normal |
| Viscosity delay | 0 |
| Pre-injection solvent washes | 3 |
| Post-injection solvent washes (A) | 6 |
| Injection volume | 2.0 µl |
| Pre-injection sample washes | 3 |
| Sample pumps | 6 |
Preparation of Solutions:
Diluent: Methanol
Blank Preparation: Use Methanol diuent as blank
Preparation of Standard stock solution:
Accurately weight about 0.1 g of N-methyl-3-pyrrolidinol(NMP)impurity working standard into a 100 ml of volumetric flask containing about 20 ml of Methanol. Mix and Make up to the volume with Methanol(1000 ppm).pipette out 10.0 ml of this solution into 50 ml volumetric flask containing 20 ml of methanol. mix make up to the mark with methanol(200 ppm).
Preparation of standard solution
Transfer 5.0 mL standard stock solution(200 ppm) in to a 10 ml volumetric flask and Make up to the volume with Methanol.
Sample preparation:
Accurately weigh and transfer about 1.0 g of sample into 10 mL volumetric flask. Add about 3.0 ml of Methanol.sonicate to dissolve and make up to the mark with Methanol.
Simlarly prepare one more test solution
Procedure:
Equilibrate the column with prescribed chromatographic condition and follow the injection.
sequence as given below:-
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | Blank | 1 |
| 4 | Test Solution | 2 |
| 5 | Blank | 1 |
| 6 | Bracketing standard | 1 |
Elution pattern:
| S. No | Peak | RT/RRT |
| 1 | N-methyl-3-pyrrolidinol | |
| 2 | methanol |
Calculation:
AT x Ws x 10 x 5 x10 x P
%N-methyl-3-pyrrolidinol content = ———————————— x 100
AS x 100 x 50 x10 x WT x 100
AT = Peak area of N-methyl-3-pyrrolidinol observed in test sample solution
AS = Average Peak area of N-methyl-3-pyrrolidinol observed in the standard solution
WS =Weight of N-methyl-3-pyrrolidinol in standard solution in mg
WT =Weight of N-methyl-3-pyrolidinol in test sample in g
P = Purity of standard
System suitability
The test is not valid, if it fails to meet the following acceptance criteria for system suitability
- Relative standard deviation of the peak area obtained from six injections of standard preparation should not more than 15.0 %
9. VALIDATION PARAMETERS TO BE VALIDATED
Note: More than one parameter can be performed in a sequence under valid system suitability.
9.1 Specificity:
Specificity of test method shall be established by injecting all impurity to be examined simultaneously. Prepare blank solution, standard solutions individually, mixed standard and spiked test sample as per method of analysis. Retention time observed for each solvent shall be recorded
Acceptance Criteria:
Retention times of each impurity should match in individual, mixed and spiked solution and there should be no interference at the retention time of the solvents.
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | Blank | 1 |
| 4 | N-methyl-3-pyrrolidinol | 1 |
| 5 | methanol | 1 |
| 6 | Spike Test sample | 1 |
| 7 | Blank | 1 |
| 8 | Bracketing Standard | 1 |
9.2 Determination of LOD and LOQ:
LOD and LOQ will be estimated based on signal to noise ratio values. Determine the LOD and LOQ of N-methyl-3-pyrrolidinol(NMP) theoretically having S/N ratio ≥ 3 and 10 respectively. These value shall be calculated from the signal to noise ratio of 100% specification level standard.
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | LOQ/LOD | 1 |
| 4 | LOQ/LOD | 1 |
| 5 | LOQ/LOD | 1 |
Confirmation of LOD and LOQ:
Prepare the solution of N-methyl-3-pyrrolidinol standard as determined from the above and confirm by injecting solution in to the Gas Chromatograph.
Acceptance Criteria:
The peak area of LOD solution should be consistent and the S/N ratio of the peak should be ≥ 3.0.
The peak area of LOQ solution should be consistent and the S/N ratio of the peak should be ≥ 10.
Precision at LOQ:
Prepare a solution containing N-Methyl-3-pyrrolidinol standard at LOQ concentration and analyze six times. Determine the % RSD of peak area.
Acceptance Criteria:
The % RSD for the peak area should be NMT 15.0 and the S/N ratio should be ≥ 15.
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | Blank | 2 |
| 4 | LOD solution | 3 |
| 5 | Bracketing Standard | 1 |
| 6 | LOQ Solution | 6 |
| 7 | Bracketing Standard | 1 |
9.3 PRECISION:
System suitability shall be demonstrated by injecting standard solution as per described test procedure. Standard solution will be injected six times and the precision will be evaluated by computing the relative standard deviation for area response of N-methyl-3-pyrrolidinol in standard solution.
System Precision:
Prepare N-methyl-3-pyrrolidinol standard solution as per test method and inject for six replicates.
Acceptance criteria:
The % RSD for six replicate injections of standard solution should be NMT 10.0.
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | Blank | 2 |
| 4 | Standard Bracket | 1 |
Method Precision:
Prepare test sample six times and analyze as per the method and report the content of N-methyl-3-pyrrolidinol in the sample.
Acceptance Criteria:
% RSD of content in six sample preparations should be NMT 10.0.
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | Blank | 1 |
| 4 | Test Sample-1 | 1 |
| 5 | Test Sample-2 | 1 |
| 6 | Test Sample-3 | 1 |
| 7 | Test Sample-4 | 1 |
| 8 | Test Sample-5 | 1 |
| 9 | Test Sample-6 | 1 |
| 10 | Blank | 1 |
| 11 | Bracketing standard | 1 |
Note: If the content of N-methyl-3-pyrrolidinol is below LOQ, spike N-methyl-3-pyrrolidinol at specification level in the sample solution and inject the spiked sample by preparing six preparations.
9.4 Intermediate Precision:
To demonstrate intermediate precision of the test method, conduct analyst-to-analyst variability by two analysts under similar conditions on different days using same column . Analyst-2 shall prepare six individual sample preparations separately of the same batch as described in the method. Analyst-1 data shall be used from the method precision study. Calculate the % RSD for individual analyst results and combined results of both analysts.
Prepare test sample six times from the same batch and analyze, and report the content in the samples.
Acceptance Criteria:
% RSD of content of N-methyl-3-pyrrolidinol in six sample preparations should be NMT 10.0.
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | Blank | 1 |
| 4 | Test Sample-1 | 1 |
| 5 | Test Sample-2 | 1 |
| 6 | Test Sample-3 | 1 |
| 7 | Test Sample-4 | 1 |
| 8 | Test Sample-5 | 1 |
| 9 | Test Sample-6 | 1 |
| 10 | Blank | 1 |
| 11 | Bracketing standard | 1 |
9.5 Linearity and Range:
The linearity of response for N-Methyl-3-pyrrolidinol to be examined in Glycopyrronium bromide shall be demonstrated by injecting standard solution containing N-methyl-3-pyrrolidinol in six different concentrations from LOQ, 50%, 75% 100% 125% and 150% of the standard concentration . Each concentration shall be injected in duplicate. Calibration curves will be plotted for N-methyl-3-pyrrolidinol between concentration and the area response.
Report the range from Correlation coefficient and Precision.
Acceptance criteria:
The correlation coefficient should be NLT 0.98.
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | Blank | 1 |
| 4 | Linearity at LOQ | 2 |
| 5 | Linearity at 50% | 2 |
| 6 | Linearity at 75% | 2 |
| 7 | Linearity at 100% | 2 |
| 8 | Linearity at 125% | 2 |
| 9 | Linearity at 150% | 2 |
| 10 | Blank | 1 |
| 11 | Bracketing standard | 1 |
9.6 Accuracy/ Recovery:
Accuracy is a measurement of exactness of the analytical method. Accuracy can be accessed by spiking the standard solution of N-methyl-3-pyrrolidinol into test sample at levels LOQ, 50%, 100% and 150% with respect to the target (Specification) level and % recovery at these levels.
Analyze the solutions in triplicate preparations at each level.
Acceptance criteria:
The % mean recovery of solvent should be between 70%-130%.
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | Blank | 1 |
| 4 | Control Sample | 2 |
| 5 | Blank | 1 |
| 6 | LOQ Standard | 2 |
| 7 | Blank | 1 |
| 8 | Recovery at LOQ-1 | 1 |
| 9 | Recovery at LOQ-2 | 1 |
| 10 | Recovery at LOQ-3 | 1 |
| 11 | Recovery at 50%-1 | 1 |
| 12 | Recovery at 50%-2 | 1 |
| 13 | Recovery at 50%-3 | 1 |
| 14 | Blank | 1 |
| 15 | Bracketing standard | 1 |
| 16 | Recovery at 100%-1 | 1 |
| 18 | Recovery at 100%-2 | 1 |
| 19 | Recovery at 100%-3 | 1 |
| 20 | Recovery at 150%-1 | 1 |
| 21 | Recovery at 150%-2 | 1 |
| 22 | Recovery at 150%-3 | 1 |
| 23 | Blank | 1 |
| 24 | Bracketing standard | 1 |
9.7 Robustness:
Robustness of the method shall be investigated by varying the instrumental conditions such as
flow rate (±0.1mL/min) and split ratio.
Confirm the system suitability parameters by deliberate variations in the method parameters.
9.7.1. Variation in Carrier gas flow: ± 5.0%
9.7.2. Variation in Oven temp: ± 2.0%
Inject blank, six replicate injections of standard solution and spiked sample solutions into the GC system.
Acceptance criteria:
The system suitability and specificity should meet.
| S. No | Solution | No. of Injections |
| 1 | Blank | 1 |
| 2 | Standard solution | 6 |
| 3 | Blank | 1 |
| 4 | Spiked test solution | 1 |
| 5 | Bracketing standard | 1 |
Note: Follow the sequence for each variation
9.8 Solution stability:
To evaluate stability of standard and sample solution, solution containing N-methyl-3-pyrrolidinol shall be prepared as describe in test methodology and repeatedly injected up to 24 hrs from the time of preparation. The stability of solution shall be calculated based on %RSD of area response of repeatedly injected standard solution over a period of 24 hrs. Inject blank, six replicates of standard solution and one injection of sample solution (prepared as per the method precision) upto 24 hours time intervals.
Acceptance criteria:
The system suitability requirements should meet at all the time intervals. The content of solvents in sample solutions should be with in (80-120%) with respect to the initial content.
Note: If the solvent impurities are not detected in the sample inject spike sample solution.
| S. No | Solution | No. of Injections | |||
| 1 | Blank | 1 | |||
| 2 | Standard solution | upto 24hrs | |||
| 3 | Blank | 1 | |||
| 4 | Spiked test solution | upto 24hrs | |||
| 5 | Bracketing standard | 1 | |||
| 10. | Deviation: Any deviation observed during method validation shall be recorded in this section. | ||||
| 11. | Conclusion: |
| Final conclusion shall be drawn regarding developed Analytical method and its validation for its use to analyse N-methyl-3-pyrrolidinol in Glycopyrronium bromide by GC. |
12. Summary:
Summary of the validation report shall be provided in this column.
13. Abbreviations:
Abbreviations used shall be provided under this column.
14. Annexure(s)
Attached chromatograms will be as annexure(s)
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