SOP for Good Chromatographic Practices as per BP / USP | SOP for HPLC Integration |
- SOP covers below points:
- Chromatographic terms and their definitions with formulas
- Good Chromatographic Integration Practices
- HPLC Integration Types
- Integration parameters
- Manual Integration
1.0 OBJECTIVE :
- To lay down the guidelines for chromatographic practices in Quality Control Department.
2.0 SCOPE :
- This SOP is applicable to chromatographic analysis by High performance liquid chromatography (HPLC) carried out in Quality Control Department
3.0 RESPONSIBILITY :
- Officer and above
- Quality Control Head
5.0 PROCEDURE :
- Chromatogram: A graphical or other representation of detector response, effluent concentration or other quantity used as a measure as a measure of effluent concentration, versus time or volume.
- Peak: The portion of chromatogram recording the detector response when a single component or 2 or unresolved components is eluted from the column.
- Baseline: A horizontal line drowns from each detected peak start point to each detected peak end point.
- Peak apex: The highest point on a positive chromatographic peak or the lowest point on a negative chromatographic peak.
- Retention time: Time required for elution of a component.
- Retention time: Volume of mobile phase required for elution of a component. It is calculated by multiplying retention time and flow rate in milliliters per minute.
- Chromatographic integration: A process to detect peaks & identify the baseline in order to calculate responses of different peaks i.e. area/ height & retention time. The integration by default shall be auto integration.
- Auto integration: A mathematical process (employed by the software) directed by a defined set of integration parameters to detect peaks and to calculate peak responses.
- Manual integration: A process employed by the data user to integrate the peaks by using cursor without setting the auto integration parameters or events.
- Peak width: A parameter that specifies the minimum half height peak width (in seconds/ minutes) for detecting a chromatographic peak.
- Threshold: A parameter that specifies the minimum rate of change (in µv/ sec) of slope required for detecting a peak start and peak end.
- Reporting threshold: A limit above which an impurity is to be reported.
- Identification threshold: A limit above which an impurity is to be identified
- Disregard limit: In chromatographic testes, the nominal content at or below which peaks/ signals are not taken into account for calculating a sum of impurities.
- Positive error: The reported/observed value is greater than actual/expected value.
- Negative error: The reported/observed value is lesser than actual/expected value.
- Percentage relative standard deviation(% RSD)
- Relative response factor:
- Response factor: The response factor of the drug substance or related substances per unit weight. Typically the response factor of the drug substance (or related substances) can be calculated by the following formula,
- Construct linearity curve for both the related substance and drug substance by plotting the response verses the concentration, the relative response factor can be determined by,
- % interference
- Rf value
- Signal-to-noise ratio(S/N) for chromatographic techniques:
- The signal-to-noise ratio (S/N) influences the precision of quantification and is calculated from the equation:
- Peak to valley ratio:
- The peak-to-valley ratio (p/v) may be employed as a system suitability requirement in a test for related substances when baseline separation between 2 peaks is not reached
5.2 This procedure is applicable to chromatographic analysis by HPLC for tests such as assay, known impurities, uniformity of content, dissolution, Related substances/ chromatographic purity, and any other test as applicable.
5.3 Chromatographic Analysis
- Ensure that the specification and standard test procedure for the analysis of under test sample is valid and correct.
- The availability of column, working standard, reference standard, reagents, chemicals and any specific requirement such as filter etc. as per standard test procedure shall be verified.
- Fix the column to HPLC for washing with recommended solvents.
- Purge the system sufficiently to remove any air bubble.
- Flush the column with the respective solvent which is used for analysis followed by distilled water, wherever required.
- Prepare the mobile phase by using HPLC grade solvents and adjust pH of mobile phase, if required, as per standard test procedure.
- De-gas the mobile phase with the help of vacuum filtration method or ultra sonication method.
- Select the correct method in HPLC as per standard test procedure from the available method database in HPLC.
- If the method is not available in method database then method developer/ reviewer creates the method in HPLC as per the standard test procedure.
- While selecting or creating the method in HPLC, method developer/ reviewer conforms the correctness of column, wavelength, flow rate and column temperature (if applicable) etc. as per standard test procedure.
- Load the mobile phase and equilibrate the column with mobile phase till stable base line achieved.
- If the baseline is unstable or noisy then flush the HPLC system with hot water by attaching the union in place of the column to clean the flow cell, also check the mobile phase is prepared by using HPLC grade solvents & degassed.
- Prepare the standard solution / system suitability solution as per the standard test procedure and inject diluent and then the injection of standard solution / system suitability solution.
- The sequence is prepared by the operator and verified by method developer/reviewer.
- Start the actual sequence of analysis after authorization from the method developer / reviewer.
- The chromatogram is verified for retention time/ resolution/ tailing factor/ theoretical plate/ relative retention time/ peak shape/ scaling/ peak marking/ peak height/ run time/ base line/ drift etc. as applicable as per standard test procedure.
- If the system suitability criteria do not comply as per standard test procedure, it shall be informed to method developer/ reviewer.
- The sequence is modified by method developer to incorporate additional injection for system suitability verification. Only three injections are permitted for system suitability verification.
- If the system suitability criteria do not comply in three injections, the sequence will be discontinued. The entry of the same is taken into the log of “Unusual event observed during HPLC analysis” Annexure No. 01.
- If system suitability criteria comply, further analysis will be continued as per the sequence.
- During the analysis, any unusual events observed such as retention time change, peak shape variation, area variation, system suitability failure (% RSD of replicate standards), any system trouble shooting etc., the same is informed to reviewer/ HOD and the analysis is discontinued.
- If the sequence is run up to the standard solution injection, the entry of the same is taken into the log of “Unusual event observed during HPLC analysis” Annexure No. 01.
- The data path is mentioned in “Unusual event observed during HPLC/ analysis” to identify the chromatogram related with unusual events.
- If unusual event observed after the test solution is injected, it is triggered through incident and documented.
- Method developer/ Reviewer verifies the preparation of mobile phase, standard solution, and chromatographic conditions etc., if all the given parameters are correct as per standard test procedure then method developer/ reviewer in consultation with QC Head can allow to change the column or the chromatography condition as per Adjustments / variations of operating conditions for Isocratic/Gradient elution as per Pharmacopoeia.
- By changing the column or chromatographic condition, if system suitability complies as per standard test procedure then proceeds for further analysis.
- System suitability criteria specified in standard test procedure is ensured for chromatograms of all injections and it should be complied.
- If it is mentioned in standard test procedure/ pharmacopeia to verify in system suitability criteria for specific chromatogram, it is ensured that the system suitability criteria should be complied for that chromatogram only.
- Upon completion of analysis i.e. sample set/ batch sequence, the analyst shall verify the procedural compliance.
- Run time of the chromatogram shall be same for all injections such as blank, placebo solution, system suitability solution, other reference solution, standard solution and test solution as well as bracketing standard as per standard test procedure.
- If run time is not mentioned in standard test procedure, run time shall be kept at about 1.5 times of main analyte peak for assay and 2.5 times of main analyte peak for related substances test.
5.4 Integration for tests employing Quantification of Principal peaks(s) – (such as Assay, Preservative content, Content of uniformity/ Uniformity of dosage units and Dissolution).
- Integration parameters such as Peak width, Peak threshold, Integration time and other events shall be set appropriately to detect peak(s) of interest.
- “Base to Base” auto integration shall be the preferred type of integration for symmetrical peaks having major responses without co-elution / overlapping and where the base line is stable. However, use of “Valley to valley” integration event shall be also included, if required by the test chromatogram.
- Following is a typical example(s) for a single peak during assay test.
- Illustration of an “Acceptable practice” of doing integration
- Illustration of an “Unacceptable practice” of doing integration
- Peak enhancing: Extending/ adding the area, which does not belong to subject peak this gives +ve error.
- Peak shaving: Eliminating a part of the subject peak area this gives –ve error.
- Other type of peak shaving: Baseline above the original baseline, which gives –ve error.
- The integration parameters shall remain same for the chromatograms in the same sequence for the same type of test. However, if the standard preparation is similar i.e. assay and content of uniformity, then same integration parameters shall be set for both tests analyzed in the same sequence.
- Analyst shall integrate the principal peak of 1st chromatogram with proper integration and save integration which will be applied for all remaining chromatograms of sequence.
- Reporting: For assay, content of uniformity and dissolution, the report scaling of chromatogram(s) shall be in the auto / full scale or zoomed scale or at least 70% analyte peak height of total chromatogram to show the appropriate integration.
5.5 Integration for test samples employing Quantification of minor peaks in combination with Principle peak(s) – (such as related substances, chromatographic purity & other low content tests)
- The processing method shall be defined as per individual test procedure and specific integration events.
- Once the peaks are properly detected, they shall be identified such as blank/ diluent 1…n, Placebo 1 …n, Principal (main) peak, Known impurities 1…n, Unknown impurities 1…n.
- After completion of test analysis, the processing method shall be created for sample set in the auto integration settings.
- When the auto integration setting does not integrate all the intended peaks, and then modify the integration parameters with reasons.
- The reasons should be documented in the audit trail within the same sequence.
- Some of the typical reasons including but not limited to the below; for example: changed the peak width/ threshold and or other parameter/ event as the previously set integration parameters/ events are not able to integrate the peaks X and Y.
- Following are few examples of related substances tests,
- Unresolved peaks
- Illustration of an “Acceptable practices” of doing integration for an unresolved peak.
- Perpendicular skim: Draw a vertical line from the valley of two peaks to the original baseline. Integrate the first peak from when the baseline starts to rise & end at the vertical line. “Integrate the second peak from the vertical line upto the point where the signal returns to the original baseline. (Refer figure a)
- Illustration of an “Unacceptable practice” of doing integration for an unresolved peak.
- Tangent Skim: A tangent is drawn from when the baseline starts to rise and to end when the peak ends (Refer figure b).
- Improper integration which gives +ve error. (Refer figure c & d).
- Shoulder or Co-elution with large peak
- Illustration of an “Acceptable practices” of doing integration for a shouldering peak.
- Start integration, when the signal first begins to rise during the down curve and end when the signal drops.
- Illustration of an “Unacceptable practice” of doing integration for a shouldering peak.
- Spike or Base line ripple
- Illustration of an “Acceptable practices” of doing integration for a negative peak.
- Start integration, from the expected baseline and end when the peak drops to the baseline.
- Illustration of an “Unacceptable practice” of doing integration for a negative peak.
- Improper integration which gives +ve error.
- Reporting: The preferable report scaling of related substances chromatogram (by HPLC) shall be is -0.02 to + 0.05mV or at least 50% of the height of lowest impurity peak. Enlarged chromatograms should be used to differentiate the baseline noise and peak.
- Overlay of chromatograms: Blank and test sample or set of test samples chromatograms shall be over laid by enlarging / zooming them in such a way that the smallest peaks are visible evidently.
5.6 Handling of chromatograms with Manual Integration
- Manual integration of peak(s) is not an acceptable approach. However, to indicate the necessity of manual integration, the sample shall be processed.
- Manual integration for related substances test will be carried out by integrator.
- Manual integration other than related substances test will be carried out in consult with Head-QC and it will be triggered though incident and investigated.
- Manual integration can be allowed only in “exceptional” cases where the integration could not be performed as demonstrated (with at least two different auto integration setting) in the results from the auto integration.
- Some of the “exceptional” cases where manual integration is needed are listed below, but not limited to the following scenario’s.
- Limitations due to software integration
- Missed peak: Where auto integration does not detect a peak due to low response i.e. peak is not integrated.
- Wrong peak: Where auto integration detects a peak, which is incorrect & having marginal difference in retention time.
- Complicated chromatography due to sample and / or matrix interference
- Unresolved peak: Where auto integration integrates two peaks with closest RT as one peak or other improper integration.
- Split peaks: Where auto integration results to divide the peak at the inflection point.
- Shouldering or co-elution: Where the baseline is not dropped for tangents for unresolved peaks other improper integration of this type.
- Baseline noise: Where baseline is having substantial amount of noise, due to which auto integration results in error.
- Baseline drift: Where baseline is either rising/ falling usually due to gradient elution, due to which auto integration results in error.
- Negative peaks: Where there is sudden drop in base line, usually in refractive index due to which auto integration results in error.
- Excessive peak tailing: Where failure of instrument response to return to baseline usually due to excessive amount of sample load/ concentration.
- Fluctuations in the chromatograph
- Spike: Where a sudden dip/ shoot up of baseline due to vibration or electrical noise, usually expected in Gas chromatography
- Limitations due to software integration
5.7 Documentation and review
- Each chromatogram shall mention but not limited to the following details:
- Name of Organization
- Sample Name
- Sample ID
- Data file name
- Method file name
- Batch file name
- Date Acquired
- Data processed
- Analyst Name
- Analysed by
- Checked by
- All the auto integrated chromatograms shall be printed and documented along with record of analysis. For chromatograms that are manually integrated, print the normal and enlarged/ zoomed chromatograms to show the integration.
- Bracketing standard chromatograms shall be printed and %RSD shall be checked for the same.
- If first bracketing is not within the limit, and cause is identified, inject 2nd bracketing in the same sequence. If second bracketing standard shall be within the limit, the data shall be made valid and reported.
- Each chromatogram shall be signed with date by the analyst as Analysed by and the reviewer as checked by.
- Any unauthorized re-processing of the chromatograms shall not be done.
5.8 General Points to Consider:
- For assay test, the sample and standard concentration should be close. For reporting the results, average of two injections should be considered for assay.
- Retention time should be ± 15 %.
- If the blank & placebo peaks are appearing in the sample solution and if their response is greater in sample solution as compared to the blank & placebo solution then such peaks shall be considered as unknown peaks eluted at the same retention time by subtracting the blank & placebo peak areas.
- During related substance test, the processing methods used to process the injections of the same set shall be same for the blank, placebo, standard and the sample solution.
- Standard chromatograms can be processed by changing the height and area parameters only, but the threshold and width shall be same for all the injection of the same sample set.
- In case of related substance /Chromatographic purity /related compound test the smaller peaks or integration of the peaks is not correct then such peaks can be integrated manually with the intimation to the section in-charge or head of the department.
- In case of related substance /Chromatographic purity /related compound test by gradient method manual integration may be applied to get the proper integration for all the available peaks in the chromatograms.
- The bracketing standard/ system suitability solution can be injected in between the sequence or at the end of the sequence.
- For normal phase chromatography; the HPLC needs to be converted into normal phase before use, do no attached column during conversion.
- The procedure for converting from reverse phase to normal phase is as follows; Wash HPLC system with hot water to remove traces of any buffer. Then flush with HPLC grade methanol for about 30 minutes. Flush system with IPA followed by Hexane.
- To convert back to reverse phase above procedure shall be followed in reverse order.
- Washing of HPLC column before and after use is recommended with suitable solvent, like water: methanol:: 80:20 for C8/C18 columns.
- Use suitable solvents for seal wash/injector wash in HPLC. Replace the same regularly.
- For HPLCs if the water is used as a solvent, it needs to be changed daily to avoid any fungi growth.
- For mobile phase/diluents having only buffers shall be prepared freshly, before use.
|Standard Operating Procedure
|High Performance Liquid Chromatographic
|United State Pharmacopoeia
|Relative Standard Deviation
|Relative retention time
|Unusual event observed during HPLC analysis
8.0 SOP REFERENCES
- BP 2015, Appendix III – Chromatographic separation Techniques
- USP 38 NF 33, <621> Chromatography
END OF THE SOP
Annex. No. 01 Unusual event observed during HPLC analysis