SOP Title: Chromatographic Analysis, Trouble shooting and Documentation

SOP Title: Chromatographic Analysis, Trouble shooting and Documentation

1. Objective:

To lay down the procedure for Chromatographic Analysis, Trouble shooting and Documentation in the Quality Control Department.

 

2. Scope:

This procedure is applicable for Chromatographic Analysis, Trouble shooting and Documentation in the Quality Control Department.

 

3. Responsibility:

• Quality Control Department: To prepare and review the SOP. To follow the procedures laid down for Chromatographic Analysis, Trouble shooting and Documentation in Quality Control Department as per this SOP.
• Quality Assurance Department: To review and approve the SOP and Annexure.

 

4. Accountability:

Head Quality Control Department, Head Quality Assurance Department

5. Procedure:

5.1	Mobile Phase :
5.1.1	The mobile phase shall be prepared as per respective standard test procedure.
5.1.2	Always use HPLC grade chemicals & solvents. Always filter mobile phase through 0.45 µm filters.
5.1.3	Filter buffer and organic solvent (if required) separately with 0.45 µm filter and then mix to avoid evaporation of organic solvent.
5.1.4	If organic solvent already filtered through 0.45 µm or lesser then no need to filter.
5.1.5	Shake the mobile phase bottle till the buffer and solvents gets properly mixed.
5.1.6	Ensure that there is no phase separation.
5.1.7	Degas the mobile phase by sonicating 2 – 5 minutes or as required.
5.1.8	Mobile phase bottle should be labelled and filled as per Annexure No. 01.
5.1.9	Use freshly prepared mobile phase for HPLC analysis which will be valid for three days only or till the completion of running sequence or based on mobile phase stability data whichever is letter and in case of sensitive mobile phase use freshly prepared mobile phase.
5.1.10	Do not use any hazy mobile phase during HPLC analysis.
5.2	Standard Preparation :
5.2.1	The standard shall be prepared as per respective standard test procedure, taking into consideration the Standard solution stability and storage requirement.
5.3	Analysis :
5.3.1	Always use fresh HPLC vials & septa for analysis.
5.3.2	Prepare the sequence as per respective standard test procedure.
5.3.3	Prior to start the sequence analyst shall take the printout of sequence and shall sign with date. Reviewer shall review the sequence and put the sign and date. In case, reviewer is not available in the shift then another analyst shall review the sequence. In case, another analyst also not available then run the sequence by self-reviewing and sequence shall be reviewed by reviewer on next day.
5.3.4	Pre-Evaluation injection: A system suitability check shall be injected at the beginning to check the chromatographic system suitability, mobile phase preparation and proper saturation of the HPLC column.
5.3.5	Injection for system suitability check shall be processed and shall not be considered for calculation or reporting, it is just to ensure the system is ok. However repeat injection of standard avoided and no sample injection should be injected.
5.3.6	The system suitability shall be established as per the standard test procedures, before starting the analysis.
5.3.7	A printout of sequence shall be attached with analytical Analytical Work Report along with chromatograms.
5.3.8	Injection sequence shall be as per the standard test procedure. If no specific sequence are mentioned in the standard test procedure then follow the sequence as mentioned below.
5.4	Preparation of sample Set/ Sequence:
5.4.1	Preparation of sequence is mainly separated in to two sets, i.e. Pre-Evaluation (PE) and Main Sequence set.
5.4.2	Pre-Evaluation(PE): Sequence is prepared to check system suitability parameter by injecting, Blank, System Suitability Solution, Standard Solution
5.4.3	Pre-Evaluation(PE) (Sequence – I)   Assay/ RS Content uniformity(CU) Dissolution Blank Blank Blank Standard Solution Standard Solution Standard Solution System Suitability (if applicable) System Suitability (if applicable) System Suitability (if applicable)
5.4.4	Main Sequence set (Sequence – II):-
Assay Content uniformity Dissolution Related Substance Blank Blank Blank Blank System Suitability (if applicable) System Suitability (if applicable) System Suitability (if applicable) System Suitability (if applicable) Standard Solution-1 Standard Solution-1 Standard Solution-1 Blank (if required) Standard Solution-2 Standard Solution-2 Standard Solution-2 Standard Solution-1 Standard Solution-3 Standard Solution-3 Standard Solution-3 Standard Solution-2 Standard Solution-4 Standard Solution-4 Standard Solution-4 Standard Solution-3 Standard Solution-5 Standard Solution-5 Standard Solution-5 Standard Solution-4 Standard Solution-6 (if applicable) Standard Solution-6 (if applicable) Standard Solution-6 (if applicable) Standard Solution-5 Blank Blank Blank Standard Solution-6(if applicable) Sample Solution 1-Inj-1 Tablet-1 Tablet-7 Blank Blank Sample Solution 1-Inj-2 Tablet-2 Tablet-8 Placebo Placebo Sample Solution 2-Inj-1 Tablet-3 Tablet-9 Sample Solution 1 Sample Solution 1 Sample Solution 2-Inj-2 Tablet-4 Tablet-10 Sample Solution 2 Sample Solution 2 Sample Solution 3-Inj-1 Tablet-5   Sample Solution 3 Sample Solution 3 Sample Solution 3-Inj-2 Tablet-6   Sample Solution 4 Sample Solution 4 BKT_Standard Solution BKT_Standard Solution BKT_Standard Solution Sample Solution 5 Blank(if applicable) Blank(if applicable) Blank(if applicable) Sample Solution 6 – – – Blank – – – BKT_Standard Solution – – – System Suitability(if applicable) Note: In case for any single batch testing is performed on 12 tablets then inject bracketing standard after 12 injection in that case no need to inject bracketing standard after 6 injections.
5.5
5.5 1	In case No. of injections for standard are not mentioned in standard test procedure then inject the five replicates of the standard. In case where diluted standard has to be injected then inject six replicates for the same.
5.5 2	The system suitability shall be demonstrated throughout the run by bracketing standards RSD.
5.5 3	After every six injections or end of the sample set, a single standard (Bracketing standard) solution injection shall be made and the cumulative/overall RSD of these standards with earlier system suitability standards shall be calculated. The acceptance criteria for system suitability shall be overall RSD not more than 2.0 % for assay and 5.0 % for RS or as per standard test procedure.
5.5 4	In case, system suitability with bracketing standard is not meeting with acceptance criteria, then previous sample shall be invalidated with remarks.
5.5 5	The retention time of standard and sample peaks should be within 10 % of actual retention time throughout the analysis for same concentration solutions.
5.5 6	Resolution, tailing factor and theoretical plates shall comply with respective standard test procedure.
5.6	Adjustment of Chromatographic Condition:
5.6.1	Composition of mobile phase: The amount of the minor solvent component may be adjusted by ± 30% relative to minor component or ±2% absolute, whichever is the larger, if required to meet the system suitability criteria, No other component is altered by more than 10% absolute.
5.6.2	pH of the aqueous component of the mobile phase: ±0.2 pH, unless otherwise stated in the standard test procedure, or ± 1.0 pH when neutral substances are to be examined.
5.6.3	Concentration of salts in the buffer component of a mobile phase: In the buffer component of a mobile phase: ± 10 per cent
5.6.4	Detector wavelength: No adjustment permitted
5.7
5.7.1	Column length: ± 70 %
5.7.2	Column internal diameter:  ± 25 %
5.7.3	Particle size: Maximal reduction of 50 % of particle size, no increase permitted.
5.7.4	Flow rate: ± 50 per cent. When in a monograph the retention time of the principle peak is indicated, the flow rate has to be adjusted if the column internal diameter has been changed.
5.7.5	Injection Volume: The injection volume can be reduced as far as consistence with accepted precision and detection limits, no increase permitted.
5.7.6	Gradient Elution: The configuration of the equipment employed may significantly alter the resolution, retention time and relative retention time described in the method.  If this occurs, it may be due to excessive dwell volume, which is the volume between the point at which the 2 eluents meets and the outlet of the column.
5.8
5.8.1	Stationary phase: Column length : ± 70 % Column internal diameter: ± 50 % Particle size : Maximal reduction of 50 %, no increase permitted Film thickness: -50 % to + 100 % Flow rate : ± 50 % Temperature: ± 10 %
5.8.2	Injection volume: May be decreased, provided detection and repeatability are satisfactory.
5.9	Recording of Chromatograms for Assay/CU/ Dissolution Test:
5.4.1	Run the chromatogram as per run time given in STP.
5.4.2	Unless otherwise specified, run the chromatogram at least 1.5 times of the main Peak Retention Time (RT).
5.10	Integration, processing and printing of chromatograms for Assay/CU/ Dissolution Test:
5.10.1	Set the integration parameter i.e. Peak Width, Peak Threshold, Minimum Height, Minimum Area, appropriately for proper marking of peak.
5.10.2	Before integration, adjust the scaling of chromatogram template such a way that main peak height should more 70% of full scale chromatogram.
5.10.3	Scaling of the chromatograms should be same for all chromatograms throughout the first to last injection of that particular sequence.
5.10.4	Changing of height of in between chromatogram not allowed unless and until justified.
5.10.5	Re-processing of chromatogram is not allowed unless and until justified.
5.10.6	Take the print out of processing method with set integration parameter at the end of chromatogram processing.
5.10.7	Take the chromatogram print out of standard solution (Sequence-I), Standard solution first Replicate (Sequence-II) and last bracketing standard with system suitability parameter as per STP.
5.10.8	The system suitability Chromatogram table should contain peak name, RT, Area, Tailing Factor/ Theoretical plates/ Resolution/ Capacity Factor etc. as applicable.
5.10.9	Rest of the other chromatogram table should contain peak Name, RT and Area.
5.10.10	The chromatograms which are disregarded shall be invalidated with reason. The reason for disregarding the chromatograms may be abnormal peak shape, system suitability failure due to variation in area count/inconsistent area, faulty integration, ghost peak or any other reason.
5.10.11	The analyst shall write the reason for disregarding with “INVALID /Invalid Data” remark/stamp on the chromatogram. The disregarded chromatogram shall be filed along with the valid chromatograms.
5.10.12	Recording of chromatograms for Related Substance/ Impurity/ Chromatographic purity Test:-
5.10.13	Run the chromatogram as per run time given in STP.
5.10.14	Unless otherwise specified, Run the sample chromatograms at least 2.5 times of the main peak RT.
5.10.15	Run the 1st blank, System suitability solution, Placebo solution and last blank equal to sample run time.
5.10.16	Unless otherwise specified, Run the Standard solution replicate and other blanks chromatograms at least 1.5 times of the main peak RT.
5.11	Integration/processing/printing of chromatograms for Related Substance/ Impurity/ Chromatographic purity Test:-
5.11 1	Before integration, adjust the scaling of chromatograms such a way that smallest peak on chromatogram to be seen.
5.11 2	Set integration parameter i.e. peak width, peak threshold, Base to Base, Valley to valley, tangent peak etc.
5.11 3	Same integration parameters shall be set for Blank, Placebo and sample chromatograms.
5.11 4	Same scaling shall be set for Blank, Placebo and sample chromatograms.
5.11 5	For standard solution, system suitability solution and other blanks different integration can be set according to requirement. However for standard replicates and bracketing standard processing same integration parameter and same scaling shall be used.
5.11 6	Changing of height of in between chromatogram not allowed unless and until justified.
5.11 7	Re-processing of chromatogram is not allowed unless and until justified.
5.11 8	Take the print out of processing method with set integration parameter at the end of chromatogram processing.
5.11 9	Take the chromatogram print out of standard solution (Sequence-I), Standard solution first Replicate (Sequence-II) and last bracketing standard with system suitability parameter as per STP.
5.11 10	The system suitability Chromatogram table should contain Peak Name, RT, Area, Tailing Factor/ Theoretical plates/ Resolution/ Capacity Factor etc. as applicable.
5.11 11	Rest of the other chromatogram table should contain peak Name, RT Area and Area %.
5.11 12	Manual integration allowed for small peaks wherever auto integration not possible.
5.11 13	Blank and placebo shall be integrated as per sample solution integration.
5.11 14	Do not consider the peak for calculation from sample solution which have same RT as that of Blank and placebo solution peak. Disregard Blank and placebo peaks from sample solution.
5.11 15	Calculate the impurities as mentioned in the STP.
5.11 16	Based on the RT or RRT name the impurities in chromatograms or calculation sheet.
5.11 17	Report the impurities as per specification.
5.11 18	During system suitability only initial replicate injections shall be considered for calculation of % RSD.
5.11 19	If necessary, Reprocessing of chromatograms shall be done and shall be documented with reason(s). Invalidate previous chromatograms with remark.
5.11 20	Any unknown peak found in the chromatogram other than the diluent, placebo and impurity peaks shall be integrated and considered as unknown peak.
5.11 21	Integration parameters such as peak width, slope sensitivity, minimum peak area / height etc. shall be recorded as used for each chromatogram or print out of integration parameters shall be attached with the chromatograms.
5.11 22	Attach the sequence print and method print for the entire analysis.
5.11 23	Sequence print shall contain Vial No, Sample Name, Sample ID, Method File, Data File, Injection Volume, column ID, Analyst Name etc.
5.11 24	Analyst should sign all the chromatograms with date.
5.11 25	Enter the usage details in instruments, Columns usages record and reference / working standard usage logbooks.
5.12	Trouble Shooting in Chromatographic Analysis:-
Failing of Tailing Factor/ Theoretical Plates. Column bonding/packing detoriated due to continuous use. Flush the column with hot water, followed by 90% organic solvent, replace/ discard column if necessary. Take new column. Wrong column selection. Connect correct column as per STP and start. Not using HPLC Grade solvent in mobile phase Use of HPLC grade solvent and chemicals. Column leakage Remove the ferrules/ tube fittings and refit it tightly. Replace if necessary. Solution degradation. Prepare fresh solution and inject. Ghost peak in Blank, Standard solution and sample solution Contamination in diluents Prepare new diluent. Make all fresh preparation with new diluents. Vials Contamination or particular vial Contamination. Fill solution in new vials discard old vials. Particular flask contamination. Make fresh solution with new flask. Filtration Syringe contamination. Filter sample solution with new syringe. Syringe filter contamination Filter sample solution with new syringe filter. Mobile phase contamination Prepare new mobile phase and use. No peak observed in Chromatograms. Blank/diluent solution filled instead of standard/sample solution. Fill new vial with standard/sample solution and inject. Wrong standard solution selection. Prepare new standard and inject. Wrong wavelength selection. Select correct wavelength and start. Column leakage. Remove the ferrules/ tube fittings and refit it tightly. Replace if necessary. Injector leakage. If yes, contact service engineer. Injector needle broken. Contact service engineer. Wrong mobile phase. Check and prepare new mobile phase. Wrong column selection. Check and use proper column. No vial in a tray at given position. Check vial tray & vial position. Keep vial at correct position.
Broken/ leakage in vial. Check for vial intactness. Fill new vial. Septa fall in vial. Check vial and fill new vial. Wrong dilution to low concentration. Check dilutions. Make proper dilutions. Leakage in detector tubing. Check detector tubing, replace if necessary. Wrong instrument method selection. Check method properly selected. Sequence interrupted/ aborted. Power failure. Check power failure data. Software connectivity lost. Check connectivity. Restart the instrument and software. Vail not detected. Check vials position in tray. Computer shutdown/ malfunctioning. Check computer. Malfunctioning of HPLC parts like, injector detector etc. Check for particular part. If yes, contact service engineer. System over pressure/ Back pressure Check for Blockage in tubing/reservoir filters/ Column back pressure. Flush the tubing with hot water or replace. Sonicate reservoir filter with hot water and methanol. Wash the column with hot water. Peak split/ distorted peak. Column detoriation. Wash the column and check, replace column if necessary. Solution degradation. Check solution stability, prepare fresh solution and inject. Wrong column selection Check and use proper column. Wrong diluent used Check diluent preparation, prepare new and inject
Failing resolution. Column bonding/packing detoriated due to continuous use. Flush the column with hot water, followed by 90% organic solvent, replace/ discard column if necessary. Take new column. Wrong column selection. Connect correct column as per STP and start. Not using HPLC Grade solvent in mobile phase Use of HPLC grade solvent and chemicals. Solution degradation. Check solution stability,  prepare fresh solution and inject Impurity degradation. Check impurity solution stability. Prepare fresh solution and inject. Peak merging. Column detoriation. Wash the column and check, replace column if necessary. Wrong column selection. Check and use proper column Wrong mobile phase preparation. Check and prepare new mobile phase. Wrong sequence preparation. Typo error in Standard/ sample name. Training to Analyst/ Reviewer. Assess the impact of error on quality of product and make decision. Wrong instrument method selection. Manual error or analyst error. Training to Analyst/ Reviewer. Re-inject sequence if it is within solution stability or else prepare fresh solution Wrong injection volume. Manual error or analyst error. Training to Analyst/ Reviewer. Re-inject sequence if it is within solution stability or else prepare fresh solution
5.13	Procedure for Hypothetical Analysis:-
5.13.1	Hypothetical analysis used when there is no assignable cause is identified during investigation of lab error, incidence of testing samples.
5.13.2	Any evidence or ideas that may indicate a possible root cause can be explored or verified by hypothesis testing.
5.13.3	Design a protocol for hypothetical testing and define the tentative steps that would follow to investigate the assignable cause.
5.13.4	Hypothetical analysis protocol shall be designed by considering all possible suspected causes.
5.13.5	1) Hypothesis testing must always be defined in a pre-approved protocol. This helps assure that testing is well planned, is scientifically sound and justified and prevents inadvertent use of inappropriate ad-hoc testing. Such testing can easily be interpreted as an attempt to “test into specification” and is likely to result in unfavourable scrutiny from regulatory authorities. Hypothesis testing is essentially a process of elimination. A well designed hypothesis testing protocol explores the possibilities, eliminates those proven as not the case and works in a structured manner towards defining the root cause or potential root cause(s) based on scientific and conclusive evidence. 2) The hypothesis testing protocol must define: •A description of the hypothesis test •The potential root cause being investigated •The samples that will be tested •Exact details on how to execute the test •How the data will be evaluated 3) The results from hypotheses testing must only be used to confirm or discount a root cause. They cannot replace the original result or be reported as final analytical results.
5.13.6	Evaluate all suspected causes one by one step wise. If assignable causes found during first step then stop further testing close the investigation with proper scientific justification.
5.13.7	Recommend corrective and preventive action wherever applicable.
5.13.8	Hypothetical analysis may include but not limited to following points;
5.13.9	Investigate sample Preparation error, sample degradation, sample contamination.
5.13.10	Hypothesis testing should be restricted to original samples wherever possible. It’s also advisable that hypothesis testing gives consideration to analysis by a second analyst.
5.13.11	Re-injection of same sample, preparation of new sample, preparation of new mobile phase, changing analyst, changing column, changing chemicals and its make, Re-sampling of RM/FP/In-process sample,
5.13.12	Where an assignable cause is not determined by the laboratory investigation then manufacturing error investigation must be undertaken.
5.13.13	Investigation of manufacturing steps, handling and storage of sample in Production Area, Handling and storage of RM, Cross contamination in RM, Cross contamination in production, investigation of Packing steps, Effect of temperature on sample during packing, contamination of primary packing material, carryover of previous product manufactured.
5.13.14	Cleaning verification of previous product, cross contamination in dispensing area, Quality of water used in manufacturing process, Any suspect on calibration of Equipment used in manufacturing and testing, health and hygiene of the personal involved in manufacturing and testing of product.
5.13.15	If that investigation does not reveal a manufacturing error, and sufficient analytical data exist from the retest to decide that the original out-of-specification result arose from an unassignable laboratory error, then one may conclude that the original out-of-specification result is not representative of the batch. Product history also should be considered at this point.
5.14	HPLC System conversion for Reverse phase and Normal phase  :-
	System conversion from Reverse phase to Normal phase:-
5.14.1	Purge and flush the HPLC system with water for 30 to 35 minutes all parts.
5.14.2	Then Purge and flush the HPLC system with methanol for 30 to 35 minutes all parts.
5.14.3	Then flush the HPLC system with IPA for 30 – 35 minutes
	System conversion from Normal phase to Reverse phase:-
5.14.4	Purge and flush the HPLC system with IPA for 30 to 35 minutes all parts.
5.14.5	Then Purge and flush the HPLC system with methanol for 30 to 35 minutes all parts.
5.14.6	Then flush the HPLC system with water for 30 – 35 minutes
5.15	Logging and Reporting Lab Error Report (LER):
5.15.1	Lab error report (LER) shall be filed for chromatographic day to day activity which does not have impact on quality of the product and need immediate corrective action in chromatographic errors before injecting sample solution. For Example:- system suitability failure, column leakage etc.
5.15.2	Lab Error Report Log should be maintained as per annexure.
5.15.3	In case sample solution is injected in HPLC then file an incident and proceed as per SOP “Incident Reporting and investigation”.
5.15.4	Analyst shall report to Supervisor or Section Head immediately when error occur. The supervisor shall perform initial investigation to find out cause of error and shall suggest immediate corrective action.
5.15.5	Error shall be logged in the “Lab Error Report Log” as per define format and number shall be assigned.
5.15.6	Lab Error Report number shall be allotted to each error as mentioned below: LER/YY/NNN where, LER denotes Lab Error Report YY denotes last two digit of the current year and NNN denotes three digit serial number as (001, 002, 003….). For example: – Lab Error Report Number for first error of year 2024 shall be assigned as LER/2024/001.
5.15.7	The “Lab Error Report” shall be maintained as per define format having the details like Lab Error Report No., Date/Time, Product, Batch No./A.R.No., Description of error, Initial Investigation and cause of error, Immediate corrective action, Impact Evaluation/further course of action, Preventive action, verified by QA, Closing Date, Closing remark by HOD/Designee.
5.15.8	HOD/Supervisor shall evaluate impact of LER and based on outcome of immediate corrective action will suggest further course of action and preventive action.
5.15.9	The LER shall be send to QA Department for review before final closure.
5.15.10	If required, QA shall suggest further course of action in LER by considering regulatory aspects.
5.15.11	The maximum time for investigation and closeout of LER shall be 30 working days from the date of filing.
5.15.12	If the investigation is extended then justification shall be documented for the same.

 

 

6. Definitions / Abbreviations :

• Definitions :
• Quality Control Associate: Analyst, Supervisor, Executive of Quality Control Department or any personnel trained to perform duties as per this SOP.
• Quality Assurance Associate: Executive of Quality Assurance Department or any personnel trained to perform duties as per this SOP.
• Chromatography is defined as a procedure by which solute are separated by a dynamic migration process in a system consisting of two or more phases, one of which moves continuously in given direction and in which the individual substances exhibit different mobilities by reason of differences in adsorption, partition, solubility, vapour pressure, molecular size, or ionic change density. The individual substances thus separated can be identified or determined by analytical procedure.

 

• Abbreviations:

Abbreviation	Expansion
QC	Quality Control
e.g.	Example
No.	Number

 

*Note – Ready to use SOP available in “DOWNLOAD” Section.