SOP on Chromatographic Practices and Documentation for Instrumental analysis
Objective: To describe the procedure to be followed in Chromatographic Analysis and Documentation for instrumental analysis and to ensure that good laboratory practices are followed during instrumental analysis in the Quality Control laboratory.
Scope: This procedure is applicable to Chromatographic analysis through HPLC & GC and instrumental analysis documentation, in Quality Control laboratory..
Responsibility: Chemist or above of QC laboratory
Accountability: Head – Quality Control & Head – Quality Assurance
Procedure:
General Cleaning: Ensure that the electrical power supply to the instrument is switched off. Clean daily the outer surface of the instrument with a clean dry cloth. If necessary, clean the instrument using a cloth wetted with 70 % IPA. Remove the moisture, if any, with a tissue paper or a dry cloth.
Mobile phase / Diluent preparation: The mobile phase / diluent shall be prepared as per the procedure described in the respective STP and the same shall be recorded in the current version of mobile phase preparation record Format as per current version of SOP.
Chromatographic grade solvents shall be used for mobile phase preparation.
The Mobile phase shall be degassed (Passing through a 0.45 µm µm membrane or sonication) adequately to avoid flow rate inconsistencies, column equilibration, etc. which eventually results in change in retention time & failures in system suitability requirements.
The degassed mobile phase / diluent shall be transferred into a glass bottle closed with polypropylene (GL45) screw cap and labelling shall be done as per current version of SOP.
Shelf life of mobile phase / diluents shall be assigned as per current version of SOP
If mobile phase shelf life is more than 48 hours in STP then same shelf life shall be assigned for that mobile phase.
Analysis:
System suitability / Standard / Sample preparation control:
The standard / sample preparation shall be made as per the STP taking into consideration the analyte stability and storage requirement.
System suitability: In case where the resolution solution / peak identification solution / marker solution required for establishing system suitability is to be stored for longer duration it shall be assigned a shelf life based on the actual study conducted.
QC analyst shall download required instrument method and verify it with respective STP.
After instrument method verification a trial injection (RT check) of standard / blank / system suitability shall be injected before starting the injection sequence to check the retention time / baseline interference, if retention time is more than 10 minutes than 10 % variation is acceptable and for upto 10 minutes retention time, 1 minute variation is acceptable. If system suitability parameter fails to meet the acceptance criteria, then perform necessary adjustment in the mobile phase / cleaning / conditioning of the column to meet the requirement.
QC analyst shall prepare the sequence as per the respective STP
Sequence shall be verified by the supervisor before starting the analysis in a day shift. Incase any sequence started after day shift, analyst shall verify the sequence himself and start the sequence and attached the sequence with raw data.
QC analyst shall take print out of instrument method with processing method immediately after starting the sample set.
Note: Some of the commonly observed problems are peak tailing, front tailing, peak broadening, peak splitting, blank peaks and retention time shifting. These characteristics of chromatographic peak directly impact the quality of results.
The system suitability (once established) shall be valid for a maximum of 24 hours or 5 times the total time required for establishing the method system suitability, whichever is higher or as described in respective STP.
The system suitability shall be demonstrated throughout the run by intermittent evaluation. The intermittent system suitability shall be performed by the current version of SOP.
In case of unstable analyte, freshly prepared analyte standard solutions shall be injected and the area response (area/weight) shall be used for calculating a co-relation against the earliest standard area response.
Where STP requires use of more than one concentration of standard, the standard with the lowest concentration shall be used for demonstration of intermittent system precision.
Where the sample is required to be injected in duplicate / triplicate the area ratio of the main peak between the successive injections shall lie between 0.98 and 1.02 from the mean area.
In case of sample injections, involving concentrations of equal or less than 10 ppm the area ratio between duplicate / triplicate shall lie between 0.95 and 1.05 from their mean area.
Where the STP does not specify any RSD limits for standards. RSD limit shall not be more than 1.0 %
In case of unstable analyte, freshly prepared analyte solutions shall be injected and the area response (area/weight) shall be used for calculating the correlation against earlier standard area response.
During assay and related substances analysis involving isocratic runs, the flow rate of mobile phase shall be kept constant for the entire run, after the system suitability is established. If the flow rate of the system is changed, system suitability shall be established again.
During assay and related substances analysis involving gradient run, if there is delay (exceeding the run time) in injecting the next sample, the blank run with gradient operation shall be continued. The chromatograms for blank injections shall be recorded.
Incase whenever sample set is completed, further samples shall be analyzed after some time and within the system suitability time.
Flow rate shall be kept constant for the required time for isocratic mode. Blank injections shall be injected for gradient mode.
System suitability / standard injection shall be injected followed by blank before injecting the sample to ensure the system suitability. In case, there is delay of more than a single run time before injecting a sample, a bracketing standard is added in the sequence before injecting the sample preparation to ensure the system suitability.
In case the above mentioned acceptance criteria are not met, all the data collected during the time period shall be properly identified and reviewed by QC Supervisor. The system suitability with fresh standard preparations shall be carried out all over again before injecting any test samples.
If any sample set fails during run due to any reason i.e. system malfunctioning / instrument error / communication error / vial missing or bracketing standard does not meet acceptance criteria etc, same shall be handled by as per current version SOP.
Recording Chromatograms:
All chromatograms for the establishment of system suitability and up to entire analysis run shall be maintained properly.
The raw data emerging from such chromatograms shall be recorded as per current version of SOP on Preparation, Review & Approval of worksheet). Appropriate remarks (where required) shall be recorded on these chromatograms by the QC Chemist / QC Supervisor.
Each chromatogram shall be duly signed off by the analyst only for the work done by him / her and reviewed and signed off by the checker. No other person shall sign for the work done by others.
The name of analyst shall not be entered in lieu of the analyst’s signature for the work done.
However, in case the analyst does not turn up for duty for next few days, the supervisor can review the raw data for accuracy and compile the test report and sign on the test report. Necessary remarks shall be recorded by the supervisor about the accuracy of data. However, the raw data shall be signed by the original analyst upon resumption of duty.
In case of long absence or leaving the company by the original analyst, the supervisor shall then close the raw data after review for accuracy and necessary remarks and signatures.
No chromatogram shall be ever discarded or destroyed by anyone.
The chromatograms which are disregarded and not considered for calculations shall be stamped as “DISREGARDED”. The reasons for disregarding the chromatogram could be printing error, variation in area count / inconsistent area, faulty integration, abnormal drift in baseline, ghost peak or any other reason considered by the supervisor.
The analyst performing the analysis shall assign the reason for disregarding a chromatogram on the chromatogram itself, with signature and date. This shall also be counter-signed by the supervisor with date. The disregarded chromatogram shall be preserved along with the test chromatograms.
For the system generated chromatograms, the necessary information shall be printed on each chromatogram.
Analyst-1 performing the analysis shall process the chromatograms with suitable integration parameters and use the chromatogram for calculating / reporting results. However, in the absence of Analyst-1 for rational reasons, processing shall be done by the Analyst-2 with due signature and date. This shall also be counter-signed by the supervisor with date. Further, raw data so generated, shall not be kept unprocessed for more than two working days.
Reprocessing of chromatograms, if necessary, at a later date/time shall be documented with reason(s) for reprocessing and certified by the QC Manager. However, during reprocessing of chromatogram, no raw data shall be altered or deleted or modified or added.
The entire record of chromatograms shall be part of the sample evaluation test protocol.
Calculations: The calculations shall be performed as per the respective STP. All calculations shall be done as per the area count / values obtained for the standard injected in the beginning. Area counts of in-between injections of standard shall not be considered for calculations
Monitoring the Lamp Energy (Shimadzu / Waters HPLC):
Let the deuterium lamp initialize for 15 minutes before starting the analysis.
The energy of deuterium lamp shall be verified to ensure its performance for the analysis on weekly basis.
HPLC grade water shall be used as mobile phase and the flow cell shall be rinsed for 5 minutes at a flow of 1 ml/min.
For Shimadzu HPLC lamp energy shall be checked at 220 nm by pressing VP key on the instrument touch panel and record the lamp energy.
For Waters HPLC press diagnostic key and check the Sample Reference Energy at 220 nm and record.
The deuterium lamp energy shall be recorded in format.
Acceptance Criteria:
The lamp energy shall not be less than 800 mV for Shimadzu HPLC and 15 nanoamps for Waters HPLC.
Audit Trail:
What is an audit trail: Audit trails maintain a record of system activity both by system and application processes and by user activity of systems and applications.
Audit trail secures computer generated and time-stamped electronic record that allows reconstruction of the course of events relating to creation, modification and deletion of an electronic record.
Purpose of Audit trail: The purpose of an audit trail for Electronic Record systems is to provide assurance of the integrity of the Electronic Record and the associated Raw Data.
The administrator / manager shall hold the primary responsibility for ensuring computerized systems used in generating analytical data in the Quality Control laboratory are in compliance with applicable regulations, as regards design and validation. QC Manager shall ensure that the Instrument vendor shall provide 21 CFR Part 11 compliance certificate for the chromatographic software.
Audit trail features of the software shall be enabled at the time of preparation of project for each month of chromatographic hardware and software and never shall be disabled / blocked.
Verification of audit trail shall be done monthly by administrator shall be completed with in 10 days. Audit trail shall be verified to check whether there is any change in Instrument method parameter such as flow rate, gradient program, detector wavelength, run time, column oven temperature, sample compartment temperature, change in processed data such as alteration in processing parameter like change in peak width, threshold, peak naming, change in system date and time.
Verification if any abnormality found same shall be informed to QC head and shall be investigated through sop on deviation and a formal report shall be prepared. Verification of audit trail shall be recorded as per format.
Electronic data and user management shall be done as per current version SOP of Electronic data and user management).
Creation, modification and deletion of electronic users account shall be as per current version of SOP of Electronic data and user management)
Backup, restore and archival of analytical data from computer system of Instruments shall be as per current version of SOP of Backup, restore and archival of analytical data from computer system of Instruments).
Safety Precautions: Ensure that personal shall use appropriate personal protective equipment like hand gloves, safety shoes, safety goggle, apron etc. while working.
List of Annexure
Deuterium lamp energy record
Audit Trail Verification Record