SOP Title- Chromatographic Analysis and Documentation
- OBJECTIVE
To lay down the procedure for chromatographic analysis and documentation.
- SCOPE
This SOP is applicable for chromatographic analysis and documentation in quality control department.
- RESPONSIBILITY
3.1 QC Executive / designee shall be responsible for Implementation of this procedure.
3.2 QC Head / designee shall be responsible for compliance of this procedure.
- DEFINITION(s)
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, vapor pressure, molecular size, or ionic change density. The individual substances thus separated can be identified or determined by analytical procedure.
- PROCEDURE
- Mobile phase
- The mobile phase shall be prepared as per respective standard test procedure.
- The mobile phase bottle should be labeled as per define format.
- Use freshly prepared mobile phase for HPLC analysis which will be valid for one day only.
- Use HPLC grade solvents for mobile phase preparation.
- Always filter mobile phase through 0.45micron filter before use.
- Do not use any hazy mobile phase during HPLC analysis.
- Standard preparation
- The standard preparation shall be prepared as per respective standard test procedure, taking into consideration the analyte stability and storage requirement.
- Analysis
- Always use fresh HPLC vials & septa for analysis.
- Prepare the sample set as per respective standard test procedure.
- Mobile phase
- Prior to start the sample set analyst shall take the printout of sample set and shall make the sign with date on it. Reviewer shall also be review the sample set and put the sign and date. In case reviewer shall not be available in the shift, then another analyst shall review the sample set.
- A system suitability check shall be injected at the beginning to check the mobile phase preparation and saturation of HPLC column.
- Injection for system suitability check shall be processed, and shall kept with main sample set .
- The system suitability shall be established as per the standard test procedures, before proceeding the analysis.
- A printout of sample set has to be attached with analytical worksheet along with chromatograms.
- Injection sequence shall be as per the standard test procedure. If no specific sequence are mentioned in the standard test procedure then following the sequence flow as per below:
Blank →System suitability solution (if applicable) →Placebo (if applicable) →Impurity standard (if applicable) →StandardsàSamples →Bracketing Standard.
- 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.
- The system suitability shall be demonstrated throughout the run by bracketing standards RSD.
- 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 or as per standard test procedure.
- In case system suitability with bracketing standard not met with acceptance criteria in that case previous sample shall be invalidated with proper documents.
- Wherever system suitability fails in %RSD of replicate injections, following points shall be checked.
Injector / pump leak.
Column backpressure.
Temperature record of the working area.
Visual inspection of mobile phase and tubing for any possible air bubble.
Column saturation
- Wherever system suitability fails in case of tailing / column efficiency, regenerate / wash the column or replace the column and repeat the analysis. Submit the sample solution sequence only after passing the system suitability criteria.
- The retention time of standard and sample responses shall agree with in 10 % of actual retention time throughout the analysis for same concentration solutions.
- Resolution, tailing factor and theoretical plates shall comply with respective standard test procedure.
- Adjustment of Chromatographic Condition;
- 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.
- 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.
- Concentration of salts in the buffer component of a mobile phase: In the buffer component of a mobile phase: ± 10 per cent
- Detector wavelength: No adjustment permitted
- Stationary phase:
- Column length: ± 70 %
- Column internal diameter: ± 25 %
- Particle size: Maximal reduction of 50 %, no increase permitted.
- 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. No decrease of flow rate is permitted if the monograph uses apparent number of theoretical plates in the qualification section
- Injection volume: May be decreased, provided detection and repeatability of the peaks(s) to be determined are satisfactory.
- Gradient elution: the configuration of the equipment employed may significantly alter the resolution, retention time and relative retention time described in the method. Should this occur, it may be due to excessive dwell volume which is the volume between the point at which the 2 eluents meet and the top of the column.
- Gas chromatography
- 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 %
- Injection volume :May be decreased, provided detection and repeatability are satisfactory.
- Recording of chromatograms
- Prior to process the chromatogram, set the integration parameters (i.e. peak width, peak threshold, minimum height, minimum area) appropriately for proper marking of peaks.
- All the chromatograms for establishment of system suitability and up to entire run shall be recorded and documented. Appropriate remarks (if required) shall be recorded on these by the analyst. Each chromatogram shall be duly signed off by the analyst and reviewed and signed by the reviewer.
- The chromatograms which are disregarded and not considered for calculations shall be invalidated with reason and proper documentation. The reason for disregarding the chromatograms may be system suit check, abnormal peak shape, system suitability failure due to variation in area count/inconsistent area, faulty integration, ghost peak or any other reason.
- The analyst performing the analysis shall write the reason for disregarding with “INVALID” stamp on the chromatogram. The disregarded chromatogram shall be filed along with the valid chromatogram.
- During system suitability only initial replicate injections shall be considered for calculation of RSD.
- Reprocessing of chromatograms, if necessary, shall be documented with reason(s) & “INVALID” stamp.
- Any unknown peak found in the chromatogram other than the diluent, placebo and impurity peaks shall be integrate and considered as unknown peak.
- 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 chromatogram.
- Attached the sequence print and method print for the entire analysis.
- Sequence print shall contain Vial No, Tray No, Sample Name, Sample ID, Method File, Data File, Injection Volume.
- Analyst and reviewer shall sign all the chromatograms with date.
- Enter the usage details in instruments, Columns usages record and reference / working standard usage logbooks.
- Affix the Mobile Phase bottle status label.
- Affix the Label For all Solution as per Annexure.
- ABBREVIATION(s)
| Abbreviation | Full Description |
| QC | Quality Control |
| e.g. | Example |
| No. | Number |
| °C | Degree Centigrade |
| RH | Relative Humidity |
| % | Percent |
| N | Normal |
| DL | Detection Limit |
| QL | Quantitation Limit |
| ppm | Parts per million |
| ppb | Parts per billion |
| USP | United State of Pharmacopoeia |
*Note – Ready to use SOP available in “DOWNLOAD” Section.