To describe the procedure to be followed in HPLC analysis and documentation and to insure that Good Laboratory Practices are followed in the HPLC analysis.
This SOP is applicable for the procedure to be followed in HPLC analysis and documentation and to insure that Good Laboratory Practices are followed in the HPLC analysis.
Officer/ Executive - Quality Control
Manager – Quality Control.
5.1 There shall be sequence available for the analysis before start up on the instrument.
5.2 Mobile Phase
5.2.1 The mobile phase shall be prepared as per the composition described in the standard test procedure (STP) of respective sample.
5.2.2 The mobile phase container shall have labeling details as follows:
Mobile phase :
Analysis of :
Prepared by :
Use before : Date :
Detail like weight of buffer (s), Balance No., Observed pH, etc. for the mobile phase shall be recorded in record of analysis.
5.2.3 The mobile phase shall not be use after 7 days of its date of preparation.
5.2.4 The mobile phase shall be discarded if any haziness or precipitation is found upon visual examination.
5.3 Resolution Solution
5.3.1 In case where the resolution solution required for system suitability is to be stored for longer duration it shall be assigned a self-life based on the following.
A) The resolution solution required for system suitability parameter as required by the respective STP on each days of the study.
B) For the main analyze peak the difference in the area count on the day of preparation and at the end of the self life shall not vary by 20 % with respect to the limit area count.
C) For the impurities peak the difference in the area count on the day of preparation and at the end of the self life shall not vary more than 20 % with respect to the initial area count.
5.4 Standard Preparation
5.4.1 The standard preparation shall be prepared as per the STP, taking in consideration them stability & storages requirement.
5.5.1 The system suitability shall be established as per the STP, before proceeding with the analysis.
5.5.2 Injection sequence shall be captured from the system as per the STP of respective product and shall be recorded.
5.5.3 The injection sequence to be followed shall be as per the respective STP the following injection sequence may be used as a guidelines:
Blank →System suitability (or System Suit →Blank) → Placebo (if required) → Impurity standard (if applicable) → Standard →Sample
5.5.4 The system suitability (once established) shall be valid for a maximum period of 24 hour. After about every 24 hour system suitability (from the time when first system suitability is established).
5.5.5 The system suitability shall be demonstrated after about every 24 hour in the following manner:
5.5.6 For assay and related substances analysis after about every 24-hour standard solution in triplicate shall injected. The RSD of triplicate injection shall be NMT 1%
5.6 Acceptance Criteria
5.6.1 The store resolution solution if used, shall meet the acceptance area on all the day of its use. Otherwise a freshly prepared resolution solution shall be injected.
5.6.2 Where the sample is required to be injected in duplicate /triplicate the area ratio between the successive injections shall lie between: 0.99 to 1.01 from the mean area.
Method where the STP does not specify any RSD limit; the area ratio between the successive injection of the standard shall lie between 0.99 to 1.01 from the mean area.
5.6.3 The area ratio of the average area of the intermittent set of standard and average of initial standard, shall lie between 0.99 to 1.01 from there mean area.
5.6.4 During assay and related substance 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 change for more than 200 minute, fresh system suitability shall be established.
5.6.5 During assay and related substance analysis involving gradient run. If there is delay (exceeding the run time) in injection the next sample, the blank run with gradient operation shall be continued. The chromatogram for blank injection shall be recorded.
5.6.6 In case the above mentioned acceptance criteria are not met, all the data collected during the suspect time period shall be properly identified and reviewed by supervisor. The system suitability shall be carried by out all over again, before injecting any test samples.
5.7 Recording Chromatogram
5.7.1 All chromatogram before establishment of system suitability and up to entire run, shall be recorded and documented. Appropriate reamer (if required) shall be recorded by the analyst / supervisor.
5.7.2 In case where the chromatogram needs disregarding, the analyst shall show it to the supervisor /QAM for review and approval.
5.7.3 The disregarded chromatogram shall be checked and certified by supervisor /QAM.
5.7.4 The chromatogram, which is disregarded and not considered for calculation, shall be stamped as “DISREGARDED”. The reason for disregarding the chromatogram could be variation in the area count / inconsistent area, faulty integration, abnormal drift in baseline. Ghost peak or any other reason.
5.7.5 The analyst performing the analysis shall assign the reason of disregarding a chromatogram on the chromatogram itself. The disregarded chromatogram shall be filled along with the test chromatogram.
5.7.6 For the system generated chromatogram, the necessary information shall be printed on each chromatogram.
5.7.7 The integration parameter such as peak width, peak threshold, minimum peak area and height shall be recorded, as used for integration of chromatogram.
5.7.8 Reprocessing of chromatogram, if necessary at a letter date /time shall be documented with reason (s) for reprocessing and certified by the QAM.
5.8.1 The calculation shall be performed as per the respective STP. All calculation shall be as per the area count / value obtained from the standard injected in the beginning. Area count of the in between injection of standard shall not be considered for calculation.
Related: Calculation of Relative Response Factor (RRF)
Related: Calculation of Relative Response Factor (RRF)
6.1 SOP - Standard Operating Procedure
6.2 HPLC - High Performance Liquid Chromatography
6.3 STP - Standard testing procedure
6.4 RSD - Related Standard Deviation
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