1.0 Objective
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.
2.0 scope
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.
3.0 Responsibility
Officer/ Executive – QC
4.0 Accountability
Manager – QC
5.0 Procedure
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 describes in the standard testing procedure
(STP) of respective sample.
5.2.2 The mobile phase
container shall have labeling details as follows:
Mobile phase :
Product :
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 precipitations 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 Analysis
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 esablished).
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 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.
5.6.5 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 disregarding 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 the 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 QZM.
5.8 Calculation
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.
6.0 Abbreviations
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
6.5 QC – Quality Control
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