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HPLC methodology
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Content of HPLC test procedure
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System Suitability Testing (SST)
- Relative Response actor (RR)
case study
The prequalification programme --Assessor's training
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19-20 January 2011
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Information Sources
Chiral
3on--e-change
3on--pair<affinity
=ormal phase
Reversed phase
Si>e e-clusion
The reversed-phase HPLC %ith ?! detection is most commonly used
form of HPLC; is selected to illustrate the parameters of HPLC
method and validation:
The prequalification programme --Assessor's training
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19-20 January 2011
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A flow scheme for HPLC
The prequalification programme --Assessor's training
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19-20 January 2011
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Content of HPLC test procedure
#ny analytical procedure submitted should be described in
sufficient detail; includes@
Chromatographic condition@
7 Column@ type (e:g:; C&A or CA); dimension (length; inner
diameter); particle si>e (&1Bm; 5 Bm)
7 "etector@ %avelength
7 3nCection volume
7 column T
7 flo% rate;
The prequalification programme --Assessor's training
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19-20 January 2011
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Content of HPLC test procedure
Calculations
4*S 0:+:R:0 analytical procedures and validation summaries
The prequalification programme --Assessor's training
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19-20 January 2011
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Compendial methods
)hen claim a compendial method; there should be no change in@
"etector %avelength
Precision@
7 #ssay@ RS" F&G (#P3) or F 0G (PP); n H 5
7 3mpurities@ in general; RS" F 5G at the limit level; up to &1G or
higher at L*4; n H 2
Resolution (R)@ E0
The prequalification programme --Assessor's training
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19-20 January 2011
10
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System suitability testin! "SST#
or #ssay@
precision I one or more other parameter
or impurity test@
resolution I precision I one or more other parameter
The prequalification programme --Assessor's training
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19-20 January 2011
13
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$elati%e $esponse &actor "$$&#
'uantitation of Impurities
To revie%@
a) RR calculation; and
b) if RR is properly used in the final calculation for G
impurity
3f RR %ithin 1:A-&:0; correction may not be necessay
he preparation of solutions)
7 assay) concentration of reference standard should be close to the sample
solution
7 impurities) concentration of the reference standards should be close to
the limit
Minimum : concentrations
System precision@
7 by multiple inCections (n H5) of a homogeneous sample
(standard solution):
7 RS" F &G is recommended for assayM
7 RS" F 5G is recommended for related substances (reference
standards at the limit)
7 3ndicates the performance of the HPLC system
7 #s a system suitability test
The prequalification programme --Assessor's training
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19-20 January 2011
2"
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Precision