Method and Validation basics

HPLC case study

Hua YIN
) Assessor (
The prequalification programme --Assessor's training
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19-20 January 2011
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Outline

HPLC methodology
-
Content of HPLC test procedure
-
System Suitability Testing (SST)
- Relative Response actor (RR)

!alidation of HPLC method

case study
The prequalification programme --Assessor's training
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19-20 January 2011
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Information Sources

"# C"$R revie%er guideline for validation of

chromatographic methods (&''()

)H* TRS '+, #ppendi- ( .#nalytical /ethod !alidation

0112

3CH 40(R&) 0115

Compendial 6eneral Chapters

/ethods and !alidation presentation7Lynda Paleshnui8

The prequalification programme --Assessor's training
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19-20 January 2011
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High Performance Li9uid Chromatography (HPLC)
HPLC is a separation techni9ue based on a solid stationary phase and
a li9uid mobile phase: Separations are achieved by partition;
adsorption; or ion-e-change processes; depending upon the type of
stationary phase used:

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@

Preparation of mobile phase

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

$lution procedure@ isocratic or gradient elution

Preparation of standards and samples

*peration procedure@ se9uence of inCections

System suitability testing (SS) and criteria

Calculations
4*S 0:+:R:0 analytical procedures and validation summaries
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Compendial methods
)hen claim a compendial method; there should be no change in@

The type of column i:e the stationary phases

"etector %avelength

Components in /obile phase

System suitability testing and criteria

#dCustments to ratio of components in mobile phase; flo% rate; column
temp; dimension of column; particle si>e (reduction only); may be
necessary to achieve the system suitability criteria: The allo%able
variations for each parameter; see 3nt:Ph &:&(:( or ?SP general
chapter D20&E:
The prequalification programme --Assessor's training
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System suitability testin! "SST#

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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System suitability testin! "SST#

Tailing factor<pea8 asymmetry@ (F 0)

The prequalification programme --Assessor's training
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19-20 January 2011
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System suitability testin! "SST#

=umber of theoretical plates (=)@ column efficiency H 0111

6radient elution is one %ay to increase the =

The prequalification programme --Assessor's training
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19-20 January 2011
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System suitability testin! "SST#
# SST should contain@

or #ssay@
precision I one or more other parameter

or impurity test@
resolution I precision I one or more other parameter
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$elati%e $esponse &actor "$$&#
'uantitation of Impurities

#gainst impurity RSJs@ %hen reference standard available

#gainst #P3 itself

Relative response factor should be considered
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$elati%e $esponse &actor "$$&#

Response factor@ the response (e:g: pea8 area) of drug

substance or related substances per unit %eight:
RK pea8 area < concentration (mg<ml)

Relative response factor (RR)@

RRKR impurity < R API( O$(
RRKslope impurity < slope API
The prequalification programme --Assessor's training
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19-20 January 2011
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$elati%e $esponse &actor "$$&#
$ifampicine)
y K*+,*+- - I (:'2+
$ifampicine 'uinone)
y K -.,+/0 - I &:&5(
RRK -.,+/0 < *+,*+-
K1:A(
The prequalification programme --Assessor's training
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19-20 January 2011
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$elati%e $esponse &actor "$$&#

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

Correction factorK &<RR; the reciprocal of the RR

The prequalification programme --Assessor's training
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19-20 January 2011
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$e%ie1 points for HPLC method

is the analytical procedure described in detail includin! all the parameters 2

is SS 1ell defined to ensure the consistency of system performance2

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

he 1ay of 3uantitation of impurities

In case API is used as the reference( $$& should be used or 4ustification of
e5clusion should be pro%ided,
o chec6 the determination of $$&( chec6 the correction of calculation of
impurities

confirm7complete the 'OS -,*,$,-

The prequalification programme --Assessor's training
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19-20 January 2011
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Validation 8 compendial methods
Assay 8 API
=o validation generally re9uired: $-ception@ specificity for maCor impurities
not in the monograph:
Assay 8 &PP
Specificity; accuracy and precision (repeatability),
Purity 8 API and &PP
ull validation for specified impurities that are not included in the
monograph (specificity; linearity; accuracy; repeatability; intermediate
precision; L*"<L*4)
!alidation of the limit for individual un8no%ns; if tighter than that in the
monograph@ L*4 of the #P3 should be belo% the limit for individual
un8no%ns
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Non9compendial methods
ull validation is re9uired for purity; assay and dissolution methods
(HPLC; ?!) @
Specificity
Linearity
#ccuracy
Repeatability
3ntermediate precision
L*"<L*4 (not re9uired for assay; dissolution)
Robustness (recommended)
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Specificity

Llan8 solution to sho% no interference

Placebo to demonstrate the lac8 of interference from e-cipients

Spi8ed samples to sho% that all 8no%n related substances are

resolved from each other

Stressed sample of about &1 to 01G degradation is used to

demonstrate the resolution among degradation products
7 Chec8 pea8 purity of drug substance by photodiode array detector (P"#)@ eg
purity angle is lo%er than the purity threshold:

Representative chromatograms should be provided %ith time scale

and attenuation indicated
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Linearity 7 $an!e

The %or8ing sample concentration and samples tested for

accuracy should be in the linear range (concentrations
!s: Pea8 areas)

Minimum : concentrations

"ilute of stoc8 solution or separate %eighings

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Linearity 7 $an!e

Assay ) 0;9+-;< of the theoretical content of acti%e

Content =niformity) >;9+*;<

?issolution) @-;< of limitsA e! if limits co%er from

-;< to /;< l,c, "controlled release#( linearity should
co%er ;9++;< of l,c,

Impurities) reportin! le%el to +-;< of shelf life limit

Assay7Purity by a sin!le method) reportin! le%el of

the impurities to +-;< of assay limit
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19-20 January 2011
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Linearity 7 $an!e
Correlation coefficient "r#
API) B ;,//0
Impurities) B ;,//
y9Intercept and slope should be indicated to!ether 1ith
plot of the data
The prequalification programme --Assessor's training
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19-20 January 2011
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Accuracy
Assay
API@ against an RS of 8no%n purity; or via an alternate method of
8no%n accuracyM analysis in triplicate:
&PP@ samples<placeboes spi8ed %ith #P3; across the range of 0;9+-;<
of the tar!et concentration( * concentrations( in triplicate each:
$eport per cent reco%ery "mean result and $S?#) +;;@-<
3CH 40 states@ accuracy may be inferred once precision; linearity
and specificity have been established: ("emonstration preferred):
The prequalification programme --Assessor's training
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19-20 January 2011
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Accuracy
3mpurities@ #P3<PP spi8ed %ith 8no%n impurities
$-perienced in P4@
#cross the range of LO'9+:;< of the target concentration
(shelf life limit); +-5 concentrations; in triplicate each:
(L*4; 51G; &11G; &51G)
Per cent reco%ery@ in general; 1ithin 0;9+-;<( depends
on the level of limit
The prequalification programme --Assessor's training
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19-20 January 2011
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Precision

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
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Precision

$epeatability "method precision#

7 Multiple measurements of a sample by the same analyst
7 A minimum of . determinations at the test concentration ".
times of a sin!le batch#( or
7 * le%els "0;<( +;;<( +-;<# ( * repetitions each "combined
1ith accuracy#
7 &or Assay) $S? C -,;<
7 &or indi%idual impurity abo%e ;,;:<( in !eneral( $S? C +;<
The prequalification programme --Assessor's training
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19-20 January 2011
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Precision

Intermediate precision "part of ru!!edness#

7 est a sample on multiple days( analysts( e3uipments
7 $epeat the method precision by different analyst in
different e3uipment usin! different lot of column on
different days
7 $S? should be the same re3uirement as method precision

$eproducibility "inter9laboratory trial#

7 Not re3uested in the submission
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LO?7LO'

signal to noise ratio@ L*" +@& ; L*4 &1@&

7 /ay vary %ith lamp aging; model<manufacturer of detector; column

standard deviation of the response and the slope of the calibration

curve at levels appro-imating the L*" <L*4
N K the standard deviation of the response; base on
7 the standard deviation of the blan8
7 The calibration curve
should be validated by analysis of samples at the limits:
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LO?7LO'

LO?) belo1 the reportin! threshold

LO') at or belo1 the specified limit

=ot re9uired for assay<dissolution methods:

#pplicant should provide

7 the method of determination
7 the limits;
7 chromotograms
The prequalification programme --Assessor's training
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19-20 January 2011
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$obustness

The methodOs capability to remain unaffected by small but

deliberate variations in method parameters
7 3nfluence of variations of pH in a mobile phase
7 3nfluence of variations in mobile phase composition
7 "ifferent columns (different lots and<or suppliers)
7 Temperature
7 lo% rate

$valuate the System suitability parameters

The prequalification programme --Assessor's training
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19-20 January 2011
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$obustness
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Conclusion

HPLC methods play a critical role in analysis of

pharmaceutical product

!alidation of HPLC should demonstrate that the method

is suitable for its intended use

Revie% the information in dossier against 4*S 0:+:R:0

"ata for acceptance; release; stability %ill only be

trust%orthy if the methods used are reliable
The prequalification programme --Assessor's training
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Case Study
Case Study &--HPLC /ethod:doc
4*S 0:+:R:doc
Case Study 0 -- !alidation of HPLC /ethod:doc