2 4 Method Validation HPLC Case Study

Method and Validation basics
HPLC case study—
Hua YIN
)Assessor(
Outline
 HPLC methodology
- Content of HPLC test procedure
- System Suitability Testing (SST)
- Relative Response Factor (RRF)
 Validation of HPLC method
 case study
The prequalification programme --Assessor's training | 19-20 January 2011

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Information Sources
 FDA CDER reviewer guideline for validation of

chromatographic methods (1994)
 WHO TRS 937 Appendix 4 “Analytical Method Validation

2006
 ICH Q2(R1) 2005
 Compendial General Chapters
 Methods and Validation presentation–Lynda Paleshnuik

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High Performance Liquid Chromatography (HPLC)
HPLC is a separation technique based on a solid stationary phase and

a liquid mobile phase. Separations are achieved by partition,
adsorption, or ion-exchange processes, depending upon the type of
stationary phase used.
 Chiral
 Ion--exchange
 Ion--pair/affinity
 Normal phase
 Reversed phase
 Size exclusion
The reversed-phase HPLC with UV detection is most commonly used

form of HPLC, is selected to illustrate the parameters of HPLC
method and validation.

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A flow scheme for HPLC

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Content of HPLC test procedure
Any analytical procedure submitted should be described in
sufficient detail, includes:
 Preparation of mobile phase
 Chromatographic condition:
– Column: type (e.g., C18 or C8), dimension (length, inner
diameter), particle size (10μm, 5 μm)
– Detector: wavelength
– Injection volume
– column T
– flow rate,

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Content of HPLC test procedure
 Elution procedure: isocratic or gradient elution
 Preparation of standards and samples
 Operation procedure: sequence of injections
 System suitability testing (SST) and criteria
 Calculations
QOS 2.3.R.2 analytical procedures and validation summaries

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Compendial methods
When claim a compendial method, there should be no change in:
 The type of column i.e the stationary phases
 Detector wavelength
 Components in Mobile phase
 System suitability testing and criteria
Adjustments to ratio of components in mobile phase, flow rate, column

temp, dimension of column, particle size (reduction only), may be
necessary to achieve the system suitability criteria. The allowable
variations for each parameter, see Int.Ph 1.14.4 or USP general
chapter <621>.

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System suitability testing (SST)
 Precision:
– Assay: RSD ≤1% (API) or ≤ 2% (FPP), n ≥ 5
– Impurities: in general, RSD ≤ 5% at the limit level, up to 10% or
higher at LOQ, n ≥ 6
 Resolution (R): >2

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 Tailing factor/peak asymmetry: (≤ 2)

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 Number of theoretical plates (N): column efficiency ≥ 2000
 Gradient elution is one way to increase the N

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A SST should contain:
 For Assay:
precision + one or more other parameter
 For impurity test:
resolution + precision + one or more other parameter

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Relative Response Factor (RRF)
Quantitation of Impurities
 Against impurity RS’s: when reference standard available
 Against API itself
Relative response factor should be considered

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 Response factor: the response (e.g. peak area) of drug

substance or related substances per unit weight.
RF= peak area / concentration (mg/ml)
 Relative response factor (RRF):

RRF=RF impurity / RF API, OR,
RRF=slope impurity / slope API

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Rifampicine:
y =31.312 x + 4.963
Rifampicine Quinone:
y = 26.198 x + 1.154
RRF= 26.198 / 31.312
=0.84

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 To review:
a) RRF calculation, and
b) if RRF is properly used in the final calculation for %

impurity
If RRF within 0.8-1.2, correction may not be necessay
 Correction factor= 1/RRF, the reciprocal of the RRF

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Review points for HPLC method
 is the analytical procedure described in detail including all the parameters ?
 is SST well defined to ensure the consistency of system performance?
 The preparation of solutions:

– assay: concentration of reference standard should be close to the sample
solution
– impurities: concentration of the reference standards should be close to
the limit
 The way of quantitation of impurities

In case API is used as the reference, RRF should be used or justification of
exclusion should be provided.
To check the determination of RRF, check the correction of calculation of
impurities
 confirm/complete the QOS 2.3.R.2

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Validation – compendial methods
Assay – API
No validation generally required. Exception: specificity for major impurities
not in the monograph.
Assay – FPP
Specificity, accuracy and precision (repeatability).
Purity – API and FPP
Full validation for specified impurities that are not included in the monograph
(specificity, linearity, accuracy, repeatability, intermediate precision,
LOD/LOQ)
Validation of the limit for individual unknowns, if tighter than that in the
monograph: LOQ of the API should be below the limit for individual
unknowns

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Non-compendial methods
Full validation is required for purity, assay and dissolution methods

(HPLC, UV) :
Specificity
Linearity
Accuracy
Repeatability
Intermediate precision
LOD/LOQ (not required for assay, dissolution)
Robustness (recommended)

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Specificity
 Blank solution to show no interference
 Placebo to demonstrate the lack of interference from excipients
 Spiked samples to show that all known related substances are

resolved from each other
 Stressed sample of about 10 to 20% degradation is used to

demonstrate the resolution among degradation products
– Check peak purity of drug substance by photodiode array detector (PDA): eg
purity angle is lower than the purity threshold.
 Representative chromatograms should be provided with time scale

and attenuation indicated

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Linearity / Range
 The working sample concentration and samples tested for

accuracy should be in the linear range (concentrations
Vs. Peak areas)
 Minimum 5 concentrations
 Dilute of stock solution or separate weighings

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Linearity / Range
 Assay : 80-120% of the theoretical content of active
 Content Uniformity: 70-130%
 Dissolution: ±20% of limits; eg if limits cover from

20% to 90% l.c. (controlled release), linearity should
cover 0-110% of l.c.
 Impurities: reporting level to 120% of shelf life limit
 Assay/Purity by a single method: reporting level of

the impurities to 120% of assay limit

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Linearity / Range
Correlation coefficient (r)
API: ≥ 0.998
Impurities: ≥ 0.99
y-Intercept and slope should be indicated together with

plot of the data

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Accuracy
Assay
API: against an RS of known purity, or via an alternate method of

known accuracy; analysis in triplicate.
FPP: samples/placeboes spiked with API, across the range of 80-

120% of the target concentration, 3 concentrations, in triplicate
each.
Report per cent recovery (mean result and RSD): 100±2%
ICH Q2 states: accuracy may be inferred once precision, linearity

and specificity have been established. (Demonstration preferred).

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Accuracy
Impurities: API/FPP spiked with known impurities
Experienced in PQ:
Across the range of LOQ-150% of the target concentration

(shelf life limit), 3-5 concentrations, in triplicate each.
(LOQ, 50%, 100%, 150%)
Per cent recovery: in general, within 80-120%, depends

on the level of limit

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Precision
 System precision:
– by multiple injections (n ≥5) of a homogeneous sample
(standard solution).
– RSD ≤ 1% is recommended for assay;
– RSD ≤ 5% is recommended for related substances (reference
standards at the limit)
– Indicates the performance of the HPLC system
– As a system suitability test

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Precision
 Repeatability (method precision)

– Multiple measurements of a sample by the same analyst
– A minimum of 6 determinations at the test concentration (6
times of a single batch), or
– 3 levels (80%, 100%, 120%) , 3 repetitions each (combined
with accuracy)
– For Assay: RSD ≤ 2.0%
– For individual impurity above 0.05%, in general, RSD ≤ 10%

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Precision
 Intermediate precision (part of ruggedness)

– Test a sample on multiple days, analysts, equipments
– Repeat the method precision by different analyst in
different equipment using different lot of column on
different days
– RSD should be the same requirement as method precision
 Reproducibility (inter-laboratory trial)

– Not requested in the submission

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LOD/LOQ
 signal to noise ratio: LOD 3:1 , LOQ 10:1

– May vary with lamp aging, model/manufacturer of detector, column
 standard deviation of the response and the slope of the calibration

curve at levels approximating the LOD /LOQ
σ = the standard deviation of the response, base on

– the standard deviation of the blank
– The calibration curve
should be validated by analysis of samples at the limits.

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LOD/LOQ
 LOD: below the reporting threshold
 LOQ: at or below the specified limit
Not required for assay/dissolution methods.
 Applicant should provide

– the method of determination
– the limits,
– chromotograms

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Robustness
 The method's capability to remain unaffected by small but

deliberate variations in method parameters
– Influence of variations of pH in a mobile phase
– Influence of variations in mobile phase composition
– Different columns (different lots and/or suppliers)
– Temperature
– Flow rate
 Evaluate the System suitability parameters

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Robustness

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Conclusion
 HPLC methods play a critical role in analysis of

pharmaceutical product
 Validation of HPLC should demonstrate that the method

is suitable for its intended use
 Review the information in dossier against QOS 2.3.R.2
 Data for acceptance, release, stability will only be

trustworthy if the methods used are reliable

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Case Study
Case Study 1--HPLC Method.doc
QOS 2.3.R.doc
Case Study 2 -- Validation of HPLC Method.doc

34 |

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Method and Validation basics

HPLC case study—

- Content of HPLC test procedure

- System Suitability Testing (SST)

- Relative Response Factor (RRF)

 Validation of HPLC method

The prequalification programme --Assessor's training | 19-20 January 2011

 FDA CDER reviewer guideline for validation of

 WHO TRS 937 Appendix 4 “Analytical Method Validation

 ICH Q2(R1) 2005

 Compendial General Chapters

 Methods and Validation presentation–Lynda Paleshnuik

The prequalification programme --Assessor's training | 19-20 January 2011

HPLC is a separation technique based on a solid stationary phase and

The reversed-phase HPLC with UV detection is most commonly used

The prequalification programme --Assessor's training | 19-20 January 2011

The prequalification programme --Assessor's training | 19-20 January 2011

The prequalification programme --Assessor's training | 19-20 January 2011

 Elution procedure: isocratic or gradient elution

 Preparation of standards and samples

 Operation procedure: sequence of injections

 System suitability testing (SST) and criteria

QOS 2.3.R.2 analytical procedures and validation summaries

The prequalification programme --Assessor's training | 19-20 January 2011

When claim a compendial method, there should be no change in:

 The type of column i.e the stationary phases

 Components in Mobile phase

 System suitability testing and criteria

Adjustments to ratio of components in mobile phase, flow rate, column

The prequalification programme --Assessor's training | 19-20 January 2011

 Resolution (R): >2

The prequalification programme --Assessor's training | 19-20 January 2011

The prequalification programme --Assessor's training | 19-20 January 2011

 Gradient elution is one way to increase the N

The prequalification programme --Assessor's training | 19-20 January 2011

A SST should contain:

precision + one or more other parameter

 For impurity test:

resolution + precision + one or more other parameter

The prequalification programme --Assessor's training | 19-20 January 2011

Relative response factor should be considered

The prequalification programme --Assessor's training | 19-20 January 2011

 Response factor: the response (e.g. peak area) of drug

RF= peak area / concentration (mg/ml)

 Relative response factor (RRF):

The prequalification programme --Assessor's training | 19-20 January 2011

RRF= 26.198 / 31.312

The prequalification programme --Assessor's training | 19-20 January 2011

a) RRF calculation, and

b) if RRF is properly used in the final calculation for %

 Correction factor= 1/RRF, the reciprocal of the RRF

The prequalification programme --Assessor's training | 19-20 January 2011

 is SST well defined to ensure the consistency of system performance?

 The preparation of solutions:

 The way of quantitation of impurities

 confirm/complete the QOS 2.3.R.2

The prequalification programme --Assessor's training | 19-20 January 2011

The prequalification programme --Assessor's training | 19-20 January 2011

Full validation is required for purity, assay and dissolution methods

The prequalification programme --Assessor's training | 19-20 January 2011

 Blank solution to show no interference

 Placebo to demonstrate the lack of interference from excipients

 Spiked samples to show that all known related substances are