2 4 Method Validation HPLC Case Study

5/22/2018 2 4 Method Validation HPLC Case Study

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Method and Validation basics

HPLC case study

Hua YIN(Assessor)


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The prequalification programme --Assessor's training | 19-20 January 20112|

Outline

HPLC methodology

- Content of HPLC test procedure

- System Suitability Testing (SST)

- Relative Response Factor (RRF)

Validation of HPLC method

case study


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Information Sources

FDA CDER reviewer guideline for validation ofchromatographic methods (1994)

WHO TRS 937 Appendix 4 Analytical Method Validation

2006

ICH Q2(R1) 2005

Compendial General Chapters

Methods and Validation presentationLynda 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 detectionis most commonly usedform of HPLC, is selected to illustrate the parameters of HPLC

method and validation.


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A f low 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, innerdiameter), particle size (10m, 5 m)

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.2analytical 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, columntemp, dimension of column, particle size (reduction only), may be

necessary to achieve the system suitability criteria. The allowablevariations for each parameter, see Int.Ph 1.14.4 or USP generalchapter .


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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 RSs: 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 drugsubstance or related substances per unit weight.

RF= peak area / concentration (mg/ml)

Relative response factor (RRF):

RRF=RFimpurity/ RFAPI,OR,

RRF=slopeimpurity/ slopeAPI


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Rifampicine:

y =31.312x + 4.963

Rifampicine Quinone:

y = 26.198x + 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 proceduredescribed in detail including all the parameters ? is SSTwell defined to ensure the consistency of system performance?

The preparation of solutions:

assay: concentration of reference standard should be close to thesample solution

impurities: concentration of the reference standards should be close tothe limit

The way of quantitation of impurities

In case API is used as the reference, RRF should be used or justification ofexclusion should be provided.

To check the determination of RRF, check the correction of calculation ofimpurities

confirm/complete the QOS 2.3.R.2


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Validationcompendial methods

AssayAPI

No validation generally required. Exception: specificity for majorimpurities not in the monograph.

AssayFPP

Specificity, accuracy and precision (repeatability).PurityAPI and FPP

Full validation for specified impurities that are not included in themonograph (specificity, linearity, accuracy, repeatability,intermediate precision, LOD/LOQ)

Validation of the limit for individual unknowns, if tighter than that inthe monograph: LOQ of the API should be below the limit forindividual 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 foraccuracy 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 shouldcover 0-110% of l.c.

Impurities: reporting level to 120% of shelf life limit

Assay/Purity by a single method: reporting level ofthe 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 ofknown accuracy; analysis in triplicate.

FPP: samples/placeboes spiked with API, across the range of 80-120% of the target concentration, 3 concentrations, in triplicateeach.

Report per cent recovery (mean result and RSD): 1002%

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 ofLOQ-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 recommendedfor assay;

RSD 5% is recommendedfor related substances (referencestandards 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 calibrationcurve at levels approximating the LOD /LOQ

= 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 butdeliberate variationsin 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 ofpharmaceutical 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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The prequalification programme --Assessor's training | 19-20 January 201134 |

Case Study

Case Study 1--HPLC Method.doc

QOS 2.3.R.doc

Case Study 2 -- Validation of HPLC Method.doc
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2 4 Method Validation HPLC Case Study