Review of Analytical Methods Hua YIN. The prequalification programme--Assessor's training 25-28 May...

Review of Analytical Methods

Hua YIN

The prequalification programme--Assessor's training 25-28 May 20152 |

OutlineOutline

Standard test procedures—focus on Chromatography– Description of the methods– Review tips– Adjustment to compendial methods

Summary of the analytical procedure in QOS


SpecificationSpecification

As defined in ICH's Q6A guidance document, a specification is a list

of tests, references to analytical procedures, and appropriate

acceptance criteria, which are numerical limits, ranges, or other

criteria for the tests described.


API MethodologyAPI Methodology

Description

Identity (IR)

Related substances (Impurities)

Assay

Residual solvents

Melting point, Heavy metals, pH, Residue on ignition/sulfated ash, LOD/water content, Specific optical rotation

User parameters:

Particle size

Polymorphism

Bulk density/Tapped density


FPP MethodologyFPP Methodology

Description /appearance

Identification: API (s), chemical preservatives

Purity: degradation products, residual solvents

Assay

Physical tests: e.g. LOD/water content, pH, friability, hardness , particle size

Performance tests: e.g. dissolution, disintegration (where applicable)

Uniformity of dosage units: mass or content uniformity

Content of preservatives

Microbial contamination, Sterility, bacterial endotoxins


Analytical MethodsAnalytical Methods

Description of all analytical procedures in details

Justification: e.g. dissolution

Validation: ID, assay and purity tests– Assay, dissolution, content uniformity, content of preservatives– Impurities – Residual Solvents


DescriptionDescription

The analytical procedure should contain a complete description sufficiently detailed to enable replicate by other analyst: –Operational parameters

–Preparation of reagents, reference and test solutions

–Performance of system suitability test

–Specific instructions, precautions

–Formula for calculation


Chromatographic MethodChromatographic Method

A flow scheme for HPLC


HPLC methods- Description HPLC methods- Description

Any analytical procedure should be described in sufficient details, including:

Chromatographic condition:

– Column: type (e.g., C18 or C8), dimension (length, inner diameter), particle size (10μm, 5 μm)

– Column Temperature– Detector: wavelength– Injection volume– Flow rate


HPLC methods- DescriptionHPLC methods- Description

Mobile phase

Elution procedure: isocratic or gradient elution

Preparation of standards and samples

System suitability testing (SST) and criteria

Formula of calculation (RRF for known impurities)


Review of HPLC methods - SSTReview of HPLC methods - SST

System suitability testing (SST): an integral part of chromatographic analytical procedures – to verify the chromatographic system is adequate for the intended analysis

– System precision (RSD)– Resolution (R)– Number of theoretical plates (N) – Tailing factor (T)


System suitability testing (SST)System suitability testing (SST)

System precision– Assay: RSD≤ 2% for FPP, n ≥ 5 – using RS solution

RSD ≤1% for API (see table below)

– Impurities: in general, RSD ≤ 5% at the limit level-- typically done using a solution of the API with a concentration corresponding to the limit for unspecified impurities



Resolution (R): >2 for two closest eluting peaks



Number of theoretical plates (N):



Tailing factor/peak asymmetry:

EP: 0.8 -1.5 unless otherwise prescribed)

Number of theoretical plates (N) and tailing factor (T) can be used as additional SSTs ,or if there are no suitable impurities for the determination of resolution



A SST should contain:

For Assay:

precision + one or more other parameter (e.g. resolution, tailing factor, N,)

For impurity test:

resolution + precision. resolution may be exchanged for another parameter in some circumstances.


Review of HPLC methods Review of HPLC methods

Confirm the analytical procedure is described in detail including all the parameters

– Chromatographic condition– Mobile phase preparations– Elution procedure: isocratic or gradient elution

Check if SST is defined properly


Review of HPLC methodsReview of HPLC methods

Check the preparation of solutions:

– Assay: concentration of reference standard should be equal to the sample solution

– Impurities: concentration of RS for impurities should be at the specification limit unless justified

Method of quantification (e.g. against API or impurity reference standard(s))


Review of HPLC methodsQuantification Method of impurities

Review of HPLC methodsQuantification Method of impurities

Quantitated against impurity RS: for identified impurities, particularly toxic imps.

Quantitated against API as an external standard (for identified imp):– Response factors (RF) of impurities are close to that of the API

(0.8-1.2). – RF is not close to API - overestimated or correction factor to be

applied

Unspecified impurities: quantitated against API at a concentration corresponding to the limit established for unspecified impurities (i.e., the ICH Identification Threshold).


Relative Response Factor (RRF)Relative Response Factor (RRF)

When Quantitated against API, relative response factor (RRF) may be used to calculate the actual quantity of the impurity.

Response factor (RF): 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 at equal concentration

Normally, slope of area and concentration of impurity and standard are calculated to determine the RRF: Slope impurity / Slope API


Relative Response Factor (RRF)(to be revised with an example of RRF out of the range of 0.8-1.2)

Relative Response Factor (RRF)(to be revised with an example of RRF out of the range of 0.8-1.2)

Rifampicine Quinone:

y = 26.198 x + 1.154

Rifampicine:

y =31.312 x + 4.963

RRF= 26.198 / 31.312

=0.84


Relative Response Factor (RRF)Relative Response Factor (RRF)

To review:

a) If RRF is used (compare to the compendial method if applicable). Be cautious of any possibility of underestimate of impurity)

b) Check RRF is correctly determined and used in the calculation formula

Correction factor= 1/RRF

RRF within 0.8-1.2, correction may not be necessary


Review points for HPLC methodReview points for HPLC method

Is the analytical procedure described in sufficient detail ?

Is SST well defined to ensure the consistency of system performance?

Review quantitation of impurities, if RRF is used correctly ?

Review the Summary of the analytical procedures in QOS 2.3.R.2 (see example)


Gas Chromatography- GCGas Chromatography- GC

Chromatographic condition – Column: size (l, Ø), stationary phase, particle size (packed column),

film thickness (capillary column)

– Carrier gas: helium or nitrogen

– Flow rate

– Injection volume and split volume: e.g. 1ml, 10:1

– Temperature: injection port, column, detector

– Detection: e.g. flame ionisation detector (FID)

Test and standard solutions

System suitability testing (SST) and criteria: precision and resolution, RSD (n=6)

Calculation


Thin-Layer Chromatography - TLCThin-Layer Chromatography - TLC

TLC Plate: e.g. silica gel plate

Mobile phase

Test and standard solutions

Application volume: e.g. 2µl

Development: over 3/4 of the plate

Detection/visualization: under UV light or spray with reagents

System suitability (SST): e.g. > 2 spots to separate; spots of interest should be clearly separated and visible at the limit of detection


DissolutionDissolution

The equipment/ apparatus : Paddle, basket

Dissolution media: volume, pH, surfactant

Agitation/rotation speed

Sampling time

Test and standard solutions (filtration)

API quantitation (e.g. HPLC, UV)

Acceptance criteria

filters - in-line or at the end of the sampling probe or both


Compendial methodsCompendial methods

Compendial: reference to pharmacopoeia monograph

When a compendial method for assay, impurities or dissolution is referenced: confirm with the compendial method and clearly state in the report if the method is in line with the compendia.

Eg. “It was confirmed that the applicant’s dissolution method corresponds to the method in the PhInt monograph.”

Adjustments to the specified chromatographic system maybe necessary in order to meet the SST. The compendia give guidance as to how much variation is acceptable in a chromatographic method--refer to General Chapter, USP<621>, EP method 2.2.46, Int. Ph 1.14.4



When a compendial method (HPLC) claimed, the followings must not be changed:

The chemical characteristics of column i.e the stationary phases (e.g. no replacement of C18 by C8)

Detector wavelength: ±3nm

Components in Mobile phase

System suitability tests and criteria

Changes to the above parameters require revalidation of the methods.



Various parameters of a chromatographic test may be adjusted to satisfy the SST criteria:

– mobile phase: composition (for isocratic elution, ±10% absolute ), pH (±0.2), Concentration of salts (±10%)

– Flow rate– Column parameters: particle size, dimensions (for isocratic elution)– Column Temperature (±10º)– Injection volume (precision, linearity and LOD)

Adjustment to HPLC gradient system requires greater caution.

Multiple adjustments can have a cumulative effect.

Changes out of the maximum variation as indicated in the general chapters require revalidation of the methods.


ExampleExample

pH of the buffer solution (mobile phase): 3.3 Vs 2.2 (USP)= non USP method

Ratio of the components in mobile phase 86:14 Vs 88:12 (USP) = USP method

Detector wavelength 254nm Vs 277nm (USP) = non USP method

Dissolution test uses apparatus II Vs USP apparatus I = non USP method

Non pharmacopoeial method = full validation required


Revision of the USP 37Revision of the USP 37

A guard column may be used for HPLC, meet the following criteria: – NMT 15% of the length of the analytical column– the inner diameter ≤ analytical column– the packing material must be the same (e.g. C18)– All SSTs must be met


In conclusionIn conclusion

To evaluate the clarity and completeness of the description of the analytical methods

To confirm the sameness to compendial methods if compendial standard is claimed

Review Summary of Analytical procedures QOS 3.2.R.2


Information SourcesInformation Sources

WHO TRS 937 Appendix 4 - Analytical Method Validation (2006)

ICH Q2 (R1) Validation of Analytical Procedures: Text and Methodology (2005)

Compendia General Chapter, e.g. USP<621>, EP method 2.2.46, Int. Ph 1.14.4

Methods and Validation presentation, by Lynda Paleshnuik in 2009


Thank you! Thank you!

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Review of Analytical Methods Hua YIN. The prequalification programme--Assessor's training 25-28 May...

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