Review of Analytical Methods: Hua Yin

Review of
Analytical Methods
Hua YIN
Outline
 Standard test procedures—focus on Chromatography

– Description of the methods
– Review tips
– Adjustment to compendial methods
 Summary of the analytical procedure in QOS
The prequalification programme--Assessor's training

2| 25-28 May 2015
Specification
 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.

3| 25-28 May 2015
API 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
4| 25-28 May 2015
FPP 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

5| 25-28 May 2015
Analytical 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

6| 25-28 May 2015
Description
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

7| 25-28 May 2015
Chromatographic Method
A flow scheme for HPLC

8| 25-28 May 2015
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

9| 25-28 May 2015
HPLC 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)

10 | 25-28 May 2015
Review 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)

11 | 25-28 May 2015
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

12 | 25-28 May 2015
 Resolution (R): >2 for two closest eluting peaks

13 | 25-28 May 2015
 Number of theoretical plates (N):

14 | 25-28 May 2015
 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
15 | 25-28 May 2015
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.

16 | 25-28 May 2015
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

17 | 25-28 May 2015
 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))

18 | 25-28 May 2015
Quantification 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).

19 | 25-28 May 2015
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

20 | 25-28 May 2015
(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
21 | 25-28 May 2015
 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

22 | 25-28 May 2015
Review 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)

23 | 25-28 May 2015
Gas 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
24 | 25-28 May 2015
Thin-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
25 | 25-28 May 2015
Dissolution
 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

26 | 25-28 May 2015
Compendial 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

27 | 25-28 May 2015
Compendial methods
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.

28 | 25-28 May 2015
Compendial 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.

29 | 25-28 May 2015
Example
 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

30 | 25-28 May 2015
Revision 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

31 | 25-28 May 2015
In 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

32 | 25-28 May 2015
Information 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

33 | 25-28 May 2015
Thank you!

34 | 25-28 May 2015

Review of Analytical Methods: Hua Yin

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Review of

 Standard test procedures—focus on Chromatography

 Summary of the analytical procedure in QOS

The prequalification programme--Assessor's training

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

The prequalification programme--Assessor's training

The prequalification programme--Assessor's training

 Description of all analytical procedures in details

The prequalification programme--Assessor's training

The analytical procedure should contain a complete description

The prequalification programme--Assessor's training

The prequalification programme--Assessor's training

The prequalification programme--Assessor's training

 Elution procedure: isocratic or gradient elution

 Preparation of standards and samples

 System suitability testing (SST) and criteria

 Formula of calculation (RRF for known impurities)

The prequalification programme--Assessor's training

 System suitability testing (SST): an integral part of chromatographic

The prequalification programme--Assessor's training

– Impurities: in general, RSD ≤ 5% at the limit level-- typically

The prequalification programme--Assessor's training

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

The prequalification programme--Assessor's training

The prequalification programme--Assessor's training

EP: 0.8 -1.5 unless otherwise prescribed)

A SST should contain:

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

 For impurity test:

resolution + precision. resolution may be exchanged for another

The prequalification programme--Assessor's training

 Check if SST is defined properly

The prequalification programme--Assessor's training

 Check the preparation of solutions:

The prequalification programme--Assessor's training

 Quantitated against API as an external standard (for identified imp):

 Unspecified impurities: quantitated against API at a concentration

The prequalification programme--Assessor's training

When Quantitated against API, relative response factor (RRF) may be

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

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

The prequalification programme--Assessor's training

RRF= 26.198 / 31.312

a) If RRF is used (compare to the compendial method if applicable).

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

 Correction factor= 1/RRF

RRF within 0.8-1.2, correction may not be necessary

The prequalification programme--Assessor's training

 Is the analytical procedure described in sufficient detail ?

 Is SST well defined to ensure the consistency of system

 Review quantitation of impurities, if RRF is used correctly ?

 Review the Summary of the analytical procedures in QOS 2.3.R.2

The prequalification programme--Assessor's training

 TLC Plate: e.g. silica gel plate

 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