1. Review of Analytical Methods
Hua YIN

2. World Health Organization
Outline
2017年4月17日星期一
Standard test procedures—focus on Chromatography
Description of the methods
Review tips
Adjustment to compendial methods
Summary of the analytical procedure in QOS

3. 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.

4. API Methodology Related substances (Impurities) Assay
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

5. 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

6. 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

7. 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

8. Chromatographic Method
A flow scheme for HPLC

9. 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

10. 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)

11. 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)

12. System suitability testing (SST)
World Health Organization
System suitability testing (SST)
2017年4月17日星期一
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
Repeatability The ability of an operator to consistently repeat the same measurement of the same part

13. System suitability testing (SST)
Resolution (R): >2 for two closest eluting peaks

14. System suitability testing (SST)
Number of theoretical plates (N):

15. System suitability testing (SST)
Tailing factor/peak asymmetry:
EP: 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

16. System suitability testing (SST)
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.

17. 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

18. Review 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))

19. Review of HPLC methods 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 ( ).
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).

20. 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

21. Relative Response Factor (RRF) (to be revised with an example of RRF out of the range of 0.8-1.2)
Rifampicine Quinone: y = x Rifampicine: y = x RRF= / =0.84

22. 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 , correction may not be necessary

23. 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)

24. 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

25. 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

26. 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

27. 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 , Int. Ph

28. 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.

29. 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.

30. 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

31. 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

32. 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

33. 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 , Int. Ph
Methods and Validation presentation, by Lynda Paleshnuik in 2009

34. Thank you!