Review of Analytical Methods Hua YIN

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

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.

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

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

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

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

Chromatographic Method A flow scheme for HPLC

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- 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 - 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) 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

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

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

System suitability testing (SST) 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

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.

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 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 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 (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) 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) 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) 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 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- 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 - 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

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

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

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.

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.

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

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

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

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

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