Analytical Method Development Process for New Products

1.0  Objective:

The purpose of the study is to develop analytical method for determination of Assay / Related Substances of new product by HPLC or UV-Vis Spectrophotometer as applicable.

2.0  Introduction and Overview:

This guideline provides detailed information about analytical development to be carried out on all the aspects of the method of analysis.

All the instruments/ equipment used to carry out this validation exercise should be qualified and validated. Instrument calibration status should be verified.

3.0  Scope:

This guideline provides detailed information about analytical method development to be carried out as per ICH Guidelines.

4.0  Physico-Chemical Details Related to API:

IUPAC name
INN name
Molecular formula
CAS No.
Chemical structure 1:1 Racemate Mixture
Molecular weight
Solubility
Appearance
Pka
LogP
BCS class
pH solubility profile
pH-stability profile
Polymorphism
Isomerism
Photostability
Melting point (oC)
Density
Hygroscopicity
Impurities
Monograph
Official formulation

5.0 Details of the Product:

Name
Molecular Weight
Molecular Formula
IUPAC Name
Chemical Structure
Status of Molecule:  USP / BP/ EP/ In-house
Dosage Form  Tablet/capsule/liquid orals/injection
Dosage Form Strengths
Maximum Daily Dose
Reference/ basis for Maximum Daily Dose
Reporting Thresholds
Identification Thresholds
Quantification Thresholds
Literature on Analytical Profiles
Solubility
pKa
Information on Metabolites

5.1 Literature Review

5.2 Pharmacopoeial Reference Correlating API and Product Impurities

6.0 Synthetic Scheme for API:

7.0 Structures of Impurities:

7.1 Polarities of Impurities compared with Analyte

{Draw the basic conclusions on polarities (less polar or more polar when compared with analyte) based on structures}

8.0 Chromatographic Conditions:

8.1 Selection of detector:

(Based on the structure of analyte and impurities/degradants, justify the basis for selection of detector.

If multiple detectors are used for estimation of some of the impurities, it needs to be specified)

8.2 Basis for initial wavelength selection (in case of UV, RI & ELSD):

(Should be based on spectra of the Analyte and or impurities/degradants)

8.3 Buffer & pH selection:

(Discuss the basis for finalization of pH based on pKa, buffering range of buffer, based on separation of impurities)

Document the separation of impurities at different pH conditions and conclude why a particular pH is chosen. Paste the relevant chromatograms.

Finalization of the pH shall be based on robustness data (at target pH and at ± 0.2 of the target pH)

8.4 Column Selection:

Initial column selection is based on Analyte and impurities polarity assessment based on functional groups.

8.5 Elution mode:

(Run these default gradients using the samples which contain all possible impurities to make the exact assessment of polarity range of the impurities)

9.0 Samples Used for Method Development:

Program 1:
Time	Buffer	Organic Phase
0.01	50	50
60	5	95
70	5	95
71	50	50
75	50	50

Observations:

Program 2:
Time	Buffer	Organic Phase
0.01	95	5
60	5	95
70	5	95
71	50	50
75	50	50

Observations:

(Draw the conclusions based on the elution patterns obtained for most polar and most non-polar impurities based on the gradient design with more aqueous portion and more solvent portion)

10.0 Optimization of Chromatographic Conditions:

(Document conditions of mobile phase, column, flow rate, column temperature and gradient programme which has led to finalization of chromatographic condition.)

(Document the basis for finalization of column by comparing the separation characteristics in different columns)

(Document the Column description & characteristics like Length, Internal diameter, particle size, % carbon loading, Pore size, Surface area, end capping etc)

(Document the alternate conditions used to demonstrate that there is no possibility of missing estimation of some of the impurities)

10.1 Diluent Selection:

(Should be based on solubility (extraction capability in case of formulations), peak symmetry and stability of solution)

10.2 Solubility:

10.3 Assay (%) of Analyte Peak from Sample Matrix:

(For formulations, document the procedure & diluent in which >95% assay is achieved for analyte peak of sample)

10.4 Stability of Solutions:

(Estimate the stability of solution at least for a period of 12 hours initially and update the data upon subsequent data generation)

11.0 Interference Study:

(Document the interference from blank, placebo and filter.) Attach specimen chromatograms.

12.0 Establishment of Method Ruggedness to HPLC System & Column:

12.1 Two Different HPLC Systems (like Waters / Agilent / Shimadzu):

(Document the separation of impurities in two different make of HPLC systems).

12.2 Two Different Columns (one preferably new column):

(Document the separation of impurities in two different make of columns).

12.3 Establishment of Method Sensitivity towards Chromatographic Parameters:

(Document the robustness data on pH, flow rate, column temp & mobile phase composition).

Document RT, RRT, Name of the impurity, tailing factor, Resolution for each condition along with specimen chromatograms)

12.4 Test Concentration & Injection Volume:

(Document the basis for finalization of the test concentration and injection volume based on meeting the criteria of LOQ ≤ reporting threshold)

Related: Theoretical Plates and their Determination in HPLC Analysis

13.0 Forced Degradation and Establishment of Stability Indicating Nature of the Method:

Document the degradation conditions, degradation details along with chromatograms & purity plots:

Typically the following format can be used.OR (Depend upon the nature of Molecule)

Type of Stress	Stress Condition
Thermal	105°C for 12/24 hours
Water	Refluxed for 12 hours
Acid	Refluxed for 12 hours
Base	Refluxed for 12 hours
Oxidation	bench top for 24 hours.
light	200 watt and 1.2 million lux hr
Humidity	90% RH for 7 days

RRT	% Degradation							Name (known/ unknown	Peak purity Passes	Remarks
	Thermal	Water	Acid	Base	Oxidation	light	humidity		Y/N	Process/Degradant

(Capture all known and major unknowns’ degradant peaks).

Conclusions:

All known and unknown impurities/degradant are separating from each other and from Analyte peak.

All peaks are found to be pure.

13.1 Mass Spectral Study:

(Document the Mass spectral study done on the Major degradant).

(Attach the LC-MS method and specimen chromatograms as annexure).

13.2 Mass Balance Study:

Document the % Assay of the Stressed samples (to be calculated against a standard of API with a peak height of less than 1AU).

Document the Total % of degradation and do the Mass balance.

Type of Stress	% Degradation	%Assay	Total	Remarks
Thermal
Water
Acid
Base
Oxidation
light
Humidity

13.3 Basis for Finalization of Wavelength:

(Should be based on the Spectral overlay of impurity peaks in the forced degradation samples and stability samples, if any)

(Attach the spectral overlay of the forced degradation / stability samples)

14.0 Establishment of Relative Response Factor (RRF) & Recovery:

(Document the RRF’s calculated using at least two different weights & the corresponding Recovery studies to confirm the RRF’s)

API standard lot No:                          Potency:

Name of the known impurity	Impurity lot No.	% Purity	RRT	RRF	% Recovery Calculated using RRF	Remarks

Related: Relative Response Factor (RRF) in HPLC Analysis

15.0 Comparison of API and Formulation Methods:

(Document the RRT and % of impurities on one of the API sample analysed using both the methods if methods are different and compare. The number and % of impurities shall match)

16.0 Comparison of Pharmacopoeial and In-house Methods:

(In case any methods are official for the analyte under consideration, establish and document the equivalency or superiority of the methods)

(Incase any Pharmacopoeial methods are found to be deficient, communicate the same to the concerned Pharmacopoeial authorities and attach the communication made along with the response)

17.0 Justification for Selection of Quantification Method:

(Document the basis for selection of quantification methods like area normalization or diluted standard or external standard or internal standard methods)

(Document the exclusion criteria if any along with the basis for the same)

18.0 Selection of System Suitability Criteria:

(Document the basis for selection of system suitability criteria like, resolution/ h/v ratio, tailing factor, %RSD / peak response ratio, Theoretical plates, capacity factor etc)

19.0 Analytical Methods for Different Stages of API Synthetic Route Prior to Final Formulation Stage:

(Document the reaction scheme for each stage along with reactants, intermediates, solvents, catalysts and possible byproducts.

Document the method used for estimation of purity / impurities present at different stages of API manufacturing along with specimen chromatograms.

Document or attach a report on elimination of various impurities stage wise).

20.0 Conclusions:

Document the conclusions by stating the stability indicating nature of the method.

Comment on any specific sensitivities of the method.

Comment on any special precautions to be taken while using the method

Document specific handling instructions, if any.

Document the hygroscopicity /specific storage condition of the standard(s), if any.

Document / attach the justification for finalization of specification for impurities.

21.0 Annexure:    

Annexure should be attached to the Method Development Report.

22.0 Reference:

23.0 Abbreviations:

No.	Number
+	Plus or minus
i.e. RRT	That is Relative Retention Time
API %	Active Pharmaceutical Ingredient Percentage
UV	Ultra-Violet
RI	Refractive Index
ELSD	Evaporative Light Scattering Detector

24.0  Revision History:

S. No.	Revision No.	Effective Date	Change Control No.

Also see: Analytical Method Validation Protocol

Submitted By:
Dipak Dhote

Assistant Manager (Analytical R&D)

Blue Cross Laboratories Ltd
Nashik-422010,
Maharashtra, INDIA.
Email: dipak.d@bluecrosslabs.com

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