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Analytical Method Development Process for New Products


Learn how to develop an analytical method for the new drug 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)


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



















































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
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question


5 Comments so far : Add yours...

Dinesh Cowdary said...

thanq

Subbu Nuthanki said...

Is there any specific or standard flow rate is available for water plat continuous loop circulation system

Ankur Choudhary said...

Yes it should be 1.2 - 1.5 m/sec

kaushal patel said...

plz add topic about Diffusion study for topical formulation using franz cell with calculation

Thanks

Unknown said...

Good efforts dear ,

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