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Analytical Method Development

Process for New Products
Learn how to develop an analytical method for the new
drug products.

Ankur Choudhary  5 comments

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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
Literat
ure
Revie
w
5.2
Pharm
acopo
eial
Refere
nce
Correl
ating
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)

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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 Condition

Stress
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

Thermal Water Acid Base Oxidation

(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 % %Assay Total Remark

Stress Degradation
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)
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API standard lot No: Potency:

Name of Impurity % RRT RRF %

the lot No. Purity Recove
known Calculat
impurity using RR

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)

Willow Glen
FDA Registration & US Agent
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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. That is
RRT Relative Retention Time
API Active Pharmaceutical Ingredient
% Percentage
UV Ultra-Violet
RI Refractive Index
ELSD Evaporative Light Scattering
Detector

24.0 Revision History:

S. Revision Effective Change Control

No. No. Date 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: [email protected]

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Read Comment Policy ▼

5 comments: Post Yours!

durga 24 March
thanq
Reply

Unknown 26 March
Is there any specific or standard flow rate is
available for water plat continuous loop
circulation system
Reply

Ankur Choudhary 27 March

Yes it should be 1.2 - 1.5 m/sec
Reply

Unknown 13 May
plz add topic about Diffusion study for
topical formulation using franz cell with
calculation

Thanks
Reply

Unknown 21 October
Good efforts dear ,
Reply

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