EFFECTIVE
EFFECTIVE ANALYTICAL
ANALYTICAL
METHOD
METHOD DEVELOPMENT
DEVELOPMENT
BY
BY HPLC
HPLC
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Choice of Method
It is important to appreciate the difference
between an ‘analytical method’ (combination
of steps illustrated by the ‘analytical process’)
and an ‘analytical technique’ (chemical or
instrumental procedure by which analytical Always use a
data is eventually obtained). Statistical and standard method if
documentation one is available as
this will save on
development time.
However the method
must be checked to
prove that it suitable
for your
laboratory/situation.
Modification may
well be required.
Importance of Analytical Methods
Without analytical methods it is not possible to
know what has happened during Process, trial
batch, stability, quality of products up to shelf
life.
Assay
Impurities/Degradation Products
Dissolution
Chiral Purity
Preservative Content
Most of analytical test can be evaluated by using
chromatographic technique.
TYPE OF CHROMATOGRAPHY
Chromatography – Chrome means color, graphic
means measurements or write
Preparative & Analytical chromatography
TLC – Thin layer chromatography
IEC – Ion exchange chromatography
Size exclusion chromatography
Ion chromatography
Gas chromatography
HPLC – High performance liquid chromatography
Prior to Start analytical activities
Qualified (IQ & OQ) instruments
Calibrated instruments
Documented methods
Reliable reference standards
Qualified analysts
Sample selection and integrity
Preliminary Information
pH & pK of molecule
Molecular weight
Solubility of molecule
Stability nature of molecule
Polymorph
Salt & hydrate form
Literature survey about molecule
Collect available information from vendor
All pharmacopeia information
To collect information of Analyte
Literature survey
Description of sample
Number of Analytes present
Chemical structure and molecular weight of analytes
Type of dosage form and strength
Spectral data
Solubility of analyte (Different solvent & different PH)
Sample stability data
Impurity profile (Deg, process, genotoxic, formulation
related,metabolite)
General literaure information about drug excipient
compatibility
Initial method development
Start with preliminary method based on what is
known of drug substance
Can determine content of drug substance
Known or expected degradants are separated
Use preliminary method to analyze stress stability
studies
Stress deliberately creates significant degradation
Can evaluate ability of method to separate degradants
Use this information to modify/improve method
Critical components – Sample pre, HPLC analysis
THERMAL-HUMIDITY ACID-BASE
STRESSING SOLUTION STRESSING
* as is or * various pH
in solution conc. of acid
* various temp. or base
and humidity * w/o heat
conditions. * duration
* duration
OXIDATIVE PHOTOSTABILITY
SOLUTION STRESSING
* oxididizing agents: * as is or
H2O2; O2; air; in solution
radical initiator; * ICH Option
metal catalyst * Intensity
* conc. of oxidizer * duration
* duration
Identifying Degradants
Predicting routes of degradation
Acid/base hydrolysis of esters and amides
Oxidation of thiols, alcohols and amines
Loss of methyl groups
Synthesizing possible degradants
Prepare possible degradant
Known Structure
Test it in potential analytical method
Detectability
Separation from parent peak
Basic ideas about method development
If molecule is polar nature, select non polar stationary phase
If molecule is Non polar nature, select polar stationary phase
Non –polar stationary phase: C8, C-18, with coated
Polar – Silica, CN,
The sample is Acid, Base, Neutral – C8, C18
Mobile phase: Water –Organic solvent
Selectivity: Use organic modifier
Low pH: C8 or C18 column, Acidic mobile phase
High pH: C8 or C18 column, Basic mobile phase
Most of pharmaceutical products are polar nature, hence RP-
HPLC is more suitable
Approch-1
Adopt with available reference (Phar, Lit, DMF, Other)
Method to be verified:
Specificity,(FD)
Method Precision,
System Suitability,
Recovery,
LOD,
LOQ
If required change method and justify
Comparative data (ME)
RF- Pahrmaco to be referred
Approch-2
Analytical method needs to be developed which
includes detection limit, specificity, recovery, linear
and precise.
To separate active, impurities, synthetic impurities ,
degradents,
To consider limitation of inst, analysis time,
All the basic information need to collect which is useful
to initiate method development
Literature survey & Past experience in similar
compound, type of formulation, excipients used in
formulation which is help to develop method
HPLC - RP
Column selection:
stationary phase,
carbon loading,
particle size,
length,
end cape,
surface area,
PH range
HPLC - RP
Mobile phase:
UV cut off, solubility, miscibility, compatibility,
polarity, HPLC grade. ACN, MeOH commonly used
solvents.
Initially start with water, ACN, MeOH as binary or
tertiary mixture. Use buffer phosphate or other (need to
check microbial growth which leads to impact column)
Greater then pH-7 phosphate buffer affect silica based
column
Buffered filtered and degassed
pH close to Pka
HPLC - RP
Buffer selection:
Buffer should have buffering capacity.
Buffer, ion pair reagents, organic modifiers – Enhance
reproducibility, selectivity, peak shape. Regulate the
pH and differ RT of ionized compounds
Buffer miscible and compatible with detector
Hygroscopic buffer – impact of wt +/- 20% need to
check
10 to 50 mM can be used
HPLC - RP
Mode of elution:
Isocratic preferable, 5:95 (Aquous:org),
Avoid ppt,
Back pressure need to check,
Gradient least preferable
HPLC - RP
Detector selection:
UV – Chromophoric group,
Fluorescent – fluorescent nature molecule or fluorescent
derivative,
RI – Non chromophoric,
PDA,
Select wavelength where highest response obtained, some
time select suitable wavelength to prevent interference
Selected wavelength should be precise of lower quantifiable
RF & CF need to check (0.2 to 5)
Multiple dose – optimum response to be selected
HPLC - RP
Flow rate:
Desired run time,
Separate active,
Low back pressure,
Preferable range is 0.8 to 1.5ml
HPLC - RP
Column oven temp:
Symmetric peak,
Separation of active,
Reduce viscosity of mobile phase,
Range 25 to 50 C.
HPLC - RP
Auto sample temp:
25C recommended,
Sample is not stable then lower temp can be used.
Physical observation of sample need to check during
solution stability study.
HPLC - RP
Injection volume:
To be selected wrt response of analyte, sensitivity of
inst.
20micro lit is preferable.
HPLC - RP
Diluent selection
Depends on solubility of active,
Chemical interaction,
Compatibility,
Resolution,
Peak symmetry,
Solution stability,
Extraction
Selected diluents compatibility with mobile phase.
MP as diluent preferable.
HPLC - RP
Extraction procedure:
Improved by using sonication, vortex, intermittent
shaking, stirring
Filter compatibility need to check
pH of diluent need to set inherent of stability of analyte
RLD and inhouse comparison study required for
extraction of sample, preparation time, sonication time,
filter compatibility
HPLC - RP
Selection of test and standard concentration:
Assay 7 digit and check linearity and accuracy
Dissolution direct sampling. Adjust standard
concentration.
If UV, 0.5 to 0.8 AU
RS – Significant response at 0.02% accordingly set test
concentration. Do precision at 0.04% level.
Diluted standard concentration set wrt MDD or
unknown impurity level.
HPLC - RP
Over All Six major points:
Objective for AMD
Initial HPLC condition
Sample Preparation
Calculation
Final optimization
Pre validation
Pre validation
Assay:
Method precision,
Specificity
Intermediate precision,
Solution stability,
Accuracy at different level (50, 100 & 150%),
Filter compatibility
Pre validation
Related substances:
Method precision,
Specificity
Intermediate precision,
Solution stability,
LOD & LOQ
LOQ - Precision
Accuracy at different level (50, 100 & 150%),
Filter compatibility
Pre validation
Dissolution:
Method precision,
Specificity
Intermediate precision,
Solution stability,
Accuracy at different level (50, 100 & 150%),
Filter compatibility
Validation of Analytical Procedures
Types of Analytical Procedures to be Validated
Identification tests;
Quantitative tests for impurities' content;
Limit tests for the control of impurities;
Quantitative tests of the active moiety in samples of drug
substance or drug product or other selected component(s)
in the drug product.
Content uniformity
Blend uniformity
Color test
Chemical test
To be implemented
-> Method development report to be prepared
-> SOP preparation for ADL
-> Electronic signature
-> STP, Spec, format to be revised for RM & FP
-> Validation protocol and report format, acceptance criteria to be
revisited
-> Separate development Lab – IQ, OQ, PQ for transferring inst
-> Data Assurance Unit (DAU)
-> Query response team to be formed
-> ME study team
THANK YOU
