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