ADVANCES IN

PHARMACEUTICAL ANALYSIS

Date : 24th March 2007

Presented by
Manohar Sonanis
General Manager
(Analytical Research-API)
Wockhardt Research Centre, Aurangabad
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 TECHNOLOGY OVERVIEW

• Instruments have revolutionized Chemical analysis.

• Replaces “Wet Chemistry” methods

• Provides higher sensitivity and accuracy.

• Increased lab productivity due to automation.

• Minimizes operators skill requirements and errors.

• 21 CFR, Part-11 compliance requirements.

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 NEW TECHNOLOGY INSTRUMENTS

Separation Techniques
 High Performance Liquid Chromatography –
HPLC,Thin Layer Chromatography
 Gas Chromatography, Ion Chromatography
Mass Spectrometry
 LC MSMS,GC MS,Maldi,Time of Flight – ToF

Life Science Instrumentation.

 Sequencing,DNA Synthesizer, Organic and Peptide
Synthesis, Electrophoresis

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 NEW TECHNOLOGY INSTRUMENTS

Spectroscopy Techniques.
Atomic Absorption,UV and UV-Vis,ICP,ICP – MS,Fluorescence,Near
Infrared
X-Ray Fluorescence-Ray Diffraction, Inorganic Elemental Analysis,
Surface Sciences
Electron Microscopes, Light Microscopes, Scanning

Material Characterization.
Thermal Analysis,Viscometry ,PSD

Lab Automation.
Information Management,Robotics,Liquid Handling,Microplate
Reading

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 DEMAND BY INDUSTRY

Anaytical Inst. Demand by Industry

Biotech
10% Indep. Test
9%
Gov.
11%
Hosp./Clin.
5%

Metals
4%

Semi/Elec
4%
Other
Acad. 26% Polymers
17% 4%

Oil & Gas

3%

Other
6%

Chemicals
Pharma Ag/Food 5%

17% 5%

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 DEMAND BY FUNCTION IN PHARMA INDUSTRY

Analytical Inst. Demand by Function

Other Method Devel.

6% 7%
QA/QC
20%

R&D
43%
analytical Serv.
24%

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 TECHNIQUES-APPLICATIONS
• HPLC : Separation of compounds(Qualitative&Quantitative)

• GC (Gas chromatography) : Analysis of volatile components

• LC-MS (High performance liquid chromatography hyphenated to

Mass spectroscopy)
: Impurity profiling. Clinical pharmacology.
: Characterization

• PXRD (Powder x- ray diffractometer)

: Polymorphs and Pseudo-polymorphs.
: Patent protections.
: Identification and Characterization

• DSC(Differential scanning calorimeter)

: Drug development (Drug Excipient
Interaction ). Polymorphism.
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• PSD (Particle size analyzer): Particle size determination of
powder and suspension.
: Drug dissolution rate dependency.

• FTIR (Fourier transform infra red spectroscopy)

: Identification/characterization.
Polymorphism.

• Others

 CE (Capillary : Separation of compounds

electrophoresis)
 LCNMR : For impurity profiling
 NIR (Near infra : Characterization, polymorphs etc.,
red spectroscopy)
 Ion chromatography : Inorganic analysis (Cations & Anions)

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HPLC (High Performance Liquid Chromatography)

• During 1970's, most chemical separations techniques were

based on

 Open-column chromatography,
 Paper chromatography, and
 Thin-layer chromatography.

• Above techniques were inadequate in today's requirement

 Sensitivity, selectivity and reproducibility

 Quantification of compounds
 Resolution between similar compounds (Complex).

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 HPLC (High Performance Liquid Chromatography)

• Faster Analysis, Accurate quantification of compounds

 The continual refinements and innovations in HPLC instrument

and column technologies have dramatic impacts on
pharmaceutical analysis.

• The latest trends in HPLC analysis of small-molecule Drug

 Recent advances in instrumentation coupled with more

consistent and efficient columns have increased the speed,
sensitivity, and reliability of analytical methods.

Cont…

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 HPLC (High Performance Liquid Chromatography)

• Principle Of HPLC

 Chromatographic separation process based on the difference

in the surface interactions of the analyte and eluent
molecules.

Partitioning
• Separation is based on the analyte’s relative
solubility between two liquid phases

Mobile Phase Stationary Phase

Solvent Bonded Phase

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 HPLC (High Performance Liquid Chromatography)

HPLC UPLC

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HPLC (High Performance Liquid Chromatography)

• HPLC Modes:

• Normal Phase:
 Polar stationary phase : Silica, Cyanopropyl Silyl, Phenyl,
Amino
 Non-polar solvent : Dichloromethane, n-Hexane etc.

• Reverse Phase:
 Non-polar stationary : Octyl Silyl, Octadecyl Silyl
phase
 Polar solvent : Methanol, Acetonitrile,
Tetrahydrofuran, Water

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HPLC (High Performance Liquid Chromatography)

Columns:

• Solid Support : Backbone for bonded phases.

 Usually 10µ, 5µ or 3µ, 1.7µ silica or polymeric particles.

• Bonded Phase : Functional groups firmly linked (chemically

bound) to the solid support, C-8, C18, C-4 etc.
 Extremely stable
 Reproducible

• Guard : Protects the analytical column

 Particles
 Interferences
 Prolongs the life of the analytical column

• Analytical : Performs the separation.

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HPLC (High Performance Liquid Chromatography)

• Detectors

• UV
 Single wavelength (filter)
 Variable wavelength (monochromatic)
 Multiple wavelengths (PDA)

• Fluorescence

• Electrochemical

• Mass Spectrometry

• Refractive Index

• Evaporative light scattering

• Chiral
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HPLC (High Performance Liquid Chromatography)

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HPLC (High Performance Liquid Chromatography)

0.17%

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HPLC (High Performance Liquid Chromatography)

• Innovation of UPLC (Ultra High Pressure LC):

 Faster analysis
 Peak to peak resolution is improved
 High sensitivity and better S/N ratio
 Savings of time and cost per analysis

Separation of 5 compounds in < 1 min.

Fig. :UPLC stability indicating assay.
Column: 2.1 _ 30 mm 1.7μm ACQUITY
BEH C18
Peaks are in order :
1) 5-nitroso-2,4,6-triaminopyrimidine
2) 4-amino-6-chloro-1,3-
benzenesulfanamide
3) Hydrochlorthiazide
4) Triamterine
5) Methylbenzenesulfanamide

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 HPLC (High Performance Liquid Chromatography)

• Impurity profiling in drug substances: Impurity Thresholds as per

ICH (Q3A(R2))

Daily Reporting Identification Qualification

Dose1 Threshold2,3 Threshold3 Threshold3

≤ 2g/day 0.05% 0.10% or 1.0 mg per 0.15% or 1.0

day intake (whichever mg per day
is lower) intake
(whichever is
lower)

> 2g/day 0.03% 0.05% 0.05%

Cont…

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 GC (Gas Chromatography)
• Packed column : Used for conventional low sensitive GC
analysis
• Capillary Column : New technology columns used for
better sensitivity generally with head
space GC analysis.
• GC Tubing
• Column material
• Stationary phase
• Film thickness
• Inside diameter

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 GC (Gas Chromatography)
Stationary Phases
Polysiloxanes Low Bleed Phases (Arylene)

Polyethylene Glycols

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 HS GC (Head Space GC Chromatogram)

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 GC (Gas Chromatography)
ICH Q3C(R3): Impurities : Guideline for Residual Solvents
• TABLE 1. Class 1 solvents in pharmaceutical products (solvents
that should be avoided).

Solvent Concentration limit Concern

(ppm)
Benzene 2 Carcinogen
Carbon tetrachloride 4 Toxic and
environmental hazard
1,2-Dichloroethane 5 Toxic
1,1-Dichloroethene 8 Toxic
1,1,1- 1500 Environmental hazard
Trichloroethane

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 GC (Gas Chromatography)
ICH Q3C(R3): Impurities : Guideline for Residual Solvents

Class-2: solvents in pharmaceutical products to be limited.

Class-3: Solvents with Low Toxic Potential (limit = 5000ppm)
Class 3 Solvents Class 2 Solvents
Acetic acid Heptane Acetonitrile Methanol
Acetone Isobutyl acetate Chlorobenzene 2-Methoxyethanol
Anisole Isopropyl acetate Chloroform Methylbutyl ketone
1-Butanol Methyl acetate Cyclohexane Methylcyclohexane
2-Butanol 3-Methyl-1-butanol 1,2-Dichloroethene N-Methylpyrrolidone
Butyl acetate Methylethyl ketone Dichloromethane Nitromethane
tert-Butylmethyl ether Methylisobutyl ketone 1,2-Dimethoxyethane Pyridine

Cumene 2-Methyl-1-propanol N,N-Dimethylacetamide Sulfolane

Dimethyl sulfoxide Pentane N,N-Dimethylformamide Tetralin

Ethanol 1-Pentanol 1,4-Dioxane Toluene

Ethyl acetate 1-Propanol 2-Ethoxyethanol 1,1,2-Trichloroethene
Ethyl ether 2-Propanol Ethyleneglycol Xylene*
Ethyl formate Propyl acetate Formamide

Formic acid Tetrahydrofuran Hexane

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LCMS (High Performance Liquid Chromatography
Mass Spectroscopy)
• Mass spectrometry instruments:
 Single Quad LCMS
 Tandem LC MSMS
 GC MS
 Maldi
 Time of Flight – ToF

• Introduction To LCMS
 Mass Spectrometry is superior scientific tool that provides
greater specificity, sensitivity,and accuracy than
conventional Ultraviolet or Fluorescent detectors.

• Mass Spectrometry is an analytical technique used for

 Identification of unknown compounds
 Quantification of known compounds
 Structural Elucidation
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LCMS (High Performance Liquid Chromatography
Mass Spectroscopy)

• MASS Spectrometry:

 Measures the Masses of electrically charged molecules,or

ions. Once ions are in the mass spectrometer they are sorted
according to mass to charged ratio(m/z).

 A detector converts the signal to electrical signal , as a

function m/z is converted by the data system into mass
spectrum.

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LCMS (High Performance Liquid Chromatography
Mass Spectroscopy)
• API 2000 Ion Path

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LCMS (High Performance Liquid Chromatography
Mass Spectroscopy)
• Buffer and pH Control for LCMS

 Non-volatile buffers like phosphate are not recommended

 Use volatile buffers to replace phosphate such as
Ammonium acetate, Formic acid, Acetic acid etc.
 Use a lower buffer concentrations:10-50mM

• LCMS Applications

 Identification of unknown impurities in Drug and its drug

products.
 Identification of unknown impurities formed during
degradation studies.
 To check the specificity of the HPLC Methods.
 Quantification of the substances which are very low in
concentration and where it is difficult to use other
techniques.
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LCMS (High Performance Liquid Chromatography
Mass Spectroscopy)

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LCMS (High Performance Liquid Chromatography
Mass Spectroscopy)

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LCMS (High Performance Liquid Chromatography
Mass Spectroscopy)

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LCMS (High Performance Liquid Chromatography
Mass Spectroscopy)

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 ELSD (Light Scattering Evaporative Detector)

• Universal detector for HPLC

• Not dependant on the presence of chromophoric groups on the
compound
• Analysis of semi volatile to nonvolatile compounds
• Typical mobile phase recommended as volatile buffers.
• Suitable for gradient applications.

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 XRD (X- Ray Powder Difractometer)
In 1998, Abbott had to stop production of its HIV protease inhibitor,
ritonavir (Norvir), because its manufacturing process was producing
the wrong crystalline form of the compound. The new polymorphic
structure of Norvir, one which had not been used in early testing of
the drug, affected how the semisolid capsule dissolved. Patients
were offered the drug in liquid form until the company could
reformulate the product to eliminate the wrong structure.

Glaxo Wellcome was in a lengthy court case against Novopharm for

alleged patent infringement based on its right to manufacture and
market a different polymorphic form of Glaxo’s best-selling anti-ulcer
drug, Zantac. Glaxo’s patent on one polymorph of the drug expired in
1997, and Novopharm wanted to bring this form to the generic
market. Glaxo argued that the generic form would infringe on its
patent for the second crystalline form of the drug, the patent of
which will not expire until 2002. The forms are therapeutically
equivalent. Glaxo lost its case, and now Novopharm and other
companies market generic Zantac for over-the-counter sales.

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 XRD (X- Ray Powder Difractometer)

• General principles of pharmaceutical solid polymorphism

 Importance of Pharmaceutical Solid Polymorphism

 Characterization of Polymorphs
 Influence of Polymorphism On Drug Substance And Drug
Product
 Investigating the Polymorphs
 Setting Specifications for Polymorphs in Drug Substances
 Investigating the Importance of Setting Specifications for
Polymorphs in Drug Products

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 XRD (Powder X- Ray Diffractometer)

X-ray pattern of Amorphous

compound

X-ray pattern of crystalline

Dihydrate compound

X-ray powder diffraction pattern of the anhydrous and dihydrate compound.

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 XRD (Powder X- Ray Diffractometer)
Quantification of Polymorph into another morph
Fig-A Fig-B

A. Powder diffraction patterns of the mixtures of Anhydrous and

crystalline dihydrate molecules (in the amounts 0% (sample),
0.1%, 0.2%, 0.3%, 0.5% of the dihydrate form)
B. Powder diffraction patterns of the mixtures of Anhydrous and
crystalline dihydrate (in the amounts 0% (sample) and 4.0% with
placebo in Tablet).
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 XRD (Powder X- Ray Diffractometer)
Quantification of Polymorph into another morph

y = 378.84x - 24.072
Linearity Plot for 9.8 2  2
R = 0.9936
1600.0
1400.0
Average intensity

1200.0
1000.0
800.0
600.0
400.0
200.0
0.0
0 1 2 3 4 5
% Concentration

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 DSC (Differential Scanning Calorimeter)
• Applications of Thermal Analysis in Pharmaceuticals

• To measure physical and energetic properties with precision and

accuracy

• Thermal analysis is used in the following areas

 Determination of purity
 Melting point,
 Polymorphism,
 Glass transition temperature,
 Drug- excipient interactions,
 Thermal stability

• To compare the results for the individual components and the

mixture

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 DSC (Differential Scanning Calorimeter)

DSC of Monohydrate DSC of Anhydrous

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 DSC (Differential Scanning Calorimeter)
DSC of Monohydrate + Anhydrous 1:1

Monohydrate
Characteristic onset

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 DSC (Differential Scanning Calorimeter)
Quantification of Monohydrate in Anhydrous Drug Substance

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 CONCLUSION
• Technological advances are being happened in pharmaceutical
analysis as like other areas of science.

• Due to technological advances more and more pure drug is

available and will be available for patients and drug testing
becomes more regulated.

• With the new technology instruments and automation

productivity is dramatically increased and hence it benefits the
business of the company.

• Indian pharma companies are more competent to the global

market.

• With the support of quality analytical data companies are able to

protect there innovations by patenting them.

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