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Company

LOGO Recent advancement in impurity profiling

Presented bySudipkumar modh

A seminar on

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Agenda1. Definition of ICH 1. Definition of ICH

5.Isolation And Identification5.Isolation And Identification

7.Methodology7.Methodology

a)Classical approaches a)Classical approaches

2. Importance 2. Importance

3. History3. History

4. Systemic approach4. Systemic approach

6.Case study 6.Case study

b)Modern approaches b)Modern approaches

8.Conclusion8.Conclusion

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Definition of ICH (Q6A specifications) Impurity:• “Any component of the new drug substance that is not the

chemical entity defined as the new drug substance“• “Any component of the drug product which is not the

chemical entity defined as the drug substance or an Excipient in the drug product.”

Impurity profiling:• Analytical activities, aim of …• Detection, identification/structure elucidation • Quantitative determination

• Of organic and inorganic impurities and residual solvents in bulk drugs & pharmaceutical formulations

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Importance

• all phases of synthetic drug research and production from the gram scale preparation of new compounds

• For pharmacological screening up to the scaling up procedure and finally the production of bulk drugs

• Differentiate between synthesis-related

impurities and degradation products

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HISTORY of instrumental analysis

• Period following world war II,rapid development of pharmaceutical industry

• Instrumental analysis was developed with military technology

• Qualitative analysis was met by molecular spectroscopy but with complexity

• Spectroscopic method were generally complex, and there was a need of expert spectroscopist

• Separations techniques came, at the end of 19th century revolution came by development of TLC

• After world war II,GC developed and HPLC after two decades

• The fully Instrumental technique science arrived

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Systemic approach

• According to ICH Q3A(R2)

Maximum Reporting Identification Qualificationdaily dose threshold threshold threshold(g/day) (%)

≤2 0.05 0.10% or 1.0 mg/day 0.15% or 1.0 mg/day (whichever is lower) (whichever is lower) >2 0.03 0.05% 0.05%

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Systemic approach

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Systemic approach

What to be done after separation ??• Purified and sample should be split into aliquots for MS,NMR, and

vibrational spectroscopy• Repetition of MS of sample is required 1st confirm that molecular weight of sample corresponds to initial

LC/MS data 2nd determine fragmentation pathway from MS/MS data • Empirical formula of HRMS is useful, if impurity is unknown and

unrelated with drug • In this case, by FT-IR,FT-Raman or both, functional groups are

identified• Structure is eluted by MS and NMR (Homo and hetero nuclear

direct and long range chemical shift correlation experiments)

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Isolation And Identification

• Preparative chromatography

• In reversed phase chromatography good isolation can be achieved, by

using stationary non polar phase and polar mobile phase with polarity

modifiers, pairing ions, buffers

• Due to diverse nature of impurity, not only RP-HPLC , but also

Normal phase chromatography

Supercritical fluid chromatography

Gas chromatography

Capillary electrophoresis

• May be a good choice

Isolation

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Isolation And Identification

• After isolation sample may contain artifacts from the isolation process, this constituents are referred as “chemical noise”

• This may affect particularly to high sensitivity NMR probes(1.7 mm)

Isolation

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Isolation And IdentificationIdentification

MS• Molecular weight changes show gain or loss of some neutral species exp is

CO• Formation of adduct ions can be useful for preliminary identification of

molecular ion• Exp adduct ions like sodium,pottasium,acetonitrile at +23,+39,+43• Isotope patterns also useful like of Cl, Br • HRMS is useful for unrelated impurity• MS/MS provides site of chemical modification and type of modification• Not useful for detection of positional isomers NMR• Besides of convectional NMR , 1 D NMR DEPT INEPT

2d NMR like COSYTOCSYHSQCHMBCAre used now a days

HSQC (Heteronuclear Single Quantum Coherence) HMBC (Heteronuclear Multiple Bond Coherence)

Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)

Distortion less Enhancement by Polarization Transfer(DEPT)

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Isolation And Identification

Vibrational spectroscopy• With advancement in 2d NMR,vibrational spectroscopy

like FT-IR and FT-RAMAN is somewhat not acquired • But this types of spectroscopy is essential in functional

group detection,parallely with MS and NMR• Carbonyl function groups are detected efficiently in

vibrational spectroscopy• But it is transparent in COSY and HSQC• In HMBC long correlations are observed with carbonyl

moiety Data integrations

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Case study

• Colored contaminant was being formed in final step of the synthetic process

• Formed due to coupling of aniline-like derivative with pyridine being used

as acid scavenger

• Isolated by methanol stripping of silica gel chromatography

• methanol solution containing the colored impurity was bright red

• half life of about 18 h in methanol and <10 min in acetone

• By 1.7-mm 600-MHz SMIDG probe, sample was prepared and data set

consisting of a proton reference spectrum, TOCSY, HSQC, and 10-Hz

optimized HMBC spectra was acquired

Isolation And Identification

Characterization of an unstable process impurity in the protease inhibitor Tipranavir

SMIDG=sub micro-inverse-detection gradient

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Characterization of an unstable process impurity in the protease inhibitor Tipranavir

Case studyIsolation And Identification

Formed colored impurity

drugStarting material

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• in addition to the resonances normally observed for the drug, showed three new well-resolved signals,

8.25 (d) 7.82(t) 6.27(t)• The doublet (8.25) and one of the triplets (6.27 ppm) , twice the

intensity of triplet (7.82 ppm), which had an integrated intensity corresponding to half of that of a one proton signal in the drug molecule.

• Conclusion: impurity contained two molecules of drug and that the new resonances were present in a ratio of 2 : 2 : 1

• The TOCSY spectrum confirmed the coupling of the new resonances in the aromatic region

Isolation And Identification

Case studyCharacterization of an unstable process impurity in the protease inhibitor Tipranavir

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• A HRMS spectrum gave exact mass of 848.47287 Da (less the ionizing proton) corresponding to the molecular weight of two of the aniline precursor molecules and a C5H5 fragment

• consistent with the integration of the new aromatic resonances in proton spectrum.

• GHSQC data established the following direct proton–• carbon correlation: 8.25/152.7, 7.82/125.8, and 6.27/106.2 ppm

• HMBC correlations linked at 8.25/152.7 ppm resonant pair to aromatic quaternary carbon bearing aniline group resonating at 142.6 ppm.

• other correlations were internal to five-carbon fragment

Isolation And Identification

Case studyCharacterization of an unstable process impurity in the protease inhibitor Tipranavir

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HSQC spectrum

Isolation And Identification

Case studyCharacterization of an unstable process impurity in the protease inhibitor Tipranavir

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HMBC spectrum

Isolation And Identification

Case studyCharacterization of an unstable process impurity in the protease inhibitor Tipranavir

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Data from the NMR data were supported by vibrational data

• Included increase in intensity of the band at

1636 cm–1 suggesting increased double bond

1547 cm–1 consistent with C=N stretching that is not present in Tipranavir

1170 cm–1 region of C–O stretching and C–OH bending that can be accounted for by the dimeric nature of the impurity of Tipranavir

• The five carbon fragment linking the two aniline moieties arose from the pyridine ring coming apart during the reaction

Isolation And Identification

Case studyCharacterization of an unstable process impurity in the protease inhibitor Tipranavir

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Methodology

Classical approachseparation and determination of impurities with known structure

Isolation of the impurity and off-line structure elucidation

Modern approachUsing on-line hyphenated separation/spectroscopic methods

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Classical approach

separation and determination of impurities with known structure

• HPLC• TLC• Electrophoretic and Related Methods Capillary Electrophoresis Electrophoresis-related Chromatographic

Techniques• Methods not Requiring Separation Polarography & UV

Methodology….

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HPLC

• HPLC is often used with UV• Chemometric methods used for separation optimisation but also for

improving quantitative analysis• Dimension 150-250 x 4.0-4.6 mm• RP-stationary phase with particle size of 3.5-5.0 μm • Rt in range of 10-20 min• Ultra-performance liquid chromatography (UPLC) uses short and

narrow-bore columns with small particle size packing• Exp Impurities of primaquine phosphate • dimensions of the column were 50 x 2.1mm • C18 silica packing and 1.7 μm• separation was achieved within 2 minutes

Classical approach….

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• A new development in RP-HPLC is the introduction of oil-in-water emulsion into the mobile phase

• This method, named micro emulsion liquid chromatography (MELC)

• special application in field of impurity profiling by HPLC is high-speed counter-current chromatography (HSCCC)

• (semi)preparative Analysis by HPLC-DAD to determine relative purities of each fraction in course of the preparative isolation of drugs from e.g. medicinal plant matrices

HPLCClassical approach….

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TLC

• It has good ease of use, cost-effectiveness, good sensitivity, speed of separation

• capacity to analyze multiple samples simultaneously• complementary technique to HPLC.

• HPTLC method is suitable for quantitation of impurities of alprazolam

• Over pressured liquid chromatography (OPLC) a forced flow

variant of HPTLC for determination of impurities of norethisterone by fluorimetric scanning after sulphuric acid spray

Classical approach….

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Electrophoresis and Related Methods

Capillary Electrophoresis• CE is primarily suitable separation and quantification of charged

impurities• example for use detection of ammonium ion• Useful for ammonium salts as impurities not contributed by residue

of ignition and not detected by TLC or HPLC methods Electrophoresis-related Chromatographic Techniques• micro emulsion electro kinetic chromatography (MEEKC) and

capillary electro chromatography (CEC).• Exp determination of impurities in ketorolac by MEEKC• Determination of the extremely toxic N-methyl-4-phenyl- 1,2,3,6

tetrahydropyridine impurity in pethidine• Performances of HPLC, CE and MEKC compared• Shortest run time (less than 5 min) was achieved by CE• Best sensitivity by MEKC

Classical approach….

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Methods not Requiring Separation

• Electro analytical methods never played important role in drug analysis due to limited selectivity and sensitivity

• An example is the polarographic and UV methods• Exp Determination of benzophenone impurity in phenytoin

impurities• and low concentration of phenytoin do not allow direct use of

UV spectrophotometry in drug impurity profiling • UV Derivative spectrophotometry is advantageous • Used in drug degradation studies where the relative

concentration of the degradants is in per cent range• Exp Determination of omeprazole sulphone in omeprazole • monitoring of the photo degradation of amlodipine to it

pyridine degradation product

Classical approach….

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Methodology

• Isolation of the impurity and off-line structure elucidation

HPLC-UV StudiesHPLC-MS StudiesGC-MS StudiesTLC-MS StudiesCE-MS StudiesMEKC-MS and CEC-MS StudiesHPLC-NMR Studies

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Methodology

• Isolation of the impurity and off-line structure elucidation

HPLC-UV StudiesHPLC-MS StudiesGC-MS StudiesTLC-MS StudiesCE-MS StudiesMEKC-MS and CEC-MS StudiesHPLC-NMR Studies

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Modern approach

LC-MS• Very often the molecular mass of the impurities obtainable by using

the standard soft ionisation techniques• tandem mass spectrometer is available (HPLCMS/ MS) so,

fragmentation of impurity can be used for the structure elucidation• Group exchanged on-line hydrogen to deuterium by using D2O as

HPLC mobile phase component• powerful tool for identifying active hydrogen atoms (hydroxyl, thiol,

amine, acid groups)• Useful for distinguishing between isomeric structures, difficult by

product ion spectral data or accurate mass data• Separation power and usefulness of HPLC-MS as a tool for

rapid identification of several impurities with different polarities can be improved by using the “heart-cut” technique

Methodology….

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LC-MS

HPLC-APCI-MS• (HPLC – atmospheric pressure chemical ionisation – MS)• for the identification of impurities co-eluting with the main

component (acids, bases and zwitter-ions) not detectable by UV-DAD detector

Modern approach

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COMET

A Hewlett-Packard 1100 liquid chromatography system(Agilent Technologies, Palo Alto, CA) equipped with vacuum degasser quaternary pumpauto-samplerUV diode array detector HP-MSD modelD mass spectrometerthermo stated column selector six-position Cheminert valve is inserted on “D” line of

quaternary pump to extend mobile phase selection Both column switcher and valve are connected through

contact closures controlled electronically through the ChemStation software

Modern approach

fully automated Comprehensive Orthogonal Method Evaluation Technology (COMET)

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• The mobile phase components are placed in the reservoirs• as follows: line A contains tetrahydrofuran• line B contains acetonitrile• line C contains methanol • line D contains the aqueous solutions connected to the six-

position Cheminert valve.• The mobile phase valve positions 1–6 contain the aqueous solutions water,

0.1% formic acid (pH 2.1), 0.1% glacial acetic acid (pH 3.5) , 10mM ∼ ∼ammonium acetate (pH 7.0), 0.1% trifluoroacetic acid

• Extensive evaluation of 32 HPLC columns and 18 mobile• phase combinations were performed to provide a series of MS compatible

methods, each with unique selectivity• Peak tracking with DAD• Manual peak tracking with mass spectra

COMET instrumentation

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Modern approach

fully automated Comprehensive Orthogonal Method Evaluation Technology (COMET)

COMET instrumentation

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COMET instrumentationsoftware

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COMET instrumentationModern approach

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GC-MS • Advantages

high-resolution separations highly selective sensitive detection wide ionisation capabilities

• Anise oil as starting material for the synthesis of 4-methoxy amphetamine was screened by GC-MS for impurities

GC-MSModern approach

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• TLC-MS• Separated spots on the plate could be subjected

to direct matrix-assisted-laser-desorption

ionization

• Time-of-flight mass spectrometry (TLC-MALDI TOF

-MS)

no need to remove the spots from plate

• Only wetting spot with methanol required to transfer

the analyte from inside the silica gel to the surface• thus enhancing the MALDI-TOF-MS signal

TLC-MSModern approach

TLC PLATE

MEOH

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Modern approach

MS Approaches for detection

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• majority of applications are from the fields of metabolite and natural product analyses

• stopped flow HPLC-NMR technique (together with HPLC-MS) in identifying impurities in acarbose

LC-NMRModern approach

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conclusion

• Impurity profiling is very important in the field of pharmaceutical analysis

• Unidentified and potentially toxic impurities are health hazards and in order to increase safety, impurity should be identified

• That’s all!!!

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referencesAnalysis of drug impurities Edited by Richard J. smith and michael L .Webb Blackwell

publishing

ICH guidelines

Handbook of modern pharmaceutical analysis by satinder ahuja and stephan scypinski

Volume III of SEPARATION SCIENCE AND TECHNOLOGY

Recent Advances in the Impurity Profiling of Drugs Dorottya Bartos and Sándor

Görög* Current Pharmaceutical Analysis, 2008, 4, 215-230

Characterization of a trace by‐product of the synthesis of the protease inhibitor

tipranavir GE Martin, RH Robins, FW Crow… - Journal of …, 1999 - Wiley Online

Library

http://www.biolab.dk/sidevisning.asp?ID=674

www.wikipedia.org

http://www.welsch.com/e/index.php5

http://www.thefullwiki.org/Gas_chromatography-mass_spectrometry

Automated peak tracking for comprehensive impurity profiling in orthogonal liquid

chromatographic separation using mass spectrometric detection Gang Xue∗, Anne D.

Bendick Journal of Chromatography A, 1050 (2004) 159–171

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HYDERABAD

Thank you