Recent advancement in impurity profiling

A seminar on Recent advancement in impurity profiling Presented by Sudipkumar modh 1

Agenda 2 1. Definition of ICH 2. Importance 3. History 4. Systemic approach 5.Isolation And Identification 6.Case study 7.Methodology a)Classical approaches b)Modern approaches 8.Conclusion

Definition of ICH (Q6A specifications) ,[object Object],“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: ,[object Object]

Detection, identification/structureelucidation

Of organic and inorganic impurities and residual solvents in bulk drugs & pharmaceutical formulations3

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 4

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 5

Systemic approach According to ICH Q3A(R2) 6 Maximum Reporting Identification Qualification daily dose threshold threshold threshold (g/day) (%)

Systemic approach ,[object Object],Purified and sample should be split into aliquots for MS,NMR, and vibrational spectroscopy Repetition of MS of sample is required ,[object Object]

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) 8

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 ,[object Object]

Supercritical fluid chromatography

Capillary electrophoresisMay be a good choice ,[object Object],9

Isolation And Identification ,[object Object],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) 10

Isolation And Identification ,[object Object]

MSMolecular 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 ,[object Object],Besides of convectional NMR , ,[object Object]

HMBCAre used now a days 11 HSQC (Heteronuclear Single Quantum Coherence) HMBC (Heteronuclear Multiple Bond Coherence) Distortion less Enhancement by Polarization Transfer(DEPT) Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)

Isolation And Identification ,[object Object],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 ,[object Object],12

Case study Isolation And Identification Characterization of an unstable process impurity in the protease inhibitor Tipranavir 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 13 SMIDG=sub micro-inverse-detection gradient

Characterization of an unstable process impurity in the protease inhibitor Tipranavir Case study Isolation And Identification drug Formed colored impurity 14 Starting material

in addition to the resonances normally observed for the drug, showed three new well-resolved signals, ,[object Object]

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 study Characterization of an unstable process impurity in the protease inhibitor Tipranavir 15

A HRMS spectrum gave exact mass of 848.47287Da (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 study Characterization of an unstable process impurity in the protease inhibitor Tipranavir 16

Isolation And Identification HSQC spectrum Case study Characterization of an unstable process impurity in the protease inhibitor Tipranavir 17

Isolation And Identification HMBC spectrum Case study Characterization of an unstable process impurity in the protease inhibitor Tipranavir 18

Isolation And Identification ,[object Object],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 Case study Characterization of an unstable process impurity in the protease inhibitor Tipranavir 19

separation and determination of impurities with known structure

Isolation of the impurity and off-line structure elucidation

Using on-line hyphenated separation/spectroscopic methods20

Classical approach Methodology…. separation and determination of impurities with known structure HPLC TLC ,[object Object]

Electrophoresis-related Chromatographic Techniques Methods not Requiring Separation ,[object Object],21

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…. 22

Recent advancement in impurity profiling

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