Enantiomeric impurities & Separation approaches.pptx

Dept. of Pharmaceutical Analysis, NIPER-HAJIPUR, Bihar, India
NIPER-HAJIPUR
Enantiomeric impurities &
Separation approaches
Seminar presented as a part of curricular requirement for
M. Pharm-I Year – I semester
By
Efficiency Myrsing
(Reg. No.6 )
2/03/22

NIPER-HAJIPUR
Objective of the presentation
• Discuss the concept of potential impurities and their
sources.
• Categorize the impurities based on the ICH regulatory
documents.
• Discuss the concept of enantiomer and enantiomeric
impurity.
• Discuss the separation approaches of enantiomeric
impurities
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NIPER-HAJIPUR
Contents of this presentation
1. Introduction to impurity
2. Impurities Testing Guidelines
3. Classification of Impurities
4. Concept of Optical Purity & Enantiomeric Excess
5. What are enantiomeric impurities?
6. Separation approach of enantiomeric impurities
7. Conclusion
8. References
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Introduction to impurity
2/03/22
Any other organic or inorganic material
, besides the drug substance, or
ingredients, arise out of synthesis or
unwanted chemicals that remains with
API’s.
Emphasizing on:
 purity requirements
 Identification of impurities
Different pharmacopoeias: slowly incorporating
limits to allowable levels of impurities present in APIs
or formulations.

NIPER-HAJIPUR
Impurities testing guidelines
Q3A(R2)
• Impurities in New Drug Substances
Q3B(R2)
• Impurities in New Drug Products
Q3C
(R8)
• Guidelines for residual solvent
Q3C(R9)
• Guideline for Elemental Impurities
Q3E
EWG
• Impurity: Assessment and control of Extractable and Leachable for
Pharmaceuticals and Biologics.
• The ICH Q3 series are designed for impurities testing.
• The ICH Q3 topic on impurities testing is covered by eight separate
guidelines.
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Classification of impurity
 Impurities associated in with APIs like Organic,
Inorganic impurities and Residual solvent .
 Impurities forms during formulation Method related,
Environmental related, Dosage form related.
 Formation of impurities on aging mutual interaction
amongst ingredients functional group related typical
degradation .
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NIPER-HAJIPUR
Classification of impurities associated with APIs
Organic impurities
(process and drug
related)
• starting material
• by products
• Intermediates
• degradation
products
• reagents, ligands,
catalysts.
Inorganic impurities
• Reagents, ligands,
catalysts
• heavy metals
• inorganic salts
• other material (
filter aid, charcoal,
etc.)
Residual Solvent
• Class 1 solvents:
solvent to be avoided
solvents to be limited.
solvents with low
toxic
• Other solvents (“class
4”): no adequate
toxicological data
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NIPER-HAJIPUR
 According to the ICH guidelines on impurities in new drug products,
identification of impurities below 0.1% level is not considered to be
necessary, unless potential impurities are expected to be unusually potent
or toxic.
 According to the ICH, the maximum daily dose qualification threshold to
be considered is as follows; <2 g / day, 0.1 % or 1 mg per day intake
(whichever is lower) >2 g / day, 0.05%.
 Reporting threshold: A limit above which an impurity should be
analytically reported.
 Identification threshold: A limit above which an impurity should be
structurally identified.
 Qualification threshold: A limit above which an impurity in a drug product
must be quantified for safety.
ICH Limits for Impurities
2/03/22

Thresholds MDD* of API in drug
product
Threshold limit based on
TDI**
Reporting
≤ 1g/day 0.1%
> 1g/day 0.05%
Identification
< 1mg/day 1% or 5 µg TDI (whichever
is lower)
1 – 10 mg/day 0.5% or 20 µg TDI
(whichever is lower)
10mg- 2g/day 2 g/day - 0.2% or 2 mg TDI
> 2 g/day 0.1%
Qualification
< 10mg/day 1% or 50 µg TDI (whichever
is lower)
10 – 100 mg/day 0.5% or 200 µg TDI
>100mg – 2 g/day 0.2% or 3mg TDI
> 2 g/day 0.15%
*MDD: Maximum Daily Dose **TDI: Total Daily Intake
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NIPER-HAJIPUR
Class 1 Solvents: Solvents to Be Avoided:
Because of their unacceptable toxicity, Carcinogenicity and
environmental hazard.
Solvent Concentration limit (ppm)
Benzene 2
Carbon tetrachloride 4
1,2-Dichloroethane 5
1,1-Dichloroethene 8
1,1,1-Trichloroethane 1500
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NIPER-HAJIPUR
Class -2 Solvents: should be limited in pharmaceutical
products because of their toxicity.
Solvent Concentration limit (ppm)
Acetonitrile 410
Chlorobenzene 360
Chloroform 60
Cyclohexane 3880
Hexane 290
Methanol 3000
Nitromethane 50
Pyridine 200
Toluene 890
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NIPER-HAJIPUR
Class-3 Solvents: less toxic and of lower risk to human
health. Amounts should not exceed 50 mg/day or 5,000
ppm or 0.5% .
Acetic acid Heptane
Acetone Isobutyl acetate
Anisole Isopropyl acetate
1-Butanol Methyl acetate
2-Butanol 3-Methyl-1-butanol
Butyl acetate Methylethyl ketone
tert-Butylmethyl ether Methylisobutyl ketone
Cumene 2-Methyl-1-propanol
Dimethyl sulfoxide Pentane
Ethanol 1-Pentanol
Ethyl acetate 1-Propanol
Ethyl ether 2-Propanol
Ethyl formate Propyl acetate
Formic acid Tetrahydrofuran
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NIPER-HAJIPUR
Class-4 Solvents: Solvents for which No Adequate
Toxicological Data was Found
2/03/22
1,1-Diethoxypropane
Methylisopropyl ketone
1,1-Dimethoxymethane
Methyltetrahydrofuran
2,2-Dimethoxypropane
Petroleum ether
Isooctane
Trichloroacetic acid
Isopropyl ether
Trifluoroacetic acid
Examples:

NIPER-HAJIPUR
2/03/22
Enantiomers
Enantiomers are the
chiral molecules that
are mirror images of
one others i.e. it has a
non-superimposable
mirror image structure.
Enantiomers have identical
chemical and physical properties in
an achiral environment.

2/03/22
NIPER-HAJIPUR
α
Dextrorotatory
rotate the plane polarized light to the right
(+)- d-isomer
levorotatory
rotate the plane polarized light to the left
(-)- L-isomer
Optical Activity

NIPER-HAJIPUR
2/03/22
Optical purity
A sample is said to be
enantiomerically pure when
there is only one of its
enantiomer and the value
of specific rotation (α) is at
the highest.
Any contamination by the
other enantiomer lowers
the value of of specific
rotation .
Optical Purity = Enantiomeric Excess ( e.e.)

NIPER-HAJIPUR
2/03/22
Optical purity
For example :
tartaric acid with an observed rotation of +6°. And We know that
specific rotation of pure tartaric acid is +12.0°.
Optical Purity =
+6
+12
= 0.5 or 50%
O P =
[α] mixture
[α] pure
It means that 50% of the mixture is excess of (+)- isomer and 100-50= 50% is
racemic mixture .
Therefore , Total amount of (+)-Enantiomer in the mixture will be
50+50/2=50+25=75 % and (-)-Enantiomer is therefore 25%.

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WHAT ARE ENANTIOMERIC IMPURITIES?
According to the ICH Q6 “Specifications: Test procedures and acceptance
criteria for new drug substances and new drug products: Chemical
substances” for chiral drug substances which are developed as a single
enantiomer, control of the other enantiomer should be considered in the same
manner as for other impurities
As per ICH guidelines, only active enantiomer of the drug has to be marketed,
so there is attention on separation of the inactive enantiomer which acts as a
chiral impurity.
The enantiomers of chiral drugs can differ in their interactions with enzymes,
proteins, receptors.
And produces different pharmacological or pharmacokinetic property ,
metabolites which may be toxic and affect the bioavailability and efficacy of a
drugs.
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WHY IT IS IMPORTANT TO SEPARATE ENANTIOMERIC IMPURITIES?
Ibuprofen
 S- enantiomer is an anti-inflammatory
 R-enantiomer causes un-wanted side
effect.
Amphetamine
 R-enantiomer is use in asthma and
congestion
 S- enantiomer less active.
Thalidomide
 R- enantiomer used against nausea
 S-enantiomer caused teratogenic
Albuterol
 (R)-isomer or levalbuterol use
bronchial asthma
 (s)-isomer is inactive
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What are the different technique use for
separation for enantiomeric impurities?
Enantioseparation
technique
Chromatographic
method
capillary
electrophoresis
crystallization
resolution
liquid-liquid
extraction
kinetic
resolution
Membrane-based
separation
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Column: Daicel Chiral Pak AS-3R
Dimension: 150 mm × 4.6 mm i.d.,3 µm
MP:methanol/water/diethylamine
(85/15/0.1% v/v/v)
Flow rate: 1.5 ml/ min
Detector: 222 nm
 Ondansetron is 5-HT3 receptor antagonist and effective in the prevention and treatment of
nausea and vomiting
R-enantiomer, is a highly selective and more potent 5-HT3 antagonist which shows approximately
eight times higher activity than S- enantiomer.

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S- Amlodipine R- Amlodipine
 Amlodipine is a calcium channel blockers . Its S-enantiomer was reported to be 1000 times
more pharmacologically active than its R-enantiomer.
Atenolol is a β-blockers, used mainly in treatment of cardiovascular diseases where its S-
enantiomer alone is responsible for the β-adrenoceptor blocking active.
S- Atenolol
column : Lux
Cellulose-4
Dimension : 250 ×
4.6 mm, 5 µm)
MP: acetonitrile,
ethanol and DEA
(92:8:0.2% v/v/v )
flow rate : 1.2 mL
/min
Detection: λ -240
nm
(S) (R)
R- Atenolol

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chromatographic
methods
Stationary phase Mobile phase Enantiomers(analytes)
high-performance
liquid-chromatographic
(HPLC)
coated/ immobilized
cellulose and amylose chiral
SP under normal-phase
hexane–2- propanol (60 : 40
v/v)
Cefadroxil has 3 chiral centers
and the existence of eight different
stereoisomers is possible.
LC-MS/MS
Shimadzu Prominence
HPLC system and Supelco
Astec Chirobiotic V2 25 cm
x 2.1 mm, 5 µm column
held at 20 °C
methanol, 0.1 % (v/v) glacial
acetic acid and 0.02 % (v/v)
ammonium hydroxide
“d” and “l” Enantiomers of
Amphetamine and
Methamphetamine
capillary
electrochromatography
(CEC)
Hydroxypropyl-b-
cyclodextrin bonded to an
organic monolith Capillary,
100 µm I. D. × 25 cm
10 mM ammonium acetate pH
7.0-ACN (30:70, v/v)
Hexobarbital, warfarin and
phenylalanine
supercritical fluid
chromatography (SFC)
Kromasil CHI-TBB phase
Isopropanol, ethanol, benzene,
toluene, diisopropylether, and
trichlorethane of analytical
grade, and ethyl acetate and n-
hexane of Supra Solv grade
Ibuprofen D or R(2)-enantiomer
causes some un-wanted side effect.
high speed counter-
current
chromatography
hydroxypropyl-β-
cyclodextrin (HP-β-CD)
n-hexane–ethyl acetate–0.1 mol
L−1, phosphate buffer solution
with pH = 2.67 (8:2:10, v/v/v)
(R, S)-naproxen , (S)-naproxen
showed 28 times higher activity
than the (R)-isomer

capillary electrophoresis
NIPER-HAJIPUR
2/03/22
• Analyte- enantiomers of
asenapine maleat
• Cyclodextrin (CD)
derivatives are often the chiral
selector. 7 mM β- CD
• 160 mM TRIS-acetate
buffer pH = 3.5
• Temperature--20 °C
• Applied voltage- 15 kV
• detectors- NMR , ESI-MS

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crystallization resolution
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• Crystallization has been the predominant separation technique to resolve an
enantiomeric mixture into its individual isomers on the industrial scale.
• There are three primary methods of crystallization for enantiomeric
resolution.
1. Preferential crystallization: saturated solution of racemic mixture
is added by pure crystal of one enantiomer or another chiral
compound. This make to crystallize one enantiomer first while
other crystallize late and can be separated
2. Diastereoisomeric crystallization – the resolving agent binds to
enantiomers to a diastereoisomeric salt pair. These salts are
separated as a function of their phase behavior.
3. Catalytic kinetic resolution – the resolving agent reacts at a
different rate with each enantiomer.
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crystallization resolution
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Albuterol is a β-2-adrenoceptor agonist prescribed for the treatment of bronchial
asthma. And (R)-isomer bronchodilator activity resides.
2/03/22

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Separating enantiomers by formation of Diastereomers
 Since enantiomers have identical physical
properties, such as solubility and melting point,
therefore it become extremely difficult to separate
them.
 Diastereomers, on the other hand, have different
physical properties, and this fact is used to achieve
resolution of racemates.
Reaction of a racemate with an enantiomerically
pure chiral reagent gives a mixture of
diastereomers, which can be separated. Reversing
the first reaction then leads to the separated
enantiomers plus the recovered reagent.
2/03/22

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 Enantiomers of Phenylalanine are
separated by using immobilized DNA
membranes.
d-Phenylalanine preferentially
permeated through immobilized DNA
membranes with a pore size ˂2.0 nm
(molecular weight cut off MWCO <
5000)
 while l-phenylalanine preferentially
permeated through immobilized DNA
membranes with a pore size >2.0 nm
(MWCO > 5000).
 DNA binding ability is higher for l-
phenylalanine than of d-phenylalanine.
Ultrafiltration of a racemic phenylalanine
solution using DNA solution system
resulted in preferential concentration of
d-phenylalanine in the permeate
Membrane-based separation
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 This method is based on the fact that one of the enantiomer of racemic mixture reacts
faster than other with optically active compound.
 (S)-enantiomer of ibuprofen proved to be 100 times medically more active than the
(R)-form.
 (R)-Ibuprofen ester was found to be less reactive than (S)-ibuprofen in the reaction .
 The two enantiomers of an Ibuprofen racemate react at different rates with the chiral
catalyst (Lipase from Candida rugosa), in which the enzyme shows a greater
stereopreference towards (S)-enantiomer of ibuprofen.
 Thus with differences in kinetics of reaction, racemic mixture can be separated.
kinetic resolution
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Conclusion
• Most of the drug substances single enantiomer is active .
In such cases the inactive enantiomer is consider as an
enantiomer impurity.
• Separation of these impurities is mandatory for acquiring
safety and efficacy of drug product.
• Maximum drug substances are chiral in nature hence it is
necessary to develop a suitable method to separate the
active form from the toxic or inactive form.
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References
1. Pilaniya K, Chandrawanshi HK, Pilaniya U, Manchandani P, Jain P, Singh N. Recent trends in the impurity profile of pharmaceuticals. Journal of
Advanced Pharmaceutical Technology & Research. 2010 Jul;1(3):302
2. Guideline ICH. Impurities in new drug substances Q3A (R2). InProceedings of the International Conference on Harmonization of Technical
Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland 2006 Oct 25 (Vol. 25).
3. Gogoi A, Mazumder N, Konwer S, Ranawat H, Chen NT, Zhuo GY. Enantiomeric recognition and separation by chiral nanoparticles. Molecules.
2019 Jan;24(6):1007.
4. Kannappan V, Kanthiah S. Enantiopurity assessment of chiral switch of ondansetron by direct chiral HPLC. Chromatographia. 2017 Feb
1;80(2):229-36.
5. Kannappan V, Mannemala SS. Simultaneous enantioseparation and purity determination of chiral switches of amlodipine and atenolol by liquid
chromatography. Journal of pharmaceutical and biomedical analysis. 2016 Feb 20;120:221-7.
6. Szabó ZI, Tóth G, Völgyi G, Komjáti B, Hancu G, Szente L, Sohajda T, Béni S, Muntean DL, Noszál B. Chiral separation of asenapine
enantiomers by capillary electrophoresis and characterization of cyclodextrin complexes by NMR spectroscopy, mass spectrometry and molecular
modeling. Journal of pharmaceutical and biomedical analysis. 2016 Jan 5;117:398-404.
7. Palacios SM, Palacio MA. Enantiomeric resolution of albuterol sulfate by preferential crystallization. Tetrahedron: Asymmetry. 2007 Jun
11;18(10):1170-5.
8. Barksdale JM, Clark CR. Liquid chromatographic determination of the enantiomeric composition of amphetamine and related drugs by
diastereomeric derivatization. Journal of chromatographic science. 1985 Apr 1;23(4):176-80.
9. Higuchi A, Higuchi Y, Furuta K, Yoon BO, Hara M, Maniwa S, Saitoh M, Sanui K. Chiral separation of phenylalanine by ultrafiltration through
immobilized DNA membranes. Journal of membrane Science. 2003 Aug 15;221(1-2):207-18.
10. Long WS, Kow PC, Kamaruddin AH, Bhatia S. Comparison of kinetic resolution between two racemic ibuprofen esters in an enzymic membrane
reactor. Process Biochemistry. 2005 Jun 1;40(7):2417-25.
2/03/22

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