Enantiomeric impurities &

Separation approaches
 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
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
 Introduction to impurity
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.
 Impurities testing guidelines
• The ICH Q3 series are designed for impurities testing.
• The ICH Q3 topic on impurities testing is covered by eight separate
guidelines.

• Impurities in New Drug Substances

Q3A(R2)

• Impurities in New Drug Products

Q3B(R2)

Q3C • Guidelines for residual solvent

(R8)

• Guideline for Elemental Impurities

Q3C(R9)

• Impurity: Assessment and control of Extractable and Leachable for

Q3E Pharmaceuticals and Biologics.
EWG
 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 .
Classification of impurities associated with APIs

Organic impurities
(process and drug Inorganic impurities Residual Solvent
related)

• starting material • Reagents, ligands,

catalysts • Class 1 solvents:
solvent to be avoided
• by products
• heavy metals • Class 2 solvents:
• Intermediates solvents to be limited.
• inorganic salts
• Class 3 solvents:
• degradation solvents with low
products • other material ( toxic
filter aid, charcoal,
• reagents, ligands, etc.) • Other solvents (“class
4”): no adequate
catalysts. toxicological data
 ICH Limits for Impurities

 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.
Thresholds MDD* of API in drug Threshold limit based on
product TDI**
≤ 1g/day 0.1%
Reporting
> 1g/day 0.05%
< 1mg/day 1% or 5 µg TDI (whichever
is lower)
1 – 10 mg/day 0.5% or 20 µg TDI
Identification (whichever is lower)
10mg- 2g/day 2 g/day - 0.2% or 2 mg TDI
(whichever is lower)
> 2 g/day 0.1%
< 10mg/day 1% or 50 µg TDI (whichever
is lower)
10 – 100 mg/day 0.5% or 200 µg TDI
Qualification (whichever is lower)
>100mg – 2 g/day 0.2% or 3mg TDI
(whichever is lower)
> 2 g/day 0.15%
*MDD: Maximum Daily Dose **TDI: Total Daily Intake
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
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
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
Class-4 Solvents: Solvents for which No Adequate
Toxicological Data was Found

Examples:

1,1-Diethoxypropane Petroleum ether

Methylisopropyl ketone Isooctane

1,1-Dimethoxymethane Trichloroacetic acid

Methyltetrahydrofuran Isopropyl ether

2,2-Dimethoxypropane Trifluoroacetic acid

 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.
 Optical Activity

Dextrorotatory (+)- d-isomer

rotate the plane polarized light to the right

levorotatory (-)- L-isomer

rotate the plane polarized light to the left

 Optical purity

A sample is said to be Any contamination by the

enantiomerically pure when
there is only one of its
enantiomer and the value
of specific rotation (α) is at
the highest.
other enantiomer lowers
the value of of specific
rotation .

Optical Purity = Enantiomeric Excess ( e.e.)

 Optical purity
For example :
tartaric acid with an observed rotation of +6°. And We know thatspecific
rotation of pure tartaric acid is +12.0

[α] mixture
OP=
[α] pure

+6
Optical Purity = = 0.5 or 50%
+12

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%.
 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.
 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
What are the different technique use for
separation for enantiomeric impurities?
Chromatographic
method

Membrane-based
separation capillary
electrophoresis
Enantioseparation
technique

crystallization
kinetic resolution
resolution
liquid-liquid
extraction
 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
Dept. than
eight times higher activity of Pharmaceutical Analysis, NIPER-HAJIPUR, Bihar, India
S- enantiomer.
 column : Lux
Cellulose-4
Dimension : 250 ×
4.6 mm, 5 µm)
MP: acetonitrile,
S- Amlodipine R- Amlodipine ethanol and DEA
(92:8:0.2% v/v/v )
flow rate : 1.2 mL
(S) /min
(R)
Detection: λ -240
nm
R- Atenolol
S- Atenolol
 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 isDept. of Pharmaceutical
responsible Analysis, NIPER-HAJIPUR,
for the β-adrenoceptor Bihar, India
blocking active.
chromatographic
Stationary phase Mobile phase Enantiomers(analytes)
methods
high-performance coated/ immobilized Cefadroxil has 3 chiral centers
hexane–2- propanol (60 : 40 NIPER-HAJIPUR
liquid-chromatographic cellulose and amylose chiral and the existence of eight different
v/v)
(HPLC) SP under normal-phase stereoisomers is possible.

Shimadzu Prominence
HPLC system and Supelco methanol, 0.1 % (v/v) glacial “d” and “l” Enantiomers of
LC-MS/MS Astec Chirobiotic V2 25 cm acetic acid and 0.02 % (v/v) Amphetamine and
x 2.1 mm, 5 µm column ammonium hydroxide Methamphetamine
held at 20 °C

Hydroxypropyl-b-
capillary
cyclodextrin bonded to an 10 mM ammonium acetate pH Hexobarbital, warfarin and
electrochromatography
organic monolith Capillary, 7.0-ACN (30:70, v/v) phenylalanine
(CEC) 100 µm I. D. × 25 cm

Isopropanol, ethanol, benzene,

toluene, diisopropylether, and
supercritical fluid Ibuprofen D or R(2)-enantiomer
Kromasil CHI-TBB phase trichlorethane of analytical
chromatography (SFC) causes some un-wanted side effect.
grade, and ethyl acetate and n-
hexane of Supra Solv grade

high speed counter-

n-hexane–ethyl acetate–0.1 mol (R, S)-naproxen , (S)-naproxen
current hydroxypropyl-β-
L−1, phosphate buffer solution showed 28 times higher activity
chromatography cyclodextrin (HP-β-CD)
Dept. of Pharmaceutical Analysis,
with pH NIPER-HAJIPUR,
= 2.67 (8:2:10, v/v/v) Bihar, India
than the (R)-isomer
 capillary electrophoresis

• 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
 crystallization resolution

• 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.
 crystallization resolution

Albuterol is a β-2-adrenoceptor agonist prescribed for the treatment of bronchial

asthma. And (R)-isomer bronchodilator activity resides.
 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.
 Membrane-based separation
 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
 kinetic resolution
 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.

 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.