Transcript HPLC-Dr.haya

Pharmaceutical Instrumental
Analysis
PHC 427
Dr. haya Al-johar
Chief of Research and Seized Department
Saudi Food & Drug Authority
E-mail : [email protected]
LECTURES’ OUTLINE
High performance liquid chromatography (HPLC)
Analytical features of HPLC
External and internal standard methods
Stability-indicating methods of assay.
Chiral separation of pharmaceutical compounds
Chiral separation of pharmaceutical compound
Separation and quantification of related Substances
Gas Chromatographic
The thermodynamic of gas chromatography.
Instrumentation of gas chromatography.
 Application of gas chromatography.
Capillary electrophoresis
Principles and instrumentation
Choice of optimum conditions for resolution.
 Modes of electrophoretic separation
Applications of capillary electrophoresis
Atomic absorption and emission spectrophotometry
Instrumentation of atomic absorption
Quantitative analysis by of atomic absorption
 Principles of atomic emission
 Instrumentation of atomic emission.
 Applications of atomic emission
Stability Assays
The FDA Defines a Stability Assay as a:
“Validated quantitative analytical methods that can
detect the changes with time in the chemical, physical, or
microbiological properties of the drug substance and drug
product, and that are specific so that the contents of
active ingredient, degradation products, and other
components of interest can be accurately measured
without interference.”
Stability-Indicating Assays
 use before date.
 the product should remain fully effective under
normal storage conditions.
The product’s shelf life is determined using
standardized storage conditions
To determine shelf life,
 you must measure two different aspects of
the drug after it has been stressed.
 First,
- determine its potency, or the amount of active
ingredient (simple).
 Second,
- determine the degradants or impurities that
appear as a result of aging. (difficult)
Developing a stability-indicating assay
requires consideration of three aspects
obtaining a representative sample,
choosing the separation technique, and
selecting the detector
The sample
use a set of samples for method development
instead of a single sample.
For simplicity, most workers focus on the drug
substance — the pure drug compound —instead
of the drug product
Obtain all the compounds that you might
expect to be present in the drug substance
before it is formulated (synthetic Pathway).
expect that chemical degradation will occur in
the reverse order of synthesis
obtain samples under stress condetions.
This process often is called forced degradation
The drug is subjected to acid, base, heat, light,
or oxidation.
Usually, the goal is to degrade the parent drug
by 10–20% or so
The Separatione
 Reversed-phase LC is the method of choice
 The polarity of the degraded samples can vary
widely
 gradient elution
The most common separation variables
include solvent type, mobile-phase pH, column
Type.
Acetonitrile and methanol
phosphate buffer in the pH 2.5–6.5 range.
Choose two or three column types, such
as C8, embedded polar, and cyano phases
The Detector
stability-indicating assays must be able to
determine sample components within at least
a 1000-fold concentration range from 100% to
0.05% of the parent drug
Use UV detector but the diode-array detector is
an advantage during development.
There’s nothing magic about stability indicating
assays.
Stability Studies
All pharmaceutical manufacturers are
required to periodically test stored samples of
their products (sometimes they are subjected
to high temperatures and moist
environments) in order to determine their
stability over long periods of time.
five years
three years
one year
6 months 3months
Chiral separation by HPLC
Stereochemistry Terms
Isomers: Compounds with the different chemical structures
and the same molecular formula
Stereoisomers: compounds made up of the same atoms but
have different arrangement of atoms in space
Enantiomers are the 2 mirror image forms of a chiral molecule
– can contain any number of chiral centers, as long as each
center is the exact mirror image of the corresponding
center in the other molecule
– Identical physical and chemical properties, but may have
different biological profiles. Need chiral recognition to be
separated.
– Different optical rotations (One enantiomer is (+) or
dextrorotatory (clockwise), while the other is (-) or
levorotatory (counter clockwise))
Racemate: a 1:1 mixture of enantiomers.
– Separation of enantiomers occurs when mixture is reacted
with a chiral stationary phase to form 2 diastereomeric
complexes that can be separated by chromatographic
techniques
Diastereomers: stereoisomers that are not enantiomers
– Have different chemical and physical characteristics, and
can be separated by non-chiral methods.
– Has at least 2 chiral centers; the number of potential
diastereomers for each chiral center is determined by the
equation 2n, where n=the number of chiral centers
Chiral vs Achiral Compounds
Chiral Molecule:
Has one stereogenic center (typically
C, but can be N, P, etc.), which is
attached to 4 different substituents
 asymmetric
one that is not superimposable on its
mirror image (the two are not
identical)
–
i.e. hands, keys, shoes
the two mirror image forms are
called enantiomers
Optically active
Achiral Molecule:
Has no stereogenic center; the
carbon atom has less than 4 nonequivalent substituents attached
has a plane of symmetry
one that is superimposable on its
mirror image (the two are identical)
–
i.e. nail, ball, a baseball bat
Not optically active
July 24-27, 2006, San
Diego, CA
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Methods of Chiral Separation
Non-chromatographic
Techniques
Chromatographic Techniques
Polarimetry
Thin-layer chromatography
(TLC)
Nuclear Magnetic Resonance,
Gas chromatography(GC).
Isotopic dilution
High-performance liquid
chromatography (HPLC).
Calorimetry
Supercritical fluid
chromatography (SFC).
Enzyme Techniques.
Capillary electrophoresis (CE).
Why Chirality is Important ?
44%
56%
Chiral
Drugs in Therapeutic Use
12%
Pharmaceutical
Industry
Racemates
88%
Introduction
The separation of chiral drugs is of great
pharmaceutical and clinical interest, because in most
cases only one of the enantiomers exhibits
pharmacological activity, whereas the other
enantiomer may have less or no activity, unwanted
side effects, antagonistic activities or even toxic
effects.
Why Chirality is Important ?
Different pharmacodynamic effects.
O
HN
OH
S-(-)-propranolol
O
HN
OH
R-(+)-propranolol
In 1992, U.S. Food and Drug Administration
issued guideline for pharmaceutical industry:
– Only therapeutically active isomer of chiral
drugs be brought to the market
– Each enantiomer of the drug should be studied
separately for its pharmacological and
metabolic pathways.
Resolution of Enantiomers
The separation of a mixture of enantiomers is
called resolution
To perform a resolution the mixture of
enantiomers is reacted with an optically active
compound in a reversible reaction to make a pair
of diastereomers
These diastereomers have different properties
and can be separated
The reaction is the reversed (often an acid base
reaction) to produce a single enantiomer
Conversion to diastereomers
If it was desired to separate a mixture of an R and S carboxylic
acid, for example, this mixture could be reacted with a single
enantiomer of a chiral amine to make the diastereomic ammonium
salts that could then be separated. Once the diastereomic salts
have been separated, mineral acid can reprotonate the carboxylic
acid to reform the original enantiomers. This is a general, three
step, technique for separating enantiomers:
(1) React the enantiomers with a single enantiomer of another
compound to form diastereomers
(2) Separate the diastereomers by conventional means
(chromatography, recrystallization)
(3) Regenerate the original enantiomers, now separated
S-Brucine
A common amine used in these reactions with carboxylic
acids is S-Brucine, an alkaloid found in only its S
enantiomer. S-Brucine is used because it is commercially
available, although in theory any amine that is purely one
enantiomer should work just as well.
Chiral chromatography
Another technique for separating enantiomers is
chiral chromatography. While enantiomers cannot be
distinguished in achiral environments, such as a
solvent
system
or
by
normal
silica
gel
chromatography, they can be distinguished in chiral
environments,
•Chiral HPLC
Direct
Indirect
Chiral HPLC
Chiral HPLC
Chiral
stationary
phases
CSP
Chiral
mobile
Phase
additives
CMPA
Derivatization with
chiral
reagent
Chiral Stationary Phase (CSP)
 A stationary phase which
incorporates a chiral selector:
 Chemically bonded to surface
of a solid support (silica).
 Immobilised onto the surface
of a solid support (silica).
There are five types of chiral stationary phases
including
macrocyclic glycopeptides,
cyclodextrins,
cellulose/amylose,
small molecule, and
proteins,
which are typically bonded to silica. The elution order
of chiral compounds depends upon the formation of
transient diastereoisomers due to the interaction
with the column packing. The compound that forms
the less stable diastereoisomer will elute first.
Chiral Recognition
–
Ability of chiral stationary phase, CSP, to interact
differently with each enantiomer to form transientdiastereomeric complexes; requires a minimum of 3
interactions through:
– H-bonding
– π-π interactions
– Dipole stacking
– Inclusion complexing
– Steric bulk
Biphenyl derivative
CSP
In this hypothetical example of an interaction
between a chiral stationary phase (left) with an
enantiomer of a biphenyl derivative (right),
there is a three-point interaction, with the
carboxy groups aligning with the amino groups
and the aromatics lining up with each other to
form pi stacking interactions. The enantiomer of
this biphenyl would not be able to have all three
of these interactions because its groups would
not be aligned correctly, and, consequently, it
would stick less to the chiral stationary phase
and filter off the column first.
A diagram of chiral column chromatography:
the enantiomer of the biphenyl that can form
the three-point interaction with the stationary
phase (red band) sticks better and filters off
the column after its enantiomer (green band).