1. Pharmaceutical Instrumental Analysis Dr. haya Al-johar
PHC 427
Dr. haya Al-johar
Chief of Research and Seized Department
Saudi Food & Drug Authority

2. 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.

3. 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

4. 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.”

5. 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

6. 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)

7. Developing a stability-indicating assay
requires consideration of three aspects
obtaining a representative sample,
choosing the separation technique, and
selecting the detector

8. 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

9. 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

10. 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

11. The Separatione
Reversed-phase LC is the method of choice
The polarity of the degraded samples can vary widely
gradient elution

12. 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

13. 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.

14. 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.
6 months 3months one year five years three years

18. Chiral separation by HPLC

19. 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))

20. 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

21. 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 non-equivalent 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

22. Methods of Chiral Separation
Non-chromatographic Techniques
Polarimetry
Nuclear Magnetic Resonance,
Isotopic dilution
Calorimetry
Enzyme Techniques.
Chromatographic Techniques
Thin-layer chromatography (TLC)
Gas chromatography(GC).
High-performance liquid chromatography (HPLC).
Supercritical fluid chromatography (SFC).
Capillary electro­phoresis (CE).
From that aspect separation of enantiomers is very important. Although the first attempt to separate enantiomers was carried out in …. Chiral separation had come a long way.
Methods of separation can be done using either non-chromatographic techniques such as………………. ). Gas chromatography (GC).
Or using chromatographic techniques such as:………………….
The focus of this research is to utilize HPLC for chiral separation, which s one of the most commonly used methods.

23. Why Chirality is Important ?
Drugs in Therapeutic Use
Chiral
Majority of bioorganic molecules are chiral.
In the pharmaceutical industry, about 56% of drugs in current use are chiral
About 88% are used therapeutically as racemates
In 1992, U.S. Food and Drug Administration issued guideline for pharmaceutical industry:
Only therapeutically active isomer of chiral drugs be brought to market
Each enantiomer of the drug should be studied separately for its pharmacological and metabolic pathways.
Pharmaceutical Industry
Racemates

24. 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.

25. Why Chirality is Important ?
Different pharmacodynamic effects.
S-(-)-propranolol
R-(+)-propranolol
In their pharmacokinetic and pharmacodynamic effects

26. 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.

27. 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

28. 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

30. 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.

31. 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,

32. Direct Chiral HPLC Indirect Chiral HPLC Chiral stationary phases CSP
Chiral mobile
Phase additives
CMPA
Indirect
Chiral HPLC
Derivatization with
chiral
reagent
Furthermore, Chiral HPLC can be performed by Direct methods such as in CSP and CMPA
Or indirect methods through dervitatization with a chiral reagent.
In my research we used directe and indirecte technique.

33. 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).
Let me give you a brief definition of CSP

34. 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.

35. Chiral Recognition
Ability of chiral stationary phase, CSP, to interact differently with each enantiomer to form transient-diastereomeric complexes; requires a minimum of 3 interactions through:
H-bonding
π-π interactions
Dipole stacking
Inclusion complexing
Steric bulk
CSP
Biphenyl derivative

36. 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.

37. 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).