Pharmaceutical Instrumental Analysis

PHC 427

Dr. haya Al-joharChief of Research and Seized Department

Saudi Food & Drug Authority

E-mail : hijohar2@hotmail.com

High performance liquid chromatography (HPLC)

Analytical features of HPLCExternal and internal standard methods Stability-indicating methods of assay.

Chiral separation of pharmaceutical compoundsChiral separation of pharmaceutical compound Separation and quantification of related Substances

Gas ChromatographicThe thermodynamic of gas chromatography.Instrumentation of gas chromatography. Application of gas chromatography.

LECTURES’ OUTLINE

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 absorptionQuantitative analysis by of atomic absorption Principles of atomic emission Instrumentation of atomic emission. Applications of atomic emission

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 AssaysStability Assays

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

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,To determine shelf life, you must measure two different aspects ofyou must measure two different aspects of the drug after it has been stressed.the drug after it has been stressed. First,First,- determine its potency, or the amount of active - determine its potency, or the amount of active

ingredient (simple). ingredient (simple). Second,Second,- determine the degradants or impurities that - determine the degradants or impurities that

appear as a result of aging. (difficult)appear as a result of aging. (difficult)

Developing a stability-indicating assayDeveloping a stability-indicating assayrequires consideration of three aspects requires consideration of three aspects

obtaining a representative sample,obtaining a representative sample,choosing the separation technique, andchoosing the separation technique, andselecting the detectorselecting the detector

The sampleThe sample

use a set of samples for method development use a set of samples for method development instead of a single sample.instead of a single sample.For simplicity, most workers focus on the drugFor simplicity, most workers focus on the drug

substance — the substance — the pure drugpure drug compound —instead compound —instead of the of the drug productdrug product

Obtain all the compounds that you might Obtain all the compounds that you might expect to be present in the drug substance expect to be present in the drug substance before it is formulated (synthetic Pathway).before it is formulated (synthetic Pathway).

expect that chemical degradation will occur in expect that chemical degradation will occur in the reverse order of synthesisthe reverse order of synthesis

obtain samples under stress condetions.obtain samples under stress condetions. This process often is called This process often is called forced degradationforced degradation

The drug is subjected to The drug is subjected to acid, base, heat, light, acid, base, heat, light, or oxidation.or oxidation.Usually, the goal is to degrade the parent drug Usually, the goal is to degrade the parent drug by 10–20% or soby 10–20% or so

The SeparationThe Separatione e

Reversed-phase LC is the method of choiceReversed-phase LC is the method of choice The polarity of the degraded samples can vary The polarity of the degraded samples can vary

widelywidely gradient elutiongradient elution

The most common separation variablesThe most common separation variablesinclude include solvent type, mobile-phase pH, columnsolvent type, mobile-phase pH, columnType.Type. Acetonitrile and methanolAcetonitrile and methanol phosphate buffer in the pH 2.5–6.5 range. phosphate buffer in the pH 2.5–6.5 range.

Choose two or three column types, suchChoose two or three column types, such as C8, embedded polar, and cyano phasesas C8, embedded polar, and cyano phases

The DetectorThe Detectorstability-indicating assays must be able to stability-indicating assays must be able to

determine sample components within at least determine sample components within at least a 1000-fold concentration range from 100% toa 1000-fold concentration range from 100% to

0.05% of the parent drug0.05% of the parent drugUse UV detector but the diode-array detector is Use UV detector but the diode-array detector is

an advantage during development.an advantage during development.

There’s nothing magic about stability indicatingThere’s nothing magic about stability indicatingassaysassays..

Stability StudiesStability Studies

All pharmaceutical manufacturers are required All pharmaceutical manufacturers are required to periodically test stored samples of their to periodically test stored samples of their products (sometimes they are subjected to products (sometimes they are subjected to high temperatures and moist environments) in high temperatures and moist environments) in order to determine their stability over long order to determine their stability over long periods of time.periods of time.

6 months 3months one year five years three years

Chiral separation by HPLC

Stereochemistry TermsStereochemistry Terms

IsomersIsomers:: Compounds with the different chemical structures Compounds with the different chemical structures and the same molecular formulaand the same molecular formulaStereoisomers:Stereoisomers: compounds made up of the same atoms but compounds made up of the same atoms but have different arrangement of atoms in spacehave different arrangement of atoms in spaceEnantiomersEnantiomers are the 2 mirror image forms of a chiral moleculeare the 2 mirror image forms of a chiral molecule– can contain any number of chiral centers, as long as each can contain any number of chiral centers, as long as each

center is the exact mirror image of the corresponding center is the exact mirror image of the corresponding center in the other moleculecenter in the other molecule

– Identical physical and chemical properties, but may have Identical physical and chemical properties, but may have different biological profiles. Need chiral recognition to be different biological profiles. Need chiral recognition to be separated.separated.

– Different optical rotations (One enantiomer is (+) or Different optical rotations (One enantiomer is (+) or dextrorotatory (clockwise), while the other is (-) or dextrorotatory (clockwise), while the other is (-) or levorotatory (counter clockwise))levorotatory (counter clockwise))

RacemateRacemate:: a 1:1 mixture of enantiomers. a 1:1 mixture of enantiomers.– Separation of enantiomers occurs when mixture is reacted Separation of enantiomers occurs when mixture is reacted

with a chiral stationary phase to form 2 diastereomeric with a chiral stationary phase to form 2 diastereomeric complexes that can be separated by chromatographic complexes that can be separated by chromatographic techniquestechniques

Diastereomers:Diastereomers: stereoisomers that are not enantiomersstereoisomers that are not enantiomers– Have different chemical and physical characteristics, and Have different chemical and physical characteristics, and

can be separated by non-chiral methods.can be separated by non-chiral methods.– Has at least 2 chiral centers; the number of potential Has at least 2 chiral centers; the number of potential

diastereomers for each chiral center is determined by the diastereomers for each chiral center is determined by the equation 2equation 2nn, where n=the number of chiral centers, where n=the number of chiral centers

July 24-27, 2006, San July 24-27, 2006, San Diego, CADiego, CA

Chiral vs Achiral CompoundsChiral vs Achiral Compounds

Achiral MoleculeAchiral Molecule: : Has no stereogenic center; the Has no stereogenic center; the carbon atom has less than 4 non-carbon atom has less than 4 non-equivalent substituents attachedequivalent substituents attachedhas a plane of symmetryhas a plane of symmetryone that one that isis superimposable on its superimposable on its mirror image (the two are identical)mirror image (the two are identical)

– i.e. nail, ball, a baseball bat i.e. nail, ball, a baseball bat Not optically activeNot optically active

Chiral MoleculeChiral Molecule: : Has one stereogenic center (typically Has one stereogenic center (typically C, but can be N, P, etc.), which is C, but can be N, P, etc.), which is attached to 4 different substituents attached to 4 different substituents asymmetricasymmetricone that is one that is notnot superimposable on its superimposable on its mirror image (the two are not mirror image (the two are not identical)identical)

– i.e. hands, keys, shoesi.e. hands, keys, shoesthe two mirror image forms are the two mirror image forms are called called enantiomersenantiomers Optically activeOptically active

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Methods of Chiral Methods of Chiral Separation Separation

Why Chirality is Important ?Why Chirality is Important ?

Drugs in Therapeutic Use

Pharmaceutical Industry

Chiral

Racemates

IntroductionIntroduction

The separation of chiral drugs is of great The separation of chiral drugs is of great pharmaceutical and clinical interest, because in most pharmaceutical and clinical interest, because in most cases only one of the enantiomers exhibits cases only one of the enantiomers exhibits pharmacological activity, whereas the other pharmacological activity, whereas the other enantiomer may have less or no activity, unwanted enantiomer may have less or no activity, unwanted side effects, antagonistic activities or even toxic side effects, antagonistic activities or even toxic effects. effects.

Why Chirality is Important ?Why Chirality is Important ?

Different pharmacodynamic effects.Different pharmacodynamic effects.

OH

O HN

S-(-)-propranolol

OH

O HN

R-(+)-propranolol

The separation of a mixture of enantiomers is The separation of a mixture of enantiomers is called called resolution resolution To perform a resolution the mixture of To perform a resolution the mixture of enantiomers is reacted with an optically enantiomers is reacted with an optically active compound in a reversible reaction to active compound in a reversible reaction to make a pair of diastereomers make a pair of diastereomers These diastereomers have different These diastereomers have different properties and can be separatedproperties and can be separatedThe reaction is the reversed (often an acid The reaction is the reversed (often an acid base reaction) to produce a single base reaction) to produce a single enantiomerenantiomer

Resolution of EnantiomersResolution of Enantiomers

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

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.

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.

S-Brucine

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 Stationary Phase (CSP)Chiral Stationary Phase (CSP)

A stationary phase which A stationary phase which

incorporates a chiral selector: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.

– Ability of chiral stationary phase, CSP, to interact Ability of chiral stationary phase, CSP, to interact differently with each enantiomer to form transient-differently with each enantiomer to form transient-diastereomeric complexes; requires a minimum of 3 diastereomeric complexes; requires a minimum of 3 interactions through:interactions through:

– H-bondingH-bonding– ππ--ππ interactions interactions– Dipole stackingDipole stacking– Inclusion complexingInclusion complexing– Steric bulkSteric bulk

Chiral RecognitionChiral Recognition

CSP

Biphenyl derivative

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.

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).

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).