Pharmaceutical Instrumental Analysis Dr. haya Al-johar PHC 427 Dr. haya Al-johar Chief of Research and Seized Department Saudi Food & Drug Authority E-mail : hijohar2@hotmail.com

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. 6 months 3months one year five years three years

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 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 http://wps.prenhall.com/wps/media/objects/724/741576/Instructor_Resources/Chapter_05/Text_Images/FG05_01-10UN.JPG

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.

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

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. S-(-)-propranolol R-(+)-propranolol In their pharmacokinetic and pharmacodynamic effects

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,

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.

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

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 transient-diastereomeric complexes; requires a minimum of 3 interactions through: H-bonding π-π interactions Dipole stacking Inclusion complexing Steric bulk 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.

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).