ASSIGNMENT TOPIC: CHARACTERIZATION OF DRUGS SUBJECT TITLE: ORGANIC PHARMACEUTICALS COURSE NO:PHM-CHEM-7102

SUBMITTED BY: SAHAR KHAN MS/M.PHIL PHARMACEUTICAL CHEMISTRY FIRST SEMESTER ROLL NO. 10-MSPC-12
CHEMISTRY DEPARTMENT GCU, LAHORE

SUBMITTED TO: SIR MUHAMMAD ISLAM DATE OF SUBMISSION: 03-01-2013 LAST DATE OF SUBMISSION: 04-01-2013

CONTENTS DEFINATIONS OF CHARACTERIZATION CHARACTERIZATION REQUIREMENT TYPES OF CHARACTERIZATION SOURCES OF INFORMATION FOR DRUG CHARACTERIZATION PHYSICAL CHARACTERIZATION CHEMICAL CHARACTERIZATION CHARACTERIZATION OF CAFFEINE ISOLATION OF CAFFEINE PHYSIOCHEMICAL PROPERTIES OF CAFFEINE METHODS OF DETERMINATION OF CAFFEINE QUALITATIVE AND QUANTITATIVE ANALYSIS OF CAFFEINE

CHARACTERIZATION
DEFINATION No. 1 Checking composition of a given compound is called characterization. " DEFINATION No. 2 Analysis of physio chemical properties of a given compound according to WHO is called characterization. DEFINATION No. 3 Just to determine the composition of all compounds including drugs is called characterization. DEFINATION No. 4 To check the structure, formula of a compound, functional group by using different instruments e.g UV Spectroscopy, IR Spectroscopy, Mass Spectroscopy and NMR Spectroscopy all includes characterization. DEFINATION No. 5 Isolation of active ingredient from start to end is called characterization e.g isolation of caffeine from tea leaves. DEFINATION No. 6 To isolate, purify and identification of component/constituents of drug by using Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC). DEFINATION No. 7 Qualitative and quantitative tests to identify either the given plant / Drug contain, Alkaloids, Carbohydrate, Hormones, Lipids, Protein, Starch or Tannin as active ingredient. 01 DEFINATION No. 8 A group of tests to characterize drug components are also includes in characterization.

Characterization requirement
Drug characterization studies can provide information useful for drug law enforcement authorities. Chemical links between samples can be established, and material from different seizures can be classified into groups of related samples.02

It has played a major role in pharmaceutical advances, forensic science and modern agriculture. Diseases and their remedies have also been a part of human lives. Characterization plays an important role in understanding remedies, i.e. drugs. Medicines or drugs that we take for the treatment of various ailments are chemicals, either organic or inorganic. However, most drugs are organic molecules. Let us take aspirin as an example. It is probably the most popular and widely used analgesic drug because of its structural simplicity and low cost. Aspirin is chemically known as acetyl salicylic acid, an organic molecule. The precursor of aspirin is salicin, which is found in willow tree bark. However, aspirin can easily be synthesized from phenol using the Kolbe reaction.03 In order to have a proper understanding and knowledge of drugs and their behavior, there is no other alternative but to characterize drugs . Every-where, from discovery to development, from production and storage to administration, and from desired actions to adverse effects of drugs, drug characterization is involved directly. In the drug discovery stage, suitable sources are explored. Sources of drug molecules can be natural, e.g. narcotic analgesic, morphine, from Papaver somniferum (Poppy plant), synthetic, e.g. a popular analgesic and antipyretic, paracetamol, or semi-synthetic, e.g. semisynthetic penicillins.

Types of characterization
Initial Drug Substance Characterization (IDSC) Physical Characterization Chemical Characterization Qualitative Characterization Quantitative Characterization

Initial Drug Substance Characterization (IDSC) An Initial Drug Substance Characterization (IDSC) report contains physical characterization and preformulation data. Physical characterization data include form identification, solvent identification, hygroscopicity, micromeretics, and structure elucidation. Polymorph screening and form identification are also available. Preformulation data include equilibrium solubility, pH solubility, partition coefficient, pKa determination, and accelerated physical and chemical stability. Physical Characterization

x-ray powder diffraction (XRPD) optical microscopy differential scanning calorimetry (DSC) thermogravimetry (TG) melting point determination moisture sorption/desorption isotherms infrared (IR) spectroscopy Raman spectroscopy solid-state NMR spectroscopy ultraviolet (UV)spectrum

particle size distribution surface area scanning electron microscopy

Structure Elucidation

nuclear magnetic resonance (NMR) solution NMR spectroscopy sIR spectroscopy single crystal structure determination mass spectrometry Preformulation Data

equilibrium solubility pH solubility pKa determination Log P Log D accelerated physical and chemical solid and solution stability

04

Sources of information for drug profiling and characterization:

1. Whether drug characterization work is for evidential or intelligence purposes, it is advantageous to obtain as much information on a drug sample as possible. The forensic chemist, in carrying out such characterization/profiling studies, largely relies on two different sources of information Physical Analysis Chemical Analysis 2. the most appropriate analytical approach for drug characterization studies depends on the type of sample, i.e., tablets, capsules, powders, liquids and natural materials (e.g., opium, cannabis products). The most simple type of investigation is a visual inspection of the physical characteristics of the sample. In many cases differences/similarities in, for example, the color, texture or general appearance of samples can be observed. The forensic chemist, in combining this information with information from other sources, may be able to draw conclusions on whether or not two or more drug samples are connected. 3. Valuable information to show that samples may be more directly related can also be obtained by comparison of their packaging (including materials, the way of packaging, and the examination of any fingerprints on packaging), and by examination of any characteristic marks the samples may display. Different seizures of illicit tablets, for instance, may be linked to a distribution network, a single source of production, and in some cases to the actual equipment used for tableting, by examination of the defects or marks on the tablet surfaces. 4. In addition to the examination of physical characteristics, and especially where these have limited significance, samples can also be characterized by chemical analysis. The detailed chemical analysis of drug samples by modern analytical techniques assigns to every drug sample a characteristic chemical signature of major, minor and trace components. Careful examination of these impurity profiles offers a valuable means of comparing and grouping different seizures.02

Physical properties of drug molecules:

Physical state

Drug molecules exist in various physical states, e.g. amorphous solid crystalline solid hygroscopic solid liquid or gas. The physical state of drug molecules is an important factor in the formulation and delivery of drugs. Melting point and boiling point: The melting point (m.p.) is the temperature at which a solid becomes a liquid The boiling point (b.p.) is the temperature at which the vapour pressure of the liquid is equal to the atmospheric pressure For example, the melting point of water at 1 atmosphere of pressure is 0 _C (32 _F, 273.15 K; this is also known as the ice point) and the boiling point of water is 100 _C. Melting point is used to characterize organic compounds/drugs and to confirm the purity. The melting point of a pure compound is always higher than the melting point of that compound mixed with a small amount of an impurity. The more impurity is present, the lower the melting point. Finally, a minimum melting point is reached. The mixing ratio that results in the lowest possible melting point is known as the eutectic point. The melting point increases as the molecular weight increases, and the boiling point increases as the molecular size increases. The increase in melting point is less regular than the increase in boiling point, because packing influences the melting point of a compound. Polarity and solubility: Polarity Is a physical property of a compound/drug , which relates other physical Properties, e.g. melting and boiling points, solubility and intermolecular Interactions between molecules. Generally, there is a direct correlation between the polarity of a molecule and the number and types of polar ornonpolar covalent bond that are present. In a few cases, a molecule having polar bonds, but in a symmetrical arrangement, may give rise to a no polar molecule, e.g. carbon dioxide (CO2). The polarity in a bond arises from the different electro negativities of the two atoms that take part in the bond formation. The greater the difference in electro negativity between the bonded atoms, the greater is the polarity of the bond. For example, water is a polar molecule, whereas cyclohexane is non polar.

H - OH Water a polar molecule Solubility

Cyclohexane A non polar molecule

Is the amount of a solute/drug that can be dissolved in a specific solvent Under given conditions. The dissolved substance is called the solute and the dissolving fluid is called the solvent, which together form a solution. The process of dissolving is called solvation, or hydration when the solvent is water. In fact, the interaction between a dissolved species and the molecules of a solvent is solvation. The solubility of molecules/drugs can be explained on the basis of the polarity of molecules. Polar, e.g. water , ethanol , methanol Non-Polar, e.g. benzene , n-hexane , petroleum ether Solvents do not mix. In general, like dissolves like; i.e., materials with similar polarity are soluble in each other. The hydrogen bonding and other non-bonding interactions between molecules. Refractive Index The refractive index (or index of refraction) of a medium is a measure for how much the speed of light (or other waves such as sound waves) is reduced inside the medium. This is a very important property of a material or compound, that may characterize a purity or composition of a mixture. Refractive index of a drug is a very important property of a drug product or API to know for the pharmaceutical industry because in many cases the size of drug particles are regulated. Existing laser particle size counters may actually estimate particle size if known the refractive index of the particle material. Relative Density Relative density, or specific gravity, is the ratio of the density (mass of a unit volume) of a substance to the density of a given reference material. Specific gravity usually means relative density with respect to water. The term "relative density" is often preferred in modern scientific usage.If a substance's relative density is less than one then it is less dense than the reference; if greater than 1 then it is denser than the reference.

Moisture
1.0 g each of the respective powdered samples was weighed each on aluminum foil on the automated moisture analyzer pan and set at 105C for 3 h where moisture content percentage of the sample was obtained (WHO, 1998).

Total ash
2.0 g of the respective powdered samples was ignited in a previously ignited and tarred crucible at 500C for about 3 h until the sample was white, indicating the absence of carbon, and was cooled in a desiccators and was later weighed (WHO, 1998). Loss of weight Loss of weight of drug as moisture content evaporate and low volatile solvents also evaporate at given temperature.03

Physicochemical properties of drugs in solution

Activity and chemical potential Concentration units Thermodynamics Ionisation of drugs in solution05

Characterization of a drug is as under: CAFFEINE

Caffeine is a xanthine alkaloid compound that acts as a stimulant in humans. Caffeine is sometimes called guaranine when found in guarana, mateine when found in mate, and theine when found in tea. It is found in the leaves and beans of the coffee plant, in tea, yerba mate, and guarana berries, and in small quantities in cocoa, the kola nut and the Yaupon Holly. Overall, caffeine is found in the beans, leaves, and fruit of over 60 plants, where it acts as a natural pesticide that paralyzes and kills certain insects feeding upon them.06 STRUCTURE OF CAFFEINE07

CRYSTALLINE POWDER FORM OF CAFFEINE08

ISOLATION OF CAFFEINE
1) To isolate caffeine from dry tea leaves by extraction and purify the crude extract by recrystallization. 2) To determine the mass percentage of caffeine in the tea leaves09 Isolation of pure caffeine Caffeine isolation is an important industrial process and can be performed using a number of different solvents. Benzene, chloroform, trichloroethylene and dichloromethane have all been used over the years but for reasons of safety, environmental impact, cost and flavor, they have been superseded by the following main methods: Water extraction/isolation Coffee beans are soaked in water. The water, which contains not only caffeine but also many other compounds which contribute to the flavor of coffee, is then passed through activated charcoal, which removes the caffeine. The water can then be put back with the beans and evaporated dry, leaving decaffeinated coffee with a good flavor. Coffee manufacturers recover the caffeine and resell it for use in soft drinks and medicines. Supercritical carbon dioxide extraction/isolation

Supercritical carbon dioxide is an excellent nonpolar solvent for caffeine (as well as many other organic compounds), and is safer than the organic solvents that are used for caffeine extraction. The extraction process is simple: CO2 is forced through the green coffee beans at temperatures above 31.1 C and pressures above 73 atm. Under these conditions, CO2 is in a "supercritical" state: it has gaslike properties which allow it to penetrate deep into the beans but also liquid-like properties which dissolve 97-99% of the caffeine. The caffeine-laden CO2 is then sprayed with high pressure water to remove the caffeine. The caffeine can then be isolated by charcoal adsorption (as above) or by distillation, recrystallization, or reverse osmosis.10 Isolation by nonhazardous organic solvents Organic solvents such as ethyl acetate present much less health and environmental hazard than previously used chlorinated and aromatic solvents. The hydrolysis products of ethyl acetate are ethanol and acetic acid, both nonhazardous in small quantities. Another method is to use triglyceride oils obtained

Physio Chemical Characterization of Caffeine

1. Description : A white, crystalline powder or silky, white crystals, sublimes readily. 2. Solubility : Sparingly soluble in water, freely soluble in boiling water, slightly soluble in ethanol. It dissolves in concentrated solutions of alkali benzoates or salicylates. 3. Identification : A. Melting Point: 234 C to 239 C B. Infrared absorption spectrophotometry C,D. Chemical Test given by caffeine and alkaloids Test for alkaloids 1. Dragendorffs test 1 ml of extract, add 1 ml of Dragendroffs reagent (potassium bismuth iodide solution). An orange -red precipitate indicates the presence of alkaloids. 2.Mayers test 1 ml of extract, add 1 ml of Mayers reagent (potassium mercuric iodide solution). Whitish or cream colored precipitate indicates the presence of alkaloids. 3.Hagers test 1 ml of extract, add 3 ml of Hagers reagent (saturated aqueous solution of picric acid). Yellow colored precipitate indicates the presence of alkaloids 4. Wagners test 1 ml of extract, add 2 ml of Wagners reagent (iodine in potassium iodide). Reddish brown colored precipitate indicates the presence of alkaloids E. Loss on drying F. Reaction of xanthines 4. Appearance of solution : To meet the test. 5. Acidity : Not more than 0.2ml of 0.01M NaOH is required 6. Related Substances : Thin-Layer Chromatography

7. Sulphates : Not more than 500 ppm 8. Heavy Metals : Not more than 10 ppm Pb 9. Loss on drying : Not more than 0.5% 10. Sulphated Ash : Not more than 0.1% Ignition temperature >600 C Solubility 20 g/l (20 C) Melting point 235 - 239 C Molar mass 194.19 g/mol Density 1.23 g/cm3 (18 C) Bulk density 300 - 600 kg/m3 pH value 5.5 - 6.5 (10 g/l, H2O, 20 C) Vapor pressure 20 hPa (80 C)11

Physio Chemical Characterization of Caffeine in Tabulated Form12

Caffeine General Systematic 1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione name 1,3,7-trimethylxanthine, trimethylxanthine, Other theine, mateine, guaranine, names methyltheobromine Molecular C8H10N4O2 formula SMILES O=C1C2=C(N=CN2C)N(C(=O)N1C)C Molar mass 194.19 g mol1 Appearance Odorless, white needles or powder CAS [58-08-2] number

Properties Density and 1.2 g/cm, solid phase Solubility in Slightly soluble water Soluble in ethyl acetate, chloroform, pyrimidine, pyrrole, Other tetrahydrofuran solution; moderately soluble in alcohol, solvents acetone; slightly soluble in petroleum ether, ether, benzene. Melting 237 C point Boiling 178 C (sublimes) point Acidity 10.4 (40 C) (pKa) Hazards MSDS External MSDS Main May be fatal if inhaled, swallowed hazards or absorbed through the skin. Flash point N/A RTECS EV6475000 number Except where noted otherwise, data are given for materials in their standard state (at 25 C, 100 kPa) Infobox disclaimer and references

Methods of Determination of Caffeine

Several techniques have been reported for the individual quantification of caffeine as well as simultaneous determination of caffeine with different analytes in pharmaceutical preparations biological fluids and foodstuffs. One of the earliest methods of caffeine determination is based up on ultraviolet (UV) spectrophotometry. Caffeine absorbs in the ultra violet region at 276 nm 13. Although spectrophotometry is a fast and simple method, it is not possible to determine caffeine directly in coffee beans by the conventional UV absorption due to the spectral overlap. On the other hand, the derivative spectrophotometer is relatively easy; however, it requires larger concentration of caffeine in the samples13. The more recent methods of caffeine determination are based on the high performance liquid chromatography (HPLC) combined with several detection methods like UV, mass and infrared spectrometry. However, most of these reported procedures involve relatively long retention times or require lengthy sample pretreatments14. There are also other techniques reported for caffeine determination suchas Fourier Transform Infrared spectroscopy Ion chromatography liquid chromatography coupled to mass spectrometry

Nuclear magnetic resonance (NMR) spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is probably one of the most versatile analytical tools available, and has become the technique of choice for biological fluids15 and polymer tests , and for pharmaceutical analysis. The latter includes the determination of impurities, the contents of drugs and the characterization of isomeric drug mixtures 16. Generally, NMR becomes quantitative NMR (qNMR) whenever it is applied as a quantitative analytical tool. In principle, qNMR is amenable to all NMR-sensitive nuclei and unrestricted in dimensions. Quantitative proton NMR (qHNMR) is the most commonly used technique in the analysis of foods, pharmaceuticals, natural products, etc. Because the second most important studied organic NMR nucleus (13C) is less sensitive (1.6% of 1H sensitivity for an equal number of nuclei) and low natural abundance (1.1%), it is difficult to obtain quantitative information by quantitative 13C NMR technique, especially for small natural product samples. Besides qualitative information, NMR can provide quantitative information about the sample since the intensity (or the area) of a sample is directly proportional to the number of nuclei producing the signal. The precision of the integrals determines the accuracy of quantification, which depends on the noise level of the spectrum, the line shape, quality of shimming and phase-, baseline- and drift corrections16. For simple compositional analysis, integration of the spectrum or selected spectral region is performed, followed by adjustment of the integrated intensities to reflect the number of protons giving rise to the integrated signals. The individual integrated intensities are summed and then expressed as a percent of summed integrations, which represents the molar composition of the mixture (mole%). If an absolute determination of the principal component of a complex mixture is required, it is necessary to develop a weight-percent quantitative assay. This procedure would involve obtaining a weight (mg) of a sample of the crude mixture, adding a precise quantity of a known internal standard, obtaining the solution qHNMR spectrum of the sample plus internal standard and calculating the actual weight of the desired component of the crude mixture.

Advantage of using NMR Spectroscopy

The main advantage of using NMR for quantitative analysis is that it is simple and straightforward. In most applications, the sample only has to be dissolved in a deuterated solvent followed by measurement of NMR spectrum. Ideally, every signal in the NMR spectrum has the same response factor concerning the number of magnetically equivalent nuclei that constitute the signal, which make the calculation simple 16,17. Other advantages of NMR are its nondestructive nature and selectivity, together with the fact that it reveals structural information about previously known contaminants that may be present.

Thin-layer chromatography (TLC)

Thin-layer chromatography (TLC) is a very old method of analysis that has been well proven in practice. For more than thirty years, it has occupied a prominent position, especially in qualitative investigations. With the development of modern pre-coated layers and the introduction of partially or completely automated equipments for the various stages of operation of TLC, not only are highly accurate quantitative determinations now possible, but also the requirements that the work should comply with good manufacturing practice (GMP)/ good laboratory practice (GLP) guidelines can be fulfilled.

Thin layer chromatography can be applied in different areas of analysis: pharmaceuticals and drugs, clinical chemistry, forensic chemistry, biochemistry, food analysis, environmental analysis, natural products chemistry, synthetic organic chemistry and other areas.18

Quantitative TLC Analysis of Caffeine

Development of the Optimum Mobile Phase The TLC procedure was optimized with a view to quantify caffeine in the coffee, tea and soft drink extracts. The mobile phase consisting of chloroform and methanol (10:1 v/v) gave a good resolution, and a sharp and well-defined peak at Rf=0.64 for caffeine in the standard as well as in the extracts (Figure 3 ). Well-defined spots were obtained when the developing chamber was saturated for 10 minutes at room temperature. This enables us to accurately quantify caffeine in dilute solutions.

Figure 3. TLC of caffeine standard and two extracts developed with chloroform, methanol (10:1). Left to right: Tracks 1, 2, 4, 6 represent 200, 500, 800, 1100 ng caffeine standard respectively; tracks 3 and 5 represent 500 ng tea and 450 ng coffee extracts, Quantitative 1H NMR Analysis of Caffeine

Qualitative Analysis
The spectrum of caffeine (1) consists of three sharp singlets at 3.22, 3.40 and 3.83 due to the three methyl groups connected to the nitrogen atoms as well as a singlet peak at 7.78 corresponding to the =C-H proton.It should be noted that, in all spectra in Figure 2, a sharp singlet peak is observed at 4.70 corresponding to water.

Figure 2. Proton NMR spectra of caffeine

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