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EFFECT OF SURFACTANCTS ON THE SOLUBILITY OF DRUG


AIM: To determine the effect of surfactants on the solubility of drugs. REQUIREMENTS: Diclofenac sodium, Tween 80, span 80, distilled water, test tubes, volumetric flasks, pipette, UV - visible spectrophotometer, funnel, shaker bath, whatmann filter paper, electronic balance, paracetamol. THEORY: Solutes that cause a marked decrease in the surface tension of solvent are termed as surface active agents. These substances are of importance in variety of fields as emulsifying agents, antifoaming agents, and flocculants and deflocculants. All types of surfactants contain a lipophilic hydrophobic group like long hydrocarbon chain that has greater affinity for water. To have such an affinity the group must possess an appreciable polar character (large permanent dipole). A molecule / ion that possesses this type of structure is termed as amphiphatic molecule/ion. In pharmaceutical system which are heterogeneous, many problems arise because of interfacial tension between different phases, such problems can be handled by proper selection of surfactants. Surfactant tend to get absorbed at the interface between two phases. It consists of polar head and a nonpolar part and when they are placed in two phases of different polarities, the non polar part gets oriented towards the phase of low polarity while polarity increases the interfacial tension is lowered. When a surfactant is placed in a solvent its molecules are present in large numbers at the interface than in the bulk of the solution. Adsorption at the interface continues until all the available space at the surface is occupied. There after the surfactant molecules descend down into the bulk of the solution in the form of molecular aggregate (or) micelles. The molecules are arranged in a micelle that their parts correspond to the polarity of solution are oriented towards outside. The concentration of surfactant at which it begins to form micelle is known as critical micellar concentration(CMC).A suitable balance between the opposing hydrophilic and lipophillic characteristics of surfactants is necessary to ensure surface active properties (i.e., they orient at the interface). Selection of surfactant is based on HLB system (scale) given by Griffin. Every surfactant is assigned a number known as HLB value ranging from 1-40. Increase in number of HLB value indicates increase in hydrophilic properties 1

of surfactants. CLASSIFICATION OF SURFACTANTS: Based on their ionic behavior in solution, surfactants are classified into 1. ANIONIC SURFACTANTS: They ionize in aqueous media and surfactant activity of this type of surfactants is related to anionic part. Since they are negatively charged they migrate towards anode.
Ex:- Alkali soaps,organic sulphates, organic sulphonates and phosphated compounds.

2. CATIONIC SURFACTANTS: They consistof quarternary ammonium compound. Ex:- Quarternary ammonium compounds, pyridinium compounds (Cetyl trimethyl
ammonium bromide or cetrimide).

3. NON-IONIC SURFACTANTS: They are not ionized in aqueous media. These are most widely used.These are generated by reacting any hydrophobic compound having -COOH,-OH,-CONH2,-NH2 groups with ethylene oxide Ex:-Alcohol-polyethylene glycol esters, fatty acid-polyhydric alcohol esters. 4. AMPHOLYTIC SURFACTANTS: Ampholytic surfactants have amino grouping together with carboxy, sulfuric, sulphonic, phosphoric groups. Ex: Amino acids (N-dodecyl alanine) SOLUBILITY ENHANCEMENT USING SURFACTANT SYSTEM BELOW CMC: Certain drugs reduce CMC of surfactants. Thus below CMC itself, micellar solubilization is possible i.e., co-micellisation (mixed micelle formation). Some amphiphiles donot form micelle due to cosolvent action. Surfactant increases solubility of NCE by improving wettability and reduces surface tension of aqueous system and also reduce angle of contact. ABOVE CMC: Hydrophobic drugs are placed inside micelle Hydrophobic drugs are rate limiting factors in dissolution.
Ex:-Oil-soluble vitamins, steroids, hormones.

Surfactant form aggregates (or) micelles at CMC and improves the apparent solubility of hydrophobic drugs and enhances the dissolution. Above CMC micelle becomes colloid in size nature of aggregates and its properties depend on concentration of surfactant.

NOTE: Micelle remains in the bulk and interface is completely saturated with mono molecular layer of surfactant. If bulk is also saturated then the surfactant forms a layer on it (lamellar formation). PROCEDURE: CONSTUCTION OF STANDARD GRAPH: Weigh accurately 100mg of drug and dissolve in about 3/4th volume of distilled water in 100ml volumetric flask and make up the volume upto 100ml. Take 10ml of above solution and dilute to 100ml which gives 100g/ml Take 0.1, 0.2, 0.3, 0.4 and 0.5ml from the above solution and dilute each to 10ml which gives 1,2,3,4 and 5g/ml respectively. Measure the absorbance of the above solutions at the max 276nm in UV spectrophotometer using distilled water as blank. Construct the standard graph using concentration of drug on x-axis and absorbance on y-axis. STUDY OF EFFECT OF SURFACTANTS ON THE SOLUBILITY OF THE DRUG Prepare different surfactant solutions of various concentrations as given in the Table: S. No. 1 2 3 4 5 Surfactant Concentration 0 1 2 3 4 Water(ml) 25 25 25 25 25 Surfactant (mg) 0 0.25 0.50 0.75 1.00

To each of the above system add excess of drug with continuous shaking for 2 hours to form a saturated solution and keep for 24 hours. Filter the above solution through whatmann filter paper. Dilute the above filtrate with distilled water to obtain absorbance of diluted solution in the range of standard graph and find out solubility of drug in the solvent system and report. Plot a graph of solubility Vs concentration of surfactants. Absorbance of diluted solution is measured at max 276nm in UV-visible spectrometer

REPORT: As the concentration of surfactant increases, the solubility of drug also increases.

2. DEGRADATION OF ASPIRIN USING ACCELERATED STABILITY STUDIES


AIM: To determine the shelf life and energy of activation of aspirin solution in 0.1N HCl using accelerated stability studies. REQUIREMENTS: Salicylic acid, Ferric nitrate solution (4%), 0.1N Hydrochloric acid, Aspirin, Distilled water, Volumetric flasks, Conical flasks, Pipette, Test tubes, Thermostatic water bath, Calorimeter. THEORY: The drug purchased from the retail shop contains the expiry date on the label of the pack. The expiry date is an assurance given by the manufacturer that the product should be taken before the expiry date. The dosage form fulfils the specifications present on the label regarding identity, strength and purity. The drug control department ensures through regulatory control that every product released into the market should be evaluated to fix the expiry date. The technical term for expiry date is shelf life. Shelf life is defined as the time required for a drug to reduce its concentration to 90% of the labeled concentration. The evaluation of shelf life is essential because the stability of a drug in dosage form can be influenced by the normal environmental conditions of storage (temperature, light, air and humidity) and packing conditions. Drugs such as esters (e.g. aspirin and procaine) and amides (eg: chloramphenicol) undergo hydrolytic reactions during storage under normal conditions. Similarly drugs such as ascorbic acid, promethazine undergo oxidation reactions. As a result the drug may not exhibit the desired effect and may show reduced biological response. Since degradation or decomposition involves chemical alteration (reaction) of the active ingredients, the knowledge of chemical kinetics is helpful to predict the reaction rates and further to evaluate shelf life and stability considerations which include changes in physical appearance of the products. In older method, a product is placed at room temperature or in refrigerator in order to mimic the storage conditions and the stability is evaluated for a prolonged period of 25yrs. Such a design of experimentation is time consuming and uneconomical.

Carrying out the reaction at elevated temperature accelerates the reaction rate. Therefore by conducting the experiment, a factor is obtained that has a linear relationship with temperature.The analytical method is such that starting material or decomposed product alone can be analyzed while other compound does not interfere with the method of estimation. Therefore a stability indicating assay procedure is essential for conducting the stability studies. The results of accelerated decomposition are extrapolated to the room temperature (i.e. 25oc) or specific refrigerator conditions. We generally assume that the drug reaction is zero order or first order. An accelerated stability study is an experimental design to evaluate the stability of a product by accelerating the rate of reaction. A more comprehensive pharmacological approach (USP) involves the evaluation of 1) Chemical integrity 2) Physical changes Test for sterility, resistance to microbial growth, toxicity and bioavailability studies necessary are carried out when ever such studies enable the production of shelf life for each dosage form in its package under the recommended conditions. INFLUENCE OF TEMPERATURE ON DEGRADATION: In general the rate of reaction increases with rise in temperature. Arrhenius established a more quantitative relationship between temperature and rate of reaction. The experimental equation is K = A e Ea/RT Where K= specific rate constant A = frequency factor or Arrhenius factor Ea = energy of activation R = ideal gas constant T = absolute temperature Arrhenius factor or frequency factor is a measurement of frequency of collision that cannot be expected between the reacting molecules in a reaction. Energy of activation is defined as minimum energy required by the molecule so that molecular collisions give useful products Log k = log A- Ea/2.303R 1/T 6

According to equation a plot can be drawn by taking log k on y-axis and reciprocal temperature (1/T) on x-axis. The slope gives the energy of activation. Ea = slope 2.303 R Where R = 1.897 cal/deg.mol Kinetic studies are carried out at elevated temperature to accelerate the degradation. ASPIRIN DEGRADATION: During storage conditions aspirin undergoes hydrolysis to salicylic acid and acetic acid. Aspirin follows pseudo first order kinetics in the acidic medium. In the present study aspirin is hydrolyzed using 0.1N hydrochloric acid and salicylic acid is formed which is analysed at the max 547 using spectrophotometer. Since the study is carried out at room temperature 500c and 600c. Arrhenius equation K = A e
Ea/RT

is used to find out specific reaction rate constants at

this temperature. This kind of study is useful in finding half life, shelf life and energy of activation. PROCEDURE: SALICYLIC ACID STOCK SOLUTION (1MG/ML): Weigh accurately about 100mg of salicylic acid and transfer into a 100ml volumetric flask. Add distilled water and shake the solution thoroughly to dissolve salicylic acid. Finally make up the volume to the mark. FERRIC NITRATE SOLUTION (4%): Weigh 4gm of ferric nitrate and transfer into a 100ml measuring cylinder or volumetric flask. Add 1ml of concentrated HNO3 then add water and dissolve the solid. Make up the volume to the mark. Transfer the solution into an amber color bottle and label. CONSTRUCTION OF STANDARD GRAPH: Pipette out 0.2, 0.3, 0.4, 0.6, 0.8 and 1ml of the above stock solution in 20ml of test tube and dilute up to 10ml with distilled water. To each of the above solution and 5ml of ferric nitrate solution and develop a purple color. Measure the absorbance of above solution at max 547nm by colorimeter using solution prepared in the same way except blank (10ml of distilled water and 5ml of ferric nitrate solution). Draw a graph by taking absorbance on x-axis and concentration on y-axis.

ACCELERATED STABILITY STUDIES: Aspirin decomposition (hydrolysis): Switch on the water bath and adjust the temperature to 50oc. Transfer 100ml of 0.1N hydrochloric acid to conical flask and keep aside. Weigh 100mg of aspirin and transfer to 250ml conical flask. Add 1ml or 2ml of alcohol to dissolve aspirin. Add slowly 90ml of 0.1N hydrochloric acid and shake well. Finally add 10ml of 0.1N hydrochloric acid cork the flask and keep on water bath. Withdraw 10ml of sample every 10min intervals from 0min-90min (total 10 samples). Add 5ml of ferric nitrate solution to each test tube. The solute turns into purple color. Plot the graph by taking time Vs log % aspirin undecomposed. REPORT: The accelerated stability studies were performed and energy of activation of aspirin was found to be 0.4368k cal/mole.

3. EFFECT OF PH ON SOLUBILITY OF DRUG


AIM: To determine the effect of pH on solubility of drug. REQUIREMENTS: Diclofenac, HCl, KCl, distilled water, NaOH, Potassium hydrogen phthalate, Potassium dihydrogen phosphate, Boric acid. PRINCIPLE: Whether a substance dissolves in a given system or not and the extent to which it dissolves depends largely on the nature and the intensity of the forces present on the solute, the solvent and the solute solvent interaction. The nature of these interaction energies and steric factors determine the solubility of substances in various classes of solvent.A large number of approaches have been used to increase the solubility of the given drug in the solvent of our interest. They are: 1. Use of surfactant 2. Use of salt form 3. Use of PH of solvent 4. Use of metastable polymorph A large number of modern chemotherapeutic agents are either weak acid or weak bases. The solubility of these agents can be markedly influenced by pH of their environment. Through application of law of mass action, the solubility of weak acidic or basic drugs can be predicted, as a function of pH with a considerable deleterious accuracy. Consider the example of reaction involved in the dissolution of weakly acidic drug OH (solid) OH (solution)

Where OH (solution) is equal to the solubility of the undissociated acid in moles/lit and is a constant generally referred to ks. The undissociated acid is also in equilibrium with its dissociation product. OH (solution) O -+H+

Ka = [O -][H +]/ [OH] O - = ka [OH]/ [H +] 9

The total amount of drug in solution is the sum of ionized form [O -] and unionized form [OH]. The equation for total solubility can be written as St = [OH] + [O -] = [OH] +ka [OH]/ [H+]
Since OH has previously been defined as equal to ks,

St= ks + ks . ka /[H +]

ks [1+ka /[H +]]

This equation is the most useful one for determining the total solubility of a weak acid at a specific hydrogen ion concentration. To know what must be pH of the formulation to be maintained and to predict the amount of drug in the solution, the modified form of above equation is useful as follows: St - ks = ks . ka / [H +] [H+] = ks. ka / st - ks Similarly for weak bases, [OH] = ks.kb / st -ks In selecting pH of environment for adequate solubility several other factors should be considered. The pH that satisfies solubility requirement such as stability and physiological compatibility must be used. In addition pH is critical to maintain drug solubility; the system must be adequately buffered. The selection of buffer must be consistent with the following criteria: 1. The buffer must have adequate capacity in the desired pH range. 2. The buffer must be biologically safe for the intended use. 3. The buffer should have little or no effect on the stability of the final product. For many such as weak acids or bases, the required pH may be inacceptable in terms of physiological considerations or owing to effect of pH extremes on the stability of drug itself. In such case other approaches such as co-solvents, surfactant and solubilization by complexation are used to improve the solubility and stability. PROCEDURE: PREPERATION OF PH 1.2 (HCl BUFFER): 50ml of 0.2M KCl is added to 85ml of 0.2M HCl and diluted to 200 ml with water. 0.2M KCl: 14.911gm of KCl was taken and made up to 1000ml with water. 0.2M HCl: 7.292gm of HCl was taken and made up to 1000ml with water.

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PREPERATION OF PH 4.8 (POTASSIUM HYDROGEN PTHALATE) BUFFER: 50ml of 0.1M potassium hydrogen phthalate is added to 16.5ml of 0.1N NaOH .The volume was made up to 100ml with water. 0.1M potassium hydrogen phthalate: 20.4gm of potassium hydrogen phthalate was dissolved in water and volume was made up to 1000ml. 0.1M NaOH: 4gm of NaOH was dissolved in water and made up to 1000ml. PREPERATION PHOSPHATE): 25ml of potassium dihydrogen phosphate was mixed with 19.4 ml of 0.1N NaOH and volume made up to 200ml. 0.1M KH2PO4: 36.1 gm of KH2PO4 was dissolved in water and the volume was made up to 1000ml. 0.1M NaOH: 4g of NaOH was dissolved in water and the volume was made up to 1000ml. PREPERATION OF PH 8.2 (BORATE BUFFER): 50 ml of 0.8M boric acid was placed in a 200ml volumetric flask. 6ml of 0.2M NaOH was added and then water was added to make up the volume. 0.2M boric acid: 12.36gm of boric acid was dissolved in water and the volume was made up to 1000ml. CONSTRUCTION OF STANDARD GRAPH: 100mg of the drug was weighed accurately and dissolved in about 3/4th volume of distilled water in 100ml volumetric flask and the volume was made up to 100ml (1g/ml). 10ml from the above solution was pipette out and the volume was made up to 100ml which gives 100g/ml. 0.1ml, 0.2ml, 0.3ml, 0.4ml and 0.5ml was taken from the 100g/ml solution and each was diluted to 10 ml which gave 1g/ml, 2g/ml, 3g/ml, 4g/ml and 5g/ ml respectively. The absorbance of the above solutions was measured at the max 254 (diclofenac) using water as blank. A standard graph was plotted taking concentration on X-axis and absorbance on Y-axis. PREPERATION OF BUFFER DRUG SOLUTION: OF PH 7.4 BUFFER (POTASSIUM DIHYDROGEN

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10ml of various pH solutions i.e. 1.2, 4.8, 7.4 and 8.2 are taken in different test tubes respectively. To each of the above excess of drug was added with occasional shaking for 24 hrs to form a saturated solution. The above solution was filtered through whatmann paper. The absorbance of the diluted solution is measured at the max 254 using a UV spectrophotometer after necessary dilutions with distilled water. Drug content was analysed and reported as moles/lit. A graph was plotted taking different pH values on X-axis and solubility of the drug (moles/lit) on Y-axis. REPORT: The given drug (diclofenac) showed highest solubility at pH 4.8

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4.EFFECT OF SALT FORMATION ON SOLUBILITY


AIM: To study the effect of salt formation on solubility of drug. REQUIREMENTS: UV spectrophotometer, test tubes, pipettes, filter paper, funnel, shaker, balance. PRINCIPLE: Most drugs are either weak acids or bases. One of the earliest approaches to enhance the solubility and dissolution rate of such drugs is to convert them into their salt forms. Generally with weakly acidic drugs, a strong base salt is prepared such as sodium and potassium salts of barbiturates and sulfonamides. In case of weakly basic drugs, a strong acid salt is prepared like HCl or sulfate salts of several alkaloidal drugs. At a given pH the solubility of a drug whether acidic or basic or its salt form is a constant. The influence of salt formation on the solubility rate of dissolution and absorption can be explained by considering the pH of diffusion layer and not the PH of bulk of the solution. (Refer diffusion layer theory of drug) Consider the case of a salt of a weak acid. At any given pH of the bulk of the solution, the PH of the diffusion layer (saturation solubility of drug) of the salt form of a weak acid will be higher than that observed with free acid form of a drug. Owing to the increased pH of diffusion layer the solubility and dissolution rate of a weak acid in this layer is promoted. (Higher pH favors the dissolution of a weak acid) Thus if dissolution is faster, absorption is found to be rapid. In case of salts of weak bases, pH of diffusion layer will be lower in comparison to that found with free base form of drug. Consequently the solubility of basic drugs at their lower pH is enhanced. [H+] d = Hydrogen ion concentration in diffusion layer [H+] b= Hydrogen ion concentration in bulk of the solution For salts of a weak acids [H+] d < [H+] b For salts of weak bases [H+] d > [H+] b The decrease or increase in pH of diffusion layer by the salts of a weak acid and weak bases have been attributed to the buffering action of the strong base cation and strong acid anion respectively. Another reason for enhancing solubility of salts of weak 13

acid is the precipitation of drug as very fine particles. When the soluble ionic form of drug diffuses from stagnant layer into the bulk of the solution, whose pH is low, it is transferred into the free acid form having lesser aqueous solubility at the lower pH of the bulk of the solution. Consequently the free acidic form of drug is precipitated in the form of fine particles. The resultant increase in the surface area is then responsible for the rapid dissolution and absorption in comparison to drug administered in just the acidic form. The principle of insitu salt formation has been utilized to enhance the dissolution and absorption rate of certain drugs like aspirin and penicillin from buffered alkaline tablets. The approach is to increase the pH of micro emulsion of drugs by incorporating buffer agents and promote dissolution rate. Apart from enhanced bioavailability buffered aspirin tablets have two more advantages. Gastric irritation and ulcerogenic tendency of drug is greatly decreased. Problems with the use of sodium salts of aspirin (to enhance the solubility) which otherwise has poor hydrolytic stability is overcome by the insitu salt formation. The selection of appropriate salt form for better dissolution rate is also important. It has been shown that the choline and the isopropanaline salts of theophylline dissolve 34 times more rapidly than the ethylenediamine salt and show better bioavailability. A factor that influences the solubility of salt form of drug is the size of counter ion. Generally, smaller the size of the counter ion, greater the solubility of salt. Ex: Novobiocin bioavailability from its sodium salt, calcium salt and free acid form was found to be in the ratio 50:25:1 where the counter ion is very large in size and has poor ionic strength. The solubility may be lower than the free drug itself for example the stearates and palmitates of weak bases have poor aqueous solubility. These forms are however useful in several ways such as to prolong the duration of action to overcome the bad taste, to enhance GI stability or to decrease the side effects of local or systemic. There are exceptions where the so called more soluble salt form of the drug showed poor bioavailability. One such study was the comparative dissolution of sodium phenobarbital and free phenobarbital drom their tablets. Slower dissolution with sodium salt was observed and the reason attributed to it was that its tablet swelled but did not disintegrate and thus dissolved slowly. An identical result was obtained with hydrochloride salts of several tetracycline analogs and papaverine; better dissolution and bioavailability was observed with the free bases. The reason for poor solubility and dissolution tare was the suppression action of the common ion effect.

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PROCEDURE: 100mg of drug was weighed and dissolved in 100 ml of different solvents. This becomes 1g/ml. From the above solution 10 ml was pipetted out and diluted to 100ml. This becomes 100g/ml. From this 0.1, 0.2, 0.3, 0.4 and 0.5ml was pipetted out and diluted to 10 ml which gave1g/ml, 2g/ml, 3g/ml, 4g/ml and 5g/ml respectively. 0.1%, 0.2%, 0.3% of different salt solutions of Kcl, Nacl, and KBr was prepared and saturated with drug and was kept aside for 24 hrs. Then the residue was filtered and the absorbance was taken using UV spectrophotometer after suitable dilutions. Then a graph was drawn taking salt concentration on X-axis and solubility on Y-axis. REPORT: The effect of salt formation on the solubility of the drug was studied. The given drug (diclofenac) showed highest solubility at a salt concentration of 0.1% with NaCl

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5. EFFECT OF SOLID DISPERSION BY DIFFERENT TECHNIQUES ON DISSOLUTION

AIM: To prepare a solid dispersion of poorly water soluble drug and to study the effect of dissolution rate. REQUIREMENTS: Volumetric flask, Test tubes, China dish, Glass rod, Beaker, Dissolution test apparatus, U.V spectrophotometer, Balance, pH-meter. PRINCIPLE: Over the years, a variety of solubilisation techniques have been studied and widely used. By many estimates up to 40% of new chemical entities discovered by the pharmaceutical industry today are poorly soluble or lipophillic compounds. The solubility issues complicating the delivery of these new drugs also affect the delivery of many existing drugs. Various techniques are available for enhancement of solubility. Solid dispersions is one of the most promising approach for solubility enhancement. The term solid dispersions refer to a group of solid products consisting of atleast two different components generally a hydrophilic matrix and a hydrophobic drug. The matrix can be either crystalline or amorphous. TYPES OF SOLID DISPERSIONS: Simple eutectic mixture Solid solutions Glass solutions Amorphous precipitations in a crystalline carrier

DIFFERENT METHODS USED FOR PREPARING SOLID DISPERSIONS: Melting method Solvent method Melting solvent method ( Melt evaporation ) Melt extrusion method

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Lyophilisation technique Melt agglomeration process Use of surfactants Electro spinning Super critical fluid technology

The development of solid dispersions as a practically viable method to enhance bioavailability of poorly water soluble drugs overcome the limitations of previous approaches such as salt formation. Solubilisation by cosolvents, particle size reduction. Drug in solid dispersion need not necessarily exist in micronized state. A fraction drug might molecularly disperse in the matrix thereby forming a solid dispersion.When solid dispersions are exposed to aqueous media, the carrier dissolves and the drug releases as fine colloidal particles. The resulting enhanced surface area produces higher dissolution rate and bioavailability of poorly water soluble drugs.In addition, in solid dispersion a portion of drug dissolves immediately to saturate the gastrointestinal fluid and excess drug precipitates as fine colloidal particles or oily globules of submicron size. LIMITATIONS OF SOLID DISPERSIONS: Limitations of this technology have been a drawback for the commercialization of solid dispersions. The limitations include Laborious and expensive methods of preparation. Reproducibility of physiochemical characteristics. Difficulty in incorporating into formulation of dosage forms. Scale up of manufacturing process. Stability of drug and vehicle. Its method of preparation.

SUITABLE PROPERTIES OF A CARRIER FOR SOLID DISPERSIONS: Following criteria should be considered during selection of carriers High water solubility High glass transition point Minimal water uptake 17

Soluble in common solvent with drug Relatively low melting point Capable of forming a solid dispersion with the drug

FIRST GENERATION CARRIER: Crystalline carrier: Urea, sugars, organic acids. SECOND GENERATION CARRIER: Amorphous carriers: Polyethylene glycol, povidone, polyvinyl acetate,

polymethacrylate, cellulose derivatives. THIRD GENERATION CARRIER: Surface active self emulsifying carriers: Poloxamer 408, tween 80, gelusire 44/14. SOLVENT SELECTION FOR SOLID DISPERSIONS: In order to prepare solid dispersions, solvent should be selected on the basis of following criteria. Dissolve both drug and carrier Ionic solvents to be avoided Ex: dichloromethane Ethanol less toxic is alternative Water based systems preferable Use of surfactants to create carrier drug solutions but care should be taken as they reduce tg point.

Class 1 solvents (Solvents to be avoided) ex: Benzene, carbon tetra chloride, 1, 2dichloro ethane, 1, 1-dichloroethane. Class 2 solvents (Solvents to be limited) ex: Chlorobenzene, chloroform, ethylene glycol, methanol, pyridine, toluene. Class 3 solvents (Solvents with low toxic potential) ex: Acetic acid, acetone, ethanol, heptane, propanol, ethylether, formic acid, butanol Class 4 solvents (Solvents for which no adequate toxicological data found) ex: Petroleum ether, isopropyl ether. PROCEDURE: 1. FUSION METHOD: The drug (salicylic acid) and the carrier was accurately weighed, and are mixed in china dish. Heat the drug and carrier mixture on heating plate, till a homogeneous mixture was obtained.

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Excess of heat can cause charring; A tile which was previously kept in refrigerator was taken. This tile was placed on ice tray and a homogenous mixture of drug and carrier was poured onto it, which cause sudden cooling. This molten mass was stirred and scrapped with help of spatula. A solid mass was obtained. An amount equivalent to 100mg was taken. 2. MELTING METHOD: Melt 1mg of polymer in china dish with constant stirring until it melts completely. Then slowly add 1mg of drug, continue the heating till the uniform melt forms. Allow the melt to cool at room temperature. Then scratch the material and make it into a fine powder by mortar and pestle. Pass it through the sieve 80 to obtain uniform particles. 3. SOLVENT EVAPORATION: 1gm of urea and 1gm of drug are weighed and dissolved in 25 ml of 90% of ethanol. Evaporate the mixture in a water bath at 45oC until complete evaporation of solvent occurs. Further dry the solid dispersion in a desiccator for 12 hrs. Pass it through sieve 80 to obtain uniform size particles of solid dispersion. CONSTRUCTION OF STANDARD GRAPH: 100mg of the drug was weighed accurately and dissolved in about 3/4th volume of distilled water in 100ml volumetric flask and the volume was made up to 100ml (1g/ml). 10ml from the above solution was pipette out and the volume was made up to 100ml which gives 100g/ml. 0.1ml, 0.2ml, 0.3ml, 0.4ml and 0.5ml was taken from the 100g/ml solution and each was diluted to 10 ml which gave 1g/ml, 2g/ml, 3g/ml, 4g/ml and 5g/ ml respectively. The absorbance of the above solutions was measured at the max547 using water as blank. A standard graph was plotted taking concentration on X-axis and absorbance on Y-axis. DISSOLUTION PROCEDURE: Take 200 mg of prepared solid dispersions of different ratios of prepared by solvent evaporation method.

1:1, 1:3, 1:6

Set the dissolution apparatus and dissolution flask were filled with buffers of ph 7.8. Maintain the temperature of water bath at 37.5oC and system rpm at 50. 19

Start the dissolution procedure and withdraw the samples (5ml) at different time intervals and replace 5ml of buffer solution into dissolution flask. Check the absorbance of sample doing necessary dilution at the max 547 by taking buffer as blank. PHOSPHATE BUFFER PREPARATION (PH 7.8): Prepare 200ml of pH 7.8 buffer by taking 50ml of 0.2M potassium dihydrogen phosphate in a 200ml volumetric flask and 44.5 ml of 0.2 M sodium hydroxide and then add water to make up the volume. 0.2M POTASSIUM DIHYDROGEN PHOSPHATE PREPARATION: Dissolve 27.218 gm of potassium dihydrogen phosphate in water and dilute with water to 1000ml. 0.2M SODIUM HYDROXIDE PREPARATION: Dissolve 8 gm of sodium hydroxide in distilled water and dilute to 1000ml. REPORT: Solid dispersions of salicylic acid was prepared and dissolution was conducted. The dissolution profile of solid dispersions and pure salicylic acid were compared, solid dispersions dissolve to a great extent than pure drug.

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6.TESTING THE DEGRADATION OF COMPOUNDS USING TLC FOR ASCORBIC ACID

AIM: To test the degradation of compounds using TLC for ascorbic acid. REQUIREMENT: Silica gel, TLC plates, autoclave, ascorbic acid, sodium hydroxide. PRINCIPLE: Thin layer chromatography is based on the principles of column and partition chromatography. Separation of a mixture in this case is achieved over a thin layer of aluminum oxide or silica gel to which they are adsorbed by different physical forces. The resolved mixture may be extracted with suitable solvents. Thin layer chromatography is the separation technique to separate drug or mixture of drugs which is based on the theory of capillary action. Thin layer chromatography works on the principle of different compounds having different adsorption and distribution. Thin layer chromatography is the physical method of separation in which the components to be separated are distributed between two phases i.e., stationary phase and the mobile phase In thin layer chromatography if silica is used as stationary phase the principle is partition. If alumina is used ten the principle is adsorption. Separation depends upon how much the substance get adsorbed on the stationary phase versus how much it dissolves in the mobile phase. Silica gel coated onto an inert solid support such as a glass plate. Mobile phase used is solvents of different polarity. When mixture of components or sample is spotted on the prepared TLC plate and placed in mobile phase they get separated into individual components. Silica contains some free S-OH groups. These groups form H- bonds or other vanderwaal interactions with the analyte components thus adsorption takes place. The retention factor is determined, which is specific for a compound, and it is calculated by the formula. Rf = Distance traveled by the solute. Distance traveled by the solvent front Drug decomposition or degradation occurs during storage because of chemical alteration of the active ingredients or additives. Stability studies are necessary for following reasons 21

1. Product instability 2. Chemical degradation of active drug may lead to low strength or lower concentration of drug in dosage. Ex: Digoxin, theophyllin 3. Instability due to change in physical appearance. Ex: Mottling of tablets, creaming of emulsion or caking of solid dispersions. HYDROLYSIS: The principle involved is hydrolytic reaction is as follows Drug with ester and amide groups react with 1 mole of water and undergo hydrolysis. Ester groups break faster than amide groups. Hydrolytic reactions are catalyzed by H+/OH- ions. OH- ions catalyze hydrolysis by about 10-1000 times more than H+ ions. Few drugs which decompose by hydrolysis path ways are Esters Aspirin, Atropine, Procaine Amide Chloramphenicol, Cephalosporin, Ampicilin OXIDATION: Oxidation involves the removal of electrons from a molecule. Spontaneous reaction between the compounds and molecule is called auto oxidation. The general principle that governs oxidation reaction is listed as follows.

Presence of atmospheric oxygen [air] promotes the oxidation reaction. Since oxidation, frequently involves free radical chain reaction, light provide necessary energy to initiate the oxidation process. Presence of trace metals also accelerates the oxidation process. Organic peroxides initiate chain reaction and propagate the oxidation process. Drug which decompose by oxidation process are Arachis oil, Epinephrine, Vitamin A, B12 riboflavin. PHOTOLYSIS: Light energy, light activates molecules and enhance rate of reaction. Drugs which undergo light induced chemical degradation are called photo liable drugs or photosensitive drugs. Ex.: Riboflavin, tetracycline, and chlorpromazine. Few photo chemical degradation reactions are: Color developments/color fading of tablets or liquids are few examples of photo degradation. Conversion of ergosterol to vitamin D by UV light.

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Photosynthesis where carbohydrate and oxygen are produced when chlorophyll absorbs visible light. PROCEDURE: ASCORBIC ACID: Prepare mobile phase (methanol: glacial acetic acid: acetone) in 1:8:1 ratio and the chamber was saturated. TLC plate was taken and markings were made on it. Little amount of ascorbic acid was taken and dissolved in water. Immediately it was spotted by capillary tube on TLC plate. Ascorbic acid and sodium hydroxide were dissolved They were spotted on TLC plate was run, later it was dried and spots were observed and Rf value is calculated. REPORT: Degradation of compound was checked using TLC. The Rf value of the pure sample of ascorbic acid was found to be 0.5 After 10min of addition of NaoH, Rf value was found to be 0.18

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7.DISSOLUTION METHODS OF TRANSDERMAL DRUG DELIVERY SYSTEM-GEL


AIM:

To study the dissolution methods of transdermal drug delivery systems.


REQUIREMENTS:

Glassware: Beakers, Test tubes, pipettes, volumetric flask Chemicals: Diclofenac Sodium, carbapol (940 grade), triethanolamine, propylene glycol, pH 7.4 buffer. Apparatus: Magnetic Stirrer. Instrument: Single beam UV-spectrophotometer
PRINCIPLE:

Transdermal drug delivery system aims to achieve the objective of systemic mediation through topical application to the skin surface. Transdermal drug delivery system bypass the hepatic first pass elimination and maintains the constant, prolonged and therapeutically effective drug levels in the body. For a systemically active drug to reach a target tissue remote from the site of drug administration on the skin surface, it must possess physico-chemical properties that facilitate the sorption of drug by the stratum corneum, the penetration of drug through the viable epidermis and also the uptake of drug by microcirculation in the dermal papillary layer. The rate of permeation dQ/dt across various layers of skin tissues can be expressed mathematically as dQ/dt = s (Cd - Cr) where Cd and Cr Concentrations of skin penetrate in the donor phase and in the receptor phase s Overall permeability coefficient of the skin tissues to the penetrate The kinetics of skin permeation can be more precisely analysed by studying the permeation profiles of drug across a freshly excised skin specimen mounted on the diffusion cell, such as Franz diffusion cell to gain a fundamental understanding of the skin permeation kinetics of drugs and to assist the formulation development of transdermal drug delivery systems.

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In-vitro skin permeation studies using a freshly excised skin sample mounted in a hydrodynamically in a well calibrated skin permeation cell is considered a cost and time saving approach. TECHNOLOGIES FOR DEVELOPING TRANSDERMAL DRUG DELIVERY SYSTEMS: They provide rate control over the skin permeation of drugs 1. Polymer membrane permeation controlled TDDS 2. Polymer matrix diffusion controlled TDDS 3. Drug reservoir gradient controlled TDDS 4. Micro reservoirs dissolution controlled TDDS EVALUATION OF TRANSDERMAL DRUG DELIVERY KINETICS The release and skin permeation kinetics of drug from these technologically different transdermal drug delivery systems can be evaluated using a two compartment diffusion cell assembly under identical conditions. Diclofenac gels, marketed and prepared are subjected for dissolution. This is carried out by individually mounting gelatin sheet on a vertical diffusion cell, such as franz diffusion cell and its modification, or a horizontal diffusion cell, such as valio-chien skin permeation cell. The unit is then applied with drug releasing surface in intimate contact with gelatin sheet.Permeation profile of the drug is followed by sampling the receptor solution at pre-determined intervals until steady state flux is established and assaying drug concentration in the sample. The rates of drug release from these marketed diclofenac gel and prepared gel were evaluated and compared. ADVANTAGES Transdermal rate controlled drug delivery offers one or more of the following potential biomedical benefits 1. Avoid the risks and inconvenience of intravenous therapy. 2. Bypass the variation in the absorption and metabolism associated with oral administration. 3. Increase the bioavailability and efficacy of drugs through the bypass of hepatic first pass elimination 4. Reduce the chance of over or under dosing through the prolonged, pre programmed delivery of drug at the required therapeutic rate. 5. Provide a simplified therapeutic regimen leading to better patient compliance.

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6. Permits a rapid termination of the medication if needed by simply removing the TDDS from the skin surface.

PROCEDURE: PREPARATION OF PH 7.4 PHOSPHATE BUFFER SOLUTION 50ml of 0.2M potassium dihydrogen phosphate was taken in 200ml volumetric flask, then 39.1ml of 0.2M NaOH was added. volume was made upto 200ml with distilled water PREPARATION OF 0.2 M POTASSIUM DIHYDROGEN PHOSPHATE SOLUTION 27.2g of potassium dihydrogen phosphate was taken and dissolved in small quantity of distilled water and the final volume was made upto 100ml with water. PREPARATION OF 0.2M SODIUM HYDROXIDE SOLUTION 8g of sodium hydroxide was accurately weighed and dissolved in the little quantity of water and then the final volume was made upto 1000ml. STANDARD GRAPH FOR DICLOFENAC SODIUM 100mg of diclofenac sodium pure drug was dissolved in 5ml of alcohol and remaining amount of 95ml made up with 7.4 pH buffer. This is stock 1 with concentration 1mg/ml (1000 g/ml). Subsequent dilutions are made to get concentrations of 1,2,3,4,5 g/ml by taking 0.1,0.2,0.3,0.4,0.5 from stock 2 and made upto 10ml and absorbance was measured at max of 276 nm DISSOLUTION STUDIES OF TRANSDERMAL GEL Dissolution studies of transdermal gel of diclofenac sodium marketed OMNI gel 30g and prepared diclofenac sodium gel were conducted. Procedure for preparing transdermal gel Carbapol (940 grade)- 1% Propylene glycol- 2ml Triethanol amine- 2ml 1g of carbapol (940 grade) was taken in beaker and 100ml water was added and made homogenized by homogenizer for 30mins. Drug solution (1%) was prepared by dissolving in alcohol i.e., 5mg diclofenac sodium was dissolved completely by adding alcohol. This solution was added to homogenized gel and made dispersed for 30mins. 3-4 drops of propylene glycol which acts as humectants was added to the above mixture and triethanolamine was added (3-4 drops) The mixture was homogenized well for 1hour. 26

In Vitro drug release studies were performed by using a Franz diffusion cell with a receptor compartment capacity of 50 mL. The artificial semi permeable membrane was used for the determination of drug from the prepared transdermal matrix-type patches. The membrane having a pore size 0.45 was mounted between the donor and receptor compartment of the diffusion cell. The prepared transdermal film was placed on the membrane and tied with donar compartment of franz diffusion cells.The receptor compartment of the diffusion cell was filled with phosphate buffer pH 7.4. This whole assembly was placed on a hot plate magnetic stirrer, and the solution in the receptor compartment was constantly and continuously stirred using magnetic beads, and the temperature was maintained at 37 0.5C, because the normal skin temperature of human is 32C. The samples were withdrawn at different time intervals and analyzed for drug content spectrophotometrically. The receptor phase was replenished with an equal volume of phosphate buffer at each sample withdrawal. The rates of drug release from these marketed diclofenac gel and the prepared gel were evaluated and compared.
REPORT: Invitro drug release of transdermal drug delivery system of diclofenac sodium gel

was conducted Percentage drug release of marketed diclofenac sodium gel (OMNI-30g ) after 150mins was found to be 69% and that of diclofenac sodium gel prepared was found to be 62%

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8. DISSOLUTION METHODS OF TRANSDERMAL PATCH


AIM: To study the dissolution method of transdermal drug delivery systems. REQUIREMENTS: Beakers, Test tubes, pipettes, volumetric flask Diclofenac Sodium,PVP, triethanolamine, propylene glycol, pH 7.4 buffer. Magnetic Stirrer Single beam UV-spectrophotometer
PRINCIPLE:

Transdermal drug delivery system aims to achieve the objective of systemic mediation through topical application to the skin surface. Transdermal drug delivery system bypass the hepatic first pass elimination and maintains constant, prolonged and therapeutically effective drug levels in the body. For a systemically active drug to reach a target tissue remote from the site of drug administration on the skin surface, it must possess physico-chemical properties that facilitate the sorption of drug by the stratum corneum, the penetration of drug through the viable epidermis and also the uptake of drug by microcirculation in the dermal papillary layer The rate of permeation dQ/dt across various layers of skin tissues can be expressed mathematically as dQ/dt = s (Cd - Cr) where Cd and Cr concentration of skin penetrates in the donor phase and in the receptor phase s Overall permeability coefficient of the skin tissues to the penetrate The kinetics of skin permeation can be more precisely analysed by studying the permeation profiles of drug across a freshly excised skin specimen mounted on the diffusion cell, such as franz diffusion cell to gain a fundamental understanding of the skin permeation kinetics of drugs and to assist the formulation development of transdermal drug delivery systems. In-vitro skin permeation studies using a freshly excised skin sample mounted in a hydrodynamically well calibrated skin permeation cell are considered a cost and time saving approach.

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PROCEDURE: PREPARATION OF PH 7.4 PHOSPHATE BUFFER 50ml of 0.2M potassium dihydrogen phosphate was added in 200ml volumetric flask, then 39.1ml of 0.2M NaOH was added. Volume was made upto 200ml with distilled water PREPARATION OF 0.2M POTASSIUM DIHYDROGEN PHOSPHATE 27.2g of potassium dihydrogen phosphate was taken and dissolved in small quantity of distilled water and the final volume was made upto 100ml with water. PREPARATION OF 0.2M SODIUM HYDROXIDE SOLUTION 8g of sodium hydroxide was accurately weighed and dissolved in the little quantity of water and then the final volume was made upto 1000ml. STANDARD GRAPH FOR DICLOFENAC SODIUM 100mg of the drug was weighed accurately and dissolved in about 3/4th volume of distilled water in 100ml volumetric flask and the volume was made up to 100ml (1g/ml). 10ml from the above solution was pipette out and the volume was made up to 100ml which gives 100g/ml. 0.1ml, 0.2ml, 0.3ml, 0.4ml and 0.5ml was taken from the 100g/ml solution and each was diluted to 10 ml which gave 1g/ml, 2g/ml, 3g/ml, 4g/ml and 5g/ml respectively. The absorbance of the above solutions was measured at the max547 using water as blank. A standard graph was plotted taking concentration on X-axis and absorbance on Y-axis. PREPARATION OF TRANSDERMAL PATCH 100 mg of the drug is weighed accurately and transferred to a beaker. A solution of 1.25% of HPMC is added to the above beaker. A solution of 0.75% PVP is added to the above mixture. A mercury plate and a rim (whose volume is found to be 4ml) were taken. The rim is placed on the surface of the mercury. The drug mixture is poured on the surface of the mercury inside the rim slowly and evenly. We must ensure that there is no leakage from the rim and the drug mixture doesnt spread on the surface.

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This is kept undisturbed for 24hrs. The rim is removed carefully from the surface of the mercury. The transdermal patch is separated from the rim.

DISSOLUTION STUDIES OF TRANSDERMAL PATCH: In Vitro drug release studies were performed by using a Franz diffusion cell with a receptor compartment capacity of 50mL. The artificial semi permeable membrane was used for the determination of drug from the prepared transdermal matrix-type patches. The membrane having a pore size 0.45 was mounted between the donor and receptor compartment of the diffusion cell. The prepared transdermal film was placed on the membrane and tied with donar compartment of Franz diffusion cells. The receptor compartment of the diffusion cell was filled with phosphate buffer pH 7.4. This whole assembly was placed on a hot plate magnetic stirrer, and the solution in the receptor compartment was constantly and continuously stirred using magnetic beads, and the temperature was maintained at 370.5C, because the normal skin temperature of human is 32C. The samples were withdrawn at different time intervals and analyzed for drug content spectrophotometrically. The receptor phase was replenished with an equal volume of phosphate buffer at each sample withdrawal. REPORT: Invitro drug release of transdermal drug delivery system of diclofenac sodium patch was conducted. Percentage drug release of diclofenac sodium patch after 5 hours was found to be 47%

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9. DISSOLUTION STUDIES OF DICLOFENAC IN BIORELEVANT DISSOLUTION MEDIA


AIM: To conduct dissolution studies of drug (paracetamol) in biorelevant dissolution media. REQUIREMENTS: Paracetamol, Sodium chloride, Hydrochloric acid. Volumetric flask, pipettes, test tube, beaker and funnel, Dissolution apparatus: USP-1 rotating paddle, UV spectrophotometer. THEORY: Dissolution is a process in which a solid substance solubilises in a given solvent i.e., mass transfer from the solid surface to the liquid phase. Pharmaceutical solid dosage from and solid-liquid dispersed dosage from on administration undergo dissolution in biological media, followed by absorption of the drug entity into the systemic circulation. DISSOLUTION RATE: Dissolution rate is defined as the amount of solid substance that goes into the solution per unit under standard condition of temperature, pH, solvent composition and constant solid surface area. MECHANISM OF DISSOLUTION: Dissolution process in a solid dosage form is presented by two models Wagners model Cartensens model

THEORIES OF DISSOLUTION: Dissolution is explained by 3 theories Diffusion layer model/film theory. Danckwerts model/penetration/surface renewal theory. Interfacial barrier model/double barrier/limited salvation theory.

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FACTORS AFFECTING DRUG DISSOLUTION TESTING: Dissolution rate can be influenced by many factors. The various factors affecting the dissolution rate of the drug from a dosage form fall in six main classes. Factors related to Physico chemical properties of the drug Drug product formulation Dosage form Dissolution testing device Dissolution test parameters Miscellaneous IMPORTANCE OF DISSOLUTION TESTING: Dissolution testing provides the mean to evaluate critical parameters such as adequate bioavailability and provide information necessary to the formulator in product development of more efficacious and therapeutically optimal dosage forms. When carried out appropriately, dissolution analysis of pharmaceutical dosage forms will ensure the quality of the product. Invitro dissolution testing of pharmaceutical dosage form, whether in development or in circulation is imperative not only ensures batch to batch quality equivalence in both invitro and invivo, but also to screen formulation during product development to arrive optimal effective products. To do so, the test should be designed so as to mimic closely the biological environmental conditions. OFFICIAL DISSOLUTION APPARATUS: USP states 7 apparatus for dissolution testing of different dosage form. Apparatus is selected for a dissolution process mentioned in individual monograph. Apparatus 1 - Rotating basket Apparatus 2 - Rotating paddle Apparatus 3 - Reciprocating cylinder Apparatus 4 - Flow through cell Apparatus 5 - Paddle over disk Apparatus 6 - Cylinder Apparatus 7 - Reciprocating holder Apparatus 1 and 2 Conventional dosage forms. Apparatus 3 and 4 Extended release dosage forms. Apparatus 5, 6 and 7 Transdermal drug delivery systems.

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INTERPRITATION OF DISSOLUTION DATA: Usually the overall dissolution of tablet and capsule dosage forms has been presented in one of the following ways. Cumulative amount of the drug dissolved as a function of time. Cumulative percent of the drug dissolved as a function of time. Amount of drug remaining to be dissolved as a function of time.

PRINCIPLE: Simulation of gastrointestinal condition is essential to adequately predict the invivo behavior of poorly soluble drugs. Simulating small intestinal condition with biorelevant media such as fasted state simulated intestinal fluid has become standard practice in many dissolution laboratories. However due to their complex composition, these media are expensive and need to be prepared on the day of the experiment. Developing simplified test media include cost effective and the ability to adequately reflect the physico chemical properties of the biorelevant media FaSSIF or FeSSIF. Another objective was to create mixed micelles like those formed by natural components. Simplified media with corresponding physico chemical properties were used for dissolution experiments. Oral bioavailability and dissolution rate performance depends on the presence of dissolution enhancers. Drug release from the formulation would be sensitive to the composition of the test media. Dissolution testing is frequently used to determine the rate and extent at which a drug is released from a dosage form and it plays many important roles throughout drug product development. However the traditional dissolution approach often emphasized its application in quality control testing and usually strives to obtain 100% drug release. As a result, dissolution methods are not necessarily biorelevant and meaningful application of traditional dissolution methods in the early phases of drug product development is very limited. IMPORTANCE: The use of biorelevant media has got application in early phase formulation development by demonstrating food effect, lot to lot variability, areas of formulation selection.

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Biorelevant dissolution media, method provides a valuable tool to predict certain aspects of invivo drug release used to facilitate the formulation development, selection of pharmacokinetic and clinical studies. It is also potentially been used to minimize the number of pharmacokinetic studies and to aid in the design of more efficient pharmacokinetic and clinical studies. BIORELEVANT MEDIA SIMULATING THE ENVIRONMENT IN STOMACH: Stomach is a part of entry into the gastro intestinal tract for orally administered drug products, immediate release dosage forms should disintegrate enabling the active pharmaceutical ingredient (API) to dissolve in a timely manner before it reaches the absorptive site in small intestine. Under fasting conditions it is known that PH in a healthy human stomach is acidic, ranging from 1 to 3 (2.6). A medium representing the fasting condition in human stomach (FaSSGF) was proposed by vertzone. This medium was designed to embrace the important aspects of human basal gastric juice plus a glass of water normally given with dosage form. It is desirable to have a global medium reflecting the changes in the stomach after meal intake to make an estimate of food effects on drug dissolution and release. Milk has been treated as a good starting point for a medium design because its ratio of carbohydrate/ protein/fat is similar to that observed in stomach after meal. FeSSGF has recently been applied to predict oral absorption of an experimental compound RZ 50, a poorly soluble weakly acidic drug formulated as a lipid based dosage form. PROCEDURE: PREPERATION OF BIORELAVENT MEDIA: Fed state stimulated gastric fluid of ph 1.2 is used as biorelavent media whose composition is as follows NaCl 2g HCl 7g Deionized water up to 1 liter. STANDARD GRAPH OF PARACETAMOL IN BIORELAVENT MEDIA: 100mg of paracetamol pure drug was taken and dissolved in alcohol and the final volume was made up to 100ml with biorelavent media whose concentration is 1000g/ml. This is stock 1. From stock 1, concentration of 100g/ml is prepared which is stock 2.

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From stock 2, serial dilutions of 1,2,3,4,5 g/ml are prepared by taking 0.1, 0.2, 0.3, 0.4,0.5 ml from stock 2 and diluting to 10ml. The absorbances are obtained spectrophotometrically at the max 243nm. DISSOLUTION STUDIES: Dissolution studies were carried out in rotating paddle apparatus, the RPM was set to 100 and the temperature was adjusted to 370c. The test time was set to 1hr. Two dissolution vessels are taken and fixed with 900ml of biorelavent media. Marketed tablet was added to one vessel and prepared tablet to another vessel. The dissolution apparatus was started and for every 10 minutes samples of 5ml were withdrawn and replaced with 5ml fresh media. The absorbances for these samples were noted using UVvisible spectrophotometer at the max 243nm. REPORT: The dissolution studies are performed for the marketed and prepared tablets, After 60min of dissolution the %drug release of the marketed product and prepared product was found to be 77.9% and 64.24% respectively.

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10. DISSOLUTION OF DRUGS IN DIFFERENT pH MEDIA FOR COMPARISSION OF PERFORMANCE WITH INNOVATOR

AIM: To study the dissolution of drugs in different pH media for comparison of performance of the prepared drug with the innovator. REQUIREMENTS: Volumetric flasks, pippettes, test tubes, beakers, funnels.0.1N HCl, 0.1N NaOH, methanol, diclofenac, water, dissolution apparatus: USP 2,single beam UV visible spectrophotometer. PRINCIPLE: Dissolution is a process in which a solid substance solubilises in a given solvent i.e mass transfer from the solid surface to the liquid phase. Pharmaceutical solid dosagae forms on administration undergo dissolution in biological media, followed by absorbtion of drug entity in to the systemic circulation. DISSOLUTION RATE: Dissolution rate is defined as the amount of solid substance that goes in to the solution per unit time under standard conditions of temperature, solvent composition and constant solid surface area. MECHANISM OF DISSOLUTION: Dissolution process in a solid dosage form is presented in 2 models.
Wagner's model Cartensen's model

THEORIES OF DISSOLUTION: Dissolution is explained by 3 theories 1. Diffusions layer model/film theory 36

2. Dankwert's model/penetration/surface renewal theory 3. Interfacial barrier model/double barrier/limited solvation theory. FACTORS EFFECTING DRUG DISSOLUTION TESTING:Dissolution rate can be influenced by many factors. The various factors that affect dissolution rate of drug form a dosage form fall in 6 main classes. 1. Physicochemical properties of drug 2. Drug product formulation 3. Dosage form 4. Dissolution testing device 5. Dissolution test parameters 6. Miscellaneous. IMPORTANCE OF DISSOLUTION TESTING: 1. Dissolution testing provides the mean to evaluate critical parameters such as adequate bioavailability and provides information necessary for formulation in product development of more efficacious and therapeutically optimal dosage forms. When carried out appropriate, dissolution analysis of pharmaceutical dosage form will ensure the quality of product. 2. Invitro dissolution of pharmaceutical dosage form, whether in development or in circulation is imperative not only ensure batch to batch quality equivalance in both in vitro and invivo but also to screen formulation during product development to achieve an optimal effective products. To do so, the test should be designed so as to mimic closely he biological environmental conditions. OFFICIAL DISSOLUTION APPARATUS: USP states 7 apparatus for dissolution testing of different dosage forms. Apparatus is selected for a dissolution process mentioned in the individual monograph. 1. Apparatus I Rotating basket method 2. Apparatus II Rotating paddle 3. Apparatus III reciprocating cylinder 4. Apparatus IV Flow through cell 5. Apparatus V Paddle over disk 37

6. Apparatus VI Cylinder 7. Apparatus VII reciprocating holder Apparatus I and II Conventional dosage forms Apparatus III and IV Extended release dosage forms Apparatus V, VI, VII Transdermal drug delivery systems INTERPRETATION OF DISSOLUTION DATA : Usually the overall dissolution of tablet and capsule dosage form has presented in one of the following ways. Cumulative amount of drug dissolved as a function of time. Cumulative percent of drug dissolved as a function of time. Amount of drug remaining to be dissolved as a function of time.

SIMILARITY FACTOR: In recent years, FDA had placed more emphasis on dissolution profile comparison in area of post approval changes and bio wavers under appropriate test conditions, a dissolution can characterise the product more precisely than single point dissolution test. A dissolution profile comparison between pre change and post change products for SUPAC related changes, or with different strengths helps assure similarity in product performance and signal's bio in equivalence. Among several methods investigated for dissolution profile comparison, f2 is the simplest. Moore and Flanner proposed a model independent mathematical approach to compare the dissolution profile using two factors f1 and f2. F2 = 50 log {[1 + (1/n)(Rt Tt)2]0.5 100}

Rt and Tt cumulative percentage dissolved at each of reference and product respectively. F2 measures closeness between the profiles (similarity factor) In dissolution profile, comparisons, especially to assure similarity in product performance, regulatory interest is in knowing how similar the two curves are, and to have a measure which is more sensitive to large differences at any particular time point. When two products are identical f2 = 100. An average difference of 10 % at all measured time points results in a f2 value of 50.

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FDA has set a public standard of f2 value, 50-100 to indicate similarity between two dissolution profiles. DISSOLUTION PROFILE COMPARISON: 1. Atleast two units must be used for each profile determination. 2. Mean dissolution values can be used to estimate the f2. To use the mean data, percentage coefficient of variation at the earlier point should not be more than 10%. 3. For circumstances where wide variability is observed, or a statistical evaluation of f 2 metric is derived, a bootstrap approach to calculate CI can be performed. 4. The dissolution measurements of two products (test and reference, pre and post, two strengths) should be mad under same test conditions. 5. The dissolution points for both the profiles should be same. eg: immediate release products 15, 30, 45 and 60 minutes. For extended release products 1, 2, 3, 5 and 8 hours. 6. Because f2 are sensitive to number of dissolution time points, only one measurement should be considered after 85% dissolution of the product. 7. For the products which are rapidly dissolving i.e more than 85% in 15 minutes or less a profile comparison is not necessary. 8. The f2 value of 50 or greater (50 100) ensures sameness or equivalence of two curves and thus, the performance of two products. PROCEDURE: PREPARATION OF 0.1 N HCL: 8.5ml of Hcl was taken in 1000 ml volumetric flask and was made up with distilled water. PREPARATION OF 0.1 N NaOH : 4g of NaOH pellets was accurately weighed and dissolved in little quantity of water. After completely dissolving, volume is made up to 1000 ml in volumetric flask. STANDARD GRAPH OF PARACETAMOL: 100mg of paracetamol pure drug was taken and dissolved in alcohol and the final volume was made up to 100ml with distilled water whose concentration is 1000g/ml. This is stock 1. From stock 1, concentration of 100g/ml is prepared which is stock 2. From stock 2, serial dilutions of 1,2,3,4,5 g/ml are prepared by taking 0.1, 0.2, 0.3, 0.4,0.5 ml from stock 2 and diluting to 10ml. 39

The absorbances are obtained spectrophotometrically at the max 243nm. DISSOLUTION STUDIES: v The dissolution studies were carried out in the rotating paddle apparatus. v The RPM was set to 100. v The temperature was adjusted to 37oc v Four dissolution vessels were taken and two of them were filled with 900 ml of 0.1 N HCl and other two of them were filled with 0.1N NaOH. v In two vessels with 0.1 N NaOH, marketed and prepared tablets were added, similarly also in the vessels containing 0.1 N HCl. v The dissolution apparatus was started and for every 10 min samples were taken and absorbance was noted using U.V Spectrophotometer at 243 for HCl and 257 for NaOH.

REPORT:
Dissolution study of the given drug (marketed and test) was conducted in different pH media (0.1N HCl, 0.1N NaOH). The percentage drug release of 0.1N HCL in marketed and prepared product was found to be 83.3% and 77.4% repectively. The percentage drug release 0.1N NaOH in marketed and prepared product was found to be 75.6% and 76.5% respectively

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11. PRODUCT DEVELOPMENT AND PROTOCOL PREPARATION USING PREFORMULATION DATA

DEFINITION:
It can be defined as investigation of physical and chemical properties of a drug substance alone and when combined with excipients. Preformulation testing is the first step in the rational development of dosage form of a drug substance. OBJECTIVE: Is to generate information useful to the formulator in developing stable and bioavailable dosage forms that can be mass produced. The early data collection may include such information as gross particle size, melting point, infrared analysis, chromatographic purity. Before starting with a formal program, the preformulation scientist must consider the following. Available physicochemical data (including chemical structure, different salts available). Anticipated dose. Supply situation and development schedule (i.e, time available). Nature of information the formulator should have or would like to have. Availability of stability- Indicating Assay. I BACKGROUND: o Compound name o Chemical name 41

o o o o o o o

Chemical structure Molecular weight Lot numbers Solvents of recrystallization Purity Therapeutic category Anticipated dose

II ORGANOLEPTIC PROPERTIES: o Colour o Odour o Taste It is important to establish a standard terminology to describe these properties in order to avoid confusion among scientists using different terms to describe the same property. When the colour attributes are undesired or variable, incorporation of a dye in the body or coating of the final product could be recommended. If taste is considered as unpalatable, a less soluble form of the drug is used, provided the bioavailability is not compromised. The odour and taste may be suppressed by using appropriate flavors and excipients or by coating the final product. Many drug substances are irritating to skin, such information must be highlighted such that appropriate procedures for material handling and personal protection can be developed. III PURITY: Purity determination is often performed in an analytical research and development group. Impurity can affect stability. Metal contamination at a level of few parts per million can effect deleteriously. Appearance is another area where a slight impurity can have large effect. Presence of aromatic amines suspected of being carcinogenic. Some off-colour materials can become white in many instances. METHODS TO DETERMINE PURITY: Thin layer chromatography. High pressure liquid chromatography. Paper chromatography. Gas chromatography. Differential scanning chromatography.

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Gravimetric thermal analysis. IV PHYSICAL CHARACTERISTICS: Particle size, shape and surface area: Chemical properties, physical properties and biopharmaceutical behavior of drug substances are affected by their particle size, distribution and shape. ADVANTAGES OF PARTICLE SIZE REDUCTION: Increase in surface area leads to increase in solubility and hence increase in bioavailability. DRAWBACKS: Material loss during grinding. Static electricity develops, making the material difficult to handle. Reduction of particle size to too small dimension leads to aggregation but increase hydrophobicity that lowers the dissolution rate. TECHNIQUES TO DETERMINE PARTICLE SIZE: Microscopy (Optical Microscopy). Light scattering technique. Coulter counter technique. Sieving or screening. Anderson pipette Sedimentation Elutriation. Permeability. Centrifugal. DETERMINATION OF SURFACE AREA: BRUNAUCE EMMETT TELLER THEORY OF ADSORPTION: The theory states that most substances will adsorb a monomolecular layer of gas under certain conditions of partial pressure (of gas) and temperature. Knowing the monolayer capacity of an adsorbent and the area of the adsorbate molecule, surface area can be calculated. FLOWABILITY:

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The flow properties of powders are critical for an efficient tabletting operation. This information is used in the selection of appropriate excipients, anticipation when dose of drug is large. A good flow of the powder or granulation to be compressed is necessary to assure efficient mixing and acceptable weight uniformity for the compressed tablet. METHODS TO DETERMINE POWDER FLOW: Angle of repose. Flow through an orifice. Compressibility index. Shear cell. Incorporation of lubricants and glidants improves the flow.

COMPACTABILITY / COMPRESSIBILTY: Tablet formulations are multi component systems. The ability of such a mixture to form a good compact is dictated by compressibility and compactability characteristics of each component. Luevenburgur and Rohera defined compressibility of powder has the ability to decrease in volume under pressure and compactibility as the ability of the powdered material to be compressed into a tablet of specified tensile strength. Hydraulic press is used to obtain compressibility and compactability of powder. The compactibility of the pharmaceutical powder can be characterized by studying tensile strength, hardness etc. of compacts prepared under various pressures. For determination of tensile strengths compacts are placed radially or axially between two plates and forces required to fracture the compacts are measured. Hardness is defined as the resistance of the solid to deform and is primarily related to its plasticity. Hardness is measured by static impression method (Brinell Test). Brinell hardness number is calculated by using the following equation BHN = 2F/D(D-D2 d2 ) HYGROSCOPICITY:

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Many drug substances exhibit a tendency to adsorb a mixture. The amount of moisture adsorbed by a fixed weight of anhydrous sample in equilibrium with moisture in the air at a given temperature is referred to as equilibrium moisture content. The equilibrium moisture moisture content may influence flow and compression characteristics of powders and the hardness of final tablets and granulations. The moisture content of excipients can also influence the physicochemical properties of dosage forms. In general, hygroscopic compounds should be stored in a well closed container preferably with a desiccant. The sorption isotherms showing the equilibrium moisture contents of a drug substance and excipients as a function of a relative vapour pressure may be determined by placing samples in desiccators having different humidity conditions. Proper processing and storage conditions of drugs may be selected on the basis of sorption isotherms. The analysis of sorption isotherms of excipients such as cellulose and starch derivatives indicates that water may exists in at least two forms bound and free. These two types of water may be differentiated by measuring heat of sorption and by dsc and nuclear magnetic resonance studies. It has been suggested that serious stability problems may be avoided by minimizing free water in excipients. The removal of unbound water reduces the ability of microcrystalline cellulose and compressible sugar to act as direct compaction materials. This is because freewater is needed to provide plasticity to these systems. Free water on the external surface of products can also affect the powder flow. Hygroscopicity can be determined by Dynamic vapour sorption method. Thermogravimetric analysis. By exposing samples to different humidity conditions.

DENSITY: Density is very critical for drugs of low potency, which may constitute the bulk of the final granulation of the tablet. The density of solids also affects this flow property. In the case of physical mixture of powders, significant difference in the absolute densities of the compounds would lead to segregation.

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POLYMORPHISM: Many drug substances can exists in more than one crystalline form with different space lattice arrangements. This property is known as polymorphism. The different crystal forms are called polymorphs. Crystal habit is the description of the outer appearance of the crystals. Polymorphism describes I. Complete morphology of a material. II. Crystal habit. III. Amorphous fraction. IV. Crystallographic defects. Different polymorphs of an API have different physical and chemical properties leading to changes in its o Solubility. o Stability. o Dissolution. o Compatibility. o Bioavailability. o Formulation design. o Manufacturing process o Efficacy. Drug exists in two polymorphs. Stable form (Form I) Meta stable form (form II). Polymorphic transitions can occur during grinding granulating drying and compressing operations. VARIOUS METHODS FOR INVESTIGATIONOF CRYSTALS AND

POLYMORPHS: Microscopy. Infrared spectroscopy. Thermal analysis. Single crystal X-ray. X ray powder diffraction. Dilatometry. NMR. 46

SOLUTION PROPERTIES: PH: Many drugs are either weakly acidic or basic compounds and in solution depending on the pH value, they exist as ionized or unionised species. The unionized species are more lipid soluble and hence more readily absorbed. The gastro intestinal absorption of weakly acidic or basic drugs is thus related to the fraction of the drug in solution that is unionized. The conditions that suppress ionization favours absorption. The factors that are imported in the absorption of weakly acidic or basic compounds are pH at the site of absorption. Ionization constant. Lipid solubility of the unionized species.

The relative concentration of unionized and ionisedforms of a weakly acidic or basic drug in a solution at a given pH can be readily calculated using the Henderson-Hesselbach equation.

Enteric coatings may be applied to a drug so that coating dissolves only in basic pH environment of intestine. Isotonicity in parenteral solution is maintained by altering the pH of the solution. pH DETERMINATION METHOD: pH METER pKa: An acid dissociation constant, Ka (also known as acidity constant or acid ionization constant) is a quantitative measure of the strength of an acid in solution. It is the equilibrium constant for a chemical reaction known as dissociation in acid base reactions. HA H+ +AKa = [H+] [A-]/[HA] A logarithmic measure of an acid dissociation constant is commonly called pKa. pKa = -log 10 Ka

47

The larger the value of pKa, the smaller the extent of dissociation. pKa is determined by SOLUBILITY: Solubility is defined as the maximum quantity of solute that can dissolve in a certain quantity of solvent or solution at specified conditions. Solid drugs administered orally for systemic activity must dissolve in the gastro intestinal fluids prior to this absorption. Thus the rate of dissolution of drugs in gastro intestinal fluids could influence the rate and extent of their absorption. Solubility of the drugs can be improved by Physical modifications. Chemical modifications. Half neutralization method. Titrimetric method.

PHYSICAL MODIFICATIONS: o Particle size reduction or micronisation. o Nanosuspensions. o Modification of crystal habit. o Complexation/ solubilisation. o Use of surfactants. o Solid dispersions. o Cosolvency. CHEMICAL MODIFICATIONS: o Soluble products. o Salt forms. o Complexation. DETERMINATION OF SOLUBILITY: It can be made by adding the solute in small incremental amounts to a fixed volume of solvent. After each addition, the system is vigorously shaken and examined visually for any undissolved solute particles. The supernatant liquid is collected and concentration in the solution is determined by UV-Spectroscopy.

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Solubility of an acidic or basic drug is pH dependent and must be determined over the pH range 1-8. Compounds in solution may degrade. So accurate determination of solubility is difficult. Determination of solubility of meta stable forms is difficult because they transform to more stable forms when exposed to solvents but it can be determined by determination of intrinsic dissolution rates. PARTITION COEFFICIENT: Partition coefficient (P) is the ratio of concentrations of a compound in the two phases of mixture of two immiscible solvents at equilibrium. The solvents chosen are hydrophobic and hydrophilic liquids and the drug is distributed between these two phases. Partition coeffiecient (P) = concentration of solute in organic phase/concentration of solute in aqueous phase

METHODS OF DETERMINATION OF PARTITION COEFFICIENT: SHAKE FLASK OR TUBE METHOD: It consists of dissolving some of the solute in a volume of octanol and water. Then measuring the concentration of solute in each solvent. The most common method of measuring distribution of the solute is by UV-Visible Spectroscopy. HPLC DETERMINATION: The log P of a solute can be determined by correlating its retention time with similar compounds with known logP values. ELECTROCHEMICAL METHODS: Polarized liquid interfaces have been used to examine the thermo dynamic and kinetics of the transfer of charged species from one phase to another. Two main methods exists Interfaces between two immiscible electrolyte solutions Droplet experiment. DISSOLUTION: Dissolution is a process in which a solid substance solublizes in a given solvent i.e., mass transfer from the solid surface to the liquid phase. 49

The absorption of solid drugs administered orally can be depicted by the following chart.

Where Ka and Kd are rate constants for the adsorption and dissolution process. If Kd<<Ka the absorption is described as dissolution rate limited. INTRINSIC DISSOLUTION: Intrinsic dissolution is characteristic of each solid compound in a given solvent under fixed hydrodynamic conditions. The intrinsic dissolution rate in a fixed volume of solvent is expressed as mg. dissolved. This value helps the preformulation scientist in predicting if absorption could be dissolution rate limited. The dissolution rate of a solid in its own solution is described by Noyes-Nernst equation dc/dt = AD(Cs-C)/hv If Cs>>C and surface area (A) and volume (V) are held constant then dc/dt = KS where dc/dt = dissolution rate S = solubility K = constant From the above equation dissolution rate is directly proportional to solubility. The intrinsic dissolution is determined by using rotating-disk method.

PARTICULATE DISK METHOD: Particulate dissolution studies were performed on loosely filled capsules. If the dissolution of drug was considered too slow, attempts to improve the dissolution rate were made. These include physically admixing with a surfactant, co-precipitating with a surfactant, micronizing and granulating with SLS. Dissolution was performed and the concentration of saturated solutions determined spectrophotometrically by determining the absorbance. STABILITY: 50

In designing a solid dosage form it is necessary to know the inherent stability of the drug substance to have an idea of what excipients to use, as well as how best to put them together with the drug and to know that no toxic substances are formed. Stability test is done as per ICH guidelines includes accelerated, intermediate& long term stability studies. Stability testing is divided into 3 categories: Solid-state stability of drug done Compatibility studies (stability in presence of excipients) Solution phase stability (including stability in GI fluids and granulating solvents) SOLID STATE STABILITY: Solid state stability refers to physical and chemical stability. Physical changes caused by polymorphic transitions and hydroscopicity. Chemical changes caused by solvolysis, oxidation, photolysis and pyrolysis Investigation of stability must begin with an examination of the chemical structure which provides some indication of the chemical reactivity. The physical properties of the drug, such as its solubility, pka, melting point, crystal form and equilibrium moisture content also influence stability. For structurally related compounds, the melting point may indicate relative stabilities. For structurally related compounds, the melting point may indicate relative stabilities. The stability study should be designed to identify the factors that cause degradation of the drug.

The factors : Light Heat Oxygen Moisture In general solid state reactions are slow and it is necessary to use stress conditions in the investigation of stability. The data obtained under stress conditions are then extra-polated to make a prediction of stability under appropraite storage conditions 51

Stability study information is used in understanding reaction kinetics and also to set limits on amounts of degradants. ELEVATED TEMPERATURE STUDIES: The elevated temperatures most commonly used are 300,400,500 and 600c in conjugation with the ambient humidity. Occassionally higher temperatures are used. The samples stored at the hightest temp. should be examined for the physical and chemical changes at frequent intervals and any change when compared to an appropriate control (sample stored at 50c or-200c) should be noted. If a substantial change is seen, samples are stored at lower temp are examined. If no change is seen after 30 days at 600c, the stability prognosis is excellent. STABILITY UNDER HIGH HUMIDITY CONDITIONS: In presence of moisture many drug substance, hydrolyse react with other excipients, or oxidise. Therse reactions can be accelerated by exposing the solid drug to different relative humidity conditions. Controlled humidity environment can be readily obtained using laboratory desicators contained saturated solutions of various salts. This data is useful in determing if the material should be protected and stored in a controlled low humidity environment. They may also caution against the use of excipients that absorb moisture significantly. PHOTOSTABILITY: Many drug substances fade or darken on exposure to light. Usually the extent of degradation is small and limited to the exposed surface area. This can be readily controlled by using amber glass or an opaque container, or by incorporating a dye in the product to mask the discoloration. The progress of discoloration could be readily followed using diffuse reflectance spectroscopy. STABILITY OF DRUGS TO OXIDATION: The sensitivity of each new drug entity to atmospheric oxygen must be evaluated to establish if the final product should be packaged under inert atmospheric conditions and if it should contain an anti-oxidant. Stability indicating analytical methods:

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HPLC TLC THERMAL METHODS COMPACTIBILITY STUDIES (stability in presence of excipients) In the tablet dosage form the drug is in intimate cotact with one or more exicipients, excipients could affect the stability of drug. Knowledge of drug excipient interactions is useful to the formulator in selecting appropriate excipients A typical tablet contains binder, disintegrant, lubricant and filler, compactiblity screening for a new drug must consider two or more excipients from each class. The ratio of drug to excipient used in these tests is very much subject to the discretion of the preformulation scientist. Techniques employed in drug excipient compatibility screening are Chromatographic techniques: TLC/HPLC Thermal methods: DSC, DTA Diffuse reflectance spectroscopy FTIR The availability of pH rate profile data is sometimes useful in prediction of the solid state stability of salt forms or the stability of a drug in the presence of acidic or basic excipients. If a drug substance is judged to be physically or chemically unstable when exposed to moisture, a direct compression or non-aqueous solvent granulation procedure is to be recommended for the preparation of tablets. Before using a non-aqueous solvent for this purpose, stability of the drugs in the solvents reactions in the solutions proceeds considerably more rapidly than the corresponding solid-state reactions Degradation in solution thus offers a rapid method for the generation of degradation products. The latter are often needed for the purpose of identification and synthesis (to study their toxicity where appropriate and the development of anlalytical methods. SOLUTION PHASE STABILITY: For a drug substance intended to the formulated into a dosage form such as tablet, a limited solution phase stability must be undertaken.

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These studies are necessary to assure that the drug substance does not degrade intolerabley when exposed to gastro intestinal fluid. For lablie drugs the information is useful in selection of granulation solvents and drying conditions. Thus the stability of the dissolved drug in buffer ranging from pH 1 to 8 should be investigated. If the drug is observed to degrade rapidly in acidic solutions, a less soluble or less susceptible chemical form may show increased relative bioavailability alternatively an enteric (drug) dosage form may be recommended for such a compound RECOMMENDATIONS: The major potential problems associated with the drug on which preformulation studies were conducted are listed out. The solutions the problems associated with the drug are mentioned which favours its usage.alternative and easier methods of manufacture are also discussed in here. in invivo study comparing the different alternatives must be undertaken at the earliest opportunity to select the light form of drug. The stability prognosis for the drug selected, tablet dosage form is also mentioned

DETERMINATION OF ANGLE OF REPOSE OF PARACETAMOL


REQUIREMENTS: Paracetamol powder, funnel, stand, graph. PROCEDURE: FIXED HEIGHT METHOD: A funnel was fixed to a stand such that the tip of the funnel is about 3 cm above the base level. Paracetamol powder was poured in the funnel until the pile of powder touches the tip of the funnel. Radius of the pile was taken and the angle of repose is calculated by

= tan-1 (h/r)
REPORT: The angle of repose of paracetamol powder was found to be 53.990 as it is above 250 paracetamol is not free flowing powder.

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DETERMINATION OF BULK DENSITY, TAPPED DENSITY, CARRS INDEX OF PARACETAMOL


REQUIREMENTS: Paracetamol, tapping apparatus, measuring cylinder. PROCEDURE: 10 gms of paracetamol powder was taken in a measuring cylinder and the volume occupied by the powder was noted (bulk volume)the measuring cylinder was fixed to a tapping apparatus and tapping was done until constant volume was obtained tapped volume was measured. REPORT: The bulk density, tapped density, carrs index of paracetamol powder was determined Bulk density(d) = 37.5 mg/ml Tapped density = 60.6 Carrs index = 41 The carrs index of paracetamol is greater than 25, so it has poor flow. 55

POLYMORPHIC STUDIES
REQUIREMENTS: Beakers, slides microscope. CHEMICALS REQUIRED: Methanol, water, paracetamol PROCEDURE: Paracetamol drug was dissolved in little amount of methanol. Some amount of water was added. Recrystallization of the above mixture was achieved by rapid cooling on ice After formation of crystals, filter the solution, collect the formed crystals and dry. OBSERVATION: Shape of pure drug was observed to be needle and recrystallized product was also showed the needle shape crystals. From, this it was observed that there was no polymorphic transition.

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REPORT: Paracetamol drug did not show any polymorphic tarnsitions after recrystallization from methanol. Melting points of paracetamol pure drug and recrystallized one were found, to be 170-1750c and 170-1780c respectively.

HYGROSCOPICITY
REQUIREMENTS: APPARATUS: petriplates, beaker, dessicator CHEMICALS: calcium carbonate, potassium chloride, sodium nitrate INSTRUMENT: weighing balance PROCEDURE: PREPARATION OF SATURATED SOLUTIONS OF kcl, NaNO3, Cacl2: 100 ml of saturated solutions of potassium chloride, sodium nitrate and calcium chloride were These 3 beakers were placed in 3 essicator and labelled. Each of the saturated solutions maintain certain relative humidities. EXPERIMENT:

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3 empty petiplates were taken and their weights are noted as w0.The paracetamol drug was spread in the petri plates.Then the initial weight was noted as w1.These petri plates were labelled according to the relative humidities in which they are exposed.Petri plates were placed in the dessicators and the final weights was noted. After every 24,48 and 72 hours. Percentage weight gained by the drug can be detemined by using the formula

REPORT: The percentage of moisture gained by paracetamol at different relative humiditys is less than 0.2% hence paracetamol is a non-hydroscopic substance.

PARTICLE SIZE DISTRIBUTION

REQUIREMENTS:

Eye

piece

micrometer,

stage

micrometer,

slides,

paracetamol.
PROCEDURE: CALIBRATION: Calibrate micrometer (eye piece micrometer) using stage

micrometer
PREPARATION OF SLIDE:

Paracetamol powder was placed on slide and was placed on stage of microscope. Size of 100 particles was measured. Number of particles in each size i,e. frequency measured. Graph was plotted by taking size range on X-axis and frequency on Y-axis.
REPORT: 58

Size range of paracetamol powder was found to between 40.8m-339.3m.

DETERMINATION OF pka OF PARACETAMOL


REQUIREMENTS: Paracetamol, distilled water, pH meter, 0.1 NaOH, burette, beakers. PROCEDURE: Dissolve 9 g of paracetamol in 100 ml of distilled water in volumetric flask. Shake for 2 hours, take the filtrate or supernatant liquid approximately. The tip of the pH meter sufficiently dipped into the liquid.Calibrate the pH meter with pH 7 buffer solution Take the initial pH of solution.Add 1 ml of 0.1 NaOH until the pH of solution get a constant value.Take half volume pH of the final volume. The pH of half volume is the pka of paracetamol.

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REPORT: The pka of paracetamol by half neutralization method was found to be 10.45.

EFFECT OF PH ON SOLUBILITY OF PARACETAMOL

REQUIREMENTS: Glassware, volumetric flask, test tubes, distilled water, 7.4 pH phosphate buffer, 0.1 HCl paracetamol. PROCEDURE: PREPARATION OF 7.4 pH PHOSPHATE BUFFER: 50ml of 0.2 M potassium di hydrogen phosphate 39.1 ml of 0.2 N NaOH taken in 200 ml volumetric flask and made upto the mask with distilled water. PREPARATION OF 0.1 HCl:

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Dissolve 8.5ml of conc. HCl in 1000 ml volumetric flask and made upto 1000ml with distilled water. METHOD: 9 g of paracetamol was taken in three volumetric flasks containing water, 0.1 N Hcl, 7.4 pH buffer in each flask. After dissolving make up the volume to 100 ml with respective solutions Shake for 2 hours. Take 1 ml of supernatant liquid from each volumetric flask. Make suitable dilutions to determine the absorbance of each solution. From the standard graph of paracetamol in water, 7.4 pH buffer, 0.1N Hcl. Calculate the concentration of solute in solution and solubility of paracetamol in different solvents. PREPARATION OF STANDARD GRAPH OF PARACETAMOL IN WATER: STOCK I: 100 mg of paracetamol is dissolved in 100 ml of water in a volumetric flask whose concentration is 1mg/ml. STOCK II: From the above solution 10 ml is taken and transferred to 100 ml volumetric flask make up the volume to 100 ml with distilled water. From the above stock II different dilutions are made 2,4,6,8,10g/ml REPORT: The effect of pH on solubility of paracetamol was studied. It was observed that paracetamol was slightly soluble at pH 7, 7.4 & 1.84 (0.1 N Hcl).

PARTITION CO-EFFIECIENT OF PARACETAMOL


REQUIREMENTS: Separating funnel, conical flask, beakers, measuring cylinder, volumetric flask. EQUIPMENT: Single beam UV-Visible spectrophotometer. PROCEDURE: PREPARATION OF 7.4 pH PHOSPHATE BUFFER: 50 ML OF 0.2 M potassium dihydrogen phosphate, 39.1 ml of 0.2 M NaOH were taken in 200ml volumetric flask and made upto th mask with distilled water. 61

METHOD: 35 ml of phosphate buffer and 35ml of octanol was taken in a separating funnel and shaken for 30 min to allow both the aqueous and non-aqueous phases to saturate. After saturation of both phases, allow the separating funnel to stand undisturbed, till the phosphate buffer settles at the bottom. Now separate the buffer layer and collect it into a beaker. Measure 25 ml of this phosphate buffer(saturated) and dissolve 25 mg of paracetamol in it.10 ml of phosphate buffer (saturated with octnol) is blank.1mg/ ml solution of paracetamol and buffer are mixed with octanol in funnel.Again the mixture is well shaked for 30 min. Now collect 1 ml of saturated buffer and dilute with phosphate buffer and measure the absorbance at max= 243nm.Know the concentration of paracetamol from the standard graph of paracetamol in phosphate buffer. PREPARATION OF STANDARD GRAPH OF PARCETAMOL IN PHOSPHATE BUFFER: 100 mg of paracetamol was taken and dissolved in phosphate buffer and final volume was made upto 100 ml with phosphate buffer.this is stock I with concentration 1000g/ ml. From stock I take 10 ml and dilute it to 100 ml with phosphate buffer this gives 100g/ml solution this is stock II. From stock II, subsequent dilutions are made to get concentration of 2,4,6,8,10g/ml. From standard graph of paracetamol and phosphate buffer, find out the concentration of paracetamol in aqueous phase. Calculate the partition co-effiecient by the following formula P = C1/C2 C1 = concentration of the drug in octanol C2 = concentration of drug in phosphate buffer (from the standard graph) and calculate the values of log p. REPORT: Partition co-effiecient of paracetamol was studied in octanol and phosphate buffer 7.4 pH. P was caluculated and was found to be 8.19. Therfore paracetamol is 8.19 times more soluble in octanol and is lipophilic in nature.

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EFFECT OF TEMPERATURE ON THE STABILITY OF PARACETAMOL DRUG

REQUIREMENTS: Petri plates, volumetric flask, pipettes, test tubes. EQUIPMENT: Oven, single beam UV-VISIBLE spectrophotometer. PROCEDURE: SOLUTION FORM OF PARACETAMOL DRUG 100 mg of drug was dissolved in 100 ml of water. Three solutions 100g/ml concentration in three 100ml volumetric flasks and place them in three different ovens, set at temp. 60, 70, 80oc. Now take the pure solid paracetamol and spread it evenly on 63

three petriplates with 3mm thickness of powder place the three petriplates in ovens at temperatures of 60, 70, 80oc.The exposure time is 2 hours and sampling is done for all the samples at every 30 mins interval i.e. at 30, 60, 90 and 120 mins. At every interval draw the sample and dilute it. In case of solids take 10 mg of drug dissolve it in 100ml of water. This gives 100g/ml concentration from this take 1 ml and make upto 10 ml this gives 10g/ml. Now check the absorbance of this solution, by taking water as blank at 244 nm. In case of liquid samples, dr the samples and make necessary dilutions and check the absorbance at 244 nm. Repeat this for 2 hours for both solid and solution samples. REPORT: The amount of drug taken and the amount retained was found to be constant both in case of solution and powder, when exposed to elevated temperatures. Therefore paracetamol was found to be stable at elevated temperature.

EFFECT OF LIGHT ON STABILITY OF PARACETAMOL

REQUIREMENTS: Petridishes, volumetric flasks, pipettes EQUIPMENTS: U.V chamber, single beam U.V visible spectrophotometer PROCEDURE: Take pure powder form of paracetamol and spread it evenly on a petriplate with 3mm powder thickness. Expose it to U.V light at short wavelength in a U.V chamber. The total exposure time is 2 hrs. sampling is done at every half an hour interval i.e at 30, 60, 90 and 120 min by withdrawing 10 mg of drug. Prepare paracetamol solution in a 100 ml volumetric flask by dissolving 1 gm of paracetamol in a small quantity of water and make upto 100 ml, expose it to UV light for 2 hours. Sampling is done every half-an-hour 64

interval i.e. 30,60,90 and 120 min.After each sampling is make necessary dilutions of the solid and solutions samples and measure the absorbances at 244 nm taking distilled water as blank.Repeat this for 2 hours at half-an-hour interval from the absorbances obtained calculate the concentration of paracetamol retained in mg. REPORT: Paracetamol was exposed to U.V light and the amount of drug taken and amount retained was found to be constant. There was no degradation observed. Therefore paracetamol (solution & solid) was found to be stable in light (photo stable)

SUMMARY

S.NO 1

PROPERTY Angle of repose

INFERENCE = 53.99o, not a free flowing

Bulk density Tapped density Carrs index

35.7 mg/ml 60.6 41, poor flow 172 oc Non-hygroscopic 46.8-339.3m 10.46

3 4 5 6

Melting point Hygroscopicity Particle size distribution Pka

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7 8 9

Solubility Partition coefficient Stability studies Temperature Light

Slightly soluble P = 8.19, lipophilic

Stable Stable

12. COMPATIBILITY EVALUATION OF DURGS AND EXCIPIENTS

AIM: To evaluate compatibility of drugs and excipients by infra red spectroscopy. REQUIREMENTS: Agate mortar and pestle, hydraulic press, sample holder DRUG: ASPIRIN EXCIPIENT: MCC (micro crystalline cellulose) INSTRUMENT: FTIR-8400 (SHIMADZU) PRINCIPLE: Infra red spectroscopy of vibrational spectroscopy is concerned with the study of infra red radiation which results in vibrational transitions. In any molecule it is known that atoms or groups of atoms are connected by bonds. These bonds are analogues to springs and not rigid. Because of the continuous motion of the molecule they maintain some vibration with some frequency characteristic to every 66

portion of the molecule. this is called natural frequency of vibration. When energy in the form of infrared radiation is applied and if the frequency of IR radiation applied is equal to the natural vibrational frequency of the molecule absorption of IR radiation takes place and a peak is observed. Assessment of possible incompatibilities between an active drug substance and different excipient forms an important part of the preformulation stage during the development of solid dosage form. Infrared spectroscopy allows the evaluation of possible incompatibilities. The objective of the drug excipient compatibility studies is to identify as quickly as possible, the real and possible interaction between potential formulation excipients and the API. This is an important risk reduction excecise early in formulation development. The successful formulation of a stable and effective dosage form depends on selection of excipients. Before deciding the final formulation we must have readily available knowledge of potential physical and chemical interactions between drugs and excipients, which might effect chemical nature, stability, solubility and in vivo absorption of drugs.

These evaluations are done for Identification of compatible excipients for a suitable formulation. Identification of stable storage conditions for a drug in solid or liquid state The effect of MCC on aspirin stability has been examined recently by mroso et al who found that the effect of MCC. Rate of decomposition is directly related to stearate concentration in solid mixtures. All the decomposition reactions of aspirin in pharmaceutical system are acyl transfers. PROCEDURE: PREPARATION OF SAMPLE: Sampling of solids can be done by using different methods. They are Solid run in solution technique Solid film technique Nujol mill technique Pressed pellet technique PRESSED PELLET TECHNIQUE:

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In this technique, on part of the sample was mixed with 100 parts of KBr, potassium bromide was used, as it was transparent throughout IR region. Grinding and mixing was done in agate mortar and pestle. It was compressed into a thin transparent pellet having 1 cm diameter and 0.2 thickness using hydraulic press. The pellet obtained was placed in the sample holder carefully and was used for analysis. INTERPRETATION: In Aspirin IR spectra C = O stretching and OH stretching peaks were found at 1775.10 cm-1 and 2968.23 cm-1 respectively. When aspirin was mixed with MCC there was no change in peaks which correspond to C = O & O-H of Aspirin. Hence, it was found that Aspirin was compatible with MCC.

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