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Chapter-1

Drugs and their Uses and General Introduction Drugs and their Uses and General Introduction Drugs and their Uses and General Introduction Drugs and their Uses and General Introduction

TTTTo chromatographyo chromatographyo chromatographyo chromatography....

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1. Introduction:

A drug or pharmaceutical compound is defined as a substance used in the

prevention, diagnosis, treatment or cure of diseases in human beings or other animals.Drug

products consist of an Active pharmaceutical ingredient(API), an active substance with

pharmacological activity and the inert chemical substances without pharmacological

activity. Chemical substances without pharmacological activity in the drug product are

described as Excipients.

Safety and efficacy are the two important factors to ensure the quality of the drug

products. The quality of the drug substance or drug product in turn depends on the

potency and impurities or related compounds present in the drug products.

Related compounds are the impurities in pharmaceuticals which are unwanted chemicals

that remain with the active pharmaceutical ingredients (APIs),or develop during stability

testing, or develop during formulation process or upon aging of both API and formulated

APIs to medicines. The presence of these unwanted chemicals even in small amounts

may influence the efficacyand safety of the pharmaceutical products. Hence to ensure the

safety and efficacy of the drug products, validated analytical methods are required.

Chromatographic techniques like Liquid Chromatography (LC), Gas Chromatography

(GC), Thin Layer Chromatography (TLC), are generally used to check the quality of the

drug products. Again liquid chromatography is the most popular tool widely used across

the world over more than 25 years. Most of the drug substances or impurities are organic

compounds,and High Performance Liquid Chromatography (HPLC) is the best

chromatographic tool used for the separation, quantification and analysis of drug

compounds. In this chapter all the details related to different types of Drug products,

impurities in the drug products,chromatographic principles related to the drug products

testing and scope of the research work were discussed.

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2. Types of Drugs and its applications:

Drugs products are widely used for the treatment of different types of Diseases. These are

classified into different classes based upon the therapeutic indications.

2.1.Antibiotics:

Antibiotics are specific chemical substances derived from or produced by living

organisms, which in small concentrations are capable of inhibiting the life processes of

microorganisms.

Fig 1.1: Diagrammatic representation of Antibiotics action

Antibiotics are again divided in to three different types of classes.

2.1.1.Antibacterial agents

Antibacterial agents are the drugs used in the treatment of infections caused by

bacteria. According to the effect produced, antibacterial agents can be bacteriostatic

(inhibit growth of bacteria) or bactericidal (kill the bacteria).

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2.1.2. Antiviral agents

These drugs used for the treatment of viral infections. They are selective

inhibitors of one or more unique steps of the replicate cycle of viruses. They improve

antibody formation and activity. They are selectively active against either RNA

containing or DNA containing viruses.

To effectively treat viral infections, the drug needs to stop the viral replication without

destroying the patient’s healthy cells. Mutations and resistance are common challenges.

Antibacterial drugs are not effective with the viral infections, but may be used in cases of

accompanying secondary bacterial infection. Protease inhibitors (e.g.,saquinavar,

indinavir) have been successful in blocking the enzyme responsible for viral replication.

2.1.3.Antifungal agents

Antifungal agents are the drugs used against the infection caused by fungi. Fungi are

plant like micro-organisms commonly found as molds and yeasts. The drugs chosen to

treat these mycosis or mycotic infections are usually fungicidal. The fungal cell is

destroyed as the drug prevents cell permeability and nutrition. Common fungal infections

include candidiasis (vaginal yeast infection), ringworm, and athlete’s foot. Nystatin and

fluconazole are common antifungals.

2.2.Anti-inflammatory agents

Anti-inflammatory drugs modify the inflammatory response to diseases but are

not curative and do not remove the underlying cause of the disease. Any ideal anti-

inflammatory drug should affect only aberrant, uncontrolled inflammation and not

interfere with the normal inflammatory response, which is a part of the body’s vital

defence mechanisms to invading micro-organisms.

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2.3.Central nervous system Drugs:

Central nervous system (CNS) composed of complex network of sub units which

act as conducting pathways between peripheral nervous system, receptors and effectors.

These drugs produce depressing effect on the central nervous system as their principal

pharmacological action. These include general anaesthetics, hypnotics, sedatives and

tranquilizers. Anaesthetics, hypnotics and sedatives produce depressing effect on central

nervous system in the decreasing order. Sedatives exert milder depression on central

nervous system. Hypnotics induce sleep while anaesthetics induce different degrees of

depression finally leading to unconsciousness. Tranquilizers are the central nervous

system selective depressants having skeletal muscle relaxant properties.

2.4.Cardiovascular agents:

These are the drugs, which influence hearts mechanism (either stimulate or

depress the heart by different mechanism). They produce direct action on the heart or on

the other parts of the vascular (blood vessels) system. These drugs affect heart muscles.

Cardiovascular drugs are again divided into different sub classes:

2.4.1 Antianginals: Used to treat cardiac related chest pain(angina) resulting from

ischemic heart disease(an inadequatesupply of blood to a part of the body, caused by

partial ortotal blockage of an artery).Patients suffer a lack of oxygen and blood flow tothe

myocardium. Nitrates, beta-blockers, and calciumchannel blockers are examples of

antianginals.

2.4.2 Antiarrythmias- Used to treat irregular heart rhythms. Theyregulate the conduction

activity of the heart by inhibiting theabnormal pacemeaker cells or recurring abnormal

impulsesand restoring a normal rhythm. Antiarrythmics include betablockers and drugs

that block sodium channels, potassium ionchannels, and calcium ion channels.

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2.4.3 Antihypertensives- Used to reduce a sustained elevation inblood pressure. Factors

affecting blood pressure include stress,blood volume, arterial narrowing, age, gender, and

generalcondition of health. Common antihypertensives include beta-blockersto reduce

cardiac output, diuretics to decrease fluidvolume, ACE inhibitors to reduce salt/water

retention andinhibit vascular constriction, and calcium channel blockers torelax blood

vessels.

2.4.4 Vasopressors- These drugs act to increase blood pressure. If a patient is in a state

of shock due to decreased blood volume, inadequate cardiacoutput or severe infection,

fluids may be introduced to provide adequate blood volume. In addition to fluid

replacement, vasopressors may be used to help supply blood to the brain and kidney.

2.4.5 Antihyperlipidemics- Used to lower high levels of cholesterol that can lead top

blocked blood vessels. Cholesterol is a lipid normally present in the body that is essential

for healthy cell function. Cholesterol levels are measured as total cholesterol, LDL (low

density lipoprotein), and HDL (high density lipoprotein). Excessive amounts of LDL can

lead to blocked blood vessels and cardiovascular problems. HMG-CoA

Reductaseinhibitors(“statins”) are used to treat high LDL levels.

2.4.6 Thrombolytics/Anticoagulants- Thrombolytics are used to dissolve blood clots

and anticoagulants are used to prevent their formation. Thrombolytics can be dangerous

since blood clotting can be disturbed, resulting in profuse bleeding and even fatal

bleeding. However, in cases of impending myocardial infarction or stroke, a traveling

blood clot (embolus) can be dissolved and the stroke prevented. There has been much

success with this group of drugs inrecent years. A common thrombolytic agent is

alteplase. Common anticoagulants are warfarin and heparin.

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2.5 Anti-depressants:

These drugs used to reduce the depression.

2.5.1 Tricyclics (TCAs)-These agents inhibit(slow) the reuptake(cells taking back excess

amounts) of norepinephrine andserotonin.

2.5.2 Monoamine Oxidase Inhibitors (MAOIs)-differ from TCAsand SSRIs by their

side effects. Many food interactions.Reserved for patients who do not respond to TCAs or

SSRIs.

2.5.3 Selective Serotonin Reuptake Inhibitors (SSRIs)-actspecifically to maintain

higher levels of serotonin in the brain.When serotonin is increased, mood is elevated.

They work differently than MAOIs and TCAs with minimum side effects.

2.6.Antineoplastic agents

Antineoplastics inhibit the new growth of cancer cells or neoplasms. Cancer is a form of

abnormal development, transforming normal cells into cancerous cells.Cancer cells are

abnormal in structure and growth rate andoffer no usual function, have unusual genetic

content and oftenreproduce quickly and uncontrollably.Side effects caused by many of

these drugs are uncomfortableand serious-immunosuppression (compromising one’s

ownimmune system), anemia, hair loss, GI ulceration, anddehydration/weight loss caused

by nausea and vomiting.Due to toxicity of many antineoplastics, normal healthy cellsare

destroyed along with the cancerous cells. Anticancer drugs are used for the treatment of

cancer in combination, they interfere with cell division.

The lymphatic system is the center of thebody’s immune system. It filters fluid

fromaround cells. When people refer to swollenglands in the neck, they are

usuallyreferring to swollen lymph nodes. Commonareas where lymph nodes can be

easilyfelt, especially if they are enlarged, are:the groin, armpits, above the clavicle, inthe

neck, and the back of the head justabove hairline.Antineoplastics target rapidly growing

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cellswhich not only includes cancer cells but GItract, hair follicles, and bone marrow

aswell,because they depress the immunesystem, patients are often susceptible

toinfections.

Antineoplasics are again divided into sub classes:

2.6.1 Antimetabolites- These drugs inhibit cell growth andreplication by mimicking

natural metabolites and taking theirplace within the cells. These fake metabolites inhibit

thesynthesis of important cellular enzymes, including DNA.

2.6.2 Alkyating Agents – These drugs interfere with mitosis or celldivision by binding

with DNA and preventing cellularreplication. Although these agents will affect all cells,

those thatare growing at a more rapid rate (presumably cancerous) willbe more affected.

Nitrosureas, a type of alkylating agent arelipid soluble and pass easily into the brain

where they havesome activity in treating brain cancers.

2.7Gastrointestinal agents

These drugs are used to treat disorders of the stomachand/or intestines.Drugs include

enzymes, antidiarheals, antiemetics (anti-vomiting)antiulcer agents, laxatives and stool

softeners.

2.7.1 Enzymes-Pepsin is a normal gastric enzyme that breaks downproteins. However, in

the absence of pepsin, it is still possible for thedigestive system to break down protein

molecules into amino acidsusing proteolytic enzymes found in the small intestine. If a

patient’scondition warrants using therapy (as with cystic fibrosis and chronicpancreatitis)

products that contain pancreatin, an agent preparedfrom pork pancreas, or

pancrealipasemay be indicated.Malabsorption conditions such as steatorrhea, where fat

isinadequately digested and is excreted in large amounts in feces, maybe treated with

pancrealipase.

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2.7.2Antidiarrheals- Diarrhea is a condition of frequent watery stoolswhich results from

microorganism invasion, drug or stress reaction,chronic GI inflammation and/or other

circumstances causing adecrease in intestinal absorption of water, an increased secretion

ofelectrolytes into the intestines or an excessive amount of mucusproduction.

2.7.3Antiperistaltics- These drugs slow the movement of theintestinal contents to allow

for greater water and electrolyteabsorption. Loperamide is a common antiperistalsis agent

anddiphenoxylate plus atropine is another popular antidiarrheal agent.Bismuth

subsalicylate (Pepto-Bismol) is a secretory inhibitor thatacts to prevent organisms from

attaching to the intestinal mucosaand may deactivate certain toxins as well. In cases of

infectiousdiarrhea, antibiotics such as oral metronidazole or vancomycin mayalso be

indicated. Some antibiotics can kill normal bacterial flora orfacilitate regrowth of

resistant microorganisms, and so lead todiarrhea.

2.7.4Antiemetics- This class of drugs treats the condition of nausea andvomiting. There

are many causes of this condition which is usually asymptom or side effect of as opposed

to being the actual conditionitself: food or drug reaction or allergy, pregnancy,

anxiety,exhaustion, dehydration, and a large number of diseases or illnessessuch as

cancer, or a micro-organism related infection such as otitismedia. Often these agents are

ordered in conjunction with other drugtherapies used to treat underlying conditions.

2.7.5Antacid/antiulcer agents- These drugs generally composed of inorganic saltssuch

as calcium carbonate, aluminum hydroxide, and magnesiumhydroxide which are popular

agents used to neutralize existing acid,as opposed to inhibiting its production. Cimetidine,

Ranitidine,Famotidine are (H-1) histamine receptor antagonists that inhibit thesecretion

of gastric acid by blocking its effects.

2.7.6Laxatives and Stool Softeners- these agents are commonlyprescribed to treat

constipation, the condition of dehydrated stoolcausing bowel movements which are

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infrequent, hard, and oftenpainful. Laxatives promote defecation without stress or pain

and areoften suggested for use prior to use for certain medical proceduresrelated to the

bowel (barium enema), for constipation, and forpatients with hemorrhoids, recent hernia

surgery or heart attackwhere straining is to be avoided

2.8. Respiratory Agents:

Drugs commonly indicated in the treatment of respiratorydiseases and disorders include

antihistamines, decongestants,antitussives, and bronchodilators.

2.9Ophthalmic Agents

Ophthalmic agents are used to treat various conditions ordisorders of the eye.

Due to the special requirements for ophthalmic formulations,there are often many

ingredients in a product besides theactive ingredient-preservatives, buffers, antioxidants,

andwetting agents that control factors such as pH, sterility, andproper isotonic

percentages.

2.10Diagnostic agents

Diagnostic agents are substances used to detect abnormal conditions and

functioning of the body. Radiopaques are diagnostic aids; they are the substances that

absorb x-rays and consequently produce a shadow of positive contrast in soft tissue

structures (urinary bladder, gall bladder, stomach) during roentgenographic examination,

on the other hand, air produces a shadow negative contrast. Abnormalities or pathological

disfunction of several organs of the body are diagnosed by various agents like agents for

liver function test, kidney function test.

2.11Vitamins

They are comparatively simple organic compounds, which are required in small

quantities by animals for their maintenance and normal growth of life. Except vitamin D,

animal body cannot synthesize any other vitamin. They are mainly supplied by the food

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we take. If the diet lacks any one or more vitamins, a deficiency disease results. There are

about 25 vitamins known. Of these, vitamins B and C are water soluble while vitamin A,

D, E and K are fat soluble.

2.12Endocrine system/Hormones

Harmones are chemical substances produced in certain specific parts of the body

called ductless glands also known as endocrine glands. These glands deliver the

hormones in small amounts directly into the blood stream. These substances then exert

physiological effect at a site of action, which is remote from its origin.

2.12.1Thyroid drugs-thyroid hormones are used for supplementalor replacement needs

with hypothyroidism. Usually initiated insmall doses until an response is reached.

2.12.2 Antidiabetics- These drugs lowers blood glucose levels. Treats type I andtype II

diabetes mellitus. Insulin used for type I diabetespatients who cannot produce enough

insulin from thepancrease. Type II diabetics are treated with diet and exerciseand oral

antidiabetics

2.12.3 Glucocorticoids-Steroid hormones (such as cortisone) thatexert an anti-

inflammatory effect and influence many bodyfunctions. Controlled by release of ACTH

from pituitary gland.Prolonged use may suppress the pituitary and adrenal glandsand the

body will not produce its own hormone. Thesehormones cannot be stopped abruptly and

a step-down dosageis used to taper the amount given gradually.

Prednisolone,methylprednisolone, and prednisone are commonglucocorticoids.

2.12.4 Biophosphonates-inhibits bone resorption and treat/preventosteoporosis.

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2.13Immunosuppressant agents: These are the drug substances used to reduce the

imunity of the body.Immuno suppressants generally used during organ transplantation to

avoid organ rejection.

2.14Dermatological agents: Used to treat a condition or diseaserelated to the skin.Skin

is the largest organ and protects the other organs againstmicroorganisms, trauma, extreme

temperature, and otherharmful elements.

Common dermatological agents include steroids,antihistamines and anti-infectives.

Examples: hydrocortisone cream, diphenhydramine, silversulfadiazine cream,

doxycycline hyclate, and ofloxacin.

The drying agent zinc oxide is often seen in a combinationproduct with the local

anesthetic camphor, moisture absorbingagent kaolin, and an anti-infective such as

triclosan whentreating diaper rash.

3. Impurities in the Drug products:

An impurity as defined by the ICH (The International Conference onHarmonisation of

Technical Requirements for Registration ofPharmaceuticals for Human Use) guidelines

is “Any component of themedicinal product which is not the chemical entity defined as

the activesubstance or an excipient in the product”.

Impurities are the chemical substances similar to that of drug substance, but without any

pharmacological activity. Generally impurities are termed as related substances of Drug

product as most of the cases drug substances degrades and forms impurities.Hence

Related components, relatedsubstances, and related impurities terms are synonyms for the

impurities.

3.1 Sources of impurities in pharmaceutical substances:

The origin of impurities in drugs is from various sources and phases ofthe synthetic

process and preparation of pharmaceutical dosage forms.Majority of the impurities are

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characteristics of the synthetic route of themanufacturing process. There are several

possibilities of synthesizing adrug; it is possible that the same product of different sources

may giverise to different impurities. According to the international conference

onharmonization (ICH) of technical requirements for registration ofpharmaceuticals for

human use, impurities are classified as organicimpurities, inorganic impurities and

residual solvents. Organic impuritiesmay arise from starting materials, by products,

synthetic intermediatesand degradation products. Inorganic impurities may derived from

themanufacturing process and are normally known and identified asreagents, ligands,

inorganic salts, heavy metals, catalysts, filter aids andcharcoal etc. Residual solvents are

the impurities introduced with solvents. Of the above three types, the number of possible

inorganicimpurities and residual solvents is limited. These are easily identified andtheir

physiological effects and toxicity are well known. For this reason thelimits set by the

pharmacopoeias and the ICH guidelines can guaranteethat the harmful effects of these

impurities do not contribute to thetoxicity or the side effects of the drug substances. The

situation isdifferent with the organic impurities. Drugs synthesized by multi-stepsynthesis

results in various impurities, their number and the variety oftheir structures are almost

unlimited and highly dependent on the routeand reaction conditions of the synthesis and

several other factors such asthe purity of the starting material, method of isolation,

purification,conditions of storage etc. In addition, toxicity is unknown or not

easilypredictable. For this reason the ICH guideline set threshold limits abovewhich the

identification of the impurity is essential.

3.2 Sources of Organic Impurities:

Organic impurities may arise during the manufacturing processand/or storage of the drug

substance. These impurities are derived fromdrug substance synthetic processes or

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degradation reactions in drugsubstances and drug products. The process (synthetic

process) relatedimpurities can be derived from starting materials, intermediates, reagents,

ligands, and catalysts used in the chemical synthesis, as well as byproductsfrom the side-

reactions of the chemical synthesis.Degradation products are derived from the chemical

degradation of drugsubstances and drug products under storage or stress conditions.

Theymay be identified or unidentified, volatile or non-volatile, and include thefollowing.

3.3 Impurities Originating from Drug Substance SyntheticProcesses:

Most of the drug substances (low molecular weight) are chemicallysynthesized. Chemical

entities, other than the drug substance, that areinvolved or produced in the synthetic

process can be carried over to thefinal drug substance as trace level impurities. These

chemical entitiesinclude raw materials, intermediates, solvents, chemical reagents,

Catalysts, by-products, impurities present in the starting materials, andchemical entities

formed from those starting material impurities(particularly those involved in the last steps

of the synthesis). Theseimpurities are usually referred to as process impurities. The goal

ofprocess impurity identification is to determine the structures and originsof these

impurities. This knowledge is critical for improving the syntheticchemical process, in

order to eliminate or minimize process impurities.

3.4 Starting Materials and Intermediates:

Starting materials and intermediates are the chemical building blocksused to construct the

final form of a drug substance. Unreacted startingmaterials and intermediates, particularly

those involved in the last stepsof the synthesis, can potentially survive the synthetic and

purificationprocess and appear in the final product as impurities .

3.5 Impurities in the Starting Materials:

Impurities present in the staring materials could follow the samereaction pathways as the

starting material itself, and the reactionproducts could carry over to the final product as

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process impurities.Knowledge of the impurities in starting materials helps to identify

relatedimpurities in the final product, and to understand the formationmechanisms of

these related process impurities.

3.6 Reagents, Ligands and Catalysts:

These chemicals are less commonly found in APIs; however, in somecases they may pose

a problem as impurities. Chemical reagents,ligands, and catalysts used in the synthesis of

a drug substance can becarried over to the final products as trace level impurities.

3.7 By-Products of the Synthesis:

All chemical reactions are not 100% selective; the side-reactions arecommon during the

synthesis of drug substances. By-products from theside reactions are among the most

common process impurities in drugs. By-products can be formed through a variety of side

reactions, suchas incomplete reaction, overreaction, isomerisation,

dimerisation,rearrangement, or unwanted reactions between starting materials

orintermediates with chemical reagents or catalysts.

3.8 Products of over-reaction:

In many cases the least or previous steps of the syntheses are notselective enough and the

reagents attack the intermediate not only at thedesired site.

3.9 Products of side reactions:

Some of the frequently occurring side reactions (which are unavoidablein drug synthesis)

are well- known to the synthetic chemist; other whichlead to trace level impurities have

to be detected and elucidated duringimpurity profiling.

3.10 Impurities Originating from Degradation of the DrugSubstance:

Impurities can also be formed by degradation of the end productduring manufacturing of

bulk drugs. Degradation products resulting fromstorage or formulation to different dosage

forms or aging are commonimpurities in the medicines. The definition of degradation

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product inthe ICH guideline is a molecule resulting from a chemical change in

thesubstance brought about by overtime and/or action of e.g. Light,temperature, pH or

water or by reaction with excipient and/or theintermediate

Different types of Degradation path ways for impurities formation:

1.Hydrolysis:

2.Dehydration:

3. Isomerization

4. Racemization

5. Elimination

6. Oxidation

7. Photodegradation

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Fig 1.2: Impurity thresholds for the impurities

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Fig 1.3 :Graphical representation of Impurity thresholds limits based on the

maximum daily dose

To ensure the quality of the drug product Assay and impurities estimation methods

should be accurate with reproducibility.Chromatographic technique is the best one among

different types of analytical techniques to determine the quality of the drug substance or

drug product.

4. Chromatography:

4. 1 Introduction:

"Chromatography is a method in which the components of a mixture are separated on an

adsorbent column in a flowing system." The adsorbent material, or stationary phase, first

described by Tswett in 1906, has taken many forms over the years, including paper, thin

layers of solids attached to glass plates, immobilized liquids, gels, and solid particles

packed in columns. The flowing component of the system, or mobile phase, is either a

liquid or a gas. Concurrent with development of the different adsorbent materials has

been the development of methods more specific to particular classes of analytes.

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4.2 Different types of chromatographic separations.

4.2.1Paper Chromatography: Paper chromatography is a technique that involves

placing a small dot or line of sample solution onto a strip of chromatography paper. The

paper is placed in a jar containing a shallow layer of solventand sealed. As the solvent

rises through the paper, it meets the sample mixture which starts to travel up the paper

with the solvent. This paper is made of cellulose, a polar substance, and the compounds

within the mixture travel farther if they are non-polar. More polar substances bond with

the cellulose paper more quickly, and therefore do not travel as far.

4.2.2 Thin layer chromatography

Thin layer chromatography (TLC) is a widely-employed laboratory technique and

is similar to paper chromatography. However, instead of using a stationary phase of

paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or

cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs,

better separations, and the choice between different adsorbents. For even better resolution

and to allow for quantitation, high-performance TLCcan be used.

4.2.3 Gas chromatography

Gas chromatography (GC), also sometimes known as Gas-Liquid

chromatography, (GLC), is a separation technique in which the mobile phase is a gas.

Gas chromatography is always carried out in a column, which is typically "packed" or

"capillary" (see below).

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Gas chromatography (GC) is based on apartition equilibrium of analyte between a

solid stationary phase (often a liquid silicone-based material) and a mobile gas (most

often Helium). The stationary phase is adhered to the inside of a small-diameter glass

tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column).

It is widely used in analytical chemistry; though the high temperatures used in GC make

it unsuitable for high molecular weight biopolymers or proteins (heat will denature them),

frequently encountered in biochemistry, it is well suited for use in the petrochemical,

environmental monitoring, and industrial chemical fields. It is also used extensively in

chemistry research.

4.2.4 Supercritical fluid chromatography

Supercritical fluid chromatography is a separation technique in which the mobile phase is

a fluid above and relatively close to its critical temperature and pressure.Supercritical

fluid chromatography offers some of the advantages of GC in terms of detection and

HPLC in terms of separations, in that volatility of the sample is not of paramount

importance.

4.2.5 Liquid chromatography:

Liquid chromatography (LC) is a separation technique in which the mobile phase is a

liquid. Liquid chromatography can be carried out either in a column or a plane. Present

day liquid chromatography that generally utilizes very small packing particles and a

relatively high pressure is referred to as high performance liquid chromatography

(HPLC).

Liquid chromatography (LC), is one of the widely used analytical technique, used to

separate a mixture in solution into its individual components. As indicated by Tswett, the

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separation relies on the use of two different "phases" or "immiscible layers," one of

which is held stationary while the other moves over it. Liquid chromatography is the

generic name used to describe any chromatographic procedure in whichthe mobile phase

is a liquid. The separation occurs because, under anoptimum set of conditions, each

component in a mixture will interact withthe two phases differently relative to the other

components in the mixture.

High-performance liquid chromatography (HPLC) is the term used to

describeliquidchromatography in which the liquid mobile phase is mechanicallypumped

through a column that contains the stationary phase. AnHPLC instrument, therefore,

consists of an injector, a pump, a column,and a detector.

Fig 1.4: Agilent HPLC system

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Separation can be achieved by controlling and manipulating these interactions,

which effect the relative retention times of the various sample components.

Fig 1.5: Chromatographic separations and Chromatogram

Table: 1.1 Main components of Liquid chromatographic system

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Based on the polarity of the mobile phase and Stationary phase Liquid chromatography is

again classified as Normal phase chromatography and Reverse phase chromatography.

1) Reversed Phase Chromatography:

Mobile phase polarity is more than stationary phase polarity.In a mixture of components

to be separated those analytes which are relatively less polar will be retained by the non-

polar stationary phase longer than those analytes which are relatively more polar.The

most polar compound will elute first.

In Reversed Phase separations organic molecules are separated based on their

degree of hydrophobicity. There is a correlation between the degree of

lipophylicity and retention in the column. This is the list of mobile phase

parameters effecting retention and separation in Reversed Phase.

Fig 1.6: Chromatographic separations –Reverse phase

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2) Normal Phase Chromatography:

stationary phase polarity is more than Mobile phase polarity. In a mixture of components

to be separated those analytes which are relatively more polar will be retained by the

polar stationary phase longer than those analytes which are relatively more non polar.

The least polar compound will elute first.

Elution order in Normal Phase HPLC shows that the polar solutes elute later

than non-polar lypophilic ones.

Fig 1.7: Chromatographic separations –Normal phase

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Table 1.2: Comparison of Normal phase and Reverse phase Liquid chromatography

Evolution of the Ultra performance Liquid chromatography (UPLC).

High performance liquid chromatography(HPLC) is abest separation technique that

hasbeen used in laboratories worldwide over thepast 25 years. One of the primary

driversfor the growth of this technique has beenthe evolution of packing materials used

toeffect the separation. The underlying principlesof this evolution are governed by thevan

Deemter equation, which is an empiricalformula that describes the relationshipbetween

linear velocity (flow rate) and plateheight (HETP or column efficiency). Sinceparticle

size is one of the variables, a vanDeemter curve can be used to

investigatechromatographic performance.According to the van Deemter equation,as the

particle size decreases to less than2.5 microns, not only is there a significant gainin

efficiency, but the efficiency does notdiminish at increased flow rates or linearvelocities.

By using smaller particles, speedand peak capacity (number of peaks resolvedper unit

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time in gradient separations) can beextended to new limits, termed Ultra Performance

Liquid Chromatography, or UPLC.

This technology takes full advantage of chromatographicprinciples to run separations

using columns packed with smaller particlesand/or higher flow rates for increased speed,

with superior resolution and sensitivity.

Advantages of UPLC over HPLC.

� By using smaller particles, speed and peak capacity can be extended to new

limits, termed Ultra Performance Liquid Chromatography, or UPLC.

� UPLC is a Technique for Reducing Run Times in chromatographic Methods

by Employing New LC Column and Instrument Technologies.

� UPLC retains the practicality and principles of HPLC while increasing speed,

sensitivity and resolution.

� According to Van Deemter equation, as the particle size decreases, not only is

there a significant gain in the efficiency, but the efficiency does not diminish

at increased flow rates.

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Fig 1.8: HPLC vs UPLC comparison

5.Scope and Objectives of research work:

The present research work focuses on the development of novel stability-indicating

analytical methods for widely used selective complex drug products for which the

reported methods are not capable to check the quality of drug products. The work also

includes the validation of the developed methods as per ICH requirements and

demonstrates the suitability of developed methods to assess the stability of drug products.

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Sensitive, Simple, Accurate, Rugged, Robust and cost effective method development and

validation by using advanced technologies are the main objective of the research work.

List of Drug products selected for the research work:

5.1. SirolimusTablets :Sirolimustablets belongs to the immunosuppressant class and

indicated for the prophylaxis of organ rejection in patients receiving allogeneic renal

transplants. It is widelyused drug product after Kidney transplantation.Sirolimus is a

complex macrolide and unstable molecule. Stability indicated invitro Assay and Related

substances are not reported. Therefore, Author selected this drug product to develop and

validate stability indicating Assay and RS methods.

5.2. Metoprolol succinate ER tablets: Metoprolol ER tablets are indicated for the

treatment of hypertension and to lower the blood pressure. It is an extended release drug

product with different polymers in the formulation matrix to hold the drug substance.

Direct stability indicating Assay methods were not reported for the extraction of

metoprolol from the various types of marketed samples. No methods were reported for

the quantification of impurities and degradation products by using UPLC. Hence Author

was selected this drug product to develop and validate stability indicating Assay method

by HPLC and Related substances by UPLC.

3. Alendronate sodium and Cholecalciferol Tablets.

Alendronate sodium and Cholecalciferol Drug product is a bisphosphonate and vitamin D

combination used for the treatment of Osteophorosis.Cholecalciferol is light sensitive and

undergoes oxidation if exposed to normal environmental conditions, hence it is stabilized

with the different excipients in the formulation samples.Very few methods were reported

in the literature and all are related to the raw material analysis, but none of them

discussed about the specificity of the method and the extraction of Cholecalciferolfrom

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the stabilized formulations. Normal phase stability indicating HPLC method was

developed with liquid-liquid extraction procedure.

4.Amlodpinebesylate and Valsartan Tablets: Amlodipine and valsartan tablets are

indicated for the treatment of hypertension. Amlodpine is the widely used drug substance

to reduce the blood pressure. Few related substances by HPLC methods were reported for

Amlodpine and these methods are not capable to quantify all process related and

degradation impurities. Reported methods are not suitable for the analysis of combination

products. Hence, Author has selected this drug product to develop Simple UPLC method

to quantify Amlodipine impurities in combination formulations.