1

Chapter I: Introduction

The quality of a drug plays an important role in ensuring the safety and efficacy of the

drugs. Quality assurance and control of pharmaceutical and chemical formulations is essential

for ensuring the availability of safe and effective drug formulations to consumers. Hence

Analysis of pure drug substances and their pharmaceutical dosage forms occupies a pivotal

role in assessing the suitability to use in patients. The quality of the analytical data depends on

the quality of the methods employed in generation of the data. Hence, development of rugged

and robust analytical methods is very important for statutory certification of drugs and their

formulations with the regulatory authorities.

The quality and safety of a drug is generally assured by monitoring and controlling the

assay and impurities effectively. While assay determines the potency of the drug and

impurities will determine the safety aspect of the drug. Thus, the analytical activities

concerning impurities in drugs are among the most important issues in modern

pharmaceutical analysis. Assay of pharmaceutical products plays an important role in efficacy

of the drug in patients. The impurity profile of pharmaceuticals is of increasing importance as

drug safety receives more and more attention from the public and from the media.

The wide variety of challenges is encountered while developing the methods for

different drugs depending on its nature and properties. Depending on mechanism of action,

drugs are formulated in to different pharmaceutical dosage forms like Tablets, hard gelatin

capsules, soft gelatin capsules, injections. Depending on target site of absorption of the drug,

drugs are formulated into different dosage forms with different delivery mechanisms like

immediate release, delayed release and extended release. Depending on delivery mechanism

needed, different kinds of excipients are used in formulations to achieve target release profile

of the drug in human body. Depending on the formulation matrix chosen, the complexity of

extracting the drug and its impurities from formulations will vary. This along with the

importance of achieving the selectivity, speed, cost, simplicity, sensitivity, reproducibility and

accuracy of results gives an opportunity for researchers to come out with solution to address

the challenges in getting the new methods of analysis to be adopted by the pharmaceutical

industry and chemical laboratories.

2

The drugs may be classified according to their chemical structure or therapeutic

action, as chemotherapeutic agents and pharmacodynamic agents. Their action and

classification is the subject of any postgraduate course in pharmacy or medicinal chemistry

and forms the content of many textbooks [1-5] and hence needs no reproduction.

Drugs play a vital role in the progress of human civilisation by curing diseases. The

word drug is derived from the French word drogue, which means a dry herb. In general, a

drug may be defined as a substance used in the prevention, diagnosis, treatment or cure of

diseases in man or other animals. According to World Health Organisation (WHO), a drug

may be defined as any substance or product which is used or intended to be used for

modifying or exploring physiological systems or pathological states for the benefit (physical,

mental as well as economical) of the recipient

An ideal drug when administered to the ailing individual or host, should satisfy the

following requirements: Its action should be localised at the site where it is desired to act,

Should act on a system with efficiency and safety, Should not have any toxicity, Should have

minimum side effects, Should not injure host tissues or physiological processes, Should not

develop tolerance by the tissues even administered for long duration. Such drugs are rare and

hence the search for ideal drug continues.

Importance of Analytical methods for testing potency and impurities in drugs:

Safety and efficacy of pharmaceuticals are two fundamental issues of importance in

drug therapy. The safety of a drug is determined by its pharmacological-toxicological profile

as well as the adverse effects caused by the impurities in bulk and dosage forms. The

impurities in drugs often possess unwanted pharmacological or toxicological effects by which

any benefit from their administration may be outweighed [6]. Therefore, it is quite obvious

that the products intended for human consumption must be characterized as completely as

possible. The quality and safety of a drug is generally assured by monitoring and controlling

the impurities effectively. Thus, the analytical activities concerning impurities in drugs are

among the most important issues in modern pharmaceutical analysis. This has become quite

clear by the recent research articles on this topic [7-10].

3

Control is more important today than ever. Until the beginning of the 20th century,

drug products were produced and sold having no imposed control. Quality was generally not

verified. Many products were patent medicines of dubious value. Some were harmful and

addictive. In 1937, ethylene glycol was used as a vehicle for an elixir of sulfanilamide, which

caused more than 100 deaths [11]. Thereupon the Food, Drug and Cosmetic act was revised

requiring advance proof of safety and various other controls for new drugs. The impurities to

be considered for new drugs are listed in regulatory documents of the Food and Drug

Administration (FDA) [12], International Conference on the Harmonization of the Technical

Requirements for Registration of Pharmaceuticals for Human Use (ICH) [13] and United

States Pharmacopoeia (USP). Nevertheless, there are many drugs in existence, which have not

been studied in such detail. The USP and National Formulary (NF) are the recognized

standards for potency and purity of new drugs. These compendia have become official upon

adoption of the first food and drug act. They formulate legal standards of quality, purity and

strength of new drugs. The good manufacturing practices provide minimum quality standards

for production of pharmaceuticals as well as their ingredients [14]. The ICH, which took place

in Yokohama, Japan in 1995 has released new guidelines on impurities in new drug products

[15].

These guidelines have a number of advantages, both for the industry and the regula-

tors. The most critical aspect of the elaboration of the guidelines was the definition of the

levels of impurities for identification and qualification. Qualification is the process of

acquiring and evaluating data for establishing the biological safety of an individual impurity

or a given impurity profile at the levels specified. The level of any impurity present in a new

drug substance that has been adequately tested in safety and clinical studies is considered

qualified. A rationale for selecting impurity limits based on safety considerations has to be

provided. Analytical procedures should be able to separate all the impurities from each other

and the method should be optimized to separate and quantify them in the dosage forms. Such

methods are to be validated demonstrating the accuracy, precision, specificity, limit of

detection, quantification, linearity range and interferences.

The validation of analytical procedures, i.e., the proof of its suitability for the intended

purpose, is an important part of the registration application for a new drug [16,17]. Additional

peak tailing, peak resolution and analyte recoveries are important in case of chromatographic

4

methods. The ICH has harmonized the validation requirements in two guidelines [18,19]. The

first one summarizes and defines the validation characteristics needed for various types of test

procedures; the second one extends the previous text to include the experimental data required

and some statistical interpretation. These guidelines serve as a basis worldwide both for

regulatory authorities and industry and bring the importance of a proper validation to the

attention of all those involved in the process of submission of drug master files. The analytical

research and development units in the pharmaceutical industry are responsible for preparation

and validation of test methods. Every country has legislation [20] on bulk drugs and their

pharmaceutical formulations that sets standards and obligatory quality indices for them. These

regulations are presented in separate articles- general and specific- relating to individual

drugs, and are published in the form of book called Pharmacopoeia (e.g. Indian, IP[21],

United States, USP[22], European, EP[23], United Kingdom, BP[24], Martindale Extra

Pharmacopoeia[25], Merck Index[26], etc.).

The monitoring of in-process impurities was an obscure and unidentified field about

20 years ago. Now it has become a major factor in modern pharmaceutical industry. This is

mainly because of the pressure for product quality, and the demand for higher standards of

process reliability. Toxicological issues have also brought about a greater sensitivity to the

significance of impurities at trace levels [27]. New attention has been given to the various

classes of toxicants present as impurities in pharmaceutical products. In view of these changes

it has become necessary to pay more attention to the origins and pathways of a host of

impurities within the process. Frequently, impurities are formed as isomers of the desired

reaction products and a critical impurity can often enter with the feed. Analytical

identification of the problematic compounds is the first step towards the solution of the

problem. Different analytical techniques like preparative HPLC, liquid-liquid extraction,

Flash chromatography, Mass spectrometry, High Resolution mass spectrometry, NMR

spectroscopy for characterization of impurities. Analytical methods used for Monitoring of

the process reactions often lend valuable insight into the types of impurities that may be

present. The purity of the final product may often be aided by controlling the purity of the

materials used in its synthesis. Wherever possible, the levels of impurities originating for the

starting materials should be limited through appropriate in-process controls in order to avoid

the need for their monitoring in the drug substance. The use of chromatographic techniques

5

for monitoring the starting materials, intermediates, and the process reactions is an excellent

means for controlling the purity of the final drug and thereby protecting the patient who

ultimately receives it. The best way to characterize the quality of a bulk drug is to determine

its purity. There are two possible approaches to reach this goal. The determination of the

active ingredient content with a highly accurate and precise specific method or the

determination of its impurities. In the early years of drug analysis, when chromatographic

techniques were not yet available the characterization of the purity of drugs was based on the

determination of the active ingredient content by non-specific titrimetric and photometric

methods supported by the determination of physical constants and some limit tests for known

impurities based mainly on colour reactions. The deficiencies of this approach are well

known. In many cases even highly contaminated drug materials could meet the requirements

set in the early editions of different pharmacopoeias. As a consequence of the enormous

development of the analytical technology in the last two decades entirely new

possibilities have been created for the determination of the purity of drug materials [28-

30]. In principle, it is now possible to replace all non-specific assay methods with highly

specific and precise HPLC/UPLC methods thus greatly improving the value of the

determination of the active ingredient content of bulk materials. Nearly all organic

impurities are determined by chromatographic or related methods of which Liquid

chromatography (LC) has been the most important for over the last two decades. A

thorough literature search has revealed that different methods of estimation of drugs and

its impurities based on HPLC / UPLC, capillary electrophoresis (CE), Gas-liquid

chromatography (GLC), SFC, Thin-layer chromatography (TLC) etc. were published. LC

has been the main technique used for analysis of impurities in drugs. Most used the

reversed-phase mode with UV absorbance detection whenever appropriate, because this

provided the best available reliability, analysis time, repeatability and sensitivity. In fact,

this technique has set the standard against which others are compared. Recent advances

like UPLC, U-HPLC coupled with new advances in stationary phases like columns which

are having 1.7 µm and 1.3 µm particles has revolutionized the separation science.

Today a majority of the drugs used are of synthetic origin. These are produced in bulk

and used for their therapeutic effects in pharmaceutical formulations. These biologically

active chemical substances are generally formulated into convenient dosage forms such as

6

tablets, capsules, dry syrups, liquid orals, creams or ointments, parenterals (injections in dry

or liquid form), lotions, dusting powders, aerosols, metered dose inhalers (MDI) and dry

powder inhalers (DPI) etc. These formulations deliver the drug substances in a stable, non-

toxic and acceptable form, ensuring its bioavailability and therapeutic activity.

In tablets one or more among the diluents such as lactose monohydrate,

microcrystalline cellulose, hydroxy propyl cellulose, sodium starch glycolate, magnesium

stearate, crospovidone, calcium phosphate, mannitol, sorbitol, sucrose, aerosil, acacia, gelatin,

alginic acid, tragacanth, sodium stearyl fumarate, talc, waxes, methyl paraben, propyl

paraben, meglumine, sodium benzoate, permitted flavors and colors are added. In capsules

one or more among the excipients, certified dyes, gelatin, plasticizers, starch, lactose, talc,

preservatives are added. In dry syrups and liquid orals, sucrose, sorbitol, preservatives,

certified colors and flavors are added. In creams and ointments, waxes, carbopol, petroleum

jelly, surfactants, preservatives, permitted colors and perfumes are added. In parenterals,

water, vegetable oils, mineral oils, simulated oils, propylene glycol, dioxalamines, dimethyl

acetamide are used as vehicles. Any one or more among stabilizers, anti-oxidants, buffering

agents like citrate, acetate, phosphate, co-solvents, wetting, suspending and emulsifying

agents like tween-80, sorbital oleate and preservatives are added. In lotions, dusting powders

and aerosols, talc, silica derivatives, alcohol, preservatives are added.

In view of wide variety of excipients used in formulating drugs for administration to

patients, drug substances can undergo transformation by interacting with one or more

components of the formulation. For example, drugs which contains primary amine as function

group in their structure, can undergo Millard reaction with reducing sugars like lactose. Drug

substances can also react with trace impurities like formic acid, acetic acid, formaldehyde

present in cellulose derivatives and peroxides present in excipients like povidone, cross

povidone. Formulated drugs can degrade due to acidic or basic environments created due to

the formulation matrix. Drugs can degrade due to exposure to temperature, humidity and light

during manufacturing, transportation and storage during its shelf life. Due to this it is essential

to know the degradation pathways of the drugs in acidic, basic, neutral, oxidation conditions

and their susceptibility to temperature and humidity to formulate them in a manner in which

they are stabilized and retains its quality throughout their shelf life. As most drugs contains

functional groups which can participate in reactions in some way or the other, it is essential

7

that the analytical methods developed for estimation of the purity and impurities are capable

enough to separate all the desired and undesired components and devoid of any interferences

from the formulation matrix. When analytical methods are able to precisely and accurately

quantify without missing any impurities, without underestimation or over estimation, and

detect all possible impurities and degradants those can form during stability studies with

adequate sensitivity and exactly reflect the quality of drug substances and drug products

(formulated products of drugs), those methods are called stability indicating methods.

In view of the foregoing discussion assaying and stability testing in pharmaceutical

analysis occupies an important role to meet the requirement of statutory certification of drugs

and their formulations by the industry .The complexity of problems encountered in

pharmaceutical analysis coupled with the importance of achieving high selectivity, speed,

cost, simplicity, sensitivity, precision and accuracy, new methods of analysis are being

quickly absorbed by the pharmaceutical industry and chemical laboratories depending upon

the facilities available.

Among the several instrumental techniques [HPLC/UPLC GC, CE (Capillary

electrophoresis), Fluorimetry, NMR, mass spectroscopy, spectrophotometry covering IR,

NIR, Raman, UV and visible regions] available for the assay of drugs, usually visible

spectrophotometric technique is simple and less expensive. The selectivity and sensitivity of

the visible spectrophotometric method depends only on the nature of chemical reactions

involved in color development and not on the sophistication of the equipment.

Spectrophotometric analytical procedures are not generally stability indicating. Most widely

used methods are based on HPLC / UPLC, GC. Capillary electrophoresis and Super critical

fluid chromatography are slowly gaining ground in recent years.

A stability-indicating assay method should accurately measure the active ingredients,

without interference from degradation products, process impurities, excipients, or other

potential impurities. If an industry uses a non-stability indicating analytical procedure for

release testing, then an analytical procedure capable of qualitatively and quantitatively

monitoring the impurities, including degradation products, should complement it. Analytical

procedures for stability studies of assay should be stability indicating. As a result of stability

testing a re-test period for the active substance or a shelf life for the pharmaceutical product

can be established, and storage conditions can be recommended.

8

The ICH (International conference on Harmonization) guideline QIA on Stability

Testing of New Drug Substances and Products [31] emphasizes that the testing of those

features which are susceptible to change during storage and are likely to influence quality,

safety and/or efficacy must be done by validated stability indicating testing methods. It is also

mentioned that forced decomposition studies (stress testing) at temperatures in 10 °C

increments above the accelerated temperatures, extremes of pH, under oxidative and

photolytic conditions should be carried out on the drug substance and drug product so as to

establish the inherent stability characteristics and degradation pathways to support the

suitability of the proposed analytical procedures.

Formulations containing various drugs and combinations of drugs for potentiating or

complementing one another in therapy are available in market. Pharmaceutical equivalents

containing identical amounts of the same active ingredient(s) in the same dosage form and

targeted to give in the same route of administration are called as generics drugs. For a generic

drug to be approved it must be shown to be pharmaceutically equivalent and bioequivalent to

the Reference Listed Drug (RLD). They must also meet all relevant standards of strength,

quality, purity, and identity. In some cases, no analytical method is reported and quite often

the reported procedures need improvements or changes keeping in view the findings and

advances. Among the several instrumental techniques [HPLC, GC, NMR, mass spectroscopy,

UV-Vis Spectrophotometry] available for the assay and impurities of drugs, HPLC/UPLC is a

versatile tool for the qualitative and quantitative analysis of drugs and pharmaceuticals and

has become indispensable. HPLC/UPLC technique has been regarded as the best among

various instrumental techniques in spite of its cost and maintenance problems.

Keeping in view the above discussion, author has examined the present state of

development of such analytical methods for some of the widely used drugs. Hence, the author

has proposed to develop stability indicating methods for assay and impurities for five most

widely used drug products made up of drugs, namely, dexlansoprazole, repaglinide,

candesartan cilexetil + hydrochlorothiazide, docetaxel and paricalcitol. The above drugs are

selected for research to whom there is wide scope for the development of new analytical

methods for their assay and impurities determination by HPLC/UPLC by exploiting their

characteristics, physical and chemical properties.

9

Dexlansoprazole Capsules:

Dexlansoprazole belongs to the class of proton pump inhibitors. The drug is unstable

to heat, oxidation and acid hydrolysis conditions. During formulation development stability

studies, lot of unknown degradants are observed. Literature does not contain good stability

indicating methods which addresses the separation of all degradation products of either

lansoprazole or dexlansoprazole. When the current known methods are employed, the late

eluting degradants are found to be not separating well. Author has seen this as an opportunity

to work on identification of unknown degradants as well as a stability indicating methods for

assay and impurities.

Repaglinide tablets:

Repaglinide is used for treatment of Type-2 diabetes. When the official pharmacopeal

methods are used, they are found to be deficient in separation of some unknown degradants.

Also, literature did not give a rich information on the possible solutions. This molecule

offered an opportunity to improve the existing methodology and also scope to identify few

unknown degradation products hitherto unreported.

Candesartan cilexetil + Hydrochlorothiazide Tablets:

Candesartan cilexetil is used for treatment of hypertension. Hydrochlorothiazide is

used as diuretic as a combination therapy. Although several methods are reported for analysis

of individual drugs, there was no method which can address the impurities of combination

product. Instead of following two individual methods, author has seen an opportunity to

develop a single method for estimating impurities for this combination product.

Docetaxel Injection:

Docetaxel injection is used for the treatment of cancer. When the published

methodologies are adopted, they were found to be deficient in terms of adequacy and

accuracy. Hence author has seen an opportunity to improve and publish for the benefit of

scientific community.

10

Paricalcitol Capsules:

Paricalcitol is a drug used for treatment of secondary hyperparathyroidism. While the

Official pharmacopeal method is available for aqueous based injection formulation, there are

no published methods found in literature for oil based capsules formulation. The

pharmacopoeal method, when adopted for capsules formulation, was found to be not suitable

due to large amount of interferences from placebo matrix. Also, pharmacopeia does not

provide any structural information about degradants. Paricalcitol is administered in very low

doses like 1 µg, 2µg and 4µg capsules. Hence, it is felt that it will be challenging to develop a

suitable sensitive method for low dose oil based capsule formulation and also give an attempt

to understand the nature of degradation products of paricalcitol.

11

Section ii: Brief account on drugs selected and the therapy:

Dexlansoprazole, a proton pump inhibitor:

Dexlansoprazole belongs to the class of proton pump inhibitors (PPIs). Proton-pump

inhibitors are a group of drugs whose main action is a pronounced and long-lasting reduction

of gastric acid production. They are the most potent inhibitors of acid secretion available

today. The group followed and has largely superseded another group of pharmaceuticals with

similar effects, but different mode-of-action, called H2-receptor antagonists. These drugs are

among the most widely-selling drugs in the world [32]. The vast majority of these drugs are

benzimidazole derivatives. However, promising new research indicates that imidazopyridine

derivatives may be a more effective means of treatment[33]. The widely used drugs in this

category are omeprazole, esomeprazole, rabeprazole, pantoprazole, lansoprazole and

dexlansoprazole.

These drugs are utilized in the treatment of many conditions such as:

1. Dyspepsia

2. Peptic ulcer disease (PUD)

3. Gastro esophageal reflux disease (GERD)

4. Laryngopharyngeal reflux

5. Barrett's esophagus

6. prevention of stress gastritis

7. Gastrinomas and other conditions that cause hypersecretion of acid

8. Zollinger-Ellison syndrome.

Proton pump inhibitors act by irreversibly blocking the hydrogen/potassium adenosine

triphosphatase enzyme system (the H+/K

+ ATPase, or more commonly gastric proton pump)

of the gastric parietal cells. The proton pump is the terminal stage in gastric acid secretion,

being directly responsible for secreting H+ ions into the gastric lumen, making it an ideal

target for inhibiting acid secretion. Targeting the terminal step in acid production, as well as

12

the irreversible nature of the inhibition, results in a class of drugs that are significantly more

effective than H2 antagonists and reduce gastric acid secretion by up to 99%. ("Irreversibility"

refers to the effect on a single copy of the enzyme; the effect on the overall human digestive

system is reversible, as the enzymes are naturally destroyed and replaced with new copies.)

The lack of the acid in the stomach will aid in the healing of duodenal ulcers, and reduces the

pain from indigestion and heartburn, which can be exacerbated by stomach acid. However,

lack of stomach acid is also called hypochlorhydria, the lack of sufficient hydrochloric acid,

or HCl. Hydrochloric acid is required for the digestion of proteins and for the absorption of

nutrients, particularly of vitamin B12 and of calcium.

The proton pump inhibitors are given in an inactive form. The inactive form is

neutrally charged (lipophilic) and readily crosses cell membranes into intracellular

compartments (like the parietal cell canaliculus) that have acidic environments. In an acid

environment, the inactive drug is protonated and rearranges into its active form. As described

above, the active form will covalently and irreversibly bind to the gastric proton pump,

deactivating it. Dexlansoprazole is the first PPI introduced into the market which is

formulated as long acting. It is based on a dual release technology, with the first quick release

producing a plasma peak concentration about one hour after application, and the second

retarded release producing another peak about four hours later [34].

13

Repaglinide, an insulin Secretagogue:

Repaglinide is the first drug of the meglitinide class. Meglitinides, aka "Glinides"[35],

are a class of drugs used treat diabetes type 2. They bind to an ATP-dependent K+ (KATP)

channel on the cell membrane of pancreatic beta cells in a similar manner to sulfonylureas but

at a separate binding site. This inhibits a tonic, hyperpolarizing outflux of potassium, which

causes the electric potential over the membrane to become more positive. This depolarization

opens voltage-gated Ca2+

channels. The rise in intracellular calcium leads to increased fusion

of insulin granulae with the cell membrane, and therefore increased secretion of (pro)insulin.

Repaglinide (Prandin), gained US FDA approval in 1997. It was branded Prandin

because of its quick onset and short duration of action concentrates its effect around meal time

(the prandium was the Roman meal which is comparable to the modern lunch).Another type

of drug in this class is nateglinide (Starlix). These drugs should be taken 0-30 minutes prior to

eating.

Diabetes mellitus (DM) is a set of related diseases in which the body cannot regulate

the amount of sugar (specifically, glucose) in the blood. The blood delivers glucose to provide

the body with energy to perform all of a person's daily activities. The liver converts the food a

person eats into glucose. The glucose is then released into the bloodstream. In a healthy

person, the blood glucose level is regulated by several hormones, primarliy insulin. Insulin is

produced by the pancreas, a small organ between the stomach and liver. The pancreas also

makes other important enzymes released directly into the gut that helps digest food. Insulin

allows glucose to move out of the blood into cells throughout the body where it is used for

fuel. People with diabetes either do not produce enough insulin (type 1 diabetes) or cannot use

insulin properly (type 2 diabetes), or both (which occurs with several forms of diabetes)[36].

Type 2 diabetes is usually controlled with diet, weight loss, exercise, and oral medications.

Like Sulfonylureas, for example, glyburide (Diabeta; Glynase; Micronase), glipizide

(Glucotrol), glimepiride (Amaryl), tolbutamide (Orinase), and tolazamide (Tolinase),

repaglinide stimulates cells in the pancreas to produce insulin. Glyburide may be more potent

than repaglinide at increasing insulin release in persons with low or high blood glucose levels,

whereas repaglinide may be more potent in persons with moderate blood glucose levels.

14

Repaglinide is unusual in that it has a rapid onset of action and a short duration of action.

When taken just prior to meals, it promotes the release of insulin that normally occurs with

meals and is responsible for preventing blood glucose levels from becoming high. It has been

shown to lower hemoglobin A1c levels by 1.6% to 1.9%. (Hemoglobin A1c is a blood test

which measures the effectiveness of a drug in controlling high blood glucose levels. The

lower the hemoglobin A1c, the better the control.) Today there are various drugs available in

market for treatment of Diabetes. The below table gives various class of compounds classified

according to their mechanism of action for the treatment of diabetes.

Table 1.1: Oral anti-diabetic drugs and Insulin analogs [37].

Insulin

Sensitiz

ers

Biguanides Metformin# , Buformin

‡ , Phenformin

‡

TZDs/"glitazones" (PPAR) Pioglitazone, Rivoglitazone

†,

Rosiglitazone, Troglitazone‡

Dual PPAR agonist Aleglitazar

†, Muraglitazar

§, Tesaglitazar

§,

Ragaglitazar§

Secreta

gogues

K+ ATP -Sulfonylureas

1st generation: Acetohexamide,

Carbutamide, Chlorpropamide,

Metahexamide, Tolbutamide, Tolazamide

2nd generation: Glibenclamide

(Glyburide)# , Glibornuride, Glipizide,

Gliquidone, Glisoxepide, Glyclopyramide,

Glimepiride, Gliclazide

K+ ATP -

Meglitinides/"glinides"

Repaglinide, Nateglinide, Mitiglinide

GLP-1 agonists Exenatide, Liraglutide, Taspoglutide

†,

Albiglutide†, Lixisenatide

DPP-4 inhibitors Alogliptin

†, Gemigliptin, Linagliptin,

Saxagliptin, Sitagliptin, Vildagliptin

Analogs/other insulins fast-acting (Insulin lispro · Insulin aspart ·

Insulin glulisine) · short-acting (Regular

insulin) ·

long-acting (Insulin glargine · Insulin

detemir · NPH insulin) ·

ultra-long-acting (Insulin degludec†) ·

inhalable Exubera‡

Others

Alpha-glucosidase inhibitors Acarbose, Miglitol, Voglibose

Amylin analog Pramlintide

SGLT2 inhibitors Canagliflozin†, Dapagliflozin

†,

Remogliflozin§, Sergliflozin

§

o #WHO-Essential Medicine ,

‡Withdrawn from market , Clinical trials: (

†Phase III ·

§Never to

phase III)

15

Candesartan + Hydrochlorothiazide, an anti-hypertensive and Diuretic

Candesartan cilexetil is a drug used for treating high blood pressure (hypertension).

It is in a class of drugs called angiotensin receptor blockers (ARBs). Angiotensin, formed in

the blood by the action of angiotensin converting enzyme (ACE), is a powerful chemical that

attaches to angiotensin receptors found in many tissues but primarily on smooth muscle cells

surrounding blood vessels [38]. Angiotensin II is a very potent chemical that causes muscles

surrounding blood vessels to contract, thereby narrowing blood vessels. This narrowing

increases the pressure within the vessels and can cause high blood pressure (hypertension).

Angiotensin II receptor blockers (ARBs) are medications that block the action of angiotensin

II by preventing angiotensin II from binding to angiotensin II receptors on blood vessels. As a

result, blood vessels enlarge (dilate) and blood pressure is reduced. Reduced blood pressure

makes it easier for the heart to pump blood and can improve heart failure. In addition, the

progression of kidney disease due to high blood pressure or diabetes is slowed. ARBs have

effects that are similar to angiotensin converting enzyme (ACE) inhibitors, but ACE inhibitors

act by preventing the formation of angiotensin II rather than by blocking the binding of

angiotensin II to muscles on blood vessels [39].

ARBs are used for controlling high blood pressure, treating heart failure, and

preventing kidney failure in people with diabetes or high blood pressure. They may also

prevent diabetes and reduce the risk of stroke in patients with high blood pressure and an

enlarged heart. ARBs may also prevent the recurrence of atrial fibrillation. Since these

medications have effects that are similar to those of ACE inhibitors, they often are used when

ACE inhibitors are not tolerated by patients (for example, due to excessive coughing) [39].

Hydrochlorothiazide, is a first-line diuretic drug of the thiazide class that acts by

inhibiting the kidneys' ability to retain water. This reduces the volume of the blood,

decreasing blood return to the heart and thus cardiac output and, by other mechanisms, is

believed to lower peripheral vascular resistance [40]. Thiazides are also used in the treatment

of osteoporosis. Thiazides decrease mineral bone loss by promoting calcium retention in the

kidney, and by directly stimulating osteoblast differentiation and bone mineral formation[41].

16

Hydrochlorothiazide is frequently used for the treatment of hypertension, congestive

heart failure, symptomatic edema, diabetes insipidus, renal tubular acidosis, and the

prevention of kidney stones [42]. It is also sometimes used for hypercalciuria, Dent's disease

and Ménière's disease. Hydrochlorothiazide reduces blood volume by acting on the kidneys to

reduce sodium (Na) reabsorption in the distal convoluted tubule. The major site of action in

the nephron appears on an electroneutral Na+-Cl- co-transporter by competing for the

chloride site on the transporter. By impairing Na transport in the distal convoluted tubule,

hydrochlorothiazide induces a natriuresis and concomitant water loss. Thiazides increase the

reabsorption of calcium in this segment in a manner unrelated to sodium transport.[43]. The

candesartan + hydrochlorothiazide fixed dose combination is indicated for the treatment of

hypertension. This fixed dose combination is not indicated for initial therapy [55-56]. There

are several drugs today available in the market and in research for the treatment of

hypertension. The below table gives various class of compounds classified according to their

mechanism of action for the treatment.

Table 1.2: Antihypertensives: agents acting on the renin-angiotensin system.[44]

A II RBs/

("-sartan")

Azilsartan, Candesartan, Candesartan (+HCT), Eprosartan, Irbesartan,

Losartan (+HCT), Olmesartan, Tasosartan§, Telmisartan (+HCT),

Valsartan, Valsartan (+HCT).

ACE inhibitors

("-pril")

Sulfhydryl-containing: Captopril , Zofenopril

Dicarboxylate-containing: Enalapril#, Ramipril, Quinapril, Perindopril,

Lisinopril (+HCT), Benazepril Phosphonate-containing: Fosinopril

Other/ungrouped: Alacepril, Cilazapril, Delapril, Imidapril, Moexipril,

Rentiapril, Spirapril, Temocapril, Trandolapril

Renin inhibitors/

("-kiren")

Aliskiren (+amlodipine), Remikiren§

#WHO-Essential Medicine , ‡Withdrawn from market , Clinical trials: (†Phase III · §Never to phase III)

17

Docetaxel, a mitotic inhibitor:

A mitotic inhibitor is a drug that inhibits mitosis, or cell division. These drugs

disrupt microtubules, which are structures that pull the cell apart when it divides. Mitotic

inhibitors are used in cancer treatment, because cancer cells are able to grow and eventually

spread through the body (metastasize) through continuous mitotic division and so are more

sensitive to inhibition of mitosis than normal cells. Mitotic inhibitors are derived from natural

substances such as plant alkaloids, and prevent cells from undergoing mitosis by disrupting

microtubule polymerization, thus preventing cancerous growth. Microtubules are long,

ropelike proteins that extend through the cell and move cellular components around. One of

the important functions of microtubules is to move and separate chromosomes and other

components of the cell for cell division (mitosis). Mitotic inhibitors interfere with the

assembly and disassembly of tubulin into microtubule polymers. This interrupts cell division,

usually during the mitosis (M) phase of the cell cycle when two sets of fully formed

chromosomes are supposed to separate into daughter cells.[45,46].

Docetaxel exhibits cytotoxic activity on breast, colorectal, lung, ovarian, gastric, renal

and prostate cancer cells [48, 54]. Docetaxel does not block disassembly of interphase

microtubules and so does not prevent entry into the mitotic cycle, but does block mitosis by

inhibiting mitotic spindle assembly. Resistance to paclitaxel or anthracycline doxorubicin

does not necessarily indicate resistance to docetaxel. Microtubules formed in the presence of

docetaxel are of a larger size than those formed in the presence of paclitaxel, which may result

in improved cytotoxic efficacy . Abundant formation of microtubules and the prevention to

replicate caused by the presence of docetaxel leads to apoptosis of tumour cells and is the

basis of docetaxel use as a cancer treatment[48,49].

The cytotoxic activity of docetaxel is exerted by promoting and stabilising

microtubule assembly, while preventing physiological microtubule

depolymerisation/disassembly in the absence of GTP[50]. This leads to a significant decrease

in free tubulin, needed for microtubule formation and results in inhibition of mitotic cell

division between metaphase and anaphase, preventing further cancer cell progeny. Because

microtubules do not disassemble in the presence of docetaxel, they accumulate inside the cell

and cause initiation of apoptosis. Apoptosis is also encouraged by the blocking of apoptosis-

18

blocking bcl-2 oncoprotein. Both in vitro and in vivo analysis show the anti-neoplastic

activity of docetaxel to be effective against a wide range of known cancer cells, cooperate

with other anti-neoplastic agents activity, and have greater cytotoxicity than paclitaxel,

possibly due to its more rapid intracellular uptake. The main mode of therapeutic action of

docetaxel is the suppression of microtubule dynamic assembly and disassembly, rather than

microtubule bundling leading to apoptosis, or the blocking of bcl-2 [48, 51].

. Today there are several drugs available for treatment of cancer therapy. The below

tables gives various class of compounds classified according to their mechanism of action for

the treatment.

Table 1.3: Intracellular chemotherapeutic agents / antineoplastic agents [47].

SPs/MIs

(M

phase)

Block microtubule

assembly.

Vinca alkaloids (Vinblastine#, Vincristine

#, Vinflunine

§,

Vindesine, Vinorelbine)

Taxanes (Cabazitaxel), Docetaxel# , Larotaxel, Ortataxel

†,

Paclitaxel#, Tesetaxel). Epothilones (Ixabepilone)

DNA

replication

inhibitors

DNA

precursors

/

anti

metabolites

(S phase)

Folic acids Dihydrofolate reductase inhibitors (Aminopterin,

Methotrexate#, Pemetrexed, Pralatrexate).

Thymidylate synthase inhibitors (Raltitrexed, Pemetrexed)

Purines Adenosine deaminase inhibitors (Pentostatin).

Halogenated/ribonucleotide reductase inhibitors

(Cladribine, Clofarabine , Fludarabine, Nelarabine).

Thiopurines (Thioguanine#, Mercaptopurine

#)

Pyrimidines Thymidylate synthase inhibitor (Fluorouracil#,

Capecitabine, Tegafur, Carmofur, Floxuridine).

DNA polymerase inhibitor (Cytarabine#)

Ribonucleotide reductase inhibitor (Gemcitabine)

Hypomethylating agent (Azacitidine, Decitabine)

Deoxy

ribonucleotide Ribonucleotide reductase inhibitor (Hydroxycarbamide)

Topoisom

erase

inhibitors

(S phase)

I Camptotheca (Camptothecin, Topotecan, Irinotecan,

Rubitecan‡, Belotecan)

II Podophyllum (Etoposide# , Teniposide)

II +

Intercalation

Anthracyclines (Aclarubicin, Daunorubicin#,

Doxorubicin#, Epirubicin, Idarubicin, Amrubicin

†,

Pirarubicin, Valrubicin, Zorubicin) · Anthracenediones

(Mitoxantrone, Pixantrone)

19

Table 1.3: Intracellular chemotherapeutic agents / antineoplastic agents ( conti.,)

DNA replication

inhibitors

Crosslinking of

DNA (CCNS)

Alkylating Nitrogen mustards: Mechlorethamine,

Cyclophosphamide# (Ifosfamide,

Trofosfamide), Chlorambucil#

(Melphalan, Prednimustine),

Bendamustine, Uramustine,

Estramustine.

Nitrosoureas: Carmustine, Lomustine

(Semustine), Fotemustine, Nimustine,

Ranimustine, Streptozocin.

Alkyl sulfonates: Busulfan

(Mannosulfan, Treosulfan)

Aziridines: Carboquone, ThioTEPA,

Triaziquone, Triethylenemelamine

Platinum-

based

Carboplatin, Cisplatin, Nedaplatin,

Oxaliplatin, Satraplatin

Nonclassical Hydrazines (Procarbazine#) ·

Triazenes (Dacarbazine#,

Temozolomide), Altretamine,

Mitobronitol , Pipobroman

Intercalation Streptomyces (Actinomycin#,

Bleomycin#, Mitomycin, Plicamycin)

Photosensitizers/

PDT

Aminolevulinic acid / Methyl aminolevulinate, Efaproxiral,

Porphyrin derivatives (Porfimer sodium, Talaporfin, Temoporfin,

Verteporfin)

Others Enzyme

inhibitors

FI (Tipifarnib), CDK inhibitors (Alvocidib†,

Seliciclib†), PrI (Bortezomib), PhI (Anagrelide),

IMPDI (Tiazofurine§), LI (Masoprocol), PARP

inhibitor (Olaparib§), HDAC (Panobinostat, Vorinostat,

Romidepsin)

Receptor

antagonists

ERA (Atrasentan) , Retinoid X receptor (Bexarotene),

Sex steroid (Testolactone)

Others

/ungrouped

Amsacrine, Trabectedin, Retinoids (Alitretinoin,

Tretinoin), Arsenic trioxide, Asparagine depleters

(Asparaginase#/Pegaspargase), Demecolcine,

Elesclomol§, Elsamitrucin, Etoglucid, Lonidamine,

Lucanthone , Mitoguazone, Mitotane, Oblimersen†,

Omacetaxine mepesuccinate§, Eribulin

#WHO-Essential Medicine ,

‡Withdrawn from market , Clinical trials: (

†Phase III ·

§Never to

phase III)

20

Paricalcitol, an agonist for the vitamin D receptor :

Paricalcitol, is a drug used for the prevention and treatment of secondary

hyperparathyroidism associated with chronic renal failure[55] There are three kinds of

hyperparathyroidism, namely, primary hyperparathyroidism, secondary hyperparathyroidism

and tertiary hyperparathyroidism.

Primary hyperparathyroidism causes hypercalcemia (elevated blood calcium levels)

through the excessive secretion of parathyroid hormone (PTH), usually by an adenoma

(benign tumors) of the parathyroid glands. Signs and symptoms of primary

hyperparathyroidism are those of hypercalcemia. They are classically summarized by the

mnemonic "stones, bones, abdominal groans and psychiatric moans"[56].

Secondary hyperparathyroidism refers to the excessive secretion of parathyroid

hormone (PTH) by the parathyroid glands in response to hypocalcemia (low blood calcium

levels) and associated hypertrophy of the glands. Chronic renal failure is the most common

cause of secondary hyperparathyroidism. Failing kidneys do not convert enough vitamin D to

its active form, and they do not adequately excrete phosphate. When this happens, insoluble

calcium phosphate forms in the body and removes calcium from the circulation. Both

processes lead to hypocalcemia and hence secondary hyperparathyroidism. Secondary

hyperparathyroidism can also result from malabsorption (chronic pancreatitis, small bowel

disease, malabsorption-dependent bariatric surgery) in that the fat soluble vitamin D can not

get reabsorbed. This leads to hypocalcemia and a subsequent increase in parathyroid hormone

secretion in an attempt to increase the serum calcium levels. If left untreated, the disease will

progress to tertiary hyperparathyroidism, where correction of the underlying cause will not

stop excess PTH secretion, i.e. parathyroid gland hypertrophy becomes irreversible.

Paricalcitol acts as an agonist for the vitamin D receptor and thus lowers the blood

parathyroid hormone level [57].

Tertiary hyperparathyroidism is a state of excessive secretion of parathyroid

hormone (PTH) after a long period of secondary hyperparathyroidism and resulting

hypercalcemia[58]. Cinacalcet has greatly reduces the number of patients who ultimately

21

require surgery for secondary hyperparathyroidism [59]; however, approximately 5% of

patients do not respond to medical therapy. When secondary hyperparathyroidism is corrected

and the parathyroid glands still can remain hyperfunctioning, it becomes tertiary

hyperparathyroidism. The treatment of choice is surgical removal of three and one half

parathyroid glands.

22

Section-iii: Liquid chromatography

Different LC techniques:

The following are the most widely used chromatographic techniques for developing the

stability indicating assay and impurities methods for pharmaceutical formulations.

i). High performance liquid chromatography (HPLC),

ii). Ultra performance liquid chromatography (UPLC).

The following are the most widely used detectors using the above chromatographic

techniques.

i). UV-Vis detector, ii). Diode array detector, iii). Fluorescence detector, iv). Refractive index

detector, v). ELSD detector, vi). Mass detector.

Modes of chromatography :

Modes of chromatography are defined essentially according to the nature of the

interactions between the solute and the stationary phase, which may arise from hydrogen

bonding, vander walls forces, electrostatic forces or hydrophobic forces or basing on the size

of the particles. Most widely used modes of liquid chromatography are as follows:

i). Reversed phase chromatography, ii). Normal phase chromatography, iii). Ion-exchange

chromatography, iv). Size exclusion chromatography.

The essential components of the instruments used for the above techniques, the principles

involved and their application areas are well known and widely published and hence does not

need a reproduction.

Different stationary phases used in LC:

The following are the most widely used LC columns with stationary phases for separation and

quantification of wide variety of drugs and its impurities.

23

i). Pure silica and hybrid silica columns.

ii). Silica based columns with different bonding phases like C4, C6, C8, C18, C20 and

bonding phases having functional groups like cyano, phenyl, naphthyl and amino.

iii). Silica based columns with polar embedded phases within chains of C8, C18, NH2.

iii). Hybrid silica based columns like C4, C6, C8, C18, C20 and bonding phases having

functional groups like cyano, phenyl, naphthyl and Amino.

iv). Strong cation exchange (SCX) and strong anion exchange (SAX) columns.

v). Size Exclusion chromtography (SEC) or gel permeation chromatography (GPC) columns.

vi). Silica based monolith columns.

vii). Fused core silica columns with bonding phases like C8, C18, CN, phenyl.

viii). Metal oxide columns like zirconia based and alumina based.

ix). Chiral columns.

The LC columns with the above stationary phases are available from a large number of

suppliers. The most popular brands of LC columns are Inertsil, Hypersil, X-terra, X-bridge,

Sun-fire, Atlantis, Aquity-BEH, Zorbax, Lichrosphere, Purosphere, Sperisorb, Luna,

Kromasil, ACE, YMC, Symmetry, Chiralcel and Chiralpak. These LC columns are supplied

in different dimensions, viz., lengths of 10 mm, 50 mm, 100mm, 150mm, 250mm, 300mm,

500mm and internal diameters of 2.1mm, 3.0mm, 4.0mm, 4.6mm. LC columns with

stationary phases having different particle sizes like 5.0 µm, 4.0 µm 3.5 µm, 3.0 µm, 2.5 µm,

2.0 µm, 1.9 µm, 1.8 µm, 1.7 µm and 1.3 µm are available.

The following properties of the LC column stationary phases play an important role in giving

different selectivity for separations.

i). Particle size, ii). Particle shape, iii). Pore size / Pore volume, iv). Specific surface area,

v). End capping vi). % carbon loading.

Different manufacturers supplies same columns with different properties and hence today

analytical scientists have a wide variety of choice of columns to achieve desired separations.

The properties of above mentioned different stationary phases and their applications are well

known and widely published and hence does not need a reproduction.

24

Solvents used in HPLC / UPLC:

Chromatographers have a choice among hundreds of solvents for use as mobile-phase

components, sample solvents or in sample pretreatment. A particular selection is usually

affected by solvent characteristics that relate to detection, separation, flow resistance (column

pressure drop or mobile phase viscosity) and miscibility. The solvent properties such as UV

cutoff, refractive index, viscosity, polarity, miscibility and elutropic values described in

several text books [60] will be useful to chromatographers when it comes to selecting one or

more solvents. Most widely used solvents in reverse phase chromatography are acetonitrile,

methanol, ethanol and THF and widely used solvents in normal phase chromatography are

Hexane, methylene chloride, chloroform, ethyl acetate, isopropyl alcohol, ethanol, methanol.

Applications of LC in pharmaceutical research :

(a) Separation: This can be accomplished using HPLC/UPLC by utilizing the fact that,

certain compounds have different migration rates given a particular column and mobile

phase. The extent or degree of separation is determined by the choice of stationary phase and

mobile phase along with parameters like flow, temperature and gradient programme.

(b) Identification: For this purpose a clean peak of known sample has to be observed from

the chromatogram. Selection of column mobile phase and flow rate matter to certain level in

this process. Identification is generally by comparing with reference compound based on

retention time and also based on UV-Vis spectra in some cases. Identification can be assured

by combining two or more detection methods, where necessary.

(c) Quantification: Analyte concentrations are estimated by measuring the responses ( peak

areas) known reference standards followed by unknown samples. Quantification of known

and unknown components are done by various methods like - area normalization method,

internal standard method, external standard method and diluted standard method along with

relative response factors.

(d) Isolation : It refers to the process of isolation and purification of compounds using

analytical scale or preparative scale HPLC. Volatile buffers and solvents are preferred choice

as mobile phases as it reduces the effort on purification. Solute purity and throughput is the

key challenge in isolation and purification processes.

25

Section-iv: Method development and Validation – General methodology:

Literature collection:

Collect and review the literature for method related information already reported on

the molecule. Collect information if available on solubility profile (solubility of Drug in

different solvents and at different pH conditions), analytical profile (Physico-chemical

properties, Eg: pKa, melting point, degradation pathways, etc) and stability profile (sensitivity

of the drug towards light, heat, moisture etc).

Chemical structures :

Collect the structures of the molecule and the impurities likely to be present, starting

material, byproduct, and intermediates in the reaction and degradation products. Compare the

structures of impurities, starting material, byproduct, intermediates and degradation products

with the structure of drug substances and arrive at the polarity whether they are less polar or

more polar than the compound of interest. The below table provides the polarity index of

various functional groups.

26

Selection of stationary phase:

Bonding phase can be chosen based on the polarity of the molecule. For liquid

chromatography, a wide variety of columns are available covering a wide range of

polarity by cross-linking the Si-OH groups with alkyl chains likeC4, C6, C8, C18 and

nitrile groups (CN), phenyl groups (-C6H6) and amino groups (-NH2) etc. Silica based

columns with different cross linking in the increasing order of polarity are as follows:

<--------Non-polar--------moderately polar------------Polar----------->

C18 < C8 < C6/C4 < Phenyl < Amino < Cyano < Silica

Polarity Functional Group Structure

Bonding Types

Low

High

Methylene

Phenyl

, x

Halide

Ether

Nitro

, x

Ester

, x

Aldehyde

, x

Ketone

, x

Amino

, x

Hydroxyl

Carboxylic Acid

, x

R (CH2)2

R

R F, Cl, Br, I

R O

R

N+

O

O-

R

O

O R

R

O

R H

O

R

R

R NH2

R OH

O

OH

R

27

150mm x 4.6 mm, 5 µm column column is always a good starting point.

Detector selection:

PDA (photodiode Array) detector is useful for initial method development based

on the chromophores present in the compounds to be separated. Select the initial wavelength

analyzing the UV spectra of the compounds using UV-VIS spectrophotometer. If the

compounds are not having chromophores, other detectors like RI, ELSD/CCAD are useful.

Mobile phase selection:

Buffer pH to be used in mobile phase is based on the pKa of the analyte, which is

based on the structure of the molecule. Assess whether the compound is basic, acidic or

neutral. If compound is acidic, use acidic mobile phase. For a basic compound, use low pH or

basic mobile phase. For a neutral compound, neutral mobile phase is suitable. Assessment of

pKa values based on functional groups; select a pH, which is ± 1 from the pKa values. Buffer

strength of about 10 to 25 mM is advisable for initial experiments. Acetonitrile is the best

organic modifier (Because of favorable UV transmittance and Low viscosity). Methanol is the

second best organic solvent and Third organic solvent is THF. These three solvents are widely

used to control selectivity and separations.

Order of Polarity: Methanol>Acetonitrile >Ethanol>THF>Propanol

Order of Solvent Strength: Propanol>THF> Ethanol >Acetonitrile>Methanol.

THF has some disadvantages, higher UV absorbance, reactivity with oxygen and slower

column equilibration. But sometimes it gives very unique selectivity for closely eluting peaks.

Therefore these solvents are used for solvent type selectivity. Intermediate selectivity (if

needed for a particular sample) can be obtained by blending appropriate amounts of each of

these solvents.

When a peak tailing is observed, increasing the salt concentration can reduce peak

tailing for both bases and acids. For ionizable compounds, an increase in ionic strength can

suppress solute and silica ionization, as well as secondary interactions between them. Increase

beyond 50 mM is not recommended, due to possible solubility problems of the salt in the

Reverse phase Bonded REVERSE PHASE

Bonded

28

organic portion of the mobile phase. Triethylamine (TEA) or diethyl amine (DEA) can be

added to the mobile phase to control peak tailing for bases. TEA /DEA acts as a competing

base and minimizes solute-silanol interactions. This is usually a final step to try because

TEA/DEA will reduce retention, modify the column, and complicate the mobile phase.

Whenever TEA or modifier is used to reduce the tailing, use columns with very low level of

silanols. For reducing the peak tailing for acids, 1% acetic acid can be added to the mobile

phase to minimize solute-silanol interactions as it acts as a competing acid. The relatively

high concentration of acetic acid resulted in the noisy base line. Using 0.1% TFA as the

aqueous modifier results in a more transparent mobile phase and still reduces the tailing.

Ion pair reagents to be used when necessary, when a closely related compounds

separation is required. If a mixture of ionic and non-ionic analytes to be separated, start

by optimizing the method for non-ionic compounds. Then select the appropriate ion-pair

reagent to provide the necessary selectivity. Whenever ion pair reagent is used, preferably

HPLC column should be dedicated for that specific analysis only or a cleaning Procedure

for the method with ion pair needs to be ensured every time. Alkyl sulphonates are good

first choice for basic compounds. Quaternary amines are useful for acidic compounds.

Use ion-pair reagents in the concentration range of 0.0005M to 0.02M. Sodium per

chlorate also can be used as an ion-pair reagent to get the required selectivity for acidic

components.

Selection of Elution mode:

If isocratic elution is possible, this means conditions for 0.5<k<20; If the k range

exceeds these limits, gradient elution is necessary. When an initial chromatogram

suggests a wide retention range (0.5>k>20) for an ionic sample, the use of ion pair

reagent often permits isocratic separation with 0.5<k<20. Similar changes in retention

change can also be achieved by a change in pH, to ionize late eluting compounds (for

reduced retention) or reduce the ionization of early eluting sample components (for late

elution). Gradient elution methods are widely being used due to availability of advanced

liquid chromatographic instruments and need for achieve robust separations. It is a good

idea to start method development with the following two default gradient programmes :

29

Programme 1 Programme 2

Time Buffer

Organic

phase Time Buffer

Organic

phase

0.01 50 50 0.01 95 5

60 5 95 60 5 95

70 5 95 70 5 95

71 50 50 71 50 50

75 50 50 75 50 50

Understand elution pattern of all the peaks. Based on the elution with these gradient

programmes, further optimization can be done.

Flow rate and column temperature:

Initial flow rate of 1.0 ml-1

min or 1.5 ml-1

min and column temperature as ambient

(25 – 30°C) is preferable.

Diluent selection:

Select a diluent in which impurities, starting material, byproduct, intermediates and

degradation products and the analyte are soluble. It is advisable to check first suitability of

mobile phase as diluent. All the analytes should be completely soluble and solution should be

stable reasonably atleast until its injection into LC. For Finished dosage forms, diluents shall

be selected in such a way that the analyte(s) should be extracted > 95% for impurities and

>98% for Assay. Diluent should be compatible with the mobile phase to obtain the good peak

shape.

Selection of test concentration and injection volume :

Select a test concentration and injection volume to get an appropriate response for

the analytes for an assay method. For impurities method, test concentration and injection

volume shall be based on the sensitivity required for the method. For impurities method,

LOQ of all impurities and analyte shall be less than or equal to the reporting threshold.

30

Degradation studies:

Degradation studies or stress testing is conducted in order to investigate the likely

degradation products, which in turn helps to establish the degradation pathways and the

intrinsic stability of the drug molecule and also to provide foundation for developing a

suitable stability indicating method. Stress testing the drug molecule under particular stress

condition generate samples containing degradation products. Use these samples to develop

suitable analytical methods. The degradation products generated in the stressed samples are

termed as “potential” degradation products that may or may not be formed under relevant

storage conditions. Stress drug product, and placebo separately to understand the peaks due

to placebo components, if any. Four major forced degradation studies are (i) Thermolytic

Degradation, (ii). Hydrolytic degradation, (iii). Oxidative degradation, (iv). Photolytic

degradation. Preferably, have the degradation in the range of 1 % to 20%, in order to reflect

the true degradation pathways. Beyond 20%, sometimes secondary degradations will occur,

which may not occur in actual conditions of use. After the satisfactory separations are

achieved, evaluate the peak purity of the analyte peak using PDA detector. Conduct Mass

balance study by assaying the stressed samples apart from quantifying the impurities. Sum of

Assay and impurities obtained in stressed samples is presented as mass balance. A mass

balance of >95% is always considered satisfactory. If the mass balance is less than the

required criteria, investigation to be done inorder to correct or to justify.

The following are the reasons for not achieving the mass balance.

(1). Degradation products are not eluted from the HPLC column,

(2). Degradation products are not detected by the detector used.

(3). Degradation products lost from the sample matrix, due to insolubility, volatility.

(4). Parent compound lost from the sample matrix, due to insolubility, volatility.

(5). Degradation products are co-eluted with the parent compound.

(6). Degradation products are not integrated due to poor chromatography.

(7). Inaccurate quantification due to differences in response factors.

31

Finalisation of chromatographic conditions :

Based on the interferences, and based on the separations required the experiments are to be

planned to achieve the following separation goals. A base to base separation between all the

impurities. A base to base separation between impurities and placebo peaks (if any). A base-

to-base separation between all the impurities and Principal analyte peak. A base-to-base

separation between placebo peak(s), if any and Principal analyte peak. Good peak shapes for

all the impurities. Ensure that the separations are affected by small changes in the set

conditions.

Relative response factors :

Establish the relative response factors of all known impurities by linearity method

against the target analyte. Relative response factor is dividing the slope of impurity by slope

of active compound.

Selection of system suitability or Performance calculations:

Calculating one or more of the following values is necessary to access overall system

and method performance. (i). Relative retention times, (ii). Theoretical plates, (iii). Capacity

factor, (iv). Resolution, (v). tailing factor and (vi). % RSD of peak area of replicate standard

injections.

Validation of a stability indicating LC method:

The objective of the method of validation process is to provide evidence that the

method does what it is intended to. All the variables of the method should be considered,

including sample preparation, chromatographic separation, detection and data evaluation. For

chromatographic methods used in analytical applications method validations are to be carried

out in accordance with ICH guideline Q2 (R1) “Validation of Analytical Procedures: Text

(Q2A) and Methodology (Q2B)”. Typical validation characteristics which should be

considered are : accuracy, precision (repeatability, intermediate Precision), specificity,

detection Limit, quantitation limit, linearity, range and robustness. This list should be

considered typical for the analytical procedures cited but occasional exceptions should be

32

dealt with on a case-by-case basis. The following is the table given in ICHQ2(R1) which

describe the parameters to be validated for assay and impurities methods [61].

Type of Analytical

Procedure

IDENTIFICATION TESTING FOR IMPURITIES ASSAY

Characteristics Quantitation Limit

Dissolution

( measurement

only)

Content / potency

Accuracy - + - +

Precision

Repeatability - + - +

Intermediate

precision - + (1) - + (1)

Specificity (2) + + + +

Detection limit - - (3) + -

Quantitation limit - + - -

Linearity - + - +

Range - + - +

- Signifies that this characteristic is not normally evaluated.

+ Signifies that this characteristic is normally evaluated. (1) In cases where reproducibility has been performed, intermediate precision is not

needed.

(2) Lack of specificity of one analytical procedure could be compensated by other

supporting analytical procedure(s).

(3) May be needed in some cases.

Definitions of all validation characteristics and the methodology is given in the ICH

guidance and hence does not need a reproduction. Specific procedures followed for each of

the molecules selected is given in the individual chapters. Apart from the ICH prescribed

characteristics or parameters, and stability of mobile phases are routinely studied for

operational convenience.

33

References

1. Atherden,L.M., Edt, Bentley and Drivers, Text Book of Pharmaceutical Chemistry,

8th

Edn., Oxford University Press, 1996.

2. Melentyeva, G., Antonova, L., Pharmaceutical Chemistry, Mir Publishers, Moscow,

1988.

3. Wolff, M.E., Edt., Burger’s Medicinal Chemistry, Part IV, 4th

Edn., Wiley Interscience,

New York, 1981.

4. Deorge, R.F., Edt., Wilson and Gisvolds’s Text book of Organic and Medicinal and

Pharmaceutical Chemistry, 8th

Edn., Lippincott Company, 1982.

5. Pandeya, S.N., A Text Book of Medicinal chemistry, Vol.I and II, 2 nd

Edn., 2003.

6. Hardman, J.G., Limbird, L.E., Molinoff, P.B., Ruddon, R.W., Gilman, A.G., Edt,

Goodman and Gillman's The Pharmacological Basis of Therapeutics , 9th

Edn.,

McGraw Hill, New York, 1996.

7. Gorog, S., Edt., Determination of Impurities in Drugs, Elsevier Sciences, Amsterdam,

1999.

8. Ahuja, S., Impurities Evaluation of Pharmaceuticals, Marcel Dekker, New York, 1998.

9. Husain, S., Rao, R.N., Monitoring of process impurities in drugs. In: Deyl, Z., Miksik,

I., Tagliaro, F., Tesarova, E., Edt., Advanced Chromatographic and Electromigration

Methods in Biosciences, Elsevier Sciences, Amsterdam, 1998: 834.

10. Saranjit Singh, Tarun Handa, Mallikarjun Narayanam, Archana Sahu, Mahendra

Junwal, Ravi P. Shah1., Journal of Pharmaceutical and Biomedical Analysis 69

(2012), 148– 173.

11. Zanowiak, P., In: Kirk-Othmer, Edt, Encyclopedia of Chemical Technology 18, 4th

Edn.,

Wiley, Chichester, 1996: 480.

12. ANDAs: Impurities in drug products, Guidance for Industry, US Food and Drug

Administration, Center for Drug Evaluation and Research, Department of Health and

Human Services, 2008.

13. ICH. Q3A: Impurities in New Drug Substances (June 2006) and Q3B: Impurities in

New Drug Products, Nov 1999 to June 2006.

14. Maggon, K.K., Mechkovsi, A., Drug News Perspect., 1992; 5:261.

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