Chapter - I

Introduction
 Chapter- I

Analytical Chemistry plays an important role since the early days of

Chemistry. It provides the methods for identification, separation, quantification and
structural characteristics determining the nature of chemical elements and compounds
present in the world1. The importance of its study is imperative because it plays a
critical role in the understanding of basic science to a variety of the practical
applications in industry and medicine.

Also analytical chemistry has become an indispensable subject in the

development of nanotechnology, surface characterization instruments, electron and
scanning probe microscopy that enable Scientists to visualize atomic structure with
chemical characterizations.

The recent developments of computer automation and information

technologies have shrunk analytical techniques to chip size (Lab – on – a- chip).
Potential advantages of this development are reduction of quantity of chemicals, size
of equipment and overall cost of analysis with enhanced speed. It is called as micro
Total Analysis System (µTAS). These developments in analytical techniques led to
the successful analysis of the complex biological systems and led to the birth of a
number of the omics such as Genomics (DNA sequencing, Genetic finger printing,
human genome decoding), and Proteomics (peptide sequencing in proteins) etc.

µTAS is considered as a great promise of revolutionary analytical technology

that controls and analyses single cells and single molecules. This cutting edge
technology has a potential of leading a new revolution in science as integrated circuits
did in computer developments.

Modern analytical chemistry plays an increasingly important role in the

pharmaceutical industry where aside from quality assurance it is used in discovery of
new drug candidates and in clinical applications where understanding the interactions
between the drug and the patient are critical2-7.

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

1
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 etc. 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.

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

2
medicinal chemistry and forms the content of many textbooks8-12 and hence needs no
reproduction.

Drugs play a vital role in the progress of human civilization 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 Organization (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 localized at the site where it is
desired to act, should act on a system with efficiency and safety, should not have any
toxicity, may 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 outweighed13. 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 topic14-17. Control is
more important today than ever.

3
 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 deaths18. There upon 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)19, International Conference on the
Harmonization of the Technical Requirements for Registration of Pharmaceuticals for
Human Use (ICH)20 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 ingredients21. The ICH, which took place in
Yokohama, Japan in 1995, has released new guidelines on impurities in new drug
products 22.

These guidelines have a number of advantages, both for the industry and the
regulators. 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 rationalized 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, limit of
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

4
drug23-24. Additional peak tailing, peak resolution and analyte recoveries are important
in case of chromatographic methods.

25-26
The ICH has harmonized the validation requirements in two guidelines .
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 legislation27 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, IP28, United States, USP29, European, EP30, United Kingdom, BP31,
Martindale Extra Pharmacopoeia32, Merck Index33, 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 levels34. 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

5
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 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 materials35-37. In principle, it
is now possible to replace all non-specific assay methods with highly specific and
precise HPLC 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, 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 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.

6
 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 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, sorbitol 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 contain
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

7
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 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 GC, CE (Capillary

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

8
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.

The ICH (International conference on Harmonization) guideline QIA on

38-39
Stability Testing of New Drug Substances and Products 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 100C 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 assay and impurities of drugs, HPLC is a
versatile tool for the qualitative and quantitative analysis of drugs and
pharmaceuticals and has become indispensable. HPLC 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, this study has examined the present
state of development of such analytical methods for some of the widely used drugs.
Hence, the study has proposed to develop stability indicating methods for assay and
impurities for four most widely used drug products made up of drugs, namely,
Evacetrapib (Cardiovascular disease), Niguldipine (Calcium channel blocker),
Glisoxepide (anti-diabetic drug) and Lomitapide (anti-lower cholesterol). The above
drugs are selected for research to which there is wide scope for the development of

9
new analytical methods for their assay and impurities determination by HPLC by
exploiting their characteristics, physical and chemical properties.

Selection of reagents for organic analysis

Several papers are being published every year on the reaction and possible
applications of new and old organic reagents in organic analysis (inclusive of drugs).
The selection of an appropriate reagent for a particular analytical situation is still a
challenging problem. The choice of a particular reagent depends on careful
consideration of such factors as the scale of economies of the reaction, the presence of
other functional groups besides the chosen one that might be adversely affected by the
reagents, the deactivation of the reaction center by steric and electronic effects, the
instability or high reactivity of the desired product, the rate of the reaction, position of
equilibrium as in the case of reversible reaction and other related factors. The
objective is to get the best yields possible. The selection of a reagent for the
determination of a particular compound is made after a literature survey for methods
that have been under consideration. If much information is not found in this way, the
reagent that acts most rapidly and stoichiometrically or at least giving reproducible
results can be chosen after investigation of the performance of plausibly selected ones
on a pure sample of the compound sought. Reagent selectivity for a particular
functional group (in selected drug) is normally the minimum requirement specificity
for a single compound containing the functional group is often desirable, not only to
isolate it from other compounds containing the same functional group but also to
eliminate the effects of interfering compounds.

The general objective of reagent is the formation of one or more derivatives

having measurable chemical or physical properties as completely different as possible
from that of any of the reagents. Some examples of more specific reagents are as
follows.

1. The reagent forms or destroys an acidic, alkaline, oxidizing or reducing

property of the functional group, the amount of change being determined
titrimetrically or spectrophtometrically.

2. The reagent forms a product with a solubility product different from that of the
original sample and this property is the basis for gravimetric determination for

10
 the isolation, concentration and purification of a compound examination by
other analytical techniques.

3. The reagent generates a chromophore or reduces the concentration of the

chromophore already present with change measured by one of the
spectrophotometric techniques, ultraviolet, infrared. Many spot tests for the
functional groups or spraying agents in TLC depend on the formation of
colored derivatives. Similarly, a fluorophore may be produced or quenched
and the change measured fluorimetrically.

4. The reagents act on the sample to produce a gas measurable manometricaly

one that can be collected and determined by titrimetric, gravimetric or other
type of finish.

5. The derivatisation phenomena produces a derivative that is less polar than the
original sample, therefore, more amenable to gas and high performance liquid
chromatographic analysis. Many compounds containing polar functional
groups show unfavorable properties such as low volatility, tailing irreversible
adsorption on many column packages and thermal instability. Vast
improvement in these respects, are easily realized because the polar nature of
the compound promotes derivatisation with suitable chromogenic reagents to
replace the polar group with a less polar one, giving sometimes a more
sensitive detection response.

6. The reagent produces derivatives suitable for structural investigation or

estimation by NMR and mass spectroscopic measurements.

7. Enzymes selectively catalyze specific reaction.

8. Reagents labeled with radioisotopes transfer the isotopes to the derivatives of

the compound analyzed.

In few instances the less reactive functional group may be converted to more
reactive functional group through preliminary reaction (e.g., reduction of –NO2 to –
NH2 hydrolysis of acyl substituted functional group of amine or phenol to free amino
or phenolic hydroxyl group respectively).

11
Features of Chemical Reactions and Reagents of Interest
Knowledge of chemical reactions retains its primary importance in analytical
chemistry in spite of and in many cases because of the already impressively large and
continually growing body of instrumental and nondestructive methods of analysis.
Speculations in complex mixtures of various kinds require the most intimate
knowledge of the entire panorama of chemical transformations and the best reagents
to employ for bringing these about. Direct attention is given to categorizing and
describing the major features of chemical reactions and reagents of interest in the
proposed methods of analysis of selected drugs.

Dyes as Analytical Reagents

In the present investigation dyes have been used either freely or in
combination with an oxidant in the assay of selected drugs. Dye may be defined as a
colored substance which when applied to the fiber gives it a permanent color resistant
to the action of light, water and soap. Because of their commercial importance a very
large number of dyes have been placed in the market. The color index sponsored
jointly by the “society of dyers and colorists” lists about 4500 different dyes and
pigments. They have assigned names according to the method of application and
given a color index number according to their structures. Each manufacturer however
usually labels his products with registered trademark. The dyes are categorized
according to common parent structure40. The chemical categories of dyes are given in
Table 1.1.

12
 Table 1.1

Chemical Categories of Dyes

S.No. Category of the dye Examples
1 Nitro dyes Naphthol yellow S
2 Nitroso dyes Fast green O
Tropaeoline OO
Tropaeoline OOO*
Napthol blue black
Naphthalene blue 12 BR
Congo red
Erichrome Black T
Tartrazine
3. Thiazoles Primuline
4. Diphenyl methanes Auramtne
5. Triphenyl methane and analogus Dyes Fast green FCF*
Bromo phenol blue
Bromo cresol green
Bromothymol violet
Erioglaucine A
Rosaaaniline
Pyronine G
Eosine
6. Acridines Acridine orange NO
7. Phenazines Azocarmine G
Lissammine blue BF
Wool fast blue BL
8. Pheenoxazines Celestine blue*
Gallocyanine
Cresyl fast violet acetate
9. Thiazines Methylene blue
10. Benzoquinones and Naphthaquinones Naphthazarin
11. Anthraquinones Alizarin red S*
12. Indigoids Indigotin
Cibascarlet G
13. Solubilized vat dyes Indigosol O
14. Sulfur dyes Sulfur black T
15. Sulfurised vat dyes Hydron blue R
16. Phythalocyanines Monastrial fast blue BS
17. Cyanines Kryptocyanine
Astraphloxine FF
18. Miscellaneous dyes Quinoline yellow

13
 Recently, chemists extended their dyes study to correlate visual color with
structural features of molecule in 1976, Witt pointed out that two types of groups are
usually present in highly colored compounds (Chromophores) and color intensifying
groups (auxochromes).

Table: 1.2

List of Important Chromophores and Auxochromes

Auxochromes
Chromophores
Acidic Basic

N N  
N O 
-NH2
C O Phenolic -OH -NHR,
                
O -NR2
N

                O 

C=C, C=N, C=S -COOH

-SO3H X = N, S etc.

Subsequently it was suggested that the entire conjugate system is responsible

for color and that either nitro or amino group shifts the absorption to longer
wavelengths. The more the extension of conjugation the greater will be the number of
molecular orbital’s present and energy levels are spaced more closely. Hence less
energy required for electronic transitions Table 1.3 and the absorption is shifted to
longer wavelengths (bathochromic shift). The interaction of auxochromes with the
conjugated system not only extends the conjugation but also leads to large dipole
moments and large transition dipole moments resulting with high dyes intensity
absorption. Derivatisaion such as acylation of an amino or a hydroxyl group merely
decreases the availability of an unshared pair of electrons for interaction with
conjugated systems.

14
 Table: 1. 3

Electronic Transition in Absorbing Species

Transition in energy level
involving sigma, pi and n
electrons
σ - σ *, n- σ *, n-π*, π-π* (imp).
σ* and π* are anti bonding
orbital while ‘n’ involves non-
bonding orbital having an energy
in between bonding and anti
bonding orbitals. The polarized
force between solvent and
species lower the energy levels
of excited and unexcited states.
As (n-sigm* or sigma-sigma*
require much higher energies,
they are seen in a vacuum –UV
and are harder to observe)
Involving ‘d’ & ‘f’
electrons
Transtion metals
involve electronic
transition among
different energy levels
of d-orbitals t2g
(dxy.dyz,dzx)and eg(dx2-
dy2, dz2) are split by
delta in presence of
- - - -
ligands. I <BR <cl F
-
<OH
<Oxalate2<H2o<SCN-<
NH3<en<NO2<CN-
(crystal field theory)
Charge – transfer
spectral absorption
In charge transfer
complexes,
components should be
both electron donor and
electron acceptor which
in turn involves transfer
of electron acceptor
which in turn involves
transfer of electrons to
give absorption
radiation (longer
wavelength)

Information relating to the dyes used in the present investigation

Dyes are used as analytical reagents in two different ways depending upon the
type of their involvement.

A. Colored anionic or cationic form, which involves in ion association complex

formation with oppositely charged ion of the drug.

B. Variations in λ max and ∈ max value of the dye on treatment with an oxidizing,
reducing or complex forming agent lead to the development of visible
spectrophotometric determinations of analysts (direct: reducing agent; Indirect:
Initial oxidation of analyte with an oxidant followed by estimation of un reacted
oxidant with a dye, oxidant reacted with analyte is oxidant initially taken minus
oxidant un reacted). Information relating to the dyes used for estimation of
selected drugs are presented in the Table 1.1.

15
Oxidation followed by complex formation
Fe (III)-1, 10-Phenonthraline (M1) Fe (III) 2, 2’-Bipyridine (M2) and Fe (III)
K3Fe (CN) 6

Ferric salts (ferric chloride) play a prominent role in the colorimetric

determination of organic compounds. Many phenols, hydroxamic acid esters and
more complicate compounds containing the phenolic OH groups in their molecule
react with ferric salt in an aqueous, water alcoholic or chloroform media to give
intense coloration characteristic of each particular phenol. The color dyes to the
strongly ionized complex percolates of trivalent iron, which is formed according to
the equation81.

FeCl3 +ArOH→6H+ + [Fe (OAr) 6]3

The color intensity and stability of the complex increases when the –COOH
group is adjacent to phenolic hydroxyl (eg. Salicylicacid). Addition of acid, glycerol,
alcohol and sometimes excess ferric chloride decreases the degree of phenolate
dissociation (Hence the concentration of color decreases), and the color of the
solution vanishes. Alkalizing also destroys the color by binding the iron into
hydroxide.

The amides and esters of fatty acids are characterized by oximes with which
the -NHOH group is substituted for the -NH2 group. The substitution takes place
during boiling with solutions of hydroxylamine salts.

R COO C2H5 + NH2OH-----------→ R CO NHOH + C2H5OH

R CO NH2 + NH2OH--------------→ NH3 + R CO NHOH

Hydroximic acids that are formed in this reaction can easily be detected since
they react with anion of trivalent iron to give intensely colored complex salts. The
nitro group is strong electron acceptor produces a clear –I effect (Inductive) in an
organic molecule. Nitro methane is a pseudo acid, and displays tautomerism.

CH3NO2---------------------→CH2=NOOH (iso nitro group)

The sodium salt of nitro methane reacts with FeCl3 to give a complex iron salt,
which is intensely colored. This reaction is characteristic of primary and secondary

16
nitro compounds. FeCl3 reacts with sodium acetate to give first ferric acetate (by the
usual exchange reaction), which is immediately hydrolyzed to give a complex
compound, chlorides of used ferric hexa acetate (brown color).

[Fe (OH) 3 (CH3 COO) 6]+ Cl-

Polyhydroxy alcohols or oxy acids react with FeCl3 to give stable complexes.
Oxy acids react with FeCl3 to give complex salts of iron, which simultaneously
oxidize the oxy acids.

Ferric chloride can also oxidize phenols. It oxidizes hydroquinone to quinine,

which then gives quinhydrone. Napthols are oxidized by FeCl3 to give sparingly
soluble dinapthols, in which two naphthalene rings are combined.

3 C10 H7 OH + 2FeCl3-------------→HOC10H6C10H6OH + 2 FeCl2 + 2 HCl

Other phenols also form phenolates of iron with partial oxidation.

Acting as an oxidant a Ferric salt is converted into ferrous salt. They can
easily be detected by the usual reagent for divalent iron, potassium ferricyanide41
(given below), 1, 10-phenanthroline42, bispyridyl43 or triazine.44

1, 10-phenanthroline forms a complex of low functional value with Fe (III)

which in turn functions as a better oxidant than Fe (III) itself. The reduction product
is tris complex of Fe (II), well known as Ferroin. Based on complexing tendency and
oxidizing properties. Ferric salts were suggested in the estimation of several
drugs45-48.

2,2’-bipyridine forms a complex of low functional value with Fe(III) which in

turn functions as a better oxidant than Fe (II) itself. The reduction product is tries
complex of Fe (II). Based on its complexing tendency and oxidizing properties, ferric
salt was suggested in the estimation of several drugs.49-51

In the present investigation the drug was treated with excess ferric salt under
specified experimental conditions. Acting as an oxidant, ferric salt converts to ferrous
salt, which corresponds to the drug investigation.

17
 In the present investigation 1, 10-phenanthroline (M1) 2, 2’bipyridine (M2) and
potassium fericyanide (M3) method were used for estimation of Evacetrapib,
Niguldipine, Glisoxepide and Lomitapide.

Oxidation/Reduction reaction
FC Reagent: Folin-Ciocaltaeu Reagent
Reduction of the heteropoly acid complexes by organic reagents was utilized
as the basis for the determination of several organic compounds, particularly
phenols52-54, amines and enols55-56. The wavelength of maximum absorption and
stability and reproducibility of the reaction depends upon pH, composition of hetero
poly acidic complex, nature and concentration of the reducing agent, temperature and
time57 it may be said generally, that more the number of hetero acids in the complex,
and the more venerable it is to reduction under certain critical conditions. Among the
various hetero poly acids, phosphor molybdotungstic acid, the well-known Folin
ciocaltaeu reagent was preferred by a number of workers for the determination of
drugs containing not only phenolic or amino groups but also certain other drugs that
do not contain the groups58.

The color formation by Folin ciocaltaeu (FC) reagent59 with organic

compounds may explain in the manner based a the analogy with the reports on earlier
workers60-63.

Diazo Coupling Reactions64

The diazonium salts derived from p-amino benzoic acid65, p-nitroaniline66,
72-76
p-sulphanilic acid (Pauli reagent) , 2-aminobenzothiazole77-78, 3-pheny l-5-
nitrosamine 1,2,4 thiadiazole79-80, 4-amino 6-chloro1,3-benzenedisulphona-mide81,
benzocaine82, dapsone83 and sulphanilamide84 are commonly used reagents for direct
coupling procedures. The aryl amine coupling reagents are converted to their
diazonium salts with HCl and sodium nitrite. The reaction is usually carried out in an
ice bath, the excess nitrate is removed by reaction with sulphamic acid or ammonium
sulphamate, and the PH is adjusted for the coupling reaction. The reagent is used
immediately since most diazonium salts are not stable.

The most common source of interference in analysis using the direct coupling
procedure is impurities in the sample, which also couple with the diazonium salt and

18
exhibit some absorbance at the wavelength chosen for the analysis. Interference of
this type can only be avoided by employing a separation step prior to color
development.

Several compounds of pharmaceutical importance are analyzed by direct

coupling with a diazonium salt. They include, levarterenol85, 8-hydroxy quinoline86,
isonazide, 3-amino-1H-1, 2, 4-triazole87, estraddioldipropionate88-89, tyrosine90,
thiamine91, salbutamol92, nyidrin93, pholedrin94 and terbutaline95.

Diazotization of the Analyte and Coupling

The second group of diazo coupling reactions are those in which the analyte is
converted to a diazonium salt and then coupled to a substrate. Procedures of this type
are encountered much more frequently than direct coupling procedures in
pharmaceutical analysis. The most common substrates are (1-naphthyl)
96
ethylenediamine dihydrochloride (Bratton-Marshall Reagent) and 2-naphthol.
Bratton-Marshall Reagent is preferred for quantitative work because the products are
usually soluble and have high molar absorption. 2-naphthol often forms insoluble
coupling products and is more frequently used for qualitative identification tests97.
When the substance being analyzed is diazotized, optimization of the reaction
conditions and time becomes especially important because the diazonium salts are
generally unstable and any loss through decomposition or side reactions will
decrease the sensitivity and precision of the analysis. The reaction of aromatic amines
with nitrous acid to form diazonium salts is very general and is carried out regardless
98-99
of other ring substituents. The mechanism of the reaction is outlined in the
following scheme.

H+ + HONO→H2O- NO+ → NOx + NO2

NOX + ArNH2→X-+Ar N+H2-N=O→H+ + Ar-N-N=O→
H+ + Ar-N=N-OH→Ar-N+ ≡N + H2O
Formation of diazonium salt is usually fast enough that any convenient PH
between 0 and 3 can be used. In case where the reaction is unusually slow or where
the diazonium salt is unusually labile, it might be well to optimize the PH in order to
increase the reaction rate and minimize the effects of decomposition. The reaction
rate is also increased if the PH is adjusted with hydrochloric acid rather than sulphuric

19
acid, since NOCl is a better nitrosating agent than NOHSO4. Adding NaBr or KBr can
increase the rate even further, presumably due to the formation of NOBr, which is a
better nitrosating than NOCl100.

The coupling reaction requires a polar solvent to accommodate the ionic

intermediates and water and ethanol are most frequently used. Careful control of
solvent PH is very important in achieving rapid, quantitative reaction coupling to
amine substrates should be carried out between PH 5 and 9, and phenols between PH 9
and 10. The exact PH must be determined experimentally in each case.

The spectrophotometric measurement of the coupling products of diazotized

sulphanamides with Bratton-Marshall Reagent has been studied in detail101-102. The
absorption maximum is near 545 nm for coupled sulphanamides and maximum
absorption intensity is achived in the PH range 1 to 2. A few examples of the utility of
this reaction for the analysis of drugs containing an aromatic amino group include
bendrooflumethiazide, sulphodaxime103, and sulphamerazine104-108, sulphamethazine
109
, and procainnamide 110-111.

A number of drugs have also been determined by first converting them to a

diazotisable species (either by hydrolysis or reduction) which is then coupled in the
usual manner. Chloramphenicol contains a nitro group, which can be reduced to an
amine with a suitable reducing agent, (metallic zinc/acid, stannous chloride or sodium
dithionate). Reduction to the amine followed by the Bratton Marshall reaction has
been used for the analysis of this drug112-114. Using it to diazotize an aromatic amine,
coupling the diazotized amine with a substrate and measuring the product
spectrophotometrically can determine nitrous acid. The Griess method for nitrite
employs sulphanailic acid as the source of coupling agent and α-naphthyl amine as the
substrate115. This reaction has been used for the analysis of pharmaceutical
preparations including gelatin capsules and can also be used for nitrate after a
reduction step. A typical procedure will consist of an alkaline hydrolysis, followed by
acidification, diazotization of coupling agent and finally coupling with the
116
substrate . Isosorbide dinitrate has been determined using sulphanilic acid and
α-naphthyl amine (Griess method), and also using p-nitroaniline as the coupling agent
and aniline as the substrate117.The later combination has also been used to analyze
pentaerythitol tetranitrate118.

20
 In the present investigation diazotization methods M1, M2 have been used for
the determination of NGD.

Dyes in Ion Association Complex Formation:

The term molecular complex is used to describe a variety of association
products of two or more molecules. In recent years, extensive attention has been given
to a large number of complexes formed by weak interaction of certain classes’ of
organic compounds functioning as electron donors (bases), while others act as
electron acceptors, (acids) 119-121. The forces, which lead to the formation of molecular
complex, include forces such as dipole and induced dipole interactions, London
dispersion forces, hydrogen bonding and dative bonding interactions. The donor-
acceptor complexes (where composition can be represented by integral ratios of the
components) are in many instances so unstable that they cannot be isolated in the pure
state at ordinary temperatures but exist only in solutions in equilibrium with their
components. They can however, usually be detected readily because of differences in
the physical properties (e.g. absorption spectra, solubility in organic solvents) from
those of the pure components. The ion association complex or adduct is a special form
of molecular complex resulting from two components extractable into organic
solvents from aqueous phase at suitable PH. One component is a chromate (dye or
metal complex) possessing charge (cationic or anionic nature) and so insoluble in
organic solvents. The other is colorless, possessing opposite charge (anionic or
cationic) to that of chromate.

The ion association complex extraction has been applied to the estimation of
numerous compounds, possessing basic moieties (secondary or tertiary aliphatic
amino groups) by using an acid dye as a reagent and a chlorinated solvent as an
extractant. The structure of the species formed may depend upon the experimental
conditions122-123 (Concentration of the components, pH of the aqueous phase). The
color can be altered or intensified upon acidification or re extracted into a buffer. The
presence of hydrophilic substituents such as –OH or –COOH often prevents
extraction of the complex into organic solvent as an extractant, which then depends
upon parameters such as the polarities of the amine and of the dye. Relatively poor
amines such as aryl amines, diamines and many alkaloids react with bromothymol
blue poorly or not at all with bromocresol purple. Contrary wise, this second dye

21
behaves like the first one with weakly polar amines such as amine derivatives of
biphenyl methane or phenothiazone.

Therefore, compounds may be determined in binary mixtures, the less polar

amine is estimated with bromophenol blue, that the sum of the two amines is given by
bromocresol purple. Mixtures of diphenyl hydramine and ephedrine were also
124
analyzed .

According to the same principle, acid dyes can be colorimetrically determined

with basic dyes. P-toluene-sulfonic acid, camphosulfonic acid and bromocampho-
sulfonic acid were estimated with fuchsin125, chlorotoluenesulfonic acid with acridine
orange, rhodamine S or chrysoidine126 and phenylbutazone, which develops a slightly
acidic reaction, with Gentian violet127.

The same principle has been applied for the determination of secondary and
tertiary aliphatic amines with tropaeolinOO and of tertiary aliphatic amine with
erythrosine128. Several dyes belonging to different chemical classes have been used
129-134
for the determination of basic drugs . Few dyes have been utilized for the
development of new extractive spectrophometric methods for the determination of
selected drugs in the present work. The acidic dyes have been summarized in
Table1.1.  

PRESENT STUDY:
The Spectrophotometer and HPLC techniques are most widely using techniques in
drug and pharmaceutical industry. It is very important to develop better sensitive
methods with simplification to use for routine pharmaceutical analysis. In this present
study the author is focused on development of some of new spectrophotometric and
HPLC methods for four durg products named by Niguldipine (Calciumchannel
blocker) Evacetrapib (Cardiovascular disease), Glisoxepide (anti diabetic) Lomitapide
(anti lower cholesterol) with better sensitivity and accuracy.

22
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