CHAPTER I - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/8233/6/06... · 2015-12-04 ·...
Transcript of CHAPTER I - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/8233/6/06... · 2015-12-04 ·...
CHAPTER – I
INTRODUCTION ON APPLICATION OF PHYICO-CHEMICAL METHODS
(SPECTROPHOTOPMETRY AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY)
FOR THE ASSAY OF DRUGS
1
1.01.A: DRUGS AND FORMULATIONS:
Any substance that is carefully used for diagnosis, cure and prevention for
altering the structure and function of the body is called drug1. In the modern era,
drugs play an important role in the progress of human civilization. While primitive
man depended mainly on plant product and metal salts to cure diseases, modern
man uses a wide range of synthetic organic compounds and biotechnology- derived
antibiotics, vaccines, etc. There are many important stages before a compound is
used as a drug. The three important stages in the use of a drug as a medicine, i.e., the
conversion of a drug into a formulation are
i. The discovery of the drug.
ii. The manufacture of the drug in bulk form.
iii. The formulation of a drug into different dosage forms like tablet, capsule,
injection, syrup etc.
First stage is the drug discovery, where the compounds are screened for
biological activities. Second stage is the manufacture of the drug using well
understood chemistry and adapting safe and proper manufacturing and analytical
practices and the third stage is the formulation of the drug in a convenient dosage.
Chemists play an important role in pharmaceutical research, as they synthesize,
purify and analyze the drugs. The study of conversion of drugs into medicine and its
manufacture, stability and the effectiveness of the drug dosage form is termed as
pharmaceutics. The preparation, chemical and physical composition, reactive nature,
geometry, influence on an organism, quality control methods, storage conditions are
pre-requisites in the study of drugs which fall under pharmaceutical chemistry,
2
a potential field, based on the general laws of chemistry2-7. The family of drug could
be either chemotherapeutic or pharmacodynamic agents.
Pharmacodynamic are a group of drugs, which depress or stimulate various
functions of the body, providing some relief by mitigating any abnormality in the
body. Though they are not likely to cure the diseases, they may provide temporary
relief. Depressants, stimulants towards central nervous system, adrenergic, blocking,
cholinergic, cardiovascular, diuretics, antihistaminic, anticoagulating agents belong
to this group. These have no action on infective organisms.
Chemotherapeutic agents are selectively more toxic to the invading
organisms. They cause no harm to the host. Antimalarials, antibacterial,
antiprotozoals, organometallic agents belong to this group.
Every bulk drug and corresponding pharmaceutical formulations have to
follow the set standards by each country through legislation8. Several
pharmacopoeia publications do furnish these regulations9-12. Pharmaceutical
analysis13,14 deals not only with the purity of drugs and their formulations but also
with their precursors. However, degree of purity and the quality of medicament is a
must before releasing into the market. The quality of a drug is decided only after its
authenticity is tested, both in the drug and its formulations. Quality is paramount as
it is more vital in the field of medicine as the target is life15. One has to consider the
process of production of a drug and meticulously prevent impurities and toxic
elements which may peep in. The whole operation from raw material to the final
product in the form of a drug or formulations must go through a quality control unit.
This hinges on good laboratory practice. Here the analyst do both qualitative and
quantitative determination of not only the raw material but also the drug in bulk and
3
their pharmaceutical formulations. Modern pharmaceutical analytical techniques
need the following requirements.
i. Minimal time for analysis.
ii. Analysis accuracy should satisfy the demands of pharmacopoeia.
iii. Analysis should be economical.
iv. The selected method should be precise and selective.
v. The above requirements are met by the physico-chemical methods of analysis.
Several methods for the estimation of drugs are classified into physical,
chemical, physico-chemical and biological ones. Physical methods involve the study
of the physical properties such as solubility, transparency or degree of turbidity,
color density, specific gravity etc. The chemical methods include the titrimetry,
gravimetric and volumetric procedures which are based on complex formation,
redox reactions etc. Physico-chemical methods involve the study of the physical
phenomena that occurs as a result of chemical reactions16-18. These include optical
and chromatographic methods.
The growth of pharmaceutical industry, increase in the number and variety of
drugs and availability of sophisticated instruments have paved way for rapid
progress in providing simple analytical procedures for the analysis of complex
formulations also. The availability of new techniques with improved equipments has
made the latest techniques attractive.The latest knowledge has thrown open the
possibility of adopting unique techniques for assaying a single drug alone or a
number of drugs in a formulation at one stroke. Separation techniques, particularly
chromatographic methods are valuable in analysis of pharmaceuticals. Modern
spectrophotometer which incorporates features such as microprocessor control,
diode array detector has become an essential tool for analysis. Assay methods based
4
on absorption in the ultraviolet and visible region of electromagnetic spectrum are
used extensively. Some colorless substances required to be analyzed are converted
to a derivative having color, the intensity of color measured at suitable wavelength
and compared with that of known amount of reference substance of known purity.
1.02:A:INTRODUCTION TO SPECTROPHOTOMETRY:
One of the quantitative procedures for formulations is the
spectrophotometric method, which utilizes the measurement of intensity of
electromagnetic radiation emitted or absorbed by the analyte. The
spectrophotometer has become a useful instrument for drug analysis. Now it is the
instrument of choice in conducting quantitative estimation of colored and colorless
solutions. The spectrophotometers are based on the principle of over determination,
where the number of observation wavelengths may exceed the number of
components present. The spectrophotometers have an inbuilt microprocessor
system that process and prints for spectral data. This instrument computes accurate
results within minimal time. The concentrations of each of the component in the
mixtures are printed through an inbuilt system. Of the spectrophtometric techniques
available, UV-Visible spectrophotometry is generally preferred especially by small
scale industries as the cost of the equipment is less and the maintenance problems
are minimal.
1.02. A.i: UV-VISIBLE SPECTROPHOTOMETER:
The ultraviolet-visible (UV-Vis) spectrophotometer is an instrument that
analyzes the absorbance of compounds in the ultraviolet and visible regions of the
5
electromagnetic spectrum. Unlike infrared spectroscopy which detects vibrational
motions of molecules, ultraviolet-visible spectroscopy detects electronic transitions.
In general, a hydrogen or deuterium lamp is the light source for the ultraviolet
region from 200-400 nm, and a tungsten or halogen lamp is the light source for the
visible region of 400-800 nm. The solvent and sample solutions are placed in the
reference and sample cells, respectively. Monochromatic light from the source is
allowed to pass through the cells, and the transmitted light is measured by a
detector the basic design of a UV-Vis spectrophotometer is illustrated in Figure 1.01,
P.5. Ultraviolet and visible light are energetic enough to promote valence electrons
to higher energy levels. The UV-Vis spectra have broad features that do not limit
their use only for sample identification but are also very useful for quantitative
measurements.
Fig: 1.01. UV-Visible spectrometer Basic Instrumentation
The electronic transitions that are detected by the UV-Visible spectrometer
result in absorption maxima in the spectra, which occur due to the excitation of
valence electrons of a compound from the ground level to higher energy levels. In
other words, it can be described as the excitation of electrons from bonding and
nonbonding orbitals to the antibonding or nonbonding orbitals.
6
1.02.A.ii:THEORY OF SPECTROPHOTOMETRY:
i) Wavelength and Energy:
Absorption and emission of radiant energy by molecules and atoms is the
basis for optical spectroscopy.
The absorption and the emission of energy in the electro-magnetic spectrum
occur in discrete packets of photons. The relation between the energy of a photon
and the frequency appropriate for the description of its propagation is
E = hϑ
Where, E = Energy in ergs; ϑ = Represents frequency in cycles per second; h
= Plank's constant (6.6256 x 10-27 erg-sec)
The data obtained from a spectroscopic measurement are in the form of a
plot of radiant absorbed or emitted as a function of position in the electromagnetic
spectrum. This is known as a spectrum and the position of absorption or emission is
measured in units of energy, wavelength or frequency.
ii)Beer-Lambert’s law:
The basic principle in analyzing the absorbance by UV-Vis spectrophotometer is
the Beer-Lambert law. This law states that the absorbance of a solution is directly
proportional to the concentration of solution and the thickness of the medium(cuvette-
1cm)
A = € b c
7
Where, A = absorbance of the solution; € = molar absorptivity coefficient;
c = concentration; b = path length.
The assay of an absorbing substance may be quickly carried out by preparing
a solution in a transparent solvent and measuring its absorbance at a suitable
wavelength. The concentration of the absorbing substance is calculated from the
measured absorbance.
1.02.A.iii: CRITERIA FOR SPECTROPHOTOMETRIC
ASSAYOF PHARMACEUTICAL FORMULATIONS:
Even though spectrophotometric methods are versatile in nature, in order to
have successful and satisfactory result, the process of analysis needs careful
operations. Since the color development in spectrophotometry involves diverse type
of reactions, a number of points need to be ensured before applying the method for a
particular application which includes the following.
I) METHOD DEVLOPMENT:
The first step in spectrophtometric assay is the method development it
should be to set with minimum requirements, which are essentially in acceptance
specifications for the method. A complete list of criteria should be agreed on by the
developer and the end users before the method is developed. During the actual
studies and in the final validation report, these criteria will allow clear judgment
about the acceptability of the analytical method. The statistics generated for making
comparisons are similar to what analysts will generate later in the routine use of the
8
method and therefore can serve as a tool for evaluating later questionable data.
More rigorous statistical evaluation techniques are available and should be used in
some instances, but these may not allow as direct a comparison for method trouble
shooting during routine use.
i) Choice of solvent: The solvent which is to be used in colorimetric or
spectrophotometric determinations must meet certain requirements. It must be a
good solvent for the substance under determination. Before using a particular
solvent, it must be ensured that it does not interact with the solute. The solvent must
not show significant absorption at the wavelength to be employed in the
determination. For inorganic compounds, water normally meets these requirements,
but for majority of organic compounds, it is necessary to use an organic solvent. All
solvents show absorption at some point in the ultraviolet region and care must be
taken to choose a solvent for a particular determination which does not absorb in
the requisite wavelength region. Any impurities present in the solvents may affect
the absorption at certain wavelength and it is therefore, essential to employ solvents
of the highest purity.
ii) Choice of chemical reactions and reagents of interest:
A Knowledge of Chemical reaction retains its primary importance in
analytical chemistry because of continually growing body of instrumental and
nondestructive methods of analysis.
9
TABLE –1.01
LIST OF PROPOSED AND REPORTED VISIBLE SPECTROPHOTOMETRIC METHODS
Type of Reaction Reagent used Method Drugs responded Chapter No in which the method
incorporated
Oxidative Coupling Reaction
MBTH –NaIO4
M1a
Abacavir Sulfate[ACS] Chapter -II
Milnacipran [MCN] Chapter -III
Tanofavir Disproxil Fumarate[TDF] Chapter -V
MBTH – Ce(IV) M1b Abacavir Sulfate[ACS] Chapter -II
MBTH – Fe(III) M1c Abacavir Sulfate[ACS] Chapter -II
Tanofavir Disproxil Fumarate[TDF] Chapter -V
MBTH - IBDA M1d Hydralizine HCl [HZH] Chapter -IV
Brucine – NaIO4 M2 Hydralizine HCl [HZH] Chapter -IV
NaIO4 – PHH - K3Fe(CN)6 M3 Tanofavir Disproxil Fumarate[TDF] Chapter -V
Redox Reaction
FC M4 Abacavir Sulfate[ACS] Chapter -II
AV –H2SO4 M5 Abacavir Sulfate[ACS] Chapter -II
Milnacipran [MCN] Chapter -III
FGFCF –KMnO4 M6 Abacavir Sulfate[ACS] Chapter -II
10
TABLE –1.01
LIST OF PROPOSED AND REPORTED VISIBLE SPECTROPHOTOMETRIC METHODS
Type of Reaction Reagent used Method Drugs responded Chapter No in which the method
incorporated
Charge Transfer Reaction
DDQ M7 Abacavir Sulfate[ACS] Chapter -II
CA
M8
Abacavir Sulfate[ACS] Chapter -II
Milnacipran [MCN] Chapter -III
Hydralizine HCl [HZH] Chapter -IV
Tanofavir Disproxil Fumarate[TDF] Chapter -V
Ion Association Reaction
BCG M9a
Milnacipran [MCN] Chapter -III
Hydralizine HCl [HZH] Chapter -IV
BCP M9b
Milnacipran [MCN] Chapter -III
Hydralizine HCl [HZH] Chapter -IV
BTB M9c
Milnacipran [MCN] Chapter -III
Hydralizine HCl [HZH] Chapter -IV
TPooo
M9d
Abacavir Sulfate[ACS] Chapter -II
Tanofavir Disproxil Fumarate[TDF] Chapter -V
ARS
M9e
Abacavir Sulfate[ACS] Chapter -II
Tanofavir Disproxil Fumarate[TDF] Chapter -V
Diazocoupling Reaction
PGNL –NaNO2 M10a
Abacavir Sulfate[ACS] Chapter -II
Tanofavir Disproxil Fumarate[TDF] Chapter -V
RSNL –NaNO2 M10b
Abacavir Sulfate[ACS] Chapter -II
Tanofavir Disproxil Fumarate[TDF] Chapter -V
11
Speciation 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 (briefly in
TABLE.1.01, P.9-10 and more details in the following text).
I) Oxidative coupling reactions (Method- M1a, M1b, M1c, M1d, M2 and M3):
Oxidative coupling procedures involve the coupling of drugs [possessing
functional groups such as phenolic hydroxyl, aldehyde, amine or diol] with various
reagents such as 3-methyl-2-benzothiazolinone hydrazone (MBTH), p-N,N-
dimethylphenylenediamine(DMPD), 4-aminophenazone(4-AP), phenyl hydrazine
hydrochloride (PHH), p-methylamino phenol sulfoanate(PMAP) and Brucine in the
presence of an appropriate oxidant under slightly acidic, neutral or slightly alkaline
conditions forming highly colored species. Among these reagents the author had
used 3-methyl-2-benzothiazolinone hydrazone (MBTH), phenyl hydrazine
hydrochloride (PHH) and Brucine for the visible spectrophtometric determination of
the selected drugs.
A) MBTH as reagent in Oxidative coupling reactions:
3-Methyl-2-benzothiazolinone hydrazone Hydrochloride (MBTH) which was
synthesized by Besthorn19 is one of the widely used chromogenic reagents for
spectrophotometric analysis of various organic compounds. Sawicki20 determined
aldehydes, using MBTH as reagent with appropriate oxidant and this procedure was
12
later improved, and used for sensitive determinations of aliphatic aldehydes21-24 in
tissues and collagens 25.
It is the widly used chromogenic reagents for spectrophotometric analysis of
phenols26. It undergoes an interesting reaction with phenolic, amino, ketonic and
aldehydic compounds in the presence of oxidizing agent such as H2O2, cerium (IV),
iron (III), chromium (VI) yielding a highly colored reaction products26. MBTH had
been used for spectrophotometric determination of caffeine and theophylline27,
cefprozil28 , amoxicillin29 and certain 4-quinolones in drug formulations30.
Under reaction conditions, MBTH loses two electrons and one proton on
oxidation in the presence of oxidizing agents [H2O2, cerium (IV), iron (III), chromium
(VI), IBDA and NaIO4], forming the electrophilic intermediate [active coupling
species]. This electrophilic intermediate reacts with most nucleophilic site on the
aromatic ring of amine/phenol/aldehyde (i.e., para or ortho position) of various
organic substances by electrophilic attack that spontaneously forms colored species
that can be read colorimetrically. There are several oxidizing agents [H2O2, cerium
(IV), iron (III), chromium (VI), IBDA and NaIO4] which give color with MBTH
[Universal reagent] for the determination of various drugs.
i) MBTH with Sodium meta periodate [NaIO4] (Method- M1a):
Periodic acid oxidation31-41 is applicable to compounds having two hydroxyl
groups or a hydroxyl and an amino group attached to adjacent carbon atoms and are
characterized by the cleavage of the carbon-carbon bond. Periodate oxidation can be
applied in aqueous solution over a very wide range of pH to small amounts of
material in a fairly simple and straightforward fashion. The rapid and generally
13
quantitative nature of the reaction recommends it for a very wide variety of
analytical applications. Sodium metaperiodate (IO4-) is considerably soluble in water
(12.62g/100mL, 25oC). The solubility of sodium metaperiodate is greatly reduced in
alkaline solution because of the formation of disodium metaperodate (Na2H3IO6)42.
This effect occurs at pH>5.0.Aqueous solution of sodium metaperiodate at pH 4.0 or
below is the most suitable one as the oxidant 43.
The oxidation reaction with periodate are quantitative. Certain analytical
procedures have been developed for the determination of aldehydes utilizing
periodate oxidation44,45.
Under the reaction conditions, on oxidation with NaIO4, the reagent MBTH
loses two electrons and one proton forming an electrophilic intermediate, which is
the active coupling species. This intermediate undergoes electrophilic substitution
with the selected drug to form the colored product.
The author had attempted to develop new visible spectrophotometic
methods for the selected drugs ACS, MCN and TDF, which possesses secondary
amino group, involving oxidative coupling reaction with MBTH in the presence of
NaIO4[Method M1a] forming oxidative coupling products. The probable sequence of
reactions and the developed procedures for their assay are presented in
corresponding chapters II, III & V of the corresponding drugs.
ii) MBTH with Ceric ammonium sulphate [Ce IV] (Method- M1b):
E.I. Kommas46 first suggested ceric ammonium sulphate as an oxidant with
MBTH [reagent] under acidic conditions for the determination of pharmaceuticals
possessing phenol group. H.D.Revana siddappa47,48 et al reported an analytical
procedure for the estimation of ritodrine hydrochloride in bulk samples and in unit
14
dosage forms. Sastry49 et al described a sensitive kinetic method for the
determination of ketoprofen in pure form, pharmaceuticals and biological fluids.
Zaheer ahmed50 et al developed a sensitive spectrophotometric method for the
estimation of adefovir dipivoxil in bulk and pharmaceutical preparations
The method proposed by the author utilizes an oxidative- coupling reaction
based upon oxidation of 3-methyl-2-benzo-thiazolinone hydrazone hydrochloride
(MBTH) with Ce(IV)[oxidant] in presence of HCl, forming an electrophilic
intermediate (diazonium salt of the reagent) that inturn couples with the selected
drug yielding a highly colored condensation product.
iii) MBTH with Ferric chloride [FeCl3] as oxidant (Method- M1c):
Ferric chloride has been mostly used as an oxidant for the determination of
aromatic and heterocyclic amines by Sawicki51 et al (in neutral conditions) and
Pays52 (in acidic conditions). This Oxidative coupling reaction involving MBTH in
presence of ferric chloride has been used for the assay of several drugs53-56. Rekha
Rajeev kumar57 et al described a simple, economical and accurate
spectrophotometric method for the estimation of moprolol in bulk and
pharmaceutical dosage form (Tablet). Prakash S. Sarsambi58 et al reported a visible
spectrophotometric method for the estimation of ganciclovir in bulk drug or its
formulations. Malipatil S.M59 et al developed a sensitive spectrophotometric method
in visible region for the estimation of citicoline in pharmaceutical dosage forms.
Recently Malipatil S.M60 et al reported a spectrophotometric method for the
quantitative estimation of oseltamivir phosphate in bulk drug as well as formulation.
15
G.Vijaya Raja61 et al described a new spectrophotometric method for the
determination of bromhexine hydrochloride in bulk and formulations
The selected drugs Abacavir Sulfate [ACS] and Tanofavir Disproxil Fumarate
[TDF], which possesses secondary amino group in the presence of oxidant, Fe (III)
undergoes oxidative coupling reaction with MBTH forms oxidative coupling
products [colored]. The probable sequence of reactions and the developed
procedure for their assay are presented in corresponding chapters II & V of the
corresponding drugs.
B) Brucine – Periodate (Method – M2):
Brucine (2,3 – dimethoxystrychnine) under acidic conditions has been
reported to be an effective analytical reagent for spectrophotometric determination of
nitrates and nitrites62, cerium63, manganese64, cadmium and platinum65. It was also
reported that in combination with potassium persulphate, it is used for the
spectrophotometric determination of halides66 and cysteine67 and as an indicator in
redox titration68-70. Brucine forms a 1:1 colored complex with p–dimethylamino
cinnamaldehyde under acidic conditions71.
Sodium metaperiodate is an effective oxidant for converting methyl substituted
p-dihydroxy phenols to o-quinones72 and is also color stabilizer. Sastry73 et al used
brucine-periodate reagent for spectrophotometric determination of tryptophan and
some sulphur compounds74 and for tetracyclines, chlorophenicol and streptomycin75.
According to them, periodate converts most electron rich portion of the coupler
(tryptophan and other mentioned compounds) to yield 1-mono substituted
bruciquinone derivatives (colored species). Brucine reagent gave colored species with
16
the compounds containing either primary or secondary aliphatic amino and aromatic
primary amine groups upon oxidation with periodate. On the basis of this observation
the author has developed a specific method for the assay of the selected drug
hydralizine HCl (HZH) in bulk samples and dosage forms. The details of the
spectrophotometric investigations of the corresponding drug are incorporated in
chapter IV respectively.
C) NaIO4 / (PHH)/ [Fe (CN) 6]-3(Method M3):
Periodate oxidation can be applied in aqueous solution over a very wide
range of pH to small amounts of material in a fairly simple and straightforward
fashion. The rapid and generally quantitative nature of the reaction recommends it
for a very wide variety of analytical applications. Sodium metaperiodate (IO4-) is
considerably soluble in water (12.62g/100mL, 25oC). The solubility of sodium
metaperiodate is greatly reduced in alkaline solution because of the formation of
disodium metaperodate (Na2H3IO6)42. This effect occurs at pH>5.0.Aqueous solution
of sodium metaperiodate at pH 4.0 or below is the most suitable one as the
oxidant43.
The oxidation reaction with periodate are quantitative. Certain analytical
procedures have been developed for the determination of aldehydes utilizing
periodate oxidation44,45. Even though there are several procedures based on
different principles using several reagents for the determination of aldehydes in
particular formaldehyde (existing or formed through some preliminary treatment
such as periodate oxidation of compounds possessing vicinal aminol, diol or ketol),
appear to yield highly sensitive and stable chromogen with formaldehyde especially.
17
This method permit the determination of the liberated formaldehyde directly in the
reaction medium colorimetrically by oxidative coupling reaction with schryver
reaction76 with PHH and hexacyanoferrate (III) and this method has been applied
by sastry77 et al for the determination of doxorubicin in dosage formulations.
In the present investigation, Tanofavir Disproxil Fumarate [TDF] responded
to oxidative coupling reaction with PHH in the presence of hexacyanoferrate (III)
giving formazan dye. The details of the investigation of the corresponding drug
Tanofavir Disproxil Fumarate [TDF] are incorporated in chapter V.
II) Redox reactions (Methods M4, M5 and M6):
Redox reactions [oxidation-reduction reactions], are a family of reactions that
are concerned with the transfer of electrons between species. In the present
investigations the author had used Folin Ciocalteu (FC), Ammonium vanadate (AMV)
and Wool Fast Green (FGFCF) as reagents for the visible spectrophotometric assay of
selected drugs.
I) Folin Ciocalteu [FC] reagent (Method- M4):
Heteropolyacid complexes are formed by the combination of
orthophosphoric acid and periodic, molybdic, vanadic, tungstic and molybdovanadic
acids. Treatment of complexes with reducing agents result in the formation of the
corresponding reduction products, which are blue in color (eg: molybdenum blue
from phosphomolybdate, tungsten blue from phosphotungstate). This reaction was
the basis of several methods suggested for the determination of phosphate78.
Various reducing agents have been used for the reduction of heteropolyacids.
Stannous chloride was most widely used one among several reducing agents[ 1,2, 4-
18
aminonaphthol sulphonic acid79, ascorbic acid80, hydrazine81, ferrous sulphate82, p-
amino phenol hydrochloride83, thiosulphate and sulphite84, thiourea85, pyrogallol86,
and metol87]. Among the various heteropolyacids, phosphomolybdo tungstic acid,
the well-known Folin-Ciocalteu reagent 88 (F.C reagent) was preferred by a number
of workers for the determination of drugs89-93. The wavelength of maximum
absorption and stability of the blue colored reduction product and the sensitivity
and reproducibility of the reaction are dependent upon pH, composition of the
heteropolyacid complex, nature and concentration of the reducing agent,
temperature and time.
Allopurinol94, caffeine95, pentazocine96, oxymetazoline, isoxsuprine,
orciprenaline, pholedrin, vitamin–K and rutin95 are some typical examples of drugs
which were estimated in this manner. Rao et al97reviewed the applications of this
reagent and extended the use of this reagent to drugs, containing not only phenolic
groups but also amino groups. The color formation by FC reagent98 was tentatively
explained.
The above method (Method M4) has been used in the determination of
Abacavir Sulfate [ACS] and Milnacipran [MCN] in the present investigations. The
details of the investigation have been incorporated in chapters II & III.
ii)Ammonium Vanadate (AV) - H2SO4 (Method – M5):
Vanadium a soft, ductile, silver-grey metallic transition element in the
member of group Vb of the periodic table; symbol V; Atomic number 23; atomic
mass; 50.9415; melting point ca 1,8900C; boiling point ca 3,3800C; specific gravity
about 6 at 200C; valence +2, +3, +4, and maximum +5; electronic config
[Ar]3d34s2; resembles chromium in properties.
19
It dissolves in acid solutions. It reacts with bases to form vanadates.
Vanadium trioxide (V2O3) is basic in solution and dissolves in acids to give the green
hexa-aquo ion (V(H2O)6)3+. In solution, V3+ is a strong reducing agent and slowly
attacks water with the production of hydrogen. Vanadium is usually found bound to
oxygen as a negatively charged polymeric oxyanion that tends to complex to
polarizable ligands, such as phosphorus and sulfur. Vanadium has oxidation states in
its compounds of +5, +4, +3 and +2. The usual source of vanadium in the +5
oxidation state is ammonium metavanadate, NH4VO3. This isn't very soluble in water
and is usually first dissolved in sodium hydroxide solution.
The solution can be reduced using zinc and an acid [either hydrochloric acid
or sulphuric acid], usually using moderately concentrated acid.The exact vanadium
ion present in the solution is very complicated, and varies with the pH of the
solution. The reaction is done under acidic conditions when the main ion present is
VO2+ - called the dioxovanadium (V) ion. Adding nitric acid (a reasonably powerful
oxidising agent) to the original vanadium (II) solution also produces blue VO2+ ions.
The max values of reduction products vary from 600nm – 840nm depending upon
the reaction conditions (nature and strength of acid or base medium, temperature,
time) nature of poly acid (very efficient if the composition of hetero acids are more)
and nature of reducing agent (analyte). In the present investigations, the author
has developed colored product of maximum intensity with the selected two drugs
Abacavir Sulfate [ACS] and Milnacipran [MCN], under specified experimental
conditions, when treated with Ammonium Vanadate (AV) (Method M5). The details
of the investigation have been compiled in corresponding chapters of the responded
drugs chapters II and III.
20
iii) Potassium permanganate with FG FCF (Method- M6):
Oxidation with potassium permanganate (MnO4-) takes place in acidic,
alkaline and neutral solutions. Alkaline solutions are normally preferred for
quantitative oxidation because of the speed of reaction is enhanced in this medium.
However in alkaline solution at higher temperatures spontaneous partial reduction
of permanganate to manganate can cause difficulties. Permanganate is a strong
oxidizing agent and can oxidize olefins, glycols that are the products of the
reaction99.In acid and neutral media it always does so hence, it is not feasible to
prepare glycols in this manner. They can be prepared with alkaline permanganate
cleaves glycols giving carboxylic acids rather than aldehydes. Many oxidising agents,
the most common of which are neutral or acid permanganate, can cleave double
bonds. The mechanism of oxidation probably involves in most cases the initial
formation of a glycol or cyclic ester and the further oxidation to cleave the C-C bond.
Because Compounds like carboxylic acids100, esters of malic, citric and tartaric
acids101, propylene glycol102, methanol103, unsaturated compound104, terminal
methylene groups105, thiourea106 and hydrazobenzene107 were determined either
by direct titration with permanganate or determination of excess permanganate.
Very low concentrations of many oxidising agents of the order 1 to 10 µ.Mol can be
determined colorimetrically by using dye as a reagent.
Gordon108 reported an analytical method for the determination of 1 to 10
microgram amounts of many organic compounds (e.g. sorbic acid, citric acid) using
permanganate and fast green FCF. Abacavir Sulfate [ACS] (based on reducing
properties) can undergo oxidation easily with acidic permanganate and an indirect
visible spectrophotometric method has been developed for the selected drug using
MnO4- / FGFCF (Method M6) by the author in the present investigation.
21
This method involves two steps. In the first step the Abacavir Sulfate [ACS]
was treated with permanganate. In the second step the unreacted permanganate was
determined by FGFCF. The reacted permanganate corresponds to the drug
concentration (originally taken - unreacted). The probable sequence of reactions
based on analogy is presented in the chapter II.
III) Charge Transfer complex reactions (Methods M7 & M8):
The charge transfer complex forming reactions108 are based on that “π
acceptors react with the basic nitrogenous compounds as n-donors to form charge
transfer complexes or radical anions according to the polarity of the solvent used” and
these reaction has been widely studied recently. The basic mechanism involved is
the molecular interactions between electron donors and electron acceptors are
generally associated with the formation of intensely colored charge-transfer
complexes, which absorb radiation in the visible region.
Many drugs are easy to determine by spectrophotometry based on colored
charge transfer (CT) complexes formed with electron acceptors109-115. A variety of
electron donating compounds[π-acceptors] have been reported as analytical
reagents to yield charge-transfer complexes leading to numerous applications in the
development of simple and convenient spectrophotometric methods for the
determination of many drugs in pharmaceutical formulations 116-128.
It is well known that p-benzoquinones such as 7,7,7,8-
tetracyanoquinodimethane(TCNQ),2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) and 2,3,5,6-Tetrachloro-1,4-benzoquinone (p-chloranil acid ) as p -electron
acceptors often form highly colored electron- donor–acceptor (EDA) or charge
22
transfer (CT) complexes with various donors which provides the possibility of
determination of drugs by spectrophotometric methods.
Based on the above literature reviews the author has made an attempt in
developing new visible spectrophotometric methods using 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (DDQ) for the selected drug Abacavir Sulfate[ACS] [Method M7]
and 2,3,5,6-Tetrachloro-1,4-benzoquinone (p-chloranil acid ) for the selected drugs
Abacavir Sulfate[ACS], Milnacipran [MCN], Hydralizine HCl [HZH], and Tanofavir
Disproxil Fumarate[TDF] [Method M8] as selective reagents. The details of the
investigations, scheme of reactions are compiled in chapters II, III, IV and V.
IV) Ion-association complex reactions (Methods M9a, M9b, M9c,
M9d & M9e):
The ion-association complex is “a special form of molecular complex resulting
from two components extractable into organic solvents from aqueous phase at
suitable pH”. Of the two components extractable into organic solvents, one
component is a chromogen (dye or metal complex) possessing charge (cationic or
anionic in nature) which is insoluble in organic solvents and the other is a drug
which is colorless, possessing opposite charge (anionic or cationic) to that of
chromogen. The selectivity of the reaction is increased by using appropriate organic
solvent as an extractant, which depends upon the polarities of the amine and of the
dye.
Many pharmaceutical compounds have been quantitatively determined by
the formation of an ion-pair complex with several dyes129-138. These methods involve
23
the formation of ion association colored complex between drugs and reagents [dyes]
which is extracted with suitable organic solvent.
X+ (aq) + Y- (aq) ↔ X+Y- (aq) ↔ X+Y- (org)
Where, X+ and Y- represent the protonated drug and the anion of the dye,
respectively, and the subscript (aq) and (org) refer to the aqueous and organic
phases, respectively. The absorption spectra of the ion-pair complexes extracted into
chloroform.
Application of dyes as analytical reagents in ion association
reactions:
Dye may be defined as “a colored substance which when applied to the fibre,
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 synthesised
and many of them have been placed in the market. Dyes are used as analytical
reagents in two different ways depending upon the types of their involvement.
i) Colored anionic or cationic form which involves in ion-association complex
formation with oppositely charged ion of the drug.
24
TABLE 1.02
CHEMICAL FEATURES OF DYES USED IN ION ASSOCIATION COMPLEX FORMATION
Sl.No Dye name / CI No. Chemical category Structure Chemical name
1
Bromo cresol green
(BCG)
Analogous dye
SO2
O
C
CH3
Br
OH
OHBr
CH3
Br
Br
Tetrabromo meta cresol
sulphonaphthalein
2
Bromo cresol purple
(BCP)
Analogous dye
SO2
O
C
CH3
OH
Br
CHMe2
CH3
CHMe2
Br
OH
Dibromo o- cresol
sulphonaphthalein
3
Bromo Thymol Blue
(BTB)
Analogous dye
Dibromo o- thymol
sulphonaphthalein
25
TABLE.1.02
CHEMICAL FEATURES OF DYES USED IN ION ASSOCIATION COMPLEX FORMATION
S.No Dye name / CI No. Chemical category Structure Chemical name
4
Tropaeoline ooo (Tpooo) / 14600
Azodye
NNNaO3S OH
Benzenesulphonic acid,4[
(4-hydroxy-1-naphthalenyl) azo]-, mono sodium salt
5
Alizarine Red S (ARS) / 58005
Anthraquinone dye
NaO3S
HO
O
O
2-Anthrcene sulphonic acid - 9,10-dihydro-3,4-dihydroxy
-9,10-dioxo, mono sodium salt
26
ii) Dyes on treatment with an oxidizing, reducing or complex forming agent
leads to the development of visible spectrophotometric determinations of analytes
(that may be either direct reaction - reducing agent or indirect- initial oxidiation of
analyte with an oxidant followed by estimation of unreacted oxidant with a dye).
Preliminary investigations were carried out by the author using various dyes
for the assay of selected drugs. Among the dyes tried five acidic dyes [BCG, BCP, BTB,
TPooo and ARS] have been used directly in the estimations of selected drugs. The
chemical features of the acidic dyes used in the present investigation are given in
(Table.1.02, P.24,25).
Among the acidic dyes used BPB, BTB, BCG and BCP are formed to be active
reagents for the determination of different drugs139-150. Of the four selected drugs,
the drugs Hydralizine HCl [HZH] and Milnacipran [MCN] responded with the acidic
dyes (BCG, BCP and BTB)and the drugs Abacavir Sulfate[ACS] and Tanofavir
Disproxil Fumarate[TDF] have responded with acidic dyes (TPooo and ARS)
forming ion association complexes which are extractable into chloroform from the
aqueous phase and the author has successfully developed procedures for the drugs
Hydralizine HCl [HZH] and Milnacipran [MCN] with acidic dyes [BCG (M9a), BCP
(M9b)and BTB (M9c)] and for the drugs Abacavir Sulfate[ACS] and Tanofavir
Disproxil Fumarate[TDF] with the acidic dyes (TPooo and ARS) and the details of
these investigations are compiled in chapters II, III, IV & V of the individual drugs.
V) Diazo coupling reactions (Methods M10a & M10b):
The diazo coupling reaction is defined “as a proton eliminating condensation
of diazonium salt with another compound possessing an active hydrogen atom”. The
27
coupling of a diazonium salt formed from aromatic amine takes place in mild acid,
weak alkali and strong alkali conditions respectively. Diazocoupling and formation
of diazonium salts [Nitrosation reaction in which nitrous acid (formed insitu from
sodium nitrite and hydrochloric acid) reacts with primary amines (aromatic) to
diazonium salt] have opened the way to a great number of colorimetric
determinations.
Phloroglucinol Resorcinol
The formation of the diazo coupling reaction product from diazonium salt
from aromatic amine and compound having active hydrogen atom [Phloroglucinol
or Resorcinol] is the basis for the determination of several drugs151-160 in bulk and
pharmaceutical formulations.
In the present investigation the same diazocoupling reactions have been
extended by the author for the determination of the selected drugs Abacavir
Sulfate[ACS] and Tanofavir Disproxil Fumarate[TDF] [Method M10a for
Phloroglucinol & M10b for Resorcinol] and the details of these investigations have
been incorporated in chapter II.
iii) Choice of wavelength: It is important to avoid making measurements in
the region where the molar absorptivity (ε) changes rapidly with the wavelength. In
such a region even a small error in setting the wavelength scale will result in a large
28
apparent molar absorptivity. Therefore, it is necessary to select the wavelength
corresponding to maximum ε. Beer’s law will not be obeyed when the transmittance
of the solution increases continuously over the wavelength range covered by the
light filter.
II) METHOD VALIDATION STUDIES:
Method validation is the process of proving that an analytical method is
acceptable for its intended purpose. For pharmaceutical methods, guidelines from
the United States Pharmacopeia (USP)161, International Conference on
Harmonization (ICH)162, and the Food and Drug Administration (FDA)163,164 provide
a framework for performing such validations. In general, methods for regulatory
submission must include studies on specificity, linearity, accuracy, precision, range,
detection limit, quantitation limit, and robustness. Although there is general
agreement about what type of studies should be done, there is great diversity in how
they are performed165. The literature contains diverse approaches to performing
validations166-168. Validation requirements are continually changing and vary widely,
depending on the type of drug being tested, the stage of drug development, and the
regulatory group that will review the drug application. In the early stages of drug
development, it is usually not necessary to perform all of the various validation
studies. Many researchers focus on specificity, linearity, accuracy, and precision
studies for drugs in the preclinical through Phase II (preliminary efficacy) stages.
The remaining studies are performed when the drug reaches the Phase III (efficacy)
stage of development and has a higher probability of becoming a marketed product.
The process of validating a method cannot be separated from the actual
29
development of the method conditions, because the developer will not know
whether the method conditions are acceptable until validation studies are
performed. The development and validation of a new analytical method may
therefore be an iterative process. Results of validation studies may indicate that a
change in the procedure is necessary, which may then require revalidation. During
each validation study, key method parameters are determined and then used for all
subsequent validation steps. To minimize repetitious studies and ensure that the
validation data are generated under conditions equivalent to the final procedure, we
recommend the following sequence of studies.
A) Calibration: Calibration is one of the most important step in bioactive
compound analysis. A good precision and accuracy can only be obtained when a
good calibration procedure is used. In the spectrophotometric methods, the
concentration of a sample cannot be measured directly, but is determined using
another physical measuring quantity “y” (absorbance of a solution). An
unambiguous empirical or theoretical relationship can be shown between this
quantity and the concentration of analyte. The calibration between y = g (x) is
directly useful and yields by inversion of the analytical calculation function. The
calibration function can be obtained by fitting an adequate mathematical model
through the experimental data. The most convenient calibration function is linear,
goes through the origin and is applicable over a wide dynamic range. In practice,
however, many deviations from this ideal calibration line may occur. For the
majority of analytical techniques the analyst uses the calibration equation.
Y = a + bx
30
In calibration univariate regression is applied, which means that all observations are
dependent upon a single variable x.
i) Calibration curve: The common method of using the spectrophotometer
requires the construction of a calibration curve for the constituents being
determined. Calibration is one of the most important steps in drug analysis. For this
purpose, suitable quantities of the constituents are taken and treated in exactly the
same way as the sample solution for the development of color, followed by the
measurement of the absorption at the optimum wavelength. The absorbance is then
plotted against concentration of the constituents. A straight line is obtained if Beer’s
law is followed. This calibration curve may then be used to determine the
constituents under the same conditions. The calibration curves needs checking at
intervals.
a) Standard error on estimation, Se: The standard error on estimation is a
measure of the difference between experimental and computed values of the
dependent variable. It can be represented by the following equation, Yi, and yi, are
the
)2/()(1
2
nyySn
i
iie
observed and predicted values, respectively. Standard deviations on slopes (Sb) and
intercepts (Sa) are quoted less frequently, even though they are used to evaluate
proportional differences between or among methods as well as to compute the
independent variables such as concentration etc. It is important to understand how
uncertainties in the slope are influenced by the controllable properties of the data
31
set such as the number and range of data points and also how properties of data sets
can be designed to optimize the confidence in such data.
b) Standard deviation on slope, Sb: The standard deviation on slope is
proportional to standard error of estimate and inversely proportional to the range
and square root of the number of data points.
Where, Xi is the arithmetic mean of xi values.
c) Standard deviation on intercept, Sa: Intercept values of least squares fits of
data are often to evaluate additive errors between or among different methods.
Where, xi denote the arithmetic mean of xi values
d) Correlation coefficient, r: The correlation coefficient r (x, y) is more useful to
express the relationship of the chosen scales. To obtain a correlation coefficient, the
covariance is divided by the product of the standard deviation of x and y.
ii) Sensitivity: Sensitivity is often described in terms of the molar absorptivity
(ε, L mol-1 cm-1). The awareness of the sensitivity is very important in the
32
determination of pharmaceutical compounds. The objective numerical expression of
the sensitivity of spectrophotometric methods is the molar absorptivity (ε) at the
wavelength (λ max) of maximum absorbance of the colored species,Molar
absorptivity.
(ε) = A / c l
The sensitivity of spectrophotometric measurements depends on the
monochromaticity of the radiation. The molar absorptivity diminishes as the
bandwidth increases.
The molar absorptivity cannot exceed more than 1.5 × 105 L mol-1 cm-1,
according to quantum theory. Other ways of specifying sensitivity are as specific
absorptivity or Sandell’s sensitivity169. In both the methods sensitivity is expressed
in terms of amount of analyte per unit volume of solution. Such an approach is
perhaps more convenient than using molar absorptivities as a basis of comparison.
Sandell’s sensitivity is the concentration of the analyte (µg mL-1) which will give an
absorbance of 0.001 in a cell of path length 1.0cm and is expressed as µg cm-2.
Organic reagents with high molecular weights furnish maximum sensitivity if used
as chromogenic agents. Detection limits can be reduced to somewhat by solvent
selection because molar absorptivities depend on the solvent system. Another
technique used to increase the detection limit is to use indirect determinations,
where a stoichiometric gain in the number of chromophores may result or the newly
formed chromophore may have a higher molar absorptivity. Reaction rate methods
can sometimes have lower detection limits than do conventional
spectrophotometric measurements.
33
iii) Detection limit170: Detection limit is the smallest concentration of a
solution of an element that can be detected with 95% certainty. This is the quantity
of the element that gives a reading equal to twice the standard deviation of a series
of ten determinations taken with solutions of concentrations which are close to the
level of the blank. Based on the standard deviation of the reagent blank and the
slope of the calibration curve of the analyte. The detection limit (DL) may be
expressed as,
DL = (3.3 σ)/ S
Where, σ = standard deviation of the reagent blank; S = slope of the calibration
curve. The slope S may be estimated from calibration curve of the analyte. The
estimate of σ may be measured based on the standard deviation of the reagent
blank.
iv) Quantitation limit: The quantitation limit is generally determined by the
analysis of samples with known concentrations of analyte with those of blank
samples and by establishing the minimum level at which the analyte can be
quantified with acceptable accuracy and precision. Based on the standard deviation
of the reagent blank samples and the slope of the calibration curve of the analyte, the
quantitation limit (QL) may be expressed as,
QL = (10 σ)/ S
Where σ = standard deviation of the reagent blank; S = slope of the calibration
curve.
The slope S may be estimated from calibration curve of the analyte. The
estimate of σ may be measured based on the standard deviation of the reagent
blank.
34
v) Precision and Accuracy: Precision describes reproducibility of results
where accuracy denotes the nearness of a measurement to its accepted value. The
accuracy and precision of spectrophotometric method depends on three major
factors, instrumental limitations, chemical variables and operators’ skill.
Instrumental limitations are often determined by the quality of the instruments,
optical, mechanical and electronic systems. Under ideal conditions it is possible to
achieve relative standard deviation in concentrations as low as about 0.5% which
enables the determination of microquantities of components. The precision of
spectrophotometric method also depends on concentration of the determinant.
Precision is conveniently expressed in terms of the average deviation from the mean
or in terms of standard deviation. When applied to small sets of data with which the
analytical chemists work, the standard deviation is the most reliable estimate of the
indeterminate uncertainty. When the standard deviation turns out to be
approximately proportional to the amount present in the formation on the precision
can be expressed in percent by using the coefficient of variation. Mathematical
equation for the calculation of coefficient of variation is given below
Where, s = standard deviation and ͞x = arithmetic mean of a series of
measurements.
a) Comparison of the results: The comparison of the values obtained from a set
of results with either (i) the true value or (ii) other sets of data makes it possible to
determine whether the analytical procedure has been accurate or precise, or if it is
superior to another method. There are two common methods for comparing results.
35
Student’s t-test and the variance ratio test (F-test).These methods of test require
knowledge of what is known as the number of degrees of freedom.
(i) Student’s t-test : This is a test used to compare the mean from a sample with
some standard values and to express some level of confidence in the significance of
the comparison. It is also used to test the difference between the means of the two
sets of data x1 and x2.
Where, s = standard deviation, x = arithmetic mean of a series of measurements, µ is
the true value and n is the number of trials of the measurements.
It is then related to a set of t-tables in which the probability of the t-value
falling within certain limits is expressed, either as a percentage or as a function of
unity relative to the number of degrees of freedom. This method is also used to
compare the values of the mean and precision of the test method with those of the
reference method. The value of‘t’ when comparing two sample means x1 and x2 is
given by the expression,
Where, Sp is the pool standard deviation, calculated from two samples standard
deviations S1 and S2 as follows
Where, n1 and n2 the number of trials of first and second method.
36
(ii) The Variance Ratio Test (F-test): This is used to compare the precisions of
two sets of data of two different analytical methods or the results from two different
laboratories. It is calculated from the following equation.
The larger value of S is always taken in the numerator so that the value of ‘F’
is always greater than unity. The value obtained for F is then checked for its
significance against values in the F- table calculated from an F–distribution
corresponding to the numbers of degrees of freedom for the two sets of data.
37
1.03: A: INTRODUCTION TO HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY (HPLC):
Chromatography is defined as a chemical analysis separation process which
uses selective adsorption to segregate and identify components of complex mixtures
such as solutions, liquids and vapors. It involves passing a mixture dissolved in a
"mobile phase" through a stationary phase, which separates the analyte to be
measured from other molecules in the mixture based on differential partitioning
between the mobile and stationary phases. Differences in compounds partition
coefficient results in differential retention on the stationary phase and thus changing
the separation.
Different types of Chromatographic techniques were summarized in Table:
1.03,P.38. Chromatography may be preparative or analytical. The purpose of
preparative Chromatography is to separate the components of a mixture for further
use (and is thus a form of purification). Analytical Chromatography is done normally
with smaller amounts of material and is for measuring the relative proportion of
analytes in a mixture.
1.03. A.i: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Liquid Chromatography171 is an analytical Chromatographic technique that is
useful for separating ions or molecules that are dissolved in a solvent. If the sample
solution is in contact with a second solid or liquid phase to differing degrees due to
differences in Adsorption, Ion Exchange, Partitioning or Size. These differences will
38
allow the mixture components to be separated from each other by using these
differences to determine the transit time of the solutes through a column.
TABLE: 1.03
Different types of Chromatographic techniques
During 1970’s, most chemical separations were carried out using a variety of
techniques including open-Column Chromatography, Paper Chromatography and
Thin Layer Chromatography (TLC). However, these Chromatographic techniques
Sl. no Basic principle involved Type of Chromatography
1. Techniques by Chromatographic
bed shape
Column Chromatography
Paper Chromatography
Thin layer Chromatography
2 Techniques by physical state of
mobile phase
Gas Chromatography
Liquid Chromatography
3 Affinity Chromatography Supercritical fluid Chromatography
4 Techniques by separation
mechanism
Ion Exchange Chromatography
Size Exclusion Chromatography
5 Special techniques Reversed Phase Chromatography
Two-dimensional Chromatography
SimulatedMoving-Bed
Chromatography
Pyrolysis Gas Chromatography
Fast Protein Liquid
Chromatography
Countercurrent Chromatography
Chiral Chromatography
39
were inadequate for quantification of compounds and resolution between similar
compounds. During this time pressure Liquid Chromatography began to be used to
decrease flow time, thus reducing separation time of compounds being isolated by
Column Chromatography. However, flow rates were inconsistent, and the question
of whether it was better to have constant flow rate or constant pressure debated.
High-pressure Liquid Chromatography quickly improved with the development of
column packing materials. Additional convenience of on-line detectors became
rapidly a powerful separation technique and is today called as High Performance
Liquid Chromatography (HPLC). The HPLC is the method of choice in the field of
analytical chemistry and it has both advantages and disadvantages.
Advantages:
HPLC separations can be accomplished in a matter of minutes, in some cases
even in seconds.
High resolution of complex sample mixture into individual components.
Rapid growth of HPLC is also because of its ability to analyse substances that
are unsuitable for Gas Liquid Chromatographic (GLC) analysis due to non-
volatility or thermal-instability.
Quantitative analyses are easily and accurately performed and errors of less
than 1 % are common to most HPLC methods.
Dependingon sample type and detector used, it is frequently possible to
measure 10-9 g or 1 mg of sample. With special detectors, analysis down to
10-12 pg has been reported.
40
As HPLC is versatile, it can be applied to a wide variety of samples like
organic, inorganic, high molecular weight liquids, solids, ionic and non-ionic
compounds.
Disadvantages:
HPLC instrumentation is expensive and represents a major investment for many
laboratories.
It requires a proficient operator to handle the instrument.
HPLC cannot handle gas samples.
HPLC is poor identifier. It provides superior resolution but it does not provide the
information that identifies each peak.
Sample preparation is often required.
Only one sample can be analysed at a time.
Finally, at present there is no universal and sensitive detector.
1.03.A.ii: CLASSIFICATION OF HPLC 172 - 175 :
There are four main types of HPLC techniques. They are:
1. Normal Phase Liquid Chromatography.
2. Reverse Phase Liquid Chromatography.
3. Ion Exchange Liquid Chromatography.
4. Size Exclusion Liquid Chromatography.
There are three basic types of molecular forces: ionic forces, polar forces and
dispersive forces. Each specific technique capitalizes on each of these specific
forces.
41
Polar forces are the dominant type of molecular interactions employed in Normal
Phase- HPLC.
Dispersive forces are employed in Reversed Phase-HPLC.
Ionic forces are employed in Ion Exchange HPLC.
The fourth type of HPLC technique, Size Exclusion HPLC is based on the absence of
any specific analyte interactions with the stationary phase (no force employed in
this technique).
1. Normal Phase - High Performance Liquid Chromatography (NP-HPLC):
NP-HPLC explores the differences in the strength of the polar interactions of
the analytes in the mixture with the stationary phase. The stronger the analyte-
stationary phase interaction, the longer the analyte retention. Analyte molecules
compete with the mobile phase molecules for the adsorption sites on the surface of
the stationary phase. The stronger the mobile phase interactions with the stationary
phase, the lower the difference between the stationary phase interactions and the
analyte interactions, and thus the lower the analyte retention. Mobile phases in NP-
HPLC are based on nonpolar solvents (such as Hexane, Heptane, etc.) with the small
addition of polar modifier (i.e., Methanol, Ethanol). Packing materials traditionally
used in NP-HPLC are usually porous oxides such as Silica (SiO2) or Alumina (Al2O3).
Surface of these stationary phases is covered with the dense population of OH
groups, which makes these surfaces highly polar. Chemically modified stationary
phases can also be used in NP-HPLC. Silica modified with Trimethoxy
Glycidoxypropyl Silanes (common name: diol-phase) is typical packing material
with decreased surface polarity. Since NP-HPLC uses mainly nonpolar solvents, it is
the method of choice for highly hydrophobic compounds (which may show very
42
stronger interaction with non polar mobile phases), which are insoluble in polar or
aqueous solvents.
2. Reversed Phase - High Performance Liquid Chromatography (RP-HPLC):
As opposed to NP-HPLC, RP-HPLC employs mainly dispersive forces
(hydrophobic or vander wal’s interactions). The polarities of mobile and stationary
phases are reversed, such that the surface of the stationary phase in RP-HPLC is
hydrophobic and mobile phase is polar, where mainly water-based solutions are
employed. RP-HPLC is by far the most popular mode of chromatography. Almost 90
% of all analyses of low-molecular-weight samples are carried out using RP-HPLC.
Dispersive forces employed in this separation mode are the weakest intermolecular
forces, thereby making the overall background interaction energy in the
chromatographic system very low compared to other separation techniques. This
low background energy allows for distinguishing very small differences in molecular
interactions of closely related analytes. Adsorbents employed in this mode of
chromatography are porous rigid materials with hydrophobic surfaces. The majority
of packing materials used in RP-HPLC are chemically modified porous silica.
3. Ion-Exchange Chromatography (IEC):
IEC is based on the differences in affinities of the analyte ions for the
oppositely charged ionic centers in the resin or adsorbed counter ions in the
hydrophobic stationary phase. Consider the exchange of two ions A+ and B+ between
the solution and exchange resin E:
A·E + B+ ↔B·E + A+
The equilibrium constant for this process is shown in Equation below:
K =
43
This essentially determines the relative affinity of both cations to the
exchange centers on the surface. If the constant is equal to 1, no discriminating
ability is expected for this system. The higher the equilibrium constant (provided
that it is greater than 1.0), the greater the ability of cation B+ to substitute A on the
resin surface. Depending on the charge of the exchange centers on the surface, the
resin could be either anion-exchanger (positive ionic centers on the surface) or
cation-exchanger (negative centers on the surface). Cross linked styrene-
divinylbenzene is the typical base material for ion exchange resin. Exchange groups
are attached to the phenyl rings in the structure and the degree of cross linkage is
between 5 % and 20 %. The higher the cross linkage, the harder the material and the
less susceptible it is to swelling, but the material usually shows lower ion-exchange
capacity. Four major types of ion-exchange centers are usually employed:
SO3-—strong cation-exchanger
CO2-—weak cation-exchanger
Quaternary Amine—strong anion-exchanger
Tertiary Amine—weak anion-exchanger
Analyte retention and selectivity in Ion Exchange Chromatography are strongly
dependent on the pH and ionic strength of the mobile phase.
4. Size Exclusion Chromatography (SEC):
SEC is the method for dynamic separation of molecules according to their
size. The separation is based on the exclusion of the molecules from the porous
space of packing material due to their steric hindrance. Hydrodynamic radius of the
analyte molecule is the main factor determining its retention. This is the only
44
chromatographic separation method where any positive interaction of the analyte
with the stationary phase should be avoided.
In SEC, the higher the molecular weight of the molecule, the greater its
hydrodynamic radius results in faster elution. At the same time, if an analyte
molecule interacts (undesired) with the stationary phase, thus increasing the
retention of larger molecules, which may confound separation of molecules based
solely on their hydrodynamic radius. Obviously, these two processes produce
opposite effects and analysis of the polymer molecular weight and molecular weight
distribution would be impossible. This brings specific requirements to the selection
of the column packing material and the mobile phase, where the mobile phase
molecules should interact with the surface of the stationary phase stronger than the
polymer, thus preventing its interaction with the surface. The radius is roughly
proportional to the cubic root of the molecular weight, thus giving the impression
that cubic root of the molecular weight should be proportional to the analyte
retention volume.
The adsorbent pore size distribution plays the dominant role in the
adsorbent ability to discriminate molecules according to their molecular weight.
Hydrodynamic radius of the polymer is also dependent on the analyte interaction
with the solvent. Polymer conformation and degree of the salvation varies with the
variation of the solvent properties.
45
1.03. A.iii. INSTRUMENTATION OF HPLC:
HPLC is a special branch of column chromatography in which the mobile
phase is forced through the column at high speed. As a result, the analysis time is
reduced by 1-2 orders of magnitude relative to classical column chromatography
and the use of much smaller particles of the absorbent or support becomes possible
increasing the column efficiency substantially. The Basic HPLC Instrumentation was
shown in the Fig: 1.02,P.45
Fig: 1.02. HPLC Basic Instrumentation
46
i) Solvent delivery system:
The most important component of HPLC in solvent delivery system is the
pump, because its performance directly effects the retention time, reproducibility
and detector sensitivity. Among the several solvent delivery systems, (direct gas
pressure, pneumatic intensifier, reciprocating etc.) reciprocating pump with twin or
triple pistons is widely used, as this system gives less baseline noise, good flow rate
reproducibility etc.
The pumping systems used in HPLC can be categorized in three different ways.
The first classification is according to the eluent flow rate that the pump is capable of
delivering. The second classification is according to the construction materials, and
the final classification is according to the mechanism by which the pump delivers the
eluent. Each of these classifications is considered below.
Pump Classification According to Flow Rate:
When classified in terms of flow rate, pumps may be defined as microbore or
preparative. Standard bore systems are the most commonly used pumping systems
for analytical HPLC because they provide reliable operation at flow rates ranging
from 100 µL / min to 10 µL / min. Microbore systems are intended for use with
column diameters ranging up to 2 mm. The narrow column diameter and small size
of the packing material causes relatively low flow rates for the pumping system,
from 1 to 250 µL / min as the minimum head size for reciprocating pumps is around
25 µL, smooth, reliable operation at flow rates less than 10 µL / min is difficult.
47
Pump Classification According to Materials of Construction:
Pumps may also be classified according to the primary construction
materials. The pumps are classified as metallic or non-metallic, depending on the
material used for the eluent flow path. The most commonly used material for HPLC
pumping systems is No 316 stainless steel, because of its mechanical strength,
corrosion resistance, good thermal stability and malleability. Only a handful of
HPLC solvents such as Hydrochloric acid will cause damage to No316 stainless
steel.
Therefore pumps are also constructed from non-metallic materials such as
PEEK (Poly Ethyl Ethyl Ketone), Teflon (Poly Tetra Fluoro Ethylene) and Ceramics.
Pump Classification According to Mechanism of Eluent Displacement:
The third classification of pumps is according to the mechanism by which the
liquid is forced through the Chromatograph. The pumps are classified into two types.
They are syringe pumps and reciprocating-piston pump.
Solvent degassing system:
The constituents of the mobile phase should be degassed and filtered before
use. Several methods can be applied to remove the dissolved gases in the mobile
phase. They include heating and stirring, vacuum degassing with an aspirator,
filtration through 0.45 μm filters, vacuum degassing with an air-soluble membrane,
Helium purging ultra sonification or purging or combination of these methods. HPLC
systems are also provided an online degassing system which continuously removes
the dissolved gases from the mobile phase.
48
Sample introduction system:
Two means for analyte introduction on the column are injection into a
flowing stream and a stop flow injection. These techniques can be used with a
syringe or an injection valve. Automatic injector is a microprocessor-controlled
version of the manual universal injector. Usually up to 100 samples can be loaded in
to the auto injector tray. The system parameters such as flow rates, gradient, run
time, volume to be injected etc. are chosen, stored in memory and sequentially
executed on consecutive injections.
ii) Injector:
Injectors should provide the possibility of injecting the liquid sample within
the range of 0.1 to 100 mL of volume with high reproducibility and under high pressure
(up to the 4000 psi). They should also produce minimum band broadening and
minimize possible flow disturbances. The most useful and widely used sampling
device for modern HPLC is the micro sampling injector valve. With these sampling
valves, samples can be introduced reproducibly into pressurized columns without
significant interruption of flow even at elevated temperatures.
iii) Columns:
The heart of the system is the column. Analytical column is the most
important part of the HPLC which decides the efficiency of separation. The choice of
common packing material and mobile phases depends on the physical properties of
the drug.
49
Column-packing materials
Silica is the most widely used substance for the manufacture of packing
materials it consists of a network of Siloxane linkages(Si-O-Si) in a rigid three
dimensional structure containing inter connected pores. Thus a wide range of
commercial products are available with surface areas ranging from 100 to 800 m2/g
and particle sizes from 3 to 50 µm.
The Silonol groups on the surface of silica give it a polar character, which is
exploited in adsorption chromatography using non polar organic elutents. Silica can
be drastically altered by reaction with organo chloro silanes or organo Alkoxy
Silanes giving Si-O-Si-R linkages with the surface. The attachment of hydrocarbon
chain to silica produces a non polar surface suitable for reversed phase
chromatography where mixtures of Water and organic solvents are used as eluents.
The most popular material is Octa Decyl Silica (ODS) which contains C18 chains, but
material with C2, C6, C8 and C22 chains are also available. During manufacture, such
materials can be reacted with a small mono functional Silane (eg: Trimethyl
Chlorosilane) to reduce further number of Silanol groups remaining on the surface
(End -Capping). There is a vast range of materials which have intermediate surface
polarities arising from the bonding to silica of other organic compounds which
contain groups such as phenyl, nitro, amino and hydroxyl. Strong ion exchangers are
also available in which Sulphonic acid groups ard Quaternary Ammonium groups
are bonded to silica. The useful pH range for columns is 2 to 8, since Siloxane
linkages are cleaved below pH 2 while at pH values above 8 Silica may dissolve.
In HPLC, generally two types of columns are used, Normal Phase column and
Reversed Phase column. Using normal phase chromatography, particularly of non
polar and moderately polar drugs can make excellent separation and was originally
50
believed that separation of compounds in mixtures takes place slowly by differential
adsorption on a stationary Silica phase. However, it now seems that partition plays
an important role, with the compounds interacting with the polar Silonol groups on
the Silica or with bound water molecules.
While in normal phase, seems the passage of a relatively non polar mobile
phase over a polar stationary phase, reversed phase chromatography is carried out
using a polar mobile phase such as methanol, acetonitrile, water, buffer etc. over a
non polar stationary phase.
A range of stationary phases (C18, C8, -NH2, -CN, -Phenyl etc.) are available
and very selective separation can be achieved. The pH of mobile phase can be
adjusted to suppress the ionization of the drug and thereby increase retention in the
column. For highly ionizing drugs ion-pair chromatography is used.
iv) Mobile Phase: Mobile phases used for HPLC are typically mixtures of Organic
solvents and Water or aqueous buffers. Physical properties of some HPLC solvents
were summarized in TABLE.1.04,P.51.
v) Detectors: The detection of UV light absorbance offers both convenience and
sensitivity for molecules. When a chromophore is present, the wavelength of
detection for a drug should be based on its UV Spectrum in the mobile phase and not
in pure solvents. The most selective wavelength for detecting a drug is frequently
the longest wavelength maximum to avoid interference from solvents, buffers and
excipients. Other method of detection can be useful are required in some instances.
51
1. Solute specific detectors (UV-Vis, Fluorescence, Electrochemical, Infra-red, Radio
activity)
2. Bulk property detectors (Refractive index, Viscometer, Conductivity)
3. Desolvation detectors (Flame ionization etc.)
4. LC-MS detectors.
5. Reaction detectors.
TABLE: 1.04
PHYSICAL PROPERTIES OF COMMON HPLC SOLVENTS
Performance calculations:
Calculating the following values (which can be included in a custom report) used to
access overall system performance.
Solvent MW BP RI (25oC)
UVa Cut-off (nm)
Density g / mL (25oC)
Viscosity cP
(25oC)
Dielectric Constant
Acetonitrile 41.0 82 1.342 190 0.787 0.358 38.8
Dioxane 88.1 101 1.420 215 1.034 1.26 2.21
Ethanol 46.1 78 1.359 205 0.789 1.19 24.5
Ethyl acetate 88.1 77 1.372 256 0.901 0.450 6.02
Methanol 32.0 65 1.326 205 0.792 0.584 32.7
CH2Cl2 84.9 40 1.424 233 1.326 0.44 8.93
Isopropanol 60.1 82 1.375 205 0.785 2.39 19.9
n-propanol 60.1 97 1.383 205 0.804 2.20 20.3
THF 72.1 66 1.404 210 0.889 0.51 7.58
Water 18.0 100 1.333 170 0.998 1.00 78.5
a: The wavelength at which the absorbance of 1cm cell is 1.0
52
1. Relative retention.
2. Theoretical plates.
3. Capacity factor.
4. Resolution.
5. Peak asymmetry.
6. Plates per meter.
The following information furnishes the parameters used to calculate these system
performance values for the separation of two Chromatographic components. (Note:
Where the terms w and t both appear in the same equation they must be expressed
in the same units).
Relative retention (selectivity):
α = (t2-ta) / (t1-ta)
Where, α = Relative retention; t1 = Retention time of the first peak measured from
point of injection; t2 = Retention time of the second peak measured from point of
injection; ta = Retention time of an inert peak not retained by the column, measured
from point of injection.
Theoretical plates:
n = 16 (tR / w) 2
Where, n = Number of Theoretical plates; tR = Retention time of the component; W
= Width of the base of the component peak using tangent method
53
Capacity factor:
K1 = (t2 / ta) – 1
Where, K1 = Capacity factor; ta = Retention time of an inert peak not retained by the
column, measured from point of injection.
Resolution:
R = 2 ( t2 - t1 ) / ( w2 + w1 )
Where, R = Resolution between a peak of interest (peak 2) and the peak preceding it
(peak1); W2 = Width of the base of component peak 2; W1 = Width of the base of
component peak 1
Peak asymmetry:
T = W0.05 / 2f
Where,T = Peak asymmetry, or tailing factor; W0.05 = Distance from the leading edge
to the tailing edge of the peak, measured at a point 5 % of the peak height from the
baseline; f = Distance from the peak maximum to the leading edge of the peak.
Plate per meter:
N = n / L
Where, n = Number of Theoretical plates; L = Column length in meters.
54
Height equivalent to theoretical plate (HETP):
HETP=L / n
Where, n = Number of Theoretical plates; L = Column length in meters.
Linear fit: A linear calibration fit determines the best line (linear regression) for a
series of calibration points. A minimum of two calibration points are required to
determine a linear fit. The equation for calibrating the uncorrected amount is:
Y = m X + c
Where, Y = Component area or height; m = Slope of the calibration line;
X = Uncorrected amount; c = Y- axis intercept of the calibration line; this equation is
helpful for external and internal standard method.
1.03. A.iv: HPLC METHOD VALIDATION 176-185:
Method validation can be defined as (ICH) “Establishing documented
evidence, which provides a high degree of assurance that a specific activity will
consistently produce a desired result or product meeting its predetermined
specifications and quality characteristics”.
An assay for a major component requires a different approach and
acceptance criteria than a method for a trace impurity. A final method may be
performed at different sites around the world. Differences in HPLC instrumentation,
laboratory equipment and reagent sources and variations in the skills and
background of personnel may require specific features in the HPLC method. In
55
addition, the development of different formulations of the same drug with varying
strengths or physical forms may require flexibility in method procedures.
Method validation study include system suitability, linearity, precision,
accuracy, specificity, ruggedness, robustness, limit of detection, limit of
quantification and stability of samples, reagents, instruments.
1. System Suitability: Prior to the analysis of samples of each day, the operator
must establish that the HPLC system and procedure are capable of providing data of
acceptable quality. This is accomplished with system suitability experiments, which
can be defined as tests to ensure that the method can generate results of acceptable
accuracy and Precision. The requirements for system suitability are usually
developed after method development and validation have been completed.
2. Linearity: The linearity of a method is a measure of how well a calibration plot
of response vs. concentration approximates a straight line. Linearity can be assessed
by performing single measurements at several analyte concentrations. The data is
then processed using a linear least-squares regression. The resulting plot slope,
intercept and correlation coefficient provide the desired information on linearity.
3. Precision: Precision can be defined as “The degree of agreement among
individual test results when the procedure is applied repeatedly to multiple
samplings of a homogenous sample”. A more comprehensive definition proposed by
the International Conference on Harmonization (ICH) divides precision into three
types:
56
1.Repeatability
2.Intermediate precision and
3.Reproducibility
Repeatability is the precision of a method under the same operating conditions
over a short period of time.
Intermediate precision is the agreement of complete measurements (including
standards) when the same method is applied many times within the same
laboratory.
Reproducibility examines the precision between laboratories and is often
determined in collaborative studies or method transfer experiments.
4. Accuracy: The accuracy of a measurement is defined as the closeness of the
measured value to the true value. In a method with high accuracy, a sample (whose
“true value” is known) is analyzed and the measured value is identical to the true
value. Typically, accuracy is represented and determined by recovery studies. There
are three ways to determine accuracy:
1. Comparison to a reference standard
2. Recovery of the analyte spiked into blank matrix or
3. Standard addition of the analyte.
It should be clear how the individual or total impurities are to be determined. e.g.,
Weight / weight or area percent in all cases with respect to the major analyte.
57
5. Specificity / selectivity: The terms selectivity and specificity are often used
interchangeably. According to ICH, the term specific generally refers to a method
that produces a response for a single analyte only while the term selective refers to
a method which provides responses for a number of chemical entities that may or
may not be distinguished from each other. If the response is distinguished from all
other responses, the method is said to be selective. Since there are very few
methods that respond to only one analyte, the term selectivity is usually more
appropriate. The analyte should have no interference from other extraneous
components and be well resolved from them. A representative Chromatogram or
profile should be generated and submitted to show that the extraneous peaks either
by addition of known compounds or samples from stress testing are baseline
resolved from the parent analyte.
6.Ruggedness: The ruggedness of an analytical method is the degree of
reproducibility of test results obtained by the analysis of the same samples under a
variety of normal test conditions such as different laboratories, different analysts,
using operational and environmental conditions that may differ but are still within
the specified parameters of the assay. The testing of ruggedness is normally
suggested when the method is to be used in more than one laboratory. Ruggedness
is normally expressed as the lack of the influence on the test results of operational
and environmental variables of the analytical method.
For the determination of ruggedness, the degree of reproducibility of test
result is determined as function of the assay variable. This reproducibility may be
compared to the precision of the assay under normal condition to obtain a measure
of the ruggedness of the analytical method.
58
7. Robustness: The concept of robustness of an analytical procedure has been
defined by the ICH as “a measure of its capacity to remain unaffected by small, but
deliberate variations in method parameters”. A good practice is to vary important
parameters in the method systematically and measure their effect on separation.
The variable method parameters in HPLC technique may involves flow rate, column
temperature, sample temperature, pH and mobile phase composition.
8. Limit of Detection: Limit of Detection (LOD) is the lowest concentration of
analyte in a sample that can be detected, but not necessarily quantitated, under the
stated experimental conditions. With UV detectors, it is difficult to assure the
detection precision of low level compounds due to potential gradual loss of
sensitivity of detector lamps with age or noise level variation by detector
manufacturer. At low levels, assurance is needed that the LOD and LOQ limits are
achievable with the test method each time. With no reference standard for a given
impurity or means to assure detectability, extraneous peak(s) could "disappear /
appear." A crude method to evaluate the feasibility of the extraneous peak detection
is to use the percentage claimed for LOD from the area counts of the analyte. Several
approaches for determining the LOD are possible, depending on whether the
procedure is a non-instrumental or instrumental.
Based on Visual Evaluation
Based on Signal-to-Noise
Based on the Standard Deviation of the Response and the Slope
The LOD may be expressed as:
LOD = 3.3 σ / S
59
Where, σ = Standard deviation of Intercepts of calibration curves; S = Mean of
slopes of the calibration curves.
9. Limit of Quantification: Limit of Quantitation (LOQ) is the lowest
concentration of analyte in a sample that can be determined with acceptable
precision and accuracy under the stated experimental conditions. Several
approaches for determining the LOQ are possible depending on whether the
procedure is a non-instrumental or instrumental.
Based on Signal-to-Noise Approach
Based on the Standard Deviation of the Response and the Slope
The LOQ may be expressed as:
LOQ = 10 σ / S
Where,σ = Standard deviation of Intercepts of calibration curves;S = Mean of slopes
of the calibration curves;The slope S may be estimated from the calibration curve of
the analyte.
10. Stability: To generate reproducible and reliable results, the samples,
standards, and reagents used for the HPLC method must be stable for a reasonable
time (e.g., one day, one week, and one month, depending on need). Therefore, a few
hours of standard and sample solution stability can be required even for short (10
min) separation. When more than one sample is analyzed (multiple lots of one
sample or samples from different storage conditions from a single lot), automated,
overnight runs often are performed for better lab efficiency. Such practices add
requirements for greater solution stability.
60
REFERENCES
1. Vasudevan, R. and Mathai, I. M., Ind. J. Chem., 1972, 10, 175.
2. Vasudevan, R., Subramanian, P. S. and Mathai, I. M., J. Ind. Chem. Soc., 1984, 61, 395.
3. Kampli, S. R., Nandibewoor, S. T. and Raju, J. R., Ind. J. Chem, 1990, 29, 908.
4. Hanumantha, R. K. and Bhaganwanth, R. M., J. Ind. Chem. Soc., 1991, 68,160.
5. Amjad, Z. and Mc Auley, A.M., J. Am. Chem. Soc., 1977, 99, 304.
6. Jwo, J. J. and Noyes, R. M., J. Am. Chem. Soc., 1975, 97, 5422.
7. Amjad, Z. and Mc Auley, A., J. Am. Chem. Soc., 1977, 99, 82.
8. Richardson, W.H., Oxidation in organic Chemistry” K .B. Wiberg, Academic Press,
New York Part-A 1965, 245.
9. Richardson, W.H., Oxidation in organic Chemistry” K .B. Wiberg, Academic Press,
New York Part-A 1965, 262.
10. Sethuram, B. and Mushamad, S. S., Acta Chim. Acad. Sci., 1965, 46, 115.
11. Guelbautt, G. C. and Mc Curdy W. R., J. Phys. Chem., 1963, 67, 283.
12. Mehrotra, R. N. and Ghos, S., Ind. J. Chem., 1976, 14, 663.
13. Khanna, P. K. and Krishna, B., Proc. Natl. Acad. Sci., 1977, 12, 478.
61
14. Pernarowski, M., Knevel, A. M., and Christian, J.E., J. Pharm. Sci., 1960, 50, 943.
15. Glenn, A.L., J. Pharm. Pharmacol., 1963, 15, 123.
16. Wahbi, A. M. and Farghaly, A. M., J. Pharm. Pharmcol. 1970, 22, 848.
17. Kartal, M. and Erk, N., J. Pharm. Biomed. Anal., 1999, 19, 477-485.
18. Erk, N., J. Pharm. Biomed. Anal., 1999, 20, 155-167.
19. Besthorn. S.L. and Kasture.A.U., “Talanta”., 1993, 40, 1525.
20. Sawicki. E, Hauser. T.R, Satnly. T.W, Elbert. W. and Fox. F.T., “Anal. Chem”., 1961,
33, 1574.
21. Hauser. T.R. and Commizs. R.L., “Anal. Chem”., 1964, 36, 679.
22. Hunig. S. and Balli. H., “Justus Leibigs Ann. Chem”., 1957, 609, 160.
23. Altshuller. A.P. and Leng. L.J., “Anal. Chem”., 1963, 35, 1541.
24. Cohen. J.R. and Altshuller. A.P., “Anal. Chem”., 1966, 38, 1418.
25. Davis. R.P. and Janis. R., “Nature”., 1966, 210, 318.
26. Pesez, M. and Batros, J. "Colorimetric and Fluorimetric analysis of Organic
Compounds and Drugs", Marcel Dekker, NY. 1974; 170, 175, 536.
62
27. Singh, D. K., Sahu, A., Anal-Biochem. 2006; 349(2):176-180.
28. El-Adl, S.M. & Saleh, H. M., Sci-Pharm. 2002; 70(67).
9.0. Sastry, C.S.P., Chintalapati, R., Prasad, A.V.S.S. & Sastry, B.S., Talanta 2001; 15(661).
30. Rizk, M., Ibrahim, F., Ahmad. S.M. and El-Enany, N.M., Sci-Pharm. 2000; 68(173).
31. Sastry.C.S.P, and Ramamohanarao.A., “Mikrochim Acta [Wein]”.,1989, 1,237-244.
32. Dryhurst.G., “Periodate oxidation of diol and other functional groups”., Ed.
Belcher.R and Anderson.D.M.W., Pergamom Press, London, 1970.
33. Sklarz., “Quarterly Reviews”., 1967,21,3.
34. Jackson. E.L., “Organic Reactions”., 1944,2,341.
35. Bobbit.J.M., “Adv. Carbohydrate Chemistry”., 1956,11,1.
36. Hugh.L., “Methods In Carbohydrate Chemistry Vol.V”., 1965, (77), 272.
37. Baker.S.A, and Somers.P.J., “Carbohydrates Research”., 1966,8,220.
38. Dyer.J.R., “Methods Of Biochemical Analysis Vol.III”., Ed; Glick.D, Interscience
Publishers, New York, 1956,131.
63
39. Hay.G.H, Lewis.B.A, Smith.F. and Unrau.A.M., “Methods In Carbohydrate
Chemistry, Vol.V”., 1965,251.
40. Belcher.R., “Sub micro Methods of Organic Analysis”., Pergamon Press, London, 1967.
41. Speak.J.C., “Methods In Carbohydrate Chemistry, Vol.I.”1962,441.
42. Meyer.K.H, and Rathgels.P., “Hclv.Chemm. Acta”., 1948,31,1540.
43. Jones.J.H, and Heckman.N., “Jour.Am Chem., Soc”., 1947,69,536.
44. Nicolet and Schin.N., “Jour.Biol.Chem”., 1941, 139,687.
45. Reeves., “Jour.Am. Chem.Soc”., 1941, 63,1476.
46. Kommos, E. and Michael, E., “Arch. Pharm. Chem. Sci. Ed”, 1982, 10, 146.
47. H.D. Revanasiddappa, B. Manju and P.G. Ramappa, “Anal. Sci.”,1999,15, 661-64.
48. Revannasiddappa H.D, and Manju.B, “J. Assoc. Anal. Chem.” 2000,83, 1440-45.
49. Sastry C.S.P, R. Chintalapati .R, Prasad A.V.S.S and Sastry.B.S, “Talanta”, 2001,53, 907-14.
50. Zaheer Ahmed, Y.N.Manohara Y.N, Channabasawaraj K.P and Manish
Majumdare.,“Journal Of Chemistry”, 2008, 5(4), 713-717.
51. Sawicki. E, Hauser. T.R, Elbert. W and Noe. J.L., “Anal. Chem”, 1961, 33, 722.
64
52. Pays. M., “Ann. Pharm. Fr”., 1967, 25, 29.
53. Tonio, Rama Sarma GVS and Suresh B., “ Indian Drugs” 1999; 36(9): 572- 575.
54. Reddy MN, Murthy TK and Shantha Kumar SM.. “Indian Drugs”, 2002; 39(1): 39-40.
55. Meyyanathan S.N, Maria Tresa Tonio., Rama Sarma G.V.S and Suresh B.
IndianDrugs, 1999; 36(9): 572-575.
56. Vogel A.J. Elementary Practical Organic Chemistry and Quantitative Organic
Analysis. 2nd Edn. NewDelhi: CBS Publishers; 1987.
57. Rekha Rajeev kumar, Rajeev kumar, Nagavalli, “International Journal of
ChemTech Research”, 2009,1(4), 1068-1071.
58. Prakash S. Sarsambi, D. Gowrisankar, Abhay Sonawane, Abdul
Faheem,“International Journal of ChemTech Research”,2010,2(1), 282-285.
59. Malipatil S.M, Patil S.K, Deepti.M, Kishwar Jahan, “International Journal of Pharma
Research and Development”,2010, 2(8)27-30.
60. Malipatil S.M, Kishwar Jahan, Deepthi. M., “Research Journal of Pharmaceutical,
Biological and Chemical Sciences”, 2010, 1(4) 933- 942.
65
61. Vijaya Raja .G, Triveni.Y, K. Divya K., Naga Lakshmi, Venu Gopal G, “Der Pharma
Chemica”, 2009, 1(2): 285-291.
62. Pessey. M. and Baltos. J., “Ann. Pharm. Forance”., 1970, 28, 153.
63. Sebbort. W.S., “Photogr. Sci“., 1969, 17, 13.
64. Winkler. L.H., “Ztg”., 1901, 23, 434, 25, 586.
65. Shemyakin. F.M and Volkova. V.A.,“ Jour. Gen. Chem“., USSR,1938,6, 698.
66. Manzur-ul. H.H, Quereshi. T, Chugalal. F.R. and Sayad.M., “Mikrochim, Acta”., 1969, 4, 782.
67. Nath. S.K. and Agarwal. R.P., “Chim. Anal”., 1967, 49, 38.
68. Gilehi. S, Katenko. S. and Keh. H., “Bunsek: Kagoko”., 1971, 20, 542.
69. Baker. A.S., “Jour. Agr. Food. Chem”., 1967, 15, 802.
70. Binkley. F., “Jour. Biol. Chem”., 1948, 173, 403.
71. Nakumaro. K. and Binkley. F., “Jour. Biol. Chem”., 1948, 173, 407.
72. Sastry. T.P, and Gopala Rao. G., “Z. Anal. Chem”., 1959, 169, 422.
73. Sastry. T.P, and Gopala Rao. G., “Talanta”., 1958, 1, 213.
74. Szepesy. A, and Block.E., “Gyogyazereszet”., 1962, 6, 421.
66
75. Nabi Syed. A, Siddiqui. R, and Nizam. A.A., “Chemia. Analityezna (Warsa)”, 1980, 25, 643.
76. Perez and Bartol.J., “Colorimetric And Fluorimetric Analysis Of Organic
Compounds And Drugs”, Marcel Dekker, New York, 1974, 504.
77. C.S.P.Sastry, Jana.S.V.M.Lingeswara rao., “Talanta”.,1996, 43(11), 1827-1835.
78. Snell. F.D, Snell. P.T, and Snell. A.C., “Colourimetric methods of Analysis Vol II” A.
D. Van Nostrand Company Inc. London, 1959, 549.
79. Mitsuhashi. S, and Nakanisha. A., “Seibutsgaku (Med Biol)”., 1953, 27.
80. Chen. P.S, Toribaba. T.Y, and Warner. H., “Anal. Chem”., 1956, 28, 1756.
81. Ellington. F, and Adams. W.V., “Fuel”.,1951, 30, 272.
82. Bacon. A., “Analyst”., 1950, 75, 321.
83. Rhodes. D.N., “Nature”., 1955, 176, 215.
84. Ikada. N., “Jour. Chem. Soc. Japan”., Pure Chem Sect., 1952, 73, 549.
85. Mashcheryakov. A.M., “Pochvovidenie”., 1956, 3, 88.
86. Kata. T, and Oozumi. K., “Bunseki Toshiyaku”, 1949, 3, 45.
87. Puri. R.P, and Benerjee. S.P., “Jour. Sci. Ind. Res”., 1951, 103, 86.
67
88. Folin. O, and Ciocalteu. D., “Jour. Biol. Chem”., 1927, 73, 621.
89. Sivasubramanian Lakshmi, Kasi Sankar.V, Sivaraman.V, Senthil Kumar.K,
Muthukumaran.A, and RajaT.K., “Indian Jour.of Pharmaceutical Sciences”., 2004,
66(6),799-802.
90. Mohamed Abd El-Ghaffar, Dina El-Sherbiny, Dalia El-Wass eefand Saadia El-
Ashry., “Jour.of Food and Drug analysis”.,2008, 16(2), 26-35.
91. Murthy.T. K, Sankar Gowri. D, and Rao.Y.S., “Indian drugs”., 2002,39(4), 230-233.
92. Yuan. S.H, and Pollard. A.G., “Sci. Food Agri”., 1955, 6, 223.
93. Ramana Rao. G, Kangilala. G, and Ramamohan. K., “Ind. Jour. Pharm”., 1977, 37, 140.
94. Devi. J.G, and Khorana. M.L., “Ind. Jour. Pharm”., 1953, 15, 227.
95. Singhal. D.M, and Naik. R.R., “Indian Drugs”., 1985, 23, 124.
96. Sane. R.T, and Nayak. R.R, “Indian Drugs”., 1985, 23, 124.
97. Rao. G.R, Kanjilal. G, and Mohan. K.R., “Analyst”., 1978, 103, 993.
98. Peterson. G.L., “Anal Biochem”., 1979, 100, 201.
68
99. Wolfe. S, Ingold. C.F, and Lemieuk.R.U., “Jour. Am. Chem. Soc”., 1981, 103, 938 & 940.
100. Lauer. K, and Makar. S.M., “Anal. Chem”., 1951, 23, 587.
101.Schenter. H.A, and Riemann. W., “Anal. Chem.”., 1953, 25, 1637.
102. Evas. W.L., “Jour. Am. Chem. Soc.”., 1923, 45, 171.
103. Nanji. D.R, and Norman. A.G., “Jour. Soc. Chem. Ind”., (London), 1926, 45, 337.
104. Bell. F, and Kranty. J., “Jour. Am. Pharm. Assoc.”., 1941, 30, 50.
105. Bricker. C.E, and Roberts. K.H., “Anal. Chem”., 1949, 21, 1331.
106. Suryanarayana. C.V., “Analyst”., 1972, 96, 576.
107. Reiss. R.S., “Anal. Chem”., 1958, 164, 402.
108. Gordon. H.T, “Anal. Chem”., 1951, 23, 1853.
108. Foster R, “Organic charge transfer complexes”, Academic press, London, 1969, 51.
109. Mohamed G G, Nour El-Dien F A F and Mohamed N A, “Spectrochim Acta”,
2007,68(5), 1244-1249.
110. Hasani M and Akbari S, “Spectrochim Acta”, 2007, 68(3), 409-413.
69
111. Wu H and Du L M, “Spectrochim Acta” , 2007, 67( 3-4), 976-979.
112. Khaked E, “Talanta”, 2008, 75(5), 1167-1174.
113. El-Zaria M E, “Spectrochim Acta”, 2008, 69(1), 216-221.
114. El-Sherif, Z.-A.; Mohamed, A.-O.; Walash, M.-I.; Tarras,F.-M. “J. Pharm. Biomed.
Anal”. 2000, 22, 13.
115. Moustafa, A. A.-M. “J. Pharm. Biomed. Anal.” 2000, 22, 45.
116. Al-Sulimany, F.; Townshend, A. Anal. Chim. Acta 1973, 66,195.
117. Al-Ghabasha, T.-S.; Rahim, S.-A. “ibid”., 1976, 95, 189.
118. Sass, S.; Kaifman, J.-J.; Gardenas, A.-A.; Martin, J. “J. Anal.Chem”.,. 1958, 30, 529.
119. Brownislaw, S.; Bolelaws, “J. Acta Pol. Pharm”. 1966, 23,573.
120. Gouda. A.A, “Talanta”., 2009,80: 151-157.
121. Elmorsy.K. “Talanta”., 2008,75: 1167-1174.
122. DarwishI.A., “Anal. Chim. Acta”.,2005, 549:212-220.
123. Nafisur.R and K.Mohammad. K. “J. Anal. Chem”, 2005,60,636-643.
70
124. M. Walash, M. Sharaf-EI Din, M. E. S. Metwalli, and M.RedaShabana. “Arch.
Pharm. Res”. 2004,27, 720-726.
125. Khashaba P.Y, El-Shabouri S.R, Emara K.M and Mohamed.A.M J. Pharmaceut.
Biomed. Anal. 2000,22, 363-376.
126. Abdellatef.H.E, J. Pharmaceut. Biomed. Anal. 1998,17, 1267-1271.
127. Saleh G.A. “Talanta”, 1998, 46, 111-121.
128. El Ragehy N.A , Abbas S.S , and El-KhateebS.Z. “Anal. Lett”. 1997,30,2045-2058.
129. K. Basavaiah and S. Abdulrahman Thai “J. Pharm. Sci”. 2010,34, 134-145.
130. Onal A., Kepekci S. E., Oztunc A., “J. AOAC Int.”, 2005,88, 490-495.
131. Al-Ghannam S. M., “J. Pharm. Biomed. Anal”., 2006,40, 151-156.
132. Rahman N., Hejaz-Azmi S. N., “J. Pharm. Biomed. Anal”., 2000,24, 33- 41.
133. Ramesh K. C., Gowda B. G., Melwanki M. B., Seetharamappa J., KeshavayyaJ.,
“Anal. Sci”., 2001,17, 1101-1103.
134. Marona H. R., Schapoval E. E., “J. Pharm. Biomed. Anal”., 2001,26, 501-504.
71
135. Issa M, Abdel-Gawad FM, Abou Table MA, Hussein HM. “Anal. Lett.”, 1997; 30:
2071-77.
136. Ashour S, Al-Khalil R.. “Il Farmaco”, 2005; 60: 771-75.
137. Nour El-Dien F, Mohamed G, Mohamed N. A. “Spectr. Chim.Act. A”, 2006; 65: 20-27
138. Abdalla A. Elshanawane, Samia M. Mostafa and Mohamed S. Elgawish, “Saudi
Pharmaceutical Journal”, 2008, 16, 2.
139. Rahman N, Hejaz-Azmi S.N , “J Pharm Biomed Anal”, 2000,24: 33-41.
140. Amin A.S, Issa Y.M, “Mikrochim Acta”,1995 117: 187-194.
141. Amin A.S, Issa Y.M, “Anal Lett” 30,1997, 69-78.
142. Lahuerta Z.L, Calatayud M.J, “Anal Lett”, 1996,29: 785-792.
143. Amin A.S, Moustafa M.E, El-Dosoky R , “J AOAC Int” ,2009,92: 125-130.
144. Amin A.S, “Anal Lett”,1997,30:2503-2513.
145. Amin A.S, El-Sheikh R, Zahran F, Gouda A.A , “Spectrochim Acta A Mol Biomol
Spectrosc”,2006, 67: 1088-1093.
72
146. Amin A.S, Issa Y.M , “Mikrochim Acta”,1999, 130: 173-179.
147. Abu Zuhri A.Z, Shubietah R.M, Badah G.M , J Pharm Biomed Anal,1999, 21: 459-465.
148. Amin A.S, Dessouki H.A , “Spectrochim Acta Mol Biomol Spectrosc” ,2002,58, 2541-2546.
149. Polawar P.V, Shivhare U.D, Bhusari K.P and Mathur V.B “J. Pharm. and Tech”.
2008,1(4) 539-541.
150. Alaa S. Amin*, Ibrahim S. Ahmed and Hassan A. Mohamed, “J Chem Eng Process
Technol”., 2010,1(1),1012-1014.
151. Seviger, A.J. and Stern, E.R., “Anal. Chem”., 1951, 23, 1511.
152. Dryhurst.G., “Periodate oxidation of diol and other functional groups”., Ed.
Belcher.R and Anderson.D.M.W., Pergamom Press, London, 1970.
153. Sklarz., “Quarterly Reviews”., 1967,21,3.
154. Jackson. E.L., “Organic Reactions”., 1944,2,341.
155. Bobbit.J.M., “Adv. Carbohydrate Chemistry”., 1956,11,1.
156. Hugh.L., “Methods In Carbohydrate Chemistry Vol.V”., 1965, (77), 272. 370.
73
157. Baker.S.A, and Somers.P.J., “Carbohydrates Research”., 1966,8,220.
158. Dyer.J.R., “Methods Of Biochemical Analysis Vol.III”., Ed; Glick.D, Interscience
Publishers, New York, 1956,131.
159. Hay.G.H, Lewis.B.A, Smith.F. and Unrau.A.M., “Methods In Carbohydrate
Chemistry, Vol.V”., 1965,251.
160. Belcher.R., “Sub micro Methods of Organic Analysis”., Pergamon Press, London, 1967.
161. “United State Pharmacopeia”, 23rd ed., United States Pharmacopeial Convention,
Inc., 1994,1982-84.
162. “International Conference on Harmonisation”, Draft Guideline on Validation of
Analytical Procedures: Definitions and Terminology, Federal Register, Volume
60, March 1, 1995, 112 -160.
163. Reviewer Guidance, “Validation of Chromatographic Methods”, Center for Drug
Evaluation and Research, Food and Drug Administration, 1994.
164. “Guideline for Submitting Samples and Analytical Data for Methods Validation”,
Food and Drug Administration, 1987..
74
165. Clarke, G. S., “J. Pharm. Biomed. Anal”., 1994, 12, 643.
166. Inman, E. L., Frischman, J. K., Jimenez, P. J., Winkel, G. D., Persinger, M. L. and
Rutherford, B. S., “J. Chromatogr. Sci.”, 1987, 25, 252.
167. Wilson, T. D., “J. Pharm. Biomed. Anal”, 1990, 8, 389.
168. Hokanson, G. C., Pharm. Technol.,1994, 18, 118.
169. Sandell, E.B., “Colorimetric determination of traces of metals”, 1950, Inter
Science, New York.
170. “International Conference on Harmonization of Technical Requirements for
Registration of Pharmaceuticals for Human Use”, incorporated in November
2005, London.
171. Sethi P.D, “HPLC Quantitative Analysis of Pharmaceutical formulations”, CBS
Publisher and Distributor, New Delhi, 1996, 5.
172. Albert K. ,”On-line LC–NMR and related techniques”, Wiley, Chichester, UK-2002.
173. Kazakevich Y, Lobrutto R. “HPLC for Pharmaceutical Scientist”. 4th ed. New York:
Wiley & Sons Inc.; 2007, 10-14.
174. Lindsay S. “High Performance Liquid Chromatography”. 1st Edt, John Wiley &
Sons; 1991, 45-75.
75
175. Lough W.J, Wainer I.W. “High Performance Liquid Chromatography: fundamental
principles & practice”. Blackie Academic & Professional; 1995,2-28.
176. Snyder LR, Kirkland JJ, Joseph LG. “Practical HPLC Method Development”. 2nd ed.
New York: Wiley & sons; 1997., 46-51.
177. Loyd L,Snyder R.,Joseph.JGlajch, “Practical HPLC Method Development,” 2nd
Edn., 2004,27,29.
178. Michael E, Schartz IS, Krull,“ Analytical method development and Validation”. 3rd
ed. London: John Wiley & sons; 2004, 25-46.
179. International Conference on Harmonization, “Validation of Analytical
Procedures: Methodology”, Federal Register, 1996, 1-8.
180. International Conference on Harmonization, “Draft Guidelines on Validation of
Analytical Procedures”, Federal Register 1995, 1260.
181. ICH, Specifications: Test Procedures and Acceptance Criteria for New Drug
Substances and New Drug Products: “Chemical Substances. International
Conference on Harmonization”, IFPMA, Geneva, 1999.
182. International Conference on Harmonization (ICH), Guidance for Industry, Q1A (R2):
“Stability Testing of New Drug Substances and Products”, IFPMA, Geneva,2003.
76
183. Mulholland.M, “Trends Anal. Chem”., 1988, 7, 383 , Stability Testing of New Drug
Substances and Products (Q1AR). International Conference on Harmonisation,
IFPMA, Geneva, 2000.
184. McKillop.D, Boyle G.W, Cockshott.I.D, Jones.D.C, Yates.R.A, “Xenobiotica”,
1993,23(11), 1241-1253.
185. Cockshott.I.D, Sotaniemi .E.A, Cooper .K.J, Jones.D.C, “British Journal of Clinical
Pharmacology”, 1993,36(4), 339-343.