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.

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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

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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

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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

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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-

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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.

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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

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