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SPECTROPHOTOMETRIC DETERMINATION OF RISPERIDONE AND

MOXIFLOXACINBY CHARGE TRANSFER COMPLEXATION USING

CHLORANILIC ACID

BY

IBEZIM, AKACHUKWU

PG/M.Sc/011/59502

A PROJECT SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENT FOR THE AWARD OF THE MASTER OF SCIENCE

DEGREE (M.Sc) IN PHARMACEUTICAL AND MEDICINAL

CHEMISTRY IN THEDEPARTMENT OF PHARMACEUTICAL AND

MEDICINAL CHEMISTRY,FACULTY OF PHARMACEUTICAL

SCIENCES,UNIVERSITY OF NIGERIA, NSUKKA

DECEMBER, 2012

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DEDICATION

This work is dedicated to my parents Mr and Mrs Ibezim B. Eke.

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ACKNOWLEDGEMENT

I specially give all thanks, adoration and praisefirstly to the Almighty God, the Father of our

Lord Jesus Christ and mine as well, for His immense grace and favour in completing this work.

My profound gratitude goes to my supervisors, Prof. C. J. Mba and Dr N. J. Nwodo whose

wealth of knowledge made this work a reality speedily; as well as my lecturers, Prof. P. Osadebe, Dr.

E. O. Omeje, Dr. W. O. Obonga, Mr G. C. Ebi, Pharm. P. F. Uzor, Pharm C. O. Nnadi, and Mr M. O.

Agbo

I humbly acknowledge my parents (Mr and Mrs B. E. Ibezim) who gave me life and a good

upbringing; I also thank my siblings (Mrs J. Amadi, Mrs I. Kelechi, Mrs H. Eke, Ms Comfort, Mr Ben,

Ms Charity and Mrs Nkechi) for their moral support. I thank my uncle Engr. Dr and Mrs A. B. Eke

whose mentoring about life generally prepared me for this work. I thank my academic father Prof. and

Mrs P. O. Ukoha and his lovely family for accepting me into their family and showing me priceless

care.

I am also indebted to the following persons, Dr. J. Ihediora, Dr L. N. Obasi and

Ms K. Onyia of the Department of Pure and Industrial Chemistry and Dr O. Igwe of the department of

Geology.

It is worthy to express my sincere appreciation to:Mrs N. Uzoka and Ms P. Ibeabuchi (my

course mates), Mrs U. Obioma (the secretary of the Department of Pharmaceutical and Medicinal

Chemistry) and my children at Assemblies of God Nigeria, Onuiyi for their help. I also thank my

friends and colleagues for their support and encouragement during the course of this work.

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TABLE OF CONTENTS

Page

Title page - - - - - - - - - - - i

Certification - - - - - - - - - - - ii

Dedication - - - - - - - - - - - iii

Acknowledgement - - - - - - - - - - iv

Table of Content - - - - - - - - - - v

Abstract - - - - - - - - - - - ix

CHAPTER ONE: INTRODUCTION - - - - - - - 1

1.1.0 Spectroscopy - - - - - - - - - - 1

1.1.1 Theory and Importance of Ultraviolet - visible - - - - - 2

1.1.2 Spectrophotometric method - - - - - - - - 3

1.1.3 Principle of Spectrophotometric method - - - - - - 3

1.2.0 Charge – transfer complexation: An Overview - - - - - - 5

1.2.1 Donor – acceptor bonding - - - - - - - - 5

1.2.2 Donors and acceptors - - - - - - - - - - 6

1.2.3 Change-transfer complexes: Mulliken’s theory - - - - - 9

5

1.3.0 Methods for studying complexes in solution - - - - - - 10

1.3.1 Spectrophotometric determination of the stiochiometry of a complex - - 10

1.3.2 Spectrophotometric determination of the equilibrium constant of a Complex - 12

1.3.3 Determination of thermodynamic functions of a complex - - - - - 14

1.4.0 Solvent effects on complex formation - - - - - - 15

1.4.1 Association constants - - - - - - - - - 16

1.4.2 Heats of association - - - - - - - - - 16

1.5.0 Application of charge-transfer complexation in quantitative analysis of pharmaceutical 16

1.6.0 Others methods of analyzing donor-acceptor complexes - - - - 17

1.6.1 Infra-red (IR) Techniques - - - - - - - - 17

1.6.2 Mass spectrophotometry techniques - - - - - - - 17

1.6.3 Nuclear magnetic resonance techniques - - - - - - 18

1.7.0 Chemistry and pharmacology of drugs - - - - - - 18

1.7.1 Risperidone - - - - - - - - - - 18

1.7.1.1 Synthesis - - - - - - - - - - 18

1.7.1.2 Pharmacology - - - - - - - - - - 19

1.7.1.3 Therapeutic uses - - - - - - - - - 19

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1.7.1.4 Adverse effects - - - - - - - - - 19

1.7.2 Moxifloxacin - - - - - - - - - - 20

1.7.2.1 Synthesis - - - - - - - - - - 20

1.7.2.2 Pharmacokinetics - - - - - - - - - 21

1.7.2.3 Mechanism of action - - - - - - - - - 21

1.7.2.4 Pharmacology indication - - - - - - - - 22

1.7.2.5 Adverse effect - - - - - - - - - 22

1.7.3 Chloranilic acid as π – acceptor and its applications - - - - - 22

1.7.3.1 Mechanism - - - - - - - - - - 22

1.8.0 Applications - - - - - - - - - - 23

1.8.1 Previous methods used for analysis of both drugs - - - - - 24

1.8.2 Objectives of the study - - - - - - - - 24

CHAPTER TWO: MATERIAL AND METHODS

2.1 Drugs used and their sources - - - - - - - - 25

2.1.1 Chemicals and solvents - - - - - - - - 25

2.2 Preparation of reagents - - - - - - - - 25

2.2.1 Preparation of chloranilic acid - - - - - - - 25

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2.2.2 Preparation of risperidone solution - - - - - - - 25

2.2.3 Preparation of moxifloxacin solution - - - - - - - 26

2.3 Absorption spectra - - - - - - - - - 26

2.3.1 Absorption spectrum of chloranilic acid - - - - - - 26

2.3.2 Absorption spectrum of risperidone - - - - - - - 26

2.3.3 Absorption spectrum of moxifloxacin - - - - - - 26

2.3.4 Absorption of risperidone-chloranilic acid complex - - - - - 26

2.3.5 Absorption of moxifloxacin-chloranilic acid complex - - - - 26

2.4.0 Determination of the optimum amount of chloranilic acid - - - - 26

2.4.1 Risperidone-chloranilic acid complex - - - - - - 27

2.4.2 Moxifloxacin-chloranilic acid complex - - - - - - 27

2.5 Effect of time on the complex formation - - - - - - 27

2.5.1 Effect of time on the formation of risperidone-chloranilic acid complex - - 27

2.5.2 Effect of time on the formation of moxifloxacin-chloranilic acid complex - - 27

2.6 Determination of stoichiometry of the complexes - - - - - 27

2.6.1 Stoichiometry by mole ratio method - - - - - - - 27

2.6.2 Stoichiometry by slope ratio method - - - - - - - 28

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2.7 Beer’s law calibration plot for the complexes - - - - - 29

2.7.1 Beer’s law calibration plot for risperidone-chloranilic acid complex - - 29

2.7.2 Beer’s law calibration plot for risperidone-chloranilic acid complex - - 29

2.8 Quantitative assay of the drugs - - - - - - - 29

2.8.1 Assay of risperidone-chloranilic acid complex - - - - - 29

2.8.2 Assay of moxifloxacin-chloranilic acid complex - - - - - 29

2.9 Recovery studies of the drugs - - - - - - - - 29

2.9.1 Recovery studies on risperidone - - - - - - - 29

2.9.2 Recovery studies on moxifloxacin - - - - - - - 30

CHAPTER THREE: RESULTS AND DISCUSSION

3.1 Results - - - - - - - - - - 31

3.1.1 Absorption spectra of the complexes - - - - - - - 31

3.1.1.1 Absorption spectra of risperidone-chloranilic acid complex - - - - 31

3.1.1.2 Absorption spectra of moxifloxacin-chloranilic acid complex - - - 32

3.1.2 Optimum conditions for the complex formation - - - - - 33

3.1.3 Effect of the complex formation - - - - - - - 34

3.1.4 Stoichiometry of the complexes - - - - - - - 35

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3.1.4.1 Stoichiometry of risperidone-chloranilic acid complex - - - - 35

3.1.4.2 Stoichiometry of moxifloxacin-chloranilic acid complex - - - - 36

3.1.5 Beer’s law calibration plots of the complexes - - - - - 39

3.1.6 Limit of detection and limit of quantitation for both drugs - - - - 40

3.1.7 Assay and recovery experiments - - - - - - - 41

3.2 Discussion - - - - - - - - - - 42

3.3 Conclusion - - - - - - - - - - 44

Reference - - - - - - - - - - 45

Appendix - - - - - - - - - - 46

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ABSTRACT

A simple, accurate and sensitive spectrophotometric method for the determination of risperidone

and moxifloxacin has been developed. The method was based on the charge – transfer

complexation of the drugs with chloranilic acid to form a purple – coloured complex having

absorption maximum at 500 nm for risperidone-chloranilic acid complex and 490 nm for

moxifloxacin-chloranilic acid complex. Different variables affecting the reaction conditions such

as the concentration of chloranilic acid, and reaction time were studied and optimized. Under the

optimal conditions, linear relationships between absorbance and concentrations of the drugs

with good correlation coefficient (0.996 and 0.995) were found respectively in the range of 5 –

40 µg/ml. The assay limits of detection and quantitation were 0.550 and 1.670 µg/ml for

risperidone and 0.297 and 0.900µg/ml for moxifloxacin respectively. The precision of the

method was satisfactory and the values of relative standard deviations never exceeded 2% and

there was no interference from the excipients commonly present in dosage forms. The proposed

method was successfully applied to the analysis of risperidone and moxifloxacin in pure and

pharmaceutical dosage forms with good accuracy and precision; the recovery percentage ranged

from 96.35±1.19 to 101.88 ± 0.54 for risperidone and 98.90 ± 1.40 to 102.51± 0.10. The results

obtained by the developed spectrophotometric method were compared with those obtained by the

official method in the British Pharmacopoeia for rispridone and other methods of analysing

moxifloxacin.

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CHAPTER ONE

1.0 INTRODUCTION

1.1.0 SPECTROSCOPY

Spectroscopy involves the study of the absorption, emission of light and other radiation as

related to the wavelength of the radiation1. Spectroscopy deals with the production, measurement

and interpretation of spectra arising from the interaction of electromagnetic radiation with

matter. There are many different spectroscopic methods available for solving a wide range of

analytical problems. The methods differ with respect to the species to be analyzed (such as

molecular or atomic spectroscopy), the type of radiation - matter interaction to be monitored

(such as absorption, emission or diffraction) and the region of the electromagnetic spectrum used

in the analysis. Spectroscopic methods are very informative and widely used for both

quantitative and qualitative analysis2. It has tremendous practical applications in many technical

fields especially in the identification of the constituents of organic compounds. In typical

analysis, a known concentration of a few parts per million (ppm) of the compound can be

detected and the structure of the compound can thus be ascertained1.

Spectroscopic methods are based on the absorption or emission of radiation in the ultraviolet

(UV), visible (Vis), infrared (IR) and radio (nuclear magnetic resonance, NMR) frequency

ranges. These methods monitor different types of molecular and / or atomic transitions.

Spectroscopic method is classified into photometry and spectrophotometry. Photometry simply

means light measurement that depends upon the measurement of the amount of light absorbed by

a solution (spectrophotometry) or by a suspension (turbidimetry) or the amount of light scattered

by a suspension (nephelometry) or the intensity of light emitted by an element when subjected to

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high temperature (flame photometry). The measurement of light in the visible region

(colorimetry) may be accomplished using a colorimeter or spectrophotometer or comparison

with colour standards3. Spectrophotometry is more sophisticated and has much wider

applications than photometry. It involves the use of instruments, a spectrometer and a

photometer, both housed in one cabinet. Light of a spectrophotometer which could be described

essentially as a combination of photometer (a visual, photographic, or photoelectric

instrument for measuring absolute or relative light intensities) with a monochromator (an

instrument for isolating light of a single wavelength)4. Unlike light filter which can only isolate

the required range of wavelengths needed for an analysis, a monochromator can isolate an

extremely narrow bandwidth almost comparable to a single wavelength, thereby extending range

of application of spectrophotometers from the ultraviolet (185 – 400nm) through visible (400 –

760nm) to the infrared (>760nm) region.

1.1.1 THEORY AND IMPORTANCE OF UV-VISIBLE SPECTROPHOTOMETRY

In ultraviolet and visible spectroscopy, absorption of radiation is the result of excitation of

bonding electrons5. When a molecule absorbs ultraviolet or visible light of frequency, v and

wavelength λ, an electron undergoes a transition from a lower to a higher energy level in the

molecule6. The energy difference (∆E) is related to frequency and wavelength by the expression;

∆� � �� ………………… 1.1

whereh is Planck’s constant and c is the velocity of radiation. For the region 200-750nm, the

energy required for electron transition is in the range of 600-160KJmol-1

(multiplication of the

expression by Avogadro’s number will express the energy absorbed per mole). Energies of these

magnitude are associated with the promotion of an electron from a non-bonding (n) orbital or a π

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- orbital to an antibonding π-orbital (π*) or to an antibonding σ-orbital (σ*). It therefore, follows

that electronic transitions in organic molecules could be ascribed to a σ→π or σ→ n.

The energy required for the transitions of the σ electron is much more (usually in far UV) than

the n-electron or less tightly bonded π electrons. They are seen in the vacuum-UV and harder to

observe. The types of bonds that give rise to UV-Visible absorptions are known as

chromophores. In the ultraviolet, the electrons of the chromophores are either directly used in

bond formation or are non-bonding or unshared outer electrons of an electronegative atom such

as oxygen,nitrogen or sulphur5. The most important transitions in organic compound are as

follow:

a) π → π* transition

These are usually associated with the multiple bonds of carbon with carbon, nitrogen oxygen,

and sulphur. They generally give rise to high intensity absorptions.

b) n→ π* transition

These are usually associated with the groups such as carbonyl, thiocarbonyl, and nitroso

groups. Generally the intensity of these absorptions are very much lower and lie at longer

wavelengths than those arising from π→ π* transitions. However, σ→ σ* and n→ σ*

transitions are also known to occur.

Transition-metal ions absorb in the UV and visible region and the transitions responsible involve

4f and 5d electrons of the metals. Alternatively, in some inorganic complexes, the process of

change-transfer absorption occurs.

Most applications of ultraviolet and visible spectrophotometry to organic compounds are based

on n→ π* and π→π* transitions and hence require the presence of chromophoric groups in the

molecule. These transitions occur in the region of the spectrum (about 200 to 760nm), which is

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convenient to use experimentally. Visible light consists of electromagnetic radiation in the

wavelength range 350-760nm, to which the human eye is sensitive. The wavelength and

efficiency of absorption by a substance depend on the structure of the substance and its

environment, making it possible to measure the presence or concentration of the substance.

1.1.2 SPECTROPHOTOMETRIC METHOD

This method uses spectrophotometer which is an instrument for measuring the intensity

of light of various wavelengths transmitted by a solution. The intensity of light is determined by

electric detectors, which convert radiant energy to electrical energy and can therefore eliminate

the need of subjective measurements, by the human eye. This method has a number of

advantages over other methods these include: the limit of detection is lowered by measuring the

absorption of a solution at the wavelength of maximal absorption; the possibility to avoid or

minimize the effect of foreign coloured substances by working at a suitable wavelength and

greater precision.

1.1.3 PRINCIPLES OF SPECTROPHOTOMETRIC METHOD

The fundamental principle of UV-Visible spectrophotometry lies in that light of a definite

interval of wavelength passes through a cell with a solution or solvent and falls on the

photoelectric cell that converts radiant energy into electrical energy measured by a galvanometer.

Photometric method of analysis (photocolorimetry and spectrophotometry), based on measuring

light absorption of molecules in a solution, utilizes the principle that the amount of light

absorbed by a substance in solution is proportional to the intensity of incident light and to the

concentration or number of the absorbing species in the path of the beam. These relationships

lead to two fundamental law:

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(a) Beer’s law which relates light absorption to the concentration of the absorbing substance

and

(b) Lambert’s law which relates that the light absorbed to be dependent on the path length of

the absorbing substances.

These two laws are combined as the Beer’s-Lambert law, or simply Beer’s law, and are

expressed as the equations:

�� I� I⁄ � � �� T � �� A………….. 1.2

or I� I⁄ � I T⁄ � 10��……………………………. 1.3

where Io is the intensity of incident light, I is the intensity of transmitted light; ‘T’ is the

transmittance ‘a’ is a constant factor characteristic of a solute, ‘b’ is the path length through an

absorbing solution while ‘c’ is the concentration of the absorbing solution.

The constant a, called absorptivity, the absorption coefficient or the extinction coefficient,

specifies a characteristic property of the absorbing substance and is a function of its wavelength.

Its units depend on the concentration and path length units employed. When c is expressed in

moles per litre and b is expressed in centimetres the constant ‘a’ is known as molar absorptivity

(ε) and it is used as a physical constant for absorbing species under standard conditions. It has a

unit of litres per mole (i.e. L-1

mole-1

and designated as ε).

These expressions show that there is linear relationship between the absorbance and the

concentration of a given solution, if the path length and the wavelength of radiation are kept

constant. Thus, by measuring the transmittance or absorbance, the concentration of substance in

a solution can be calculated.

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However, Beer’s law has not been obeyed in some situations. There have been known to be

deviations from the law which are as a result of measurement conditions in which the assumption

used to derive Beer’s law are not valid. There could be positive or negative deviations from

linearity. Specific instrumental and chemical effects that cause such apparent deviation include.

i) Non-zero intercept: This is usually solved by improving blank measurements or

adjustments to non-equivalent measurement conditions for the blank and standard

solution.

ii) Non-linearity due to chemical equilibriums: This is a case where the analyte can exist in

several chemical forms in solution which may be in equilibrium such polymerization,

complex formation and dissociation. The shape of the calibration plot may therefore

depend on a particular species.

iii) Non-linearity: Due to polychromatic radiation, stray radiation, variability in pathlength;

multiple reflections circular dichroism and fluorescence and inability of fixing and

reproducing the analytical wavelength.

Non-linearity due to the other chemical effects: This occurs if the analyte’s molar absorptivity is

dependent on the analyte concentration. Such effects are usually minor and occur at relatively

high concentrations (>10-10

M). Differences in solute-solvent interactions, solute-solute

interactions or hydrogen bonding at high concentrations can change the chemical or electrostatic

environment and hence the absorptivity of the analyte7.

1.2.0 CHARGE-TRANSFER COMPLEXATION: AN OVERVIEW

The concept of molecular complexes has undergone much development over the years.

Izmail’skii was one of the first few researchers to explain electron spectra (colour spectra) of

organic compounds using the idea of electron donor - acceptor interaction. Weiss proposed that

all molecular complexes were ionic in structure

complexes was as a result of intense charge resona

complex. Brackmann, however explained that molecular complexes were formed as a result of

the “complex resonance” between a no

According to him, the colour formed was as a result of the complex and not localized in either A

or D. Combining these concepts, these workers agree

molecular complex8. Mulliken, in his theory, explained the concept of charge transfer complexes

using quantum-mechanical theory

1.2.1 DONOR-ACCEPTOR BONDING

Donor – acceptor or dative bonding is a form of covalent bonding between two molecules that

exist independently.

A characteristic example is:

NMe3+ BCl3 → Me3N→BCl

The product Me3N→BCl3 is known as an

bond. The two components of the adduct are

electrons, and an acceptor, BCl3

Fig. 1.1: Illustration diagram for donor

all molecular complexes were ionic in structure and consist of D+ A

- and that the colour of these

complexes was as a result of intense charge resonance spectra arising within the ions in the


the “complex resonance” between a non-bond structure (D, A) and a bonding structure (D

ormed was as a result of the complex and not localized in either A

or D. Combining these concepts, these workers agreed that the colour formed is due to the

. Mulliken, in his theory, explained the concept of charge transfer complexes

mechanical theory9.

ACCEPTOR BONDING

acceptor or dative bonding is a form of covalent bonding between two molecules that

→BCl3 ……………. 1.4

is known as an adduct and the arrow in its formula indicates the dative

bond. The two components of the adduct are: a donor, NMe3, which has an unshared pair of

which has a vacant orbital.

diagram for donor-acceptor bonding

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and that the colour of these

nce spectra arising within the ions in the


bond structure (D, A) and a bonding structure (D+ A

-).

ormed was as a result of the complex and not localized in either A

that the colour formed is due to the

. Mulliken, in his theory, explained the concept of charge transfer complexes

acceptor or dative bonding is a form of covalent bonding between two molecules that

adduct and the arrow in its formula indicates the dative

, which has an unshared pair of

In the above molecular orbital diagram, the bonding molecular orbital

parent orbitals, and therefore becomes more associated with the boron than the original lone pair

orbital on the nitrogen. Electron density, therefore moves from nitrogen, the donor, towards

boron, the acceptor10

. Donor-acceptor bonds are also known as charge transfer complexes.

1.2.2 DONORS AND ACCEPTORS

Mulliken10

defined donor D and acceptor A as those entities such that during t

between a particular species of D and a particular species of A entities, transfer negative charges

from D to A takes place with the formation, DA as end

or of new entities. The additive combinations

combinations11

. Charge-transfer complexation occurs from the highest occupied molecular

orbital (HOMO) of the donor to the lowest unoccupied molecular orbital (LUMO) of the

acceptor molecule. If this electron transfer

charge-transfer bond is formed.

Fig. 1.2: Charge transfer transitions for HOMOs of the donors and LUMOs of acceptor

In the above molecular orbital diagram, the bonding molecular orbital Ψ is formed from both


on density, therefore moves from nitrogen, the donor, towards

acceptor bonds are also known as charge transfer complexes.

DONORS AND ACCEPTORS

defined donor D and acceptor A as those entities such that during t


place with the formation, DA as end-products either of additive combination

or of new entities. The additive combinations may be 1:1, m:1, 1:n or in general m:n

transfer complexation occurs from the highest occupied molecular


acceptor molecule. If this electron transfer involves a decrease in the energy of the system, a


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is formed from both


on density, therefore moves from nitrogen, the donor, towards

acceptor bonds are also known as charge transfer complexes.

defined donor D and acceptor A as those entities such that during the interaction


products either of additive combination

may be 1:1, m:1, 1:n or in general m:n

transfer complexation occurs from the highest occupied molecular


involves a decrease in the energy of the system, a


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The properties and structure of the complex formed are dependent on the particular orbitals

involved in the electron transfer. Mulliken10

went further to classify donors and acceptors based

on their structure before interaction. The broad division of donors is into even lone-pair or onium

(n and n’), even bonding-electron (π and σ) and odd-electron (R) radical donors. Similarly,

acceptors are classified as: even vacant-orbitals (v and v*), even bonding electron (π, σ and σ*)

and odd-electron (Q) radical acceptors. This classification is exemplified in Table 1:1

Table 1.1: Electron-donor and acceptor types

Symbol Name Essential characteristics Examples

N Onium donor Derived by the addition of a proton to

the hydride of any element of the

nitrogen, chalcogen or halogen

families.

Amines, alcohols, ethers,

ketones, nitriles.

n' Onium anion

donor

Involves complete transfer of an

electron from donor to acceptor with

the formation of an ionic structure.

solvated anions of H-acid e.g. I-,

CH3COO-, OH

-, NH2

-

Π π donor Involves proton transfer with a dative

and a no – bond structure.

Aromatic (Ar) and un-saturated

hydrocarbons and their

substitution products with

electron releasing substituents

Σ σ donor Involves transferring of a pair of

electrons by the donor(ligand) to an

acceptor(metal) to form a co-ordinate

covalent bond with the metal.

Alkyl and aralkyl halides, esters,

especially if R+ is resonance

stabilized

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R Radical Donor

(Reducing

radical)

System with relatively easily ionized

odd electron

Univalent metal atoms, aralkyl

and other easily ionized radicals

V Vacant orbital

acceptor

Neutral even system in which an

orbital or orbitals of relatively high E

(electron affinity) are vacant

BMe3, AlMe3, RX3, ZnCl2

V* Vacant orbital

cation acceptor

Involves positively charged ions

attracting unshared electron pairs to

itself.

Ag+, NO2

+, HSO3

+

Xσ Halogeniod σ

acceptor

Involves transferring a halogen atom

from electron rich species to an

electron deficient species in the non –

covalent interaction.

Halogen molecules ( Cl2, Br2, I2)

Π π acceptor Neutral even system containing

bonding π electrons relatively

strongly held.

Aromatic or unsaturated

hydrocarbons with

electronegative or electrophilic

substituents for trinitrobenzene,

maleic anhydride

Q Radical acceptor

(oxidizing or

electrophilic

radical)

Odd-electron system with relatively

affinity

Halogen atoms, H atom, NO2

acid radicals

R+

= Aromatic ring.

21

The even electron donors and acceptors could be divided into increvalent donors and sacrificial

donors. As the names imply, the valency of the electron to donors in the case of the increvalent

type increases upon complexation. They are the ion-pair aliphatic amines, amine oxides,

phosphines, alcohol etc. Sacrificial donors are compounds which donate an electron from a

bonding orbital. They include sigma (σ) donors such as hydrocarbons, especially small cyclic

hydrocarbons. These are very weak electron donors. By contrast, sacrificial π-donors such as

aromatics, particularly polycyclic systems contain electron releasing groups. Some compounds

such as azo aromatics and aromatic may behave as lone pair donors towards some acceptors and

π− donors toward others.

Increvalent acceptors are classified into two types; σ (sigma) and π (pi). δ acceptors result when

the electron is accepted by the bands of compounds like halogen e.g. iodine can form a weak

outer complex with aniline. π - acceptors are the most common organic acceptors. They include

aromatic systems containing electron-withdrawing substituents such as nitro, cyano or halogen

groups, anhydrides, acid chlorides and quinones. Examples include 2,3,5,6- tetrachloro-4-

benzoquinone, p-chloranilic acid, p-benzoquinone and picric acid.

For each of the structure-based classes of donors and acceptors, there is at least one, and there

are often two characteristic modes of functioning or behaviour. When there are two modes of

functioning, one of these is associative and the other is dissociative. In determining interaction

strengths between donors and acceptors, some factors are considered11

. One is the importance of

low ionization potentials for strong donors and high electron affinities for strong acceptors.

Mutual approachability is another important factor. For example approachability is especially

good between n (or n') donors and V (or V') acceptors, or between π donors and π acceptors but

not between π donor and acceptors.

22

Generally π-electron donors give rise to weaker complexes (association constant 0.2 – 201mol-1

than those derived from non-bonded electron donors (association constant up to 10+4

mol-1-

). The

stronger the donor and acceptor, the higher the energy of formation of the complexes and the

stronger (more stable) the complex formed. This is in agreement with the spectral findings of

Weimer and Prausnitz12

, and Srivastava and Prasad13

.

The donor and acceptor properties of molecules can be modified by chemical substitution. The

donor ability increases with decreasing ionization releasing substituents such as hydroxyl or

amino groups. Examples of such donors include hydroquinone and dimethylquinoline. The

acceptor ability increases with increasing electron affinity and introduction of suitable

electrophilic substituents such as cyano, halogens,carbonyl groups and indeed all negative

radicals. Examples of acceptors include chloranillic acid, 2,4,7-trinitroflurenone, fluoranil

tetracyanethylene, 2,3-dicloro-5,6-dicyano-1,4-benezoquinone, bromanil, tetracyano-p-

benxoquinodinexane, etc.

1.2.3 CHANGE-TRANSFER COMPLEXES: MULLIKEN’S THEORY

According to Mulliken, in the ground state N of any molecular compound AD

ΨN = aΨo + bΨ1 …………… ……. 1.5

Where Ψo is a “no-bond” wave function Ψ(A,D) of the form

Ψo = Ψ(A,D) = aΨAΨD + ………….. 1.6

and where ΨI is a dative wave function corresponding to transfer of an electron from D to

A accompanied by the establishment of a (usually weak, because of the distance between A and

D) covalent bond between the odd electrons in A- and D

+.

23

ΨI = Ψ(A- - D

+) + …………………. 1.7

+…. Indicates additional terms cΨ + …….

Incorporating equations (1.6) and (1.7) into equation 1.5

ΨN = aΨ(A,D) + bΨ(A- − D

+) ……... 1.8

The coefficients of a and b in equation (1.8) characterize the function of the “no-bond” structures

and the structures with charge-transfer in the ground state of the complex. At low value of b, the

fraction of the state with charge-transfer is small. Hence, the system becomes stabilized mainly

as a result of classic electrostatic forces (dipole-dipole; dipole-induced dipole; ion-dipole etc). At

high values of b, the contribution of the state with charge-transfer is greater and the force

determined by the charge-transfer may be much greater than the classic intermolecular forces.

There also exists an excited state, ε of a molecular compound A.D given by:

Ψε = a*Ψ(D+ − A

−) – b*Ψ(D,A) …… 1.9

An intense absorption spectrum is associated with transition ΨN − Ψε; and since ΨN has nearly

pure no-bond character and Ψε has pure ionic character (a2>>b

2) the spectrum associated with the

transition is called an intermolecular charge-transfer spectrum. Light absorption causes an

electron to jump from D to A. Mulliken also proposed some structure for complexes formed by

halogen molecules with aromatic and oxygenated solvent9.

1.3.0 METHODS FOR STUDYING COMPLEXES IN SOLUTION

There are several known and acceptable methods for studying complexation profile in solutions.

A standard method for the determination of donor-acceptor complexation in solution is through

ultraviolet-visible spectrophotometry.

1.3.1 SPECTROPHOTOMETRIC DETERMINATION OF THE STIOCHIOMETRY

OF A COMPLEX

The spectrometric determination of

formed according to the reaction, can be performed by several methods

a) Continuous variation method

The method is both simple and widely used for the spectrophotometric determination of

formulas of metal complexes. The method assumes only a single complex is present in

the solution, a situation which is often not the case. In Job’s method of continuous

variations14

, the absorbance is measured in a series of solutions that have

volume and the total concentrations of [D] and [A]

solutions of the acceptor A and the donor D are prepared. From these solutions, a series

of solutionsare prepared by mixing V

where VT is the total volume, fixed and same for all solutions, and V

volume (O ≤ VD ≤ VT). The absorbances of all solutions of the series are measured,

usually at the same wavelength of maximum absorbance of D

absorbance versus the mole fraction of the donor,

is made. The position of the maximum absorbance, A

the axis of the molar fractions (abscissa) gives the stoichiometry of the complex. In t

region of AMax, a curvature is observed, and the stoichiometric point is located by

extrapolating the straight line portions of the curve.

SPECTROPHOTOMETRIC DETERMINATION OF THE STIOCHIOMETRY

The spectrometric determination of the donor to acceptor, m/n in the complex, DmAn which is

formed according to the reaction, can be performed by several methods

………….. 1.10

Continuous variation method




, the absorbance is measured in a series of solutions that have

concentrations of [D] and [A] are fixed. In practice, two equimolar


prepared by mixing VL ml of solution D and (VT – VL)ml of

is the total volume, fixed and same for all solutions, and V

). The absorbances of all solutions of the series are measured,

usually at the same wavelength of maximum absorbance of DmAn

absorbance versus the mole fraction of the donor,

is made. The position of the maximum absorbance, A

the axis of the molar fractions (abscissa) gives the stoichiometry of the complex. In t

, a curvature is observed, and the stoichiometric point is located by

extrapolating the straight line portions of the curve.

24

SPECTROPHOTOMETRIC DETERMINATION OF THE STIOCHIOMETRY

the donor to acceptor, m/n in the complex, DmAn which is




, the absorbance is measured in a series of solutions that have the same total

. In practice, two equimolar


)ml of solution A

is the total volume, fixed and same for all solutions, and VL is the variable

). The absorbances of all solutions of the series are measured,

n and a plot of

or the acceptor,

is made. The position of the maximum absorbance, AMax relative to

the axis of the molar fractions (abscissa) gives the stoichiometry of the complex. In the

, a curvature is observed, and the stoichiometric point is located by

25

b) Method of mole-ratio

This method is also known as titration method14

. Here, a series of solutions are prepared,

containing different ratios of compound A and D. The concentration of one of the

reactants (usually of acceptor) is kept constant while that of the other is varied, so that

[D]/[A] varies for example in the range of 0.1 – 10. At CAo = constant, the results are

plotted in absorbance versus C�� C �⁄ co-ordinates or in absorbance versus CDo co-

ordinates, usually at the wavelength of maximum absorbance of the complex DmAn.

Absorbance increases linearly until near the stoichiometric point, at which M is almost

quantitatively in the form DmAnand !D# !A#⁄ � m n⁄ . Whereas beyond this point [DmAn]

, its absorbance remains practically constant and the curve becomes parallel to the X-

axis. In the region of the stoichiometric point, a curvature is observed. Despite the

curvature, the value of the ratio, m/n and the stoichiometric formula of the complex can

be determined by extrapolating the straight-line portions of the curve.

c) Slope ratio method

This method is especially valuable since it can be applied to systems in which the

complexes have large dissociation constants and are therefore, not readily suited to the

continuous variations or mole-ratio method. This method proposed by Harvey and

Manning15

, based upon absorbance measurements of solutions in which dissociation of

the complex is repressed by a large excess of one of the reactants; say A so that the

equilibrium concentration of complex is proportional to the analytical concentration D

added in the reaction. If the complex formed in the reaction is of the form:

From equation (1.10): mD & nA ' DmAn

26

and if the concentration of A is essentially constant and in sufficient excess to make

dissociation negligible, the equilibrium concentration of the complex, [DmAn] will be

essentially proportional to the analytical concentration of D added; so

!D(A)# � C� M⁄ ……... 1.11

Where the bracket refers to the equilibrium concentration and CD is the analytical

concentration. At a wavelength where only the complex absorbs and Beer’s law is

obeyed.

A � +�!D(A)# ………... 1.12

Where A is the measured absorbance, ε is the measured absorptivity and b is the

thickness of the cell in cm. substituting the value of [DmAn] from (1.11) into (1.12).

A � +��, -⁄ ………….. 1.13

A is plotted against different analytical concentration of D, keeping the concentration of

A constant and in excess. Over the straight line portion of the curve, equation (9) is valid

and this straight line will have a slope given by

Slope � +� -⁄ ………. 1.14

Similarly if D is the component in constant excess and the concentration of A is varied,

!D(A)# � C 3⁄ ……… 1.15

And if A is plotted against CA, the slope of the straight line portion of the curve will be

Slope4 � +� 3⁄ ……...... 1.16

27

The ratio of n to m in the complex may be determined by the ratio of the two slopes.

56789:56789;

� )( …………. 1.17

The slope ratio method is reliable as long as linearity of the curves shows that absorbance

is directly proportional to the concentration, provided that the concentration of the excess

components is identical in the two series of measurements.

1.3.2 SPECTROPHOTOMETRIC DETERMINATION OF THE EQUILIBRIUM

CONSTANT OF A COMPLEX

The association constant of a complex may be obtained by the use of ultraviolet or visible

spectrophotometry. The general equation for the equilibrium involved is:

D & A < !D � �A#=>………… 1.18

Where, D is an electron pair donor and A, an electron pair acceptor.

The equilibrium constant expression for this reaction

K@ � !� #!�#! # ……… 1.19

Where; Kf = equilibrium constant for DA complex formation

[DA] = Concentration of the complex (DA) at equilibrium

[D] = Initial concentration of the donor species

[A] = Initial concentration of the acceptor species

When the donor molecule does not absorb in the region of study, the relationship of the

total absorbance [A] to the acceptor concentration and to the complex concentration, at

given wavelength for a 1cm cell is given by:

28

A = εc[DA] + εA[A] …........... ….. 1.20

εc

= Molar absorptivity of the complex

εA = Molar absorptivity of the acceptor

[A]O = Concentration of uncomplexed acceptor at equilibrium

When only one complex is formed, equation (1.21) is also characteristic of the system.

[A] = [DA] + [A]o ……. 1.21

Benesi andHildebrand16

, on combining these equations deduced their own equation as:

!�#A

BC � D ! #A

E F εGH

I & ε …….. 1.22

Where; [D]o = total concentration of donor (fixed); total D (uncomplexed and complexed)

[D]o = [D] + [DA]

[A]O = total A (uncomplexed and complexed) = [A] + [DA]

Abs = charge transfer absorption of DA complex at wavelength λ

ε = Molar absorptivity of DA complex at λ

Equation 2.20 is a straight line, whose intercept is 1 + and slope = J1 +KL⁄ M. Scott17

, however

modified the Benesi- Hildebrand equation as

!N#!,#�

N�O � !,#PQR

& SQR

. PQR

……… 1.23

A plot of [A][D]b/A against [D] gives a straight line with slope,

PQR and with intercept

SQRPQR

for

a 1:1 complex. However, as a prerequisite for the Benesi-Hildebrand and Scott equations,

29

[A]>>[D]. Nagakura’s formula18

(1.24)is used in the evaluation of Kf where there is an

overlapping absorption of base with the complex.

K� � UVJ AW :MXV:� W A�VV:� :W � Y… 1.24

Where Ao A and A1 are the absorbances measured at λDA of solutions whose concentrations of

the acceptor are equal and whose donor concentration is zero, d and d1 respectively. Drago and

Rose19

expanded on equation (2.14) to derive their own equation.

ZW � NP[

� !\]# � !\# & !,#!N#N +,N ….1.25

In this method, the points of intersections of the straight lines K-1

Vs εDA are found. The values

of K-1

and εDA corresponding to the ordinate and abscissa of the point of intersection of these

straight lines are used to determine the unknown parameters εDA and K.

1.3.3 DETERMINATION OF THERMODYNAMIC FUNCTIONS OF A COMPLEX

The equilibrium constants are related to the thermodynamic function by:

∆G� � �RT In K …………….. 1.26

∆G� � ∆H� � T∆S …………. 1.27

Uab)cad Ye � ∆f[

gd; ………………. 1.28

∆h7 � �2.303kl log Z ]\n …. 1.29

Where T = temperature (in Kelvin)

∆Go = Gibb’s free energy change

30

∆Ho = Change in enthalpy

∆So = Change in entropy

K and ∆H are experimentally determined and from these values ∆Go and ∆S

o are calculated

using equation (1.27) and (1.28). The standard change in enthalpy of formation of the complexes

∆Ho is determined from the temperature dependence of the equilibrium constants by means of

equation (1.28).

o3K � W ∆f[gd & n�3pq�3q………. 1.30

Hence, ∆Ho is calculated from the graph of InK Vs I T⁄ .

When the value of ∆Ho is large and negative, a stable/strong complex is formed but when the

value of ∆Ho is small and negative, a weak complex is formed. In effect, the value of ∆H

o

determines the extent to which the charge is transferred (that is whether it is partially or

completely transferred).

One of the characteristic features of the thermodynamics of complex formation;

\ & ] < \]is the comparatively small change in free energy, ∆Go while ∆H

o and ∆S

o are

subjected to considerable changes.

This is apparently due to the mutual compensation which can be qualitatively explained as

follows: with increase in the heat of formation of the complex, i.e. with increase in the strength

of the intermolecular bond, more degrees of freedom are lost by the system and hence the

entropy is less. As a result, the correlation between ∆Ho and ∆S

o is linear.

31

In the transformation of outer charge-transfer complexes to inner complex, the magnitude of the

change of enthalpy, ∆Ho and change in entropy, ∆S

o indicate whether there is transformation of

outer complex to inner complex or not. A stable outer complex is associated with greater charge-

transfer in the ground state as well as a high value of ∆Ho, showing a large activation energy

(EA) for the transformation than a weaker outer complex. The more negative the heat of enthalpy

∆Ho, the larger the activation energy, EA and the more stable the outer complex.

1.4.0 SOLVENT EFFECTS ON COMPLEX FORMATION

The nature of the solvent also determines whether an outer or inner complex is formed. For a

non-polar solvent, the dielectric constant is low and as such the activation energy will be high.

This invariably implies the outer complex is stable. For a polar solvent the opposite is the case20

;

the activation energy will be low and transformation of outer complex to inner complex will be

fast.

Most data in literature on charge-transfer complexation obtained using a halogenmethane as a

solvent on assumption that it is inert. On the contrary, these halogenmethanes form weak

complexes with amines. The spectral studies of this complex revealed that increasing portions of

halogenmethanes relative to a fixed concentration in the amine solution of n-hexane, led to

increase in absorption. The Benesi-Hildebrand plots for the halogenomethane / N,N-

dimethylaniline in n- hexane also gave positive, zero and negative intercepts. The effects of

solvent on complex formation are based on the followings:

1.4.1 ASSOCIATION CONSTANTS

The Benesi-Hildebrand’s graph for evaluation of Kf, the association constant, gave positive,

negative or zero intercepts as Kf. Negative and zero intercepts are anormalies for the value of Kf.

Orgel and Mulliken21

suggested that the value of zero for Kf may be seen as a result of

32

“constant” charge-transfer between molecules in collision. They concluded that provided the

integral between appropriate donor and acceptor orbitals are appreciable even for pairs of

molecules in loose contact or close to one another, then charge-transfer is formed.

Foster, et al22

attributed anomalies in K to non-obedience of Beer’s law. Deviation from

Beer’s law would have to be quite large, however to explain zero and negative intercepts in

the Benesi-Hildebrand plots. It is also noted that if a halogenmethane is used as a solvent in

the evaluation of the association constant of a molecular complex, it will compete with the

acceptor for the available donor, thus KB – H is underestimated.

1.4.2 HEATS OF ASSOCIATION

Solvents play an important role on charge-transfer complexes by decreasing the thermodynamic

properties of the complexes. Davis and Farmer in their studies23

, observed that using

tetrachloromethane as solvent and going from NNN‘N’-tetramethyl-p-phenylenediamine to NN-

dimethylaniline, which is a weaker electron donor, there was a sharp drop in – ∆H with aniline,

however the thermodynamic endothermic heat of mixing must outweigh the small exothermic

contribution due to complex formation.

1.5.0 APPLICATION OF CHARGE-TRANSFER COMPLEXATION IN

QUANTITATIVE ANALYSIS OF PHARMACEUTICALS

Two spectrophotometric methods can be used in the assay of drugs, the A and Ar methods24,25

.

The A method involves the direct method measurement of A of the complexed drug at its λmax

while the Ar method is an indirect method which involves the measurement of λmax of the

acceptor giving a direct determination of the complexed acceptor and hence a direct

measurement of the concentration of the drug base.

33

Time is an important factor in quantitative analysis based on charge-transfer complexation. This

is important to ensure maximum complexation of the components and also to minimize the

changes in absorbance with time due to conversion of outer charge-transfer complexes to inner

complexes26

. The slow formation of complexes with chromogen is explained as a formation of

another reaction product of chromogen, in addition to the complex. On increasing the

temperature there may either be an increase or decrease in absorption of the complexes, but more

stable complexes are usually obtained at lower temperature26-28

. Decrease in absorption on

increasing the temperature is ascribed to the dissociation of the complex to its individual

components, thus suggesting a reversible reaction, and also a weak complex with a low

association constant. On dilution of the complex, there is fading of the complex colour, which

can be explained as competition of the solvent with the acceptor, giving rise to contact pairs.

In quantitative assay by charge-transfer complexation, the concentrations of the donor and

acceptor should be kept as low as possible to avoid anolmies due to self-association or the

formation of termolecular complexes with consequent deviation from Beer’s law. The solvent of

choice in quantitative analysis should be such that it does not result in deviation from Beer’s law.

Dioxane has been observed to do that. Reproducibility is also very important to ensure accuracy

and inter-laboratory comparisons. Interference from drug additives such as diluent, glidants,

adhesives, colourants and preservatives should be minimal to avoid deviations from Beer’s law.

These additives, however are inert and as such are not expected to interfere.

Recovery values are based on the amounts found and those calculated to be present from the

nominal concentration of the preparations tcalculated should always be within the limits of ttheoretical

indicating the accuracy of the method.

1.6.0 OTHER METHODS OF ANALYZING DONOR-ACCEPTOR COMPLEXES.

1.6.1 INFRA-RED (IR) TECHNIQUES

This is quite similar to steps in UV and Visible. Absolute

“base line” is often used. A synthetic “base line” is constructed between the minima at the sides

of the absorption maximum and a vertical line intersecting the peak of the abscissa. The length of

the vertical line measured from the intersection of the synthetic base line and peak of the

absorption maximum is used as the absorbance in quantitative calculations.

1.6.2 MASS SPECTROPHOTOMETRY TECHNIQUES

This is very sensitive, highly selective quantitative analytical techn

usually in the nanogram (ng) range and fragmentation patterns are highly reproducible even for

multi-component mixtures.

1.6.3 NUCLEAR MAGNETIC RESONANCE TECHNIQUES

The use of NMR for quantitative analysis involves several proper

chemical shift establishes the general nature of the proton environment and the interaction of

protons. The ability to integrate areas under the resonance peaks in NMR is used and employed

for quantitative calculation.

1.7.0 CHEMICAL AND PHARMACOLOGY OF DRUGS

1.7.1 RISPERIDONE

Fig.1.3: Structure of risperidone:

methyl-2,6-diazabicyclo[4.4.0]deca

RED (IR) TECHNIQUES

This is quite similar to steps in UV and Visible. Absolute absorbance at a particular frequency



ured from the intersection of the synthetic base line and peak of the

absorption maximum is used as the absorbance in quantitative calculations.

MASS SPECTROPHOTOMETRY TECHNIQUES

This is very sensitive, highly selective quantitative analytical technique. Sample size range is


NUCLEAR MAGNETIC RESONANCE TECHNIQUES

The use of NMR for quantitative analysis involves several properties of the spectrum, the



ICAL AND PHARMACOLOGY OF DRUGS

Fig.1.3: Structure of risperidone: 4-[2-[4-(6-fluorobenzo[d]isoxazol-3-yl)-1-piperidyl]ethyl]

diazabicyclo[4.4.0]deca-1,3-dien-5-one

34

absorbance at a particular frequency



ured from the intersection of the synthetic base line and peak of the

ique. Sample size range is


ties of the spectrum, the



piperidyl]ethyl]-3-

35

Risperidone belongs to the class of atypical antipsychotics.29

It is a dopamine antagonist

possessing antiserotonergic, antiadrenergic and antihistaminergic properties.

Risperidone has an empirical formula of C23H27FN4O2 and molecular weight of 410.485.

Risperidone is a benzisoxazole derivative psychotropic agent which is lipophilic in nature. It

exhibits a pH dependent solubility. Risperidone is a white to slightly beige powder. It is

practically insoluble in water, freely soluble in methylene chloride, and soluble in methanol and

0.1 N HCl. It is, slightly soluble in pH 4.0 and sparingly soluble at pH 7.0 – 10.0.30

1.7.1.1 SYNTHESIS

2.56 g of 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride and 2.30 g of 3-(2-

chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4- -one, in a 50 ml reaction

flask. Then add a sodium carbonate solution or suspension (dissolved or suspended 4.5 g of

sodium carbonate in 25 ml water). The mixture is put into heating bath at 120-130° C. with

stirring for 60 min, then cooled with continuous stirring to the room temperature and the

precipitated solid is filtered, washed with pure water, and dried to give 3.70 g of the product in

90.2% yield. The product is purified to obtain a purity of 99.5% (determined by HPLC) with

DMF and isopropanol31

.

1.7.1.2 PHARMACOLOGY

Risperidone has been classified as a "qualitatively atypical" antipsychotic agent with a relatively

low incidence of extrapyramidal side effects (when given at low doses) that has more

pronounced serotonin antagonism than dopamine antagonism. Risperidone is unique among most

other atypicals in that it has high affinity for the D2 receptor (also known as 'tight binding')

whereas most other atypicals have 'loose binding' of the D2 receptor. It has actions at several 5-

HT (serotonin) receptor subtypes. These are 5-HT2C, linked to weight gain, 5-HT2A, linked to its

36

antipsychotic action and relief of some of the extrapyramidal side effects experienced with the

typical neuroleptics.32,33

It reaches peak plasma levels quickly, regardless of whether it is administered as a liquid or pill.

Risperidone is metabolized fairly quickly, so the potential for nausea subsides usually in two to

three hours. However, the active metabolite, 9-hydroxy-risperidone, which has similar

pharmacodynamics to risperidone, remains in the body for much longer, and has been developed

as an antipsychotic in its own right, called paliperidone. An intramuscular preparation, marketed

as Risperdal Consta, can be given once every two weeks. It is slowly released from the injection

site. This method of administration may be used on sanctioned patients who are declining, or

consenting patients who may have disorganized thinking and cannot remember to take their daily

doses.34

Doses range from 12.5 to 50 mg given as an intramuscular injection once every two

weeks.

1.7.1.3 THERAPEUTIC USES

Risperidone is used for the treatment of schizophrenia, bipolar disorder and behavior problems in

people with autism.35

In autism, however, it does not improve conversational ability or social

skills, and does not appear to reduce obsessive behavior in most autistic people.35

The main

action of an antipsychotic (regardless of typical or atypical) is to decrease the action of dopamine

and/or epinephrine and norepinephrine levels in the brain

1.7.1.4 ADVERSE EFFECTS

The severity of adverse effects often depends on the dosage. Risperidone has been associated

with weight gain.36

Other common side effects include akathisia, sedation, dysphoria, insomnia,

elevated prolactin level, low blood pressure, high blood pressure, muscle stiffness, muscle pain,

tremors, hypersalivation, constipation, and stuffy nose. In addition, risperidone treatment causes

photosensitivity, and patients should be warned to avoid prolonged exposure to the sun or to use

effective sunscreen (SPF 15+).37

1.7.2 MOXIFLOXACIN

Fig.1.4: Structure of moxifloxacin:

6-fluoro-8-methoxy-4-oxo- quinoline

Moxifloxacin is a fourth-generation synthetic

Bayer AG. Moxifloxacin, a fluoroquinolone, is available as t

cyclopropyl-7-[(S,S)-2,8-diazabicyclo[4.3.0]non

14 quinoline carboxylic acid. It is a slightly yellow to yellow crystalline substance with a

molecular weight of 437.9. Its empirical formula is

1.7.2.1 SYNTHESIS

Moxifloxacin hydrochloride is prepared from ethyl

oxo-1,4-dihydro-3-quinolinecarboxylate through novel intermediate (4aS

(2,8-diazabicyclo[4.3.0]non-8-yl)

acid-O3,O

4)bis(acyloxy-O)borate

oxo-1,4-dihydro-3-quinoline carboxylate with boric acid and acetic anhydride without using any

catalyst gives (1-cyclopropyl-6,7

, and patients should be warned to avoid prolonged exposure to the sun or to use

Fig.1.4: Structure of moxifloxacin: 1-cyclopropyl-7-[(1s, 6s)-2,8-diazabicyclo[4.3.0]

quinoline-3-carboxylic acid

generation synthetic fluoroquinoloneantibacterial agent

Bayer AG. Moxifloxacin, a fluoroquinolone, is available as the monohydrochloride salt of

diazabicyclo[4.3.0]non-8-yl]-6-fluoro-8-methoxy-1,4-dihydro


molecular weight of 437.9. Its empirical formula is C21H24FN3O438,39

Moxifloxacin hydrochloride is prepared from ethyl 1-cyclopropyl-6,7-difluoro

quinolinecarboxylate through novel intermediate (4aS-Cis)-

yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydro-3-quinoline carboxylic

O)borate. The reaction of ethyl1-cyclopropyl-6,7-difluoro

quinoline carboxylate with boric acid and acetic anhydride without using any

6,7-difluoro-8-methoxy-4-oxo-1,4-dihydro-3-quinoline carboxylic

37

, and patients should be warned to avoid prolonged exposure to the sun or to use

diazabicyclo[4.3.0] non-8-yl]-

antibacterial agent developed by

he monohydrochloride salt of 1-

dihydro-4-oxo-3


difluoro-8-methoxy-4-

-1-cyclopropyl-7-

quinoline carboxylic

difluoro-8-methoxy-4-

quinoline carboxylate with boric acid and acetic anhydride without using any

quinoline carboxylic

38

acid-O3,O

4)bis(acyloxy-O)borate which on condensation in presence of a base(s) with (S,S)-2,8-

diazabicyclo[4.3.0]nonane in organic polar solvent results the novel intermediate (4aS-Cis)-1-

cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydro-3-

quinoline carboxylic acid-O3,O

4)bis(acyloxy-O) borate. This intermediate is reacted with

hydrochloric acid in presence of solvent to give Moxifloxacin hydrochloride pseudo hydrate. The

Moxifloxacin hydrochloride pseudohydrate is converted into Moxifloxacin hydrochloride

monohydrate by treating with hydrochloric acid in presence of ethanol.40

1.7.2.2 PHARMACOKINETICS

Approximately 52% of an oral or intravenous dose of moxifloxacin is metabolized via

glucuronide and sulfate conjugation. The cytochrome P450 system is not involved in

moxifloxacin metabolism. The sulfate conjugate (M1) accounts for approximately 38% of the

dose, and is eliminated primarily in the feces. Approximately 14% of an oral or intravenous dose

is converted to a glucuronide conjugate (M2), which is excreted exclusively in the urine. Peak

plasma concentrations of M2 are approximately 40% those of the parent drug, while plasma

concentrations of M1 are, in general, less than 10% those of moxifloxacin.39

In vitro studies with cytochrome (CYP) P450 enzymes indicate that moxifloxacin does not

inhibit 80 CYP3A4, CYP2D6, CYP2C9, CYP2C19, or CYP1A2, suggesting that moxifloxacin is

unlikely to alter the pharmacokinetics of drugs metabolized by these enzymes.39

The pharmacokinetics of moxifloxacin in pediatric subjects has not been studied.39

The half-life of moxifloxacin is 11.5-15.6 hours (single-dose, oral).41

Approximately 45% of an

oral or intravenous dose of moxifloxacin is excreted as unchanged drug (~20% in urine and ~25

% in faeces). A total of 96 ± 4% of an oral dose is excreted as either unchanged drug or known

metabolites. The mean (± SD) apparent total body clearance and renal clearance are 12 ± 2 L/hr

39

and 2.6 ± 0.5 L/hr, respectively.41

The cerebrospinal fluid (CSF) penetration of moxifloxacin is

70% to 80% in patients with meningitis.

1.7.2.3 MECHANISM OF ACTION

Moxifloxacin is a broad-spectrum antibiotic that is active against both Gram-positive and Gram-

negative bacteria. It functions by inhibiting DNA gyrase, a type II topoisomerase, and

topoisomerase IV,42

enzymes necessary to separate bacterial DNA, thereby inhibiting cell

replication.

This mechanism can also affect mammalian cell replication. In particular, some congeners of this

drug family (for example those that contain the C-8 fluorine),43

display high activity not only

against bacterial topoisomerases, but also against eukaryotic topoisomerases and are toxic to

cultured mammalian cells and in vivo tumor models.44

Although quinolones are highly toxic to

mammalian cells in culture, its mechanism of cytotoxic action is not known. Quinolone-induced

DNA damage was first reported in 1986 (Hussy et al.).45

Recentstudies have demonstrated a correlation between mammalian cell cytotoxicity of the

quinolones and the induction of micronuclei.46

As such, some fluoroquinolones, including

moxifloxacin, may cause injury to the chromosome of eukaryotic cells.47-50

There continues to be considerable debate as to whether or not this DNA damage is to be

considered one of the mechanisms of action concerning the severe adverse reactions experienced

by some patients following fluoroquinolone therapy.51-53

1.7.2.4 PHARMACOLOGICAL INDICATIONS

Moxifloxacin is used to treat a number of infections including: respiratory tract infections,

cellulitis, anthrax, intra-abdominal infections, endocarditis, meningitis, and tuberculosis.54

In the

adult population its oral and intravenous are

threatening bacterial infections.

1.7.2.5 ADVERSE EFFECTS

The serious adverse effects that may occur as a result of moxifloxacin therapy include

irreversible peripheral neuropathy

or serious liver injury, QTc prolongation/

and clostridium difficile-associated disease (CDAD),

reactions. Hepatitis, pseudomembranous colitis

syndrome have also been associated with moxifloxacin therapy

1.7.3.0 CHLORANILIC ACID AS

Fig 1:5: Structure of chloranilic acid:

formula of chloranilic acid: C6H2

Chloranilic acid is a strong oxidant used in coupling, cyclization, and dehydrogenation of

alcohols, phenols and steroids in organic chemistry. This yellow

density of 1.93g/cm3 with melting

aqueous mineral acid. It is very soluble in methanol, acetonitrile,

toluene, dioxane and dichloromethane.

tion its oral and intravenous are limited to the treatment of proven serious and life


peripheral neuropathy, spontaneous tendon rupture and tendonitis, acute liver failure

or serious liver injury, QTc prolongation/torsades de pointes, toxic epidermal necrolysis

associated disease (CDAD),as well as photosensitivity/phototoxicity

pseudomembranous colitis, psychotic reactions and Stevens

have also been associated with moxifloxacin therapy.55

CHLORANILIC ACID AS π-ACCEPTOR AND ITS APPLICATIONS

Fig 1:5: Structure of chloranilic acid: 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone. Chemical

2Cl2O4, molar mass: 208.98 g/mol


in organic chemistry. This yellow-orange powder

with melting points of 300.0oC. It decomposes in water, but

aqueous mineral acid. It is very soluble in methanol, acetonitrile, ethylacetate

dichloromethane.

40

limited to the treatment of proven serious and life-


, acute liver failure

toxic epidermal necrolysis (TEN),

as well as photosensitivity/phototoxicity

Stevens–Johnson

ONS

benzoquinone. Chemical


orange powdery substance has

C. It decomposes in water, but is stable in

ethylacetate, acetic acid,

41

1.7.3.1 MECHANISM

It has been observed that the rate of the reaction with chloranilic acid is: accelerated in polar

solvents: not affected by radical-producing agents and catalyzed by proton-donor species. The

mechanism is bimolecular. In the first rate-determining step, the formation of a charge-transfer

complex occurs, according to the following equation.

RH2+

+ QH+ → RH

+ + QH2……….. 1.7

Where, Q = Quinone

R = Reacting species

RH+ = Charge transfer complex

From the charge-transfer complex, two reactions are likely to happen;

i) Elimination of a proton to give an unsaturation on the molecule.

ii) Wagner-Meerwein type of re-arrangement prior to the loss of proton may occur in

specific cases. For some special reactions, a radical mechnism may be involved.

1.8.0 APPLICATIONS

Dehydrogenation with chloranilic acid has been reported for steroid ketones, steroid pyrrazoles,

steroid lactones, alcohols and phenols. The reaction is based on an initial rate-determining

transfer of hydride ion from the molecule to chloranilic acid, leading to hydroquinone derivative.

The feasibility of the reaction depends upon the degree of stabilization of the transition-state

carbonation. It has been observed that the presence of alkenes or aromatic moieties is sufficient

to initiate hydrogen transfer in the presence of chloranilic acid. Chloranilic acid is a π-acceptor

which reacts instantaneously with basic nitrogenous compounds to form a charge-transfer

42

complex of n−π type.56

It exhibits an absorption band in the UV region in, 1,4-dioxane at a

wavelength of 437 nm. The following are few studies which have reported the use of chloranilic

acid in charge transfer complexaton: Adikwu et al57

performed spectrophotometric and

thermodynamic studies of charge – transfer interaction between diethylcarbamazine citrate and

chloranilic acid and the complex formed had maximum absorption at 540 nm. Attama et al58

spectrophotometrically determined haloperidol through charge transfer complex formation with

chloranilic acd in 1,4-dioxan. Interaction between haloperidol and chloranilic acid in 1,4-dioxan

was found to yield a purple colour at 576 nm. Adikwu et al59

determined spectrophotometrically

moclobemide through charge transfer complexation with chloranilic acid in 1,4-dioxan and the

complex formed was observed at 526 nm.

1.8.1 PREVIOUS METHODS USED FOR ANALYSIS OF BOTH DRUGS

The therapeutic importance of the drugs has promoted the development of several analytical

methods for its quantitative determination. The techniques include chemical luminescence

assay60

, chiral chromatography61

, HPLC62,63

, LC/MS and HPLC – ESI/MS64-68

, pulse

polarography69

, potentiometry70

, spectrophotometry71-73

. The chromagraphic method/MS is

tedious and difficult to perform and requires selective and expensive detectors that might not be

easily accessible to many laboratories. Spectrophotometric method is considered more

convenient alternative technique because of its inherent simplicity and availability.

1.8.2 OBJECTIVE OF THE STUDY

Since literature review does not have a report on the analysis of either of the drugs by charge

transfer compexation using chloranilic acid, the objective of the present study was to investigate

the complexation reaction of risperidone and moxifloxacin respectively with chloranilic acid as a

43

chromogenic agent. It is hoped that such a method would be of use in the routine analysis of both

drugs in quality control laboratory.

44

CHAPTER TWO

MATERIALS AND METHODS

2.1 DRUGS USED AND THEIR SOURCES

Risperidone was obtained from MA Holder: Teva, UK Ltd while moxifloxacin was obtained

from Bayer, UK Ltd.

2.1.1 CHEMICALS AND SOLVENTS

Chloranilic acid, methanol (analytical grade) and 1,4-dioxan were all obtained from Sigma-

Aldrick Chemie, Germany.

All laboratory reagents were freshly prepared. UV/Visible spectrophotometer (Jenway 6305) was

used for spectral measurements while electronic weighing balance (Adventurer – Ohaus ) was

used to weigh all samples.

2.2 PREPARATION OF REAGENTS

2.2.1 PREPARATION OF CHLORANILIC ACID

Chloranilic acid (2.39 x 10-3

M) solution was prepared by accurately weighing 0.05 g of

chloranilic acid into a 100 ml volumetric flask, dissolved in 1,4-dioxan and diluted to volume

with 1,4-dioxan.

45

2.2.2.1 PREPARATION OF RISPERIDONE SOLUTION

A pure sample of risperidone (10 mg) was dissolved in 100 ml volumetric flask with methanol

and diluted to volume with methanol (100 µg/ml). A 40 µg/ml of risperidone was prepared by

diluting 20 ml of stock A to 50 ml with methanol.

2.2.2.2 PREPARATION OF MOXIFLOXACIN SOLUTION

A pure sample of moxifloxacin (10 mg) was dissolved in 100 ml volumetric flask with methanol

and diluted to volume with methanol (100 µg/ml). A 40 µg/ml of risperidone was prepared by

diluting 20 ml of stock A to 50 ml with methanol.

2.3.0 ABSORPTION SPECTRA

2.3.1 ABSORPTION SPECTRUM OF CHLORANILIC ACID

A2 ml of chloranilic acid solution (2.39 x 10-3

M) was pipetted into 5 ml calibrated volumetric

flask and made up to mark with 1,4-dioxan. Its spectrum was taken by scanning the solution

between 200 – 700 nm against a blank (1,4-dioxan).

2.3.2 ABSORPTION SPECTRUM OF RISPERIDONE

2 ml of risperidone solution (40 µg/ml) was pipetted into 5 ml calibrated volumetric flask and

made up to mark with methanol. Its spectrum was taken by scanning the solution between 200 –

400 nm against a blank (methanol).

46

2.3.3 ABSORPTION SPECTRUM OF MOXIFLOXACIN

2 ml of moxifloxacin solution (40 µg/ml) was pipetted into 5 ml calibrated volumetric flask and

made up to mark with methanol. Its spectrum was taken by scanning the solution between 200 –

400 nm against a blank (methanol).

2.3.4 ABSORPTION OF RISPERIDONE-CHLORANILIC ACID COMPLEX

1 ml of risperidone solution (40 µg/ml) and 1 ml of chloranilic acid (2.39 x 10-3

M) solution were

pipette into 5 ml calibrated volumetric flask and made up to mark with 1,4-dioxan. The solution

was allowed to stand for 30 minutes for complexation to be completed and the solution was

scanned against the blank (mixture of 1,4-dioxan and chloranilic acid) using wavelength range of

400 – 700 nm.

2.3.5 ABSORPTION OF MOXIFLOXACIN-CHLORANILIC ACID COMPLEX

1 ml of moxifoxacin solution (40 µg/ml) and 1 ml of chloranilic acid (2.39 x 10-3

M) solution

were pipetted into 5 ml calibrated volumetric flask and made up to mark with 1,4-dioxan. The

solution was allowed to stand for 30 minutes for complexation to be completed and the solution

was scanned against the blank (mixture of 1,4-dioxan and chloranilic acid) using wavelength

range of 400 – 700 nm.

2.4.0 DETERMINATION OF THE OPTIMUM AMOUNT OF CHLORANILIC ACID

REQUIRED FOR COMPLEX FORMATION

2.4.1 RISPERIDONE-CHLORANILIC ACID COMPLEX

Into 8 different calibrated test tubes was added 1ml of risperidone solution

(40 µg/ml), followed by different volumes of chloranilic acid solution (1.91 x 10-4

M)

47

ranging from 0.5 – 4.0 ml. The volume in each flask was made up to mark with 1,4

dioxan The solutions were kept for 30 minutes at room temperature after which

absorbance readings were taken at the maximum wavelength of the complex (500 nm)

against the blank (mixture of 1,4-dioxan and chloranilic acid).

2.4.2 MOXIFLOXACIN-CHLORANILIC ACID COMPLEX

Into 8 different calibrated test tubes was added 1 ml of moxifloxacin solution

(40 µg/ml), followed by different volumes of chloranilic acid solution (2.39 x 10-3

M)

ranging from 0.5 – 4.0 ml. The solutions were kept for 30 minutes at room temperature

after which absorbance readings were taken at the maximum wavelength of the complex

(490 nm) against the blank.

2.5.0 EFFECT OF TIME ON THE COMPLEX FORMATION

2.5.1 EFFECT OF TIME ON THE FORMATION OF RISPERIDONE-CHLORANILIC

ACID COMPLEX

A 1ml of risperidone solution (40 µg/ml) and 2 ml of chloranilic acid

(1.91 x 10-4

M) were pipetted into 5 ml calibrated volumetric flask and made up to mark

with 1,4-dioxan. Absorbance readings were taken at various time intervals up to 30

minutes at room temperature against a blank.

2.5.2 EFFECT OF TIME ON THE FORMATION OF MOXIFLOXACIN-

CHLORANILIC ACID COMPLEX

A 1ml of moxifloxacin solution (40 µg/ml) and 2 ml of chloranilic acid

(2.39 x 10-3

M) were pipetted into 5 ml calibrated volumetric flask and made up to mark

48

with 1,4-dioxan. Absorbance readings were taken at various time intervals up to 30

minutes at room temperature against a blank.

2.6.0 DETERMINATION OF STOICHIOMETRY OF THE COMPLEXES

Stoichiometry of the complexes formed was determined using two methods: mole ratio

and slope ratio methods.

2.6.1 STOICHIOMETRY BY MOLE RATIO METHOD

2.6.1.1 RISPERIDONE-CHLORANILIC ACID COMPLEX

To a series (0.50, 1.00, 1.50 … 4.50 ml) of chloranilic acid solution was added

complimentary volumes (4.50, 4.00, 3.50 … 0.50 ml) of risperidone in different flasks.

Each flask was shaken and allowed to stand for 15 minutes before taking the absorbance

reading at 500 nm against the blank.

2.6.1.2 MOXIFLOXACIN-CHLORANILIC ACID COMPLEX

To a series (0.50, 1.00, 1.50 … 4.50 ml) of chloranilic acid solution was added

complimentary volumes (4.50, 4.00, 3.50 … 0.50 ml) of risperidone in different flasks.

Each flask was shaken and allowed to stand for 20 minutes before taking the absorbance

reading at 490 nm against the blank.

2.6.2 STOICHIOMETRY BY SLOPE RATIO METHOD

2.6.2.1 RISPERIDONE-CHLORANILIC ACID COMPLEX

A 9.75×10-5

M solution of risperidone in methanol and 9.75×10-5

M solution of

chloranilic acid in 1,4-dioxan were used in the study. In the first set of the risperidone –

49

chloranilic acid reaction, risperidone solution was in excess and kept constant at the

volume of 2.5 ml. Varying amount of 9.75×10-5

M chloranilic acid solution according to

the following volumes; 0.20, 0.40, 0.60, 0.80 and 1.00 mlwere added separately into each

set – up. Sufficient calculated volume of a mixture of 1,4-dioxan and methanol for 5.00

mlmark was added into each set- up. These mixtures were kept for 15 minutes before

determining the absorbance at 500.0 nm against a mixture of methanol, 1,4-dioxan and

chloranilic acid (blank). Also, in the second set; chloranilic acid was kept constant and in

excess while the risperidone varied as described above. The absorbance readings were

taken at 500.0 nm against a mixture of methanol, 1,4-dioxan and chloranilic acid (blank).

2.6.2.2 MOXIFLOXACIN-CHLORANILIC ACID COMPLEX

A 1.25×10-3

M solution of moxifloxacin in methanol and 1.25×10-3

M solution of

chloranilic acid in 1,4-dioxan were used in the study. In the first set of the moxifloxacin –

chloranilic acid reaction, moxifloxacin solution was in excess and kept constant at the

volume of 2.5 ml. Varying amount of 1.25×10-3

M chloranilic acid solution according to

the following volumes; 0.20, 0.40, 0.60, 0.80 and 1.00 mlwere added separately into each

set – up. Sufficient calculated volume of a mixture of 1,4-dioxan and methanol for 5.00

mlmark was added into each set- up. These mixtures were kept for 20 minutes before

determining the absorbance at 490.0nm against a mixture of methanol, 1,4-dioxan and

chloranilic acid (blank). Also, in the second set; chloranilic acid was kept constant and in

excess with the moxifloxacin varied as described above. The absorbance readings were

taken at 490.0 nm against a mixture of methanol, 1,4-dioxan and chloranilic acid (blank).

2.7.0 BEER’S LAW CALIBRATION PLOT FOR THE COMPLEXEX

50

2.7.1 BEER’S LAW CALIBRATION PLOT FOR RISPERIDONE-CHLORANILIC

ACID COMPLEX

Aliquots containing (5 - 40 µg/ml) from the standard risperidone stock solutions were

transferred to different 5 ml calibrated volumetric flask. A 2 ml of chloranilic acid

solution (1.91 x 10-4

M) was added to each of the test tubes and made up to mark with

1,4-dioxan. After standing for 15 minutes, absorbance readings were taken at a

wavelength of 500 nm against a mixture of 1,4-dioxan and chloranilic acid solution

(blank). The absorbances of solution of each flask were plotted against the corresponding

concentrations of risperidone.

2.7.2 BEER’S CALIBRATION PLOT FOR MOXIFLOXACIN-CHLORANILIC ACID

COMPLEX

Aliquots (5 - 40 µg/ml) from the standard moxifloxacin stock solutions were transferred

to different 5 ml calibrated volumetric flask. A 2 ml of chloranilic acid solution (2.39 x

10-3

M) was added to each of the test tubes and made up to mark with 1,4-dioxan. After

standing for 20 minutes absorbance readings were taken at a wavelength of 490 nm

against a mixture of 1,4-dioxan and chloranilic acid solution (blank). The absorbances of

solution of each flask were plotted against the corresponding concentrations of

risperidone.

2.8.0 QUANTITATIVE ASSAY OF THE DRUGS

51

2.8.1 ASSAY OF RISPERIDONE

Twenty tablets of risperidone equivalent to 4.266 g were crushed in a mortar. A 0.213 g

of the powdered risperidone tablets equivalent to 1 tablet was weighed and dissolved in

100 ml volumetric flask with methanol and diluted to volume with methanol. The mixture

was filtered to remove the drug excipients and the filtrate was used to prepare 20 µg/ml

and 40 µg/ml risperidone solution respectively. The assay of these risperidone solutions

were done by the procedure previously described for risperidone determination.

2.8.2 ASSAY OF MOXIFLOXACIN

Twenty tablets of moxifloxacin equivalent to 14.26 g were crushed in a mortar. 0.713 g

of the powdered moxifloxacin tablet equivalent to 1 tablet was weighed and dissolved in

100 ml volumetric flask with methanol and diluted to volume with methanol. The

solution was filtered to remove the drug excipients and the filtrate was used to prepare 20

µg/ml and 40 µg/ml respectively. The assay of these moxifloxacin solutions were carried

out by the procedure previously described for moxifloxacin determination.

2.9.0 RECOVERY STUDIES OF THE DRUGS

2.9.1 RECOVERY STUDIES ON RISPERIDONE

Recovery studies of risperidone were done by two methods.

A 10 mg of the pure risperidone was weighed and dissolved in 100 ml volumetric flask

with methanol and diluted to volume with methanol. 20 µg/ml and 40 µg/ml of

risperidone solutions respectively were prepared out of the stock solution. The assay of

52

these risperidone solutions were carried out by the procedure previously described for

risperidone determination.

The study was performed in triplicates and the average taken

A 40 µg/ml of risperidone sample solution (filtrate) was transferred into two different test

tubes. To each of the two calibrated tubes was added 5 and 10 µg/ml of pure risperidone

solution respectively. The assay was performed as previously described.


2.9.2 RECOVERY STUDIES ON MOXIFLOXACIN

Recovery studies of moxifloxacin were done by two methods.

2.9.2.1 A 10 mg of the pure moxifloxacin was weighed and dissolved in 100 ml volumetric flask

with methanol and diluted to volume with methanol. A 20 µg/ml and 40 µg/ml of

moxifloxacin solution were prepared out of the stock solution. The assay of these

moxifloxacin solutions were done by the procedure previously reported for moxifloxacin

determination.


2.9.2.2 A 40 µg/ml of moxifloxacin (filtrate) was transferred into two different test tubes. To

each of the two tubes was added 5 and 10 µg/ml of pure moxifloxacin solution

respectively. The assay was performed as previously described.


53

CHAPTER THREE

RESULTS AND DISCUSSIONS

3.1.0 RESULTS

3.1.1 ABSORPTION SPECTRA OF THE COMPLEXES

The absorption spectrum of risperidone is shown in figure 3.1 while the spectra of chloranilic

acid and the risperidone-chloranilic acid complex are shown in figure 3.2. The absorption

spectrum of moxifloxacin is shown in figure 3.3 while the spectra of chloranilic acid and the

moxifloxacin-chloranilic acid complex are shown in figure 3.4

54

3.1.1.1 ABSORPTION SPECTRUM OF RISPERIDONE-CHLORANILIC ACID

COMPLEX

Figure 3.1: Absorption spectrum of risperidone

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 100 200 300 400 500 600 700 800

Abs

55

Figure 3.2: Absorption spectra of chloranilic acid (a) risperidone-chloranilic acid

complex (b)

0

0.05

0.1

0.15

0.2

0.25

0 100 200 300 400 500 600 700 800

Ab

sorb

an

ce

λ

a

b

56

3.1.1.2 ABSORPTION SPECTRA OF MOXIFLOXACIN-CHLORANILIC ACID

COMPLEX

Figure 3.3: Absorption spectrum of moxifloxacin

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 100 200 300 400 500 600 700 800

Abs

Figure 3.4: Absorption spectra of chloranilic acid (a)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 100 200

Ab

sorb

an

ce

Figure 3.4: Absorption spectra of chloranilic acid (a) moxifloxacin-chloranilic acid complex (b)

200 300 400 500 600 700

wavelenght (nm)

57

chloranilic acid complex (b)

700 800

58

3.1.2 OPTIMUM AMOUNT OF CHLORANILIC ACID FOR THE COMPLEX

FORMATION

Figure 3.5: Determination of optimum amount of chloranilic acid for risperidone-chloranilic

acid complex

0

0.05

0.1

0.15

0.2

0.25

0.3

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Ab

sorb

an

ce

volume (mL)

59

Figure 3.6: Determination of optimum amount of chloranilic acid for moxifloxacin-

chloranilic acid complex

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Ab

sorb

an

ce

vol of chloranilic acid (mL)

60

3.1.3 EFFECT OF TIME ON THE COMPLEX FORMATION

Figure 3.7: Risperidone-chloranilic acid complex

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25 30 35

Ab

sorb

an

ce

time (mins)

61

Figure 3.8: moxifloxacin-chloranilic acid complex

0

0.05

0.1

0.15

0.2

0.25

0.3

0 5 10 15 20 25 30 35

Ab

sorb

an

ce

time (s)

62

3.1.4 STOICHIOMETRY OF THE COMPLEXES

3.1.4.1 STOICHIOMETRY OF RISPERIDONE-CHLORANILIC ACID COMPLEX

Figure 3.9: Job’s plot for risperidone-chloranilic acid complex

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 0.2 0.4 0.6 0.8 1 1.2

Ab

sorb

an

ce

mole ratio

63

Figure 3.10: Slope ratio plot for risperidone-chloranilic acid complex

y = 0.052x + 0.038

R² = 0.990

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0 0.2 0.4 0.6 0.8 1 1.2

Ab

s

conc µg/ml

64

Figure 3.11: Slope ratio plot for risperidone-chloranilic acid complex

y = 0.027x + 0.306

R² = 0.969

0.31

0.315

0.32

0.325

0.33

0.335

0 0.2 0.4 0.6 0.8 1 1.2

Ab

s

conc µg/ml

65

3.1.4.2 STOICHIOMETRY OF MOXIFLOXACIN–CHLORANILIC ACID

COMPLEX

Figure 3.12: Job’s plot for moxifloxacin-chloranilic acid complex

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 0.2 0.4 0.6 0.8 1

Ab

sorb

an

ce

mole ratio

66

Figure 3.13: Slope ratio plot for moxifloxacin-chloranilic acid complex

y = 0.049x + 0.037

R² = 0.974

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0 0.2 0.4 0.6 0.8 1 1.2

Ab

s

conc µg/ml

67

Figure 3.14: Slope ratio plot for moxifloxacin-chloranilic acid complex

y = 0.035x + 0.066

R² = 0.969

0

0.02

0.04

0.06

0.08

0.1

0.12

0 0.2 0.4 0.6 0.8 1 1.2

Ab

s

conc µg/ml

68

3.1.5 BEER’S CALIBRATION PLOTS OF THE COMPLEXES

Figure 3.15: calibration plot for risperidone-chloranilic acid complex

y = 0.011x - 0.004

R² = 0.996

-0.1

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30 35 40 45

Ab

sorb

an

ce

conc. µg/mL

69

Figure 3.16: calibration plot for moxifloxacin-chloranilic acid complex

y = 0.005x + 0.000

R² = 0.995

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25 30 35 40 45

Ab

s

conc µg/ml

70

3.1.6 LIMIT OF DETECTION AND LIMIT OF QUANITATION FOR BOTH DRUGS

Figure 3.17: limit of detection plot for risperidone-chloranilic acid complex

y = 0.010x + 4E-05

R² = 0.987

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Ab

s

conc µg/ml

71

Figure 3.18: limit of detection for moxifloxacin-chloranilic acid complex

y = 0.006x - 0.001

R² = 0.982

-0.005

0

0.005

0.01

0.015

0.02

0.025

0.03

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Ab

s

conc µg/ml

72

3.1.7 ASSAY AND RECOVERY EXPERIMENTS

ASSAY AND RECOVERY EXPERIMENT FOR RISPERIDONE

Tables 3.17: Recovery analysis of risperidone

Conc. (µg/ml) Abs Found (µg/ml) %Recovery±S.D RSD (%)

20.00 0.216 19.27 96.35±0.23 1.19

40.00 0.445 40.11 100.28±0.04 0.10

Tables 3.18: Spiking analysis of commercial sample of risperidone


40.00 + 5.00 0.491 44.24 98.31±0.17 0.41

40.00 + 10.00 0.549 49.00 99.00±0.11 0.26

Tables 3.16: Assay of commercial sample of risperidone


20.00 0.226 20.21 101.05±0.11 0.54

40.00 0.445 40.75 101.88±0.05 0.12

73

ASSAY AND RECOVERY EXPERIMENT FOR MOXIFLOXACIN

Tables 3.20: Recovery analysis of moxifloxacin


20.00 0.099 19.78 98.90±0.12 0.61

40.00 0.199 39.96 99.90±0.04 0.10

Tables 3.21: Spiking analysis of commercial sample of moxifloxacin


40.00 + 5.00 0.229 45.86 101.92±0.17 0.29

40.00 + 10.00 0.256 51.26 102.51±0.11 0.10

Tables 3.19: Assay of commercial sample of moxifloxacin


20.00 0.100 20.19 100.95±0.17 0.84

40.00 0.200 40.17 100.43±0.56 1.40

74

3.2 DISCUSSION

Chloranilic acid is a π-acceptor which reacts with basic nitrogenous compounds to form a charge

transfer complex of n-π type56

. The spectrum of chloranilic acid in 1,4-dioxan solution exhibited

an absorption band at 420 nm. Risperidone in methanol solution exhibited an absorption band at

300 nm (Figure 3.1). The addition of risperidone solution to that chloranilic acid caused an

immediate shift with new characteristics band at 500 nm (Figure 3.2). The addition of

chloranilic acid to risperidone (n-donor) resulted in the formation of a charge transfer complex of

the n-π type. This compound was believed to be an intermediate molecular association complex

which dissociates in 1,4-dioxan producing a chloranilic acid radical anion. Moxifloxacin showed

a characteristic band at 295 nm in methanol (Figure 3.3). The addition of chloranilic acid to

moxifloxacin (n-donor) resulted in the formation of charge transfer complex of n-π type at new

characteristics absorption band of 490 nm (Fig 3.4). At a fixed donor concentration, Figures 3.5

and 3.6 for risperidone-chloranilic acid and moxifloxacin-chloranilic acid complexes

respectively, the drugs showed that there is a direct relationship between the intensity of

absorption and the concentration of chloranilic acid until a particular concentration of the acid

where the absorbance readings become stable. A 2 ml of chloranilic acid was the optimal volume

for the formation of both risperidone-chloranilic acid and moxifloxacin – chloranilic acid

complexex at 3.82 x 10-4

M and 4.78 x 10-3

M of chloranilic acid respectively.

Although the complexes (risperidone-chloranilic acid and moxifloxacin-chloranilic acid) were

formed instantaneously, maximum absorbance readings were obtained after standing for 15

minutes (risperidone-chloranilic acid complex) and 20 minutes (moxifloxacin-chloranilic acid

complex) at room temperature. The absorbances of the solutions remained constant for at least 2

75

hours. Therefore, readings should be taken after 20 minutes but not more than two hours after

mixing.

Job’s plot and slope ratio methods were employed to determine the stoichiometry of the

complexes. The results indicated that the risperidone-chloranilic acid complex was formed at the

ratio of 1:2 (donor : acceptor) according to the graph in Figure 3.9 by Job’s method. From the

regression equations of the graphs (Figure 3.10 and 3.11 respectively) obtained using slope ratio

method, it was noted that the stoichiometry of risperidone-chloranilic acid reaction was;

r� rs = 0.052 0.027 � 1.93 w 2

wherer� is the slope of the graph for varying concontration of chloranilic acid and rs is the slope

of the graph for varying concontration of risperidone

Therefore the result of the slope ratio method confirmed the stoichiometry obtained from Job’s

method.

The Job’s plot in Figure 3.12 indicated that moxifloxacin-chloranilic acid complex was formed

in the ratio of 2:3 (donor : acceptor). From the equations of the graphs (Figures 3.13 and 3.14

respectively) obtained using slope ratio method, it was noted that the stoichiometry of

moxifloxacin-chloranilic acid reaction is;

r� r( = 0.049 0.035 � 1.4 w 1.5

Where r� is the slope of the graph for varying concentration of chloranilic acid and r( is the

slope of the graph for varying concentration of moxifloxacin

Therefore the result of the slope ratio method confirmed the stoichiometry obtained from Job’s

method.

76

Standard calibration graphs for both drugs were constructed by plotting absorbance readings

against concentrations of the drugs (µg/ml). Linear graphs passing through the origin were

obtained for the complexed drugs, indicating that Beer’s law was obeyed in the concentration

range of 5 – 40 µg/ml for either of the drugs. Regression equations derived using least square

methods are:

A500 = 0.011C + 0.004 .................... Eq. 4.1

Risperidone-chloranilic acid complex with correlation coefficient R2 of 0.9960

A490 = 0.005C + 0.000 ..................... Eq. 4.2

Moxifloxacin-chloranilic acid complex with correlation coefficient R2 of 0.9950

Where C is the concentration (µg/ml) and A is absorbance of the complexes

The linearity of the calibration graphs for the two drugs was confirmed by the high values of the

correlation coefficient (r) and the small values of the intercept of the regression equations.

The sensitivity of the proposed method was evaluated by determining the limit of detection

(LOD) and limit of quantitation (LOQ), defined as 3r\ r and 10r\ r respectively, where SD is

the standard deviation of the intercept and S is the slope of the graph. The results in Table 3.15

showed the proposed method to very sensitive.

The proposed spectrophotometric method was applied to the determination of risperidone

moxifloxacin respectively in bulk and pharmaceutical dosage forms. The results are presented in

Tables 3.16 and 3.19. The same proposed method was used in the recovery studies. The results

are shown in Tables 3.17 and 3.20.

77

The assay results of the proposed method for risperidone were compared with the official method

that employed potentiometry in the analysis63

. The Britain Pharmacopeia (BP) method employed

HPLC for the assay of moxifloxacin. The present study compared the results obtained by the

proposed method for moxifloxacin with other reported methods of analyzing moxifloxacin. No

interference was observed from the excipents present in the solid dosage forms of both drugs.

3.7.0 CONCLUSION

The formation of charge transfer complex by risperidone and moxifloxacin (n-donors) and

chloranilic acid (π-acceptor) respectively was confirmed by the colour changes. Both drugs

changed the golden yellow colouration of chloranilic acid to purple colour.

The stoichiometries of the complexes were found to be 1:2 for risperidone-chloranilic acid

complex and 2:3 for moxifloxacin-chloranilic acid complex. The complexes of risperidone-

chloranilic acid and moxifloxacin-chloranilic acid obeyed Beer’s law.

The proposed method is simple, rapid, sensitive, accurate and reproducible for the determination

of either risperidone or moxifloxacin in pure and commercial pharmaceutical dosage forms.

Hence, the proposed method could be successfully used in routine analyses of either of the drugs

in quality control laboratory especially in the underdeveloped countries of the world where

sophisticated analytical techniques like HPLC is unavailable.

78

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85

APPENDIX

Table 3.1 Optimum amount of chloranilic acid for risperidone-chloranilic acid complex

Risperidone

(ml)

1 1 1 1 1 1 1 1

C.A. (ml) 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Abs 0.057 0.098 0.163 0.264 0.259 0.262 0.260 0.263

Table 3.2: optimum amount of chloranilic acid for moxifloxacin-chloranilic acid complex

Moxifloxacin

(ml)

1 1 1 1 1 1 1 1

C.A. (ml) 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Abs 0.073 0.129 0.254 0.327 0.326 0.326 0.325 0.327

Table 3.3: Time-absorbance relationship of rispeidone-chloranilic acid complex

Time

(min)

0.00 2.00 5.00 10.00 15.00 20.00 25.00 30.00

Abs 0.076 0.101 0.164 0.201 0.200 0.197 0.202 0.200

Table 3.4: Time-absorbance relationship of moxifloxacin-chloranilic acid complex

Time

(min)

0.00 5.00 10.00 15.00 20.00 25.00 30.00

86

Abs 0.139 0.185 0.212 0.260 0.257 0.259 0.257

Table 3.5: risperidone-chloranilic acid complex for Job’s plot

Drug

vol. (ml)

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

C.A.

(ml)

4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00

Mole

ratio

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Abs 0.078 0.110 0.317 0.132 0.125 0.063 0.047 0.021 0.009 -

Table 3.6: Risperidone (constant and in excess) with varied concentration of chloranilic acid

S/N

Risp(ml)

(9.75x10-5

M in 5ml)

C.A.(ml) Solvent Absorbance

1 2.50 0.20 2.30 0.050

2 2.50 0.40 2.10 0.060

3 2.50 0.60 1.90 0.068

4 2.50 0.80 1.70 0.083

5 2.50 1.00 1.50 0.91

87

Table 3.7: Chloranilic acid (constant and excess) with varied concentration of risperidone

S/N

C.A.(ml)

(9.75x10-5

M in 5ml)

Risp. (ml) Solvent Absorbance

1 2.50 0.20 2.30 0.313

2 2.50 0.40 2.10 0.315

3 2.50 0.60 1.90 0.321

4 2.50 0.80 1.70 0.329

5 2.50 1.00 1.50 0.333

Table 3.8: moxifloxacin-chloranilic acid complex for Job’s plot

Drug vol

(ml)

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

C.A (ml) 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00

Mole ratio 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Abs 00.096 0.185 0.217 0.320 0.286 0.261 0.211 0.183 0.169 -

88

Table 3.9: Moxifloxacin (constant and excess) with varied concentration of chloranilic acid

S/N

MOX/ml

(9.75x10-5

M in 5ml)

C.A./ml Solvent Absorbance

1 2.50 0.20 2.30 0.048

2 2.50 0.40 2.10 0.059

3 2.50 0.60 1.90 0.063

4 2.50 0.80 1.70 0.076

5 2.50 1.00 1.50 0.089

Table 3.10: Chloranilic acid (constant and excess) with varied concentration of moxifloxacin

S/N

C.A.(ml)

(9.75x10-5

M in 5ml)

Mox(ml) Solvent Absorbance

1 2.50 0.20 2.30 0.075

2 2.50 0.40 2.10 0.078

3 2.50 0.60 1.90 0.089

4 2.50 0.80 1.70 0.097

5 2.50 1.00 1.50 0.101

89

Table 3.11: Calibration data for risperidone-chloranilic acid complex

risp (µg/ml) 5.00 10.00 20.00 30.00 40.00

Abs 0.059 0.102 0.209 0.315 0.454

Table 3.12: Calibration data for moxifloxacin-chloranilic acid complex

mox.(µg/ml) 5.00 10.00 20.00 30.00 40.00

Abs 0.059 0.102 0.209 0.315 0.454

Table 3.13: limit of detection of risperidone

Risp(µg/ml) 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Abs 0.009 0.017 0.021 0.025 0.031 0.034 0.042

Five Absorbance readings for the least conc. (1.00 µg/ml): 0.009, 0.01, 0.01, 0.008, 0.009

Table 3.14: data for limit of detection of moxifloxacin

mox(µg/ml) 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Abs 0.003 0.008 0.011 0.013 0.016 0.019 0.024

Five absorbance readings for the least conc. (1.00 µg/ml): 0.004, 0.002, 0.004, 0.003, 0.002.

Table 3.15: LOD, LOQ and molar absorptivities of the complexes

Complex LOD (µg/ml) LOQ (µg/ml) ε (Lmol-1

cm-1

)

Risp-C.A. 0.550 1.670 4.46 x 103

Mox-C.A. 0.297 0.900 2.05 x 103

spectrophotometric determination of ... akachukwu's m...1 spectrophotometric determination of...

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