general introduction - information and library...

Chapter-I General Introduction

1

GENERAL INTRODUCTION

1.01 Introduction to Drugs

A drug, broadly speaking, is any substance that, when absorbed into the body

of a living organism, alters normal bodily function. Pharmacology defines a drug as "a

chemical substance used in the treatment, cure, prevention, or diagnosis of disease or

used to enhance physical or mental well-being. The methods of quality control and

conditions of their storage are the important contents placed in many text books of

pharmaceutical chemistry [1-8]. Drugs are synthesized in bulk and used for their

therapeutic effects in pharmaceutical formulations. These biologically active chemical

substances are generally formulated into convenient dosage forms such as tablets,

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

or liquid forms) lotions, dusting powders, aerosols, metered dose inhalers and dry

powder inhalers etc. These formulations deliver the drug substances in a stable,

nontoxic and acceptable form, ensuing its bioavailability and therapeutic activity.

According to the chemical structure and therapeutic action, the drugs may be

classified as follows.

(i)Chemotherapeutic agents

Chemotherapeutic agents are used to kill the invading organisms without

harmful effects on the tissues of the patient. They may be sub-divided into various

classes such as antibacterial, trypanocides, antiprotozoals, antifungals, anthelmintics,

antiseptics, antitubercular, antilepral drugs, antineoplastic agents, disinfectants and

antiviral drugs. Levofloxacin antibiotic has been chosen in the present investigation.

http://en.wikipedia.org/wiki/Pharmacology

http://en.wikipedia.org/wiki/Chemical


2

(ii) Pharmacodynamic agents

Pharmacodynamic agents have certain effects on animal organs but are not

specific remedies for particular diseases. They may be further subdivided into

different classes like central nervous system modifiers, adrenergic stimulants,

blocking agents, cholinergic, anticholinergic agents, cardio-vascular agents, diuretics,

anti-inflammatory agents, immuno suppressive agents, antispasmodics,

antihypertensive, antidepressants, antihistamines, anticoagulants and antipsycotic

agents. Hormones (steroidal and nonsteroidal) production to desirable extent is also

essential.

(a) Antihypertensive agents: Antihypertensive drugs are used to control blood

pressure. These are classified into various types such as peripheral antiadrenergic,

centrally acting agents, direct vasodilators, ganglionicblocking agents, β-adrenergic

blockers, calcium channel blockers, angiotensin converting enzyme

inhibitors,antagonists and miscellaneous. Trandolapril, an antihypertensive agent is

chosen for the analysis.

(b) Antidepressants: An antidepressant is a psychiatric medication used for

alleviating major depression and dysthymia (milder depression). These are classified

as selective serotonin reuptake inhibitors, neither serotonin nor epinephrine reuptake

inhibitors, noradrenergic and specific serotonergic antidepressants [9], norepinephrine

reuptake inhibitors, nor epinephrine-dopamine reuptake inhibitors, tricyclic

antidepressants, monoamine oxidase inhibitors. Novel antidepressants specifically

affect serotonin and other neurotransmitter.


3

(c) Corticosteroids: The cortex or outer portion of the adrenal gland is one of the

endocrine structures most essential for normal metabolic function. The vital role of

the adrenal cortex is due to its ability to produce a group of steroidal hormones [10].

In addition to the naturally occurring corticosteroids many synthetic steroids with

similar properties have been introduced. The pharmaceutical properties of

corticosteroids also make them useful in the treatment of rheumatoid arthritis,

bronchial asthma and bronchial hypersensitivity [11]. The anti-inflammatory

corticoids represented by hydrocortisone and related synthetic analogs have gained an

unchallenged position in modern therapeutic practice. The therapeutic effect of

steroids depends on their stability [12, 13].

(d)Antiemetic: These drugs are suitably used to treat motion sickness and the side

effects of opioid analgesics, general anaesthetics and chemotherapy directed against

cancer. Antiemetic drugs block messages to the part of the brain that controls nausea

and vomiting. Ondansetron HCl, an antiemetic drug is chosen for the present

investigation.

(e) Antimigraine: These drugs are used for the treatment of the acute migraine

attacks. They work by narrowing blood vessels in the brain, stopping pain signals

from being sent to the brain, and stopping the release of certain natural substances that

cause pain, nausea, and other symptoms of migraine. Naratriptan hydrochloride, an

antimigraine drug is selected for the investigation.

1.02 Quantitative analysis

Chemical analysis may be stated as the application of a process or a series of

processes in order to identify, quantify a substance, the components of a solution of


4

mixture, or the determination of the structures of chemical compounds. Such methods

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

detection, quantification, linearity range and interferences. The validation of

analytical procedures is an important part of the registration application for a new

drug [14, 15]. The International Chemical Harmonization (ICH) has harmonized the

requirements in two guidelines [16, 17]. These guidelines serve as a basis worldwide

both for regulatory authorities and industry in proper validation. Quantification can be

achieved by the introduction of more refined and sensitive methods of physiochemical

analysis [18-19] such as colorimetry, spectrophotometry covering UV, visible and IR

regions, fluorimetry or turbidimetry, NMR and Mass, and chromatography [20-28]

that enables one to assay of drugs more accurately and with the smallest consumption

of the analyte, reagent and time. The good manufacturing practices provide minimum

quality standards for production of pharmaceuticals as well as their ingredients [29].

Every country has legislation [31] on bulk drugs and their pharmaceutical

formulations that sets standard and obligatory quality indices for them. These

regulations are presented in separate article general and specific relating to individual

drugs, and are published in the form of book called “Pharmacopoeia” (e.g. Indian

Pharmacopoeia, IP [32], United States Pharmacopoeia USP [33], European

Pharmacopoeia EP [34], United Kingdom, BP [35], Martindale Extra Pharmacopoeia

[36], Merck Index [37], etc.). Complete quantitative analysis of a sample actually

involves the following steps.

(a) Preparation of sample solution for analytical investigation: Sample solutions for

the analytical investigation can be prepared by dissolving finely powder of the tablets

or granules of the capsules in suitable solvent for the samples in solid state.


5

(b) Conversion of the analyte into a measurable form: This step is a vital one, in

developing any analytical method. Particularly, while dealing with the interferences in

pharmaceutical products by spectrophotometry, one should have sound knowledge

about the chemical and structural factors of the analyte and interfering molecules, and

also in the selection of appropriate chromogenic reagent, which should form the

colored product with the analyte molecule, and not with the foreign molecule. Any

possible interference from foreign matter is if expected during the course of the color

development, an appropriate clean up procedure should be adopted prior to the

analysis.

(c) Measurements: The measurement step in an analysis can be carried out by

chemical, physical or biological means. An important feature of modern

pharmaceutical chemistry is the introduction of more refined and sensitive methods of

physico-chemical analysis such as spectroscopy and chromatography that enable one

to assay the drugs more accurately and with the smallest consumption of the analyte,

reagents and time. The modern methods of choice (HPLC, GLC, NMR and Mass

spectroscopy) for assay involve sophisticated equipment, which are very costly and

pose problems of maintenance. Hence they are not in the reach of most laboratories

and small-scale industries. The visible spectrophotometric (or colorimetric) or

fluorimetric methods are very simple, cheap and easy to carry out.

(d) Calculation and Interpretation of Measurements: In spectrophotometric methods

absorbance is directly proportional to the concentration of the analyte in the solution.

Since errors can be made in any measurement, the analytical chemist must consider

this possibility in interpreting his results. The methods of statistics are commonly

used and are especially useful in expressing the significance of analytical data.

Chapter – I Part- A High Performance Liquid Chromatography

6

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

1.03 High performance liquid chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) [38-39] is a simple, fast,

specific, precise and highly accurate analytical technique that is used for the

separation and determination of organic and inorganic solutes in any samples

especially biological, pharmaceutical, food, environmental, industrial etc. In HPLC,

separations are achieved by partition, adsorption or ion exchange, according to the

nature of the interactions between the solute and the stationary phase, which may arise

from hydrogen bonding, Vander walls forces, electrostatic forces or hydrophobic

forces or basing on the size of the particles [40]. Reversed phase HPLC (RP-HPLC or

RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile

phase. One common stationary phase is silica which has been treated with RMe2SiCl,

where R is a straight chain alkyl group such as C18H37 or C8H17. With these stationary

phases, retention time is longer for molecules which are more non-polar, while polar

molecules elute more readily. An investigator can increase retention time by adding

more water to the mobile phase; thereby making the affinity of the hydrophobic

analyte for the hydrophobic stationary phase stronger relative to the now more

hydrophilic mobile phase. Similarly, an investigator can decrease retention time by

adding more organic solvent to the eluent. Structural properties of the analyte

molecule play an important role in its retention characteristics. In general, an analyte

with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic

bonds, such as S-S and others) results in a longer retention time because it increases

the molecule's non-polar surface area, which is non-interacting with the water


7

structure. On the other hand, polar groups, such as -OH, -NH2, COO- or -NH3

+ reduce

retention as they are well integrated into water.

1.04 HPLC Method Validation

Validation of an analytical method is the process by which it is established by

laboratory studies, that the performance characteristics of the method meet the

requirements for the intended analytical application. The validation of the assay

procedure is carried out using the following parameters. Analytical methods should be

used within good manufacturing practice and good laboratory practice environments,

and must be developed using the protocols set out in the International Conference on

Harmonization (ICH) guidelines [41-42]. The US Food and Drug Administration

(FDA) [43] and US Pharmacopoeia (USP) [44] both refer to ICH guidelines. The

most widely applied validation characteristics are accuracy, precision (repeatability

and intermediate precision), specificity, detection limit, quantitation limit, linearity,

range, robustness and stability of analytical solutions. Method validation must have a

written and approved protocol prior to use [45].

(a)Precision: According to the ICH guide lines, precision should be performed at two

different levels - repeatability and intermediate precision. Repeatability is an

indication of how easy it is for an operator in a laboratory to obtain the same result for

the same batch of material using the same method at different times using the same

equipment and reagents. It should be determined from a minimum of six

determinations covering the specified range of the procedure or from a minimum of

six determinations at 100% of the test or target concentration. Intermediate precision

results from variations such as different days, analysts and equipment. In determining


8

intermediate precision, experimental design should be employed so that the effects (if

any) of the individual variables can be monitored. Precision criteria for an assay

method are that the instrument precision and the intra-assay precision (RSD) will be

≤2%.

% Relative Standard Deviation= (Standard deviation / mean) x 100

(b)Accuracy: The accuracy of an analytical procedure expresses the closeness of

agreement between the value which is accepted either as a conventional true value or

an accepted reference value and the value found. Accuracy is usually determined by

measuring a known amount of standard material under a variety of conditions but

preferably in the formulation, bulk material or intermediate product to ensure that

other components do not interfere with the analytical method. For assay methods,

spiked samples are prepared in triplicate at three levels across a range of 50-150% of

the target concentration. The per cent recovery should then be calculated. The

accuracy criterion for an assay method is that the mean recovery will be 100±2% at

each concentration across the range of 80-120% of the target concentration. To

document accuracy, ICH guidelines regarding methodology recommend collecting

data from a minimum of nine determinations across a minimum of three concentration

levels covering the specified range.

% Error = [(Measured value -True value) / True value] x100

(c) Limit of Detection (LOD) & Limit of Quantitation (LOQ): The limit of detection

(LOD) is defined as the lowest concentration of an analyte in a sample that can be

detected, not quantified. It is expressed as a concentration at a specified signal: noise

ratio usually 3:1. The limit of quantitation (LOQ) is defined as the lowest


9

concentration of an analyte in a sample that can be determined with acceptable

precision and accuracy under the stated operational conditions of the method. The

ICH has recommended a signal: noise ratio 10:1. LOD and LOQ may also be

calculated based on the standard deviation of the response (SD) and the slope of the

calibration curve(s) at levels approximating the LOD according to the formulae

LOD = 3Sa / b LOQ = 10Sa / b

(d) Linearity and Range: The range of an analytical procedure is the interval between

the upper and lower concentration (amounts) of analyte in the sample (including these

concentrations) for which it has been demonstrated that the analytical procedure has a

suitable level of precision, accuracy and linearity. The range of the method is validated

by verifying that the analytical method provides acceptable precision, accuracy and

linearity when applied to samples containing analyte at the extremes of the range as well

as within the range.

(e) Ruggedness: The ruggedness of an analytical method is the degree of

reproducibility of test results obtained by the analysis of the same samples under a

variety of conditions, such as different laboratories, different analysts, different

instruments, different lots of reagents, different elapsed assay times, different assay

temperatures, different days, etc.

(f) Robustness: Robustness measures the capacity of an analytical method to remain

unaffected by small but deliberate variations in method parameters. Parameters that

should be investigated are percent organic content in the mobile phase, pH of the

mobile phase, buffer concentration, and temperature and injection volume.


10

1.05 Chromatographic Parameters

(a) Resolution: Chromatographers measure the quality of separation by resolution Rf

of adjacent bands

21

12 )(2

WW

ttRf

t1 and t2 are retention times of the first and second adjacent bands ;W1 and W2 are

base line band width.

(b) Capacity factor (k): It is the measure of how well the sample molecules are

retained by the column during an isocratic separation. It is affected by the solvent

composition, separation, aging and temperature of separation.

O

OR

t

tt'K

Where tR = Band retention time and tO = Column dead volume

(c) Column Efficiency (N): It is called as the number of theoretical plates. It

measures the band spreading of a peak. When band spread in smaller, the number of

theoretical plates is higher. It indicates a good column and system performance.

Column performance can be defined in terms of values of N

Column efficiency (N)=16 (tR/W)2

Plate height H=N/L (length)

(d) Peak Asymmetry/Peak Tailing: Peak with poor symmetry can result in (i)

Inaccurate plate number and resolution measurement (ii) imprecise quantization (iii)

Degraded resolution and undetected minor bands in the peak tail (iv) Poor retention


11

reproducibility. Increased peak asymmetry value, k>1.5 the sign that the column

should be changed

(e) Selectivity: It measures relative retention of two components. Selectivity is the

function of chromatographic surface (column), melting point and temperature.

01

02

'

1

'

2

VV

VV

k

k

(f) Quantization: A critical requirement for quantitative methods is ability to

measure wide range of sample concentration with a linear response for each analyte.

To achieve the best result with an HPLC method, it is necessary to understand and

have a control of the factors that affect quantization. Calculating the following values

are used to access overall system performance.

1. Theoretical plates : n = 16 (t / W)2

2. Plates per meter : N = n / L

3. Height equivalent to theoretical

Plate (HEPT) : L/n

4. Resolution : R = 2 (t2 – t1) / (W2 + W1)

5. Peak asymmetry : T = W 0.05 / 2f

Where α = Relative retention

t2 = Retention time of the second peak measured from point of injection

t1 = Retention time of the first peak measured from point of injection

Chapter – I Part – B Visible Spectrophotometry

12

VISIBLE SPECTROPHOTOMETRY

1.06Visible Spectrophotometry

Among the several instrumental techniques available for the assay of drugs,

usually visible spectroscopic techniques [46-49] are simple and less expensive. The

fundamental principle of visible spectrophotometry lies in that light of a definite

interval of wavelength passes through a cell with a colored solution or solvent and

falls on the photoelectric cell that converts the radiant energy into electrical energy

measured by a galvanometer. Photometric methods of analysis are based on

measuring light absorption of molecules in a solution, utilizing 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. Beer‟s-Lambert law or simply Beer‟s law is expressed as the equation.

log10 (I0/I) = -1og10 T = acl = A

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, 'l' is the path

length through an absorbing solution, „c‟ is the concentration of absorbing substance

and 'A‟ is absorbance (extinction or optical density). 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

concentration is expressed in moles per liter and path length is expressed in

centimeters, the constant is known as molar absorptivity formerly called the molecular


13

or molar extinction coefficient and is used as a physical constant for absorbing species

under standard conditions. It has a unit of liters per mole per centimeter (lt. mole-1

.

cm-1

) and designated as Є. Absorbance = Є.C.l, this expression show that there is a

linear relationship between the absorbance and the concentration of a given solution,

if the path length and the wavelength of radiation are kept constant.

The objective of the present investigation is to develop a simple, accurate,

precise, rapid, and sensitive spectrophotometric method to determine the amount of

drug in bulk and pharmaceutical formulations. Spectrophotometric methods of

analysis depend on measuring the amount of radiant energy of a particular wavelength

absorbed/emitted by the sample. The important characteristics of the developed

method should be accurate, precise, low cost and less time.

1.07 Chemistry of chromogenic reagents

Chemical methods used to prepare suitable colored solutions are usually

called chromogenic reactions and the color forming reagents are known as

chromogenic reagents. The preparation of the colored solution is as important as the

measurement; hence a careful attention is extremely important while preparing a

colored solution. In spectrophotometric analysis the use of a soluble colorless

chromogenic reagent is desirable. If the reagent possesses some self color, the

preferential solvent extraction of the colored constituent by an immiscible solvent

eliminates the additive effect of the reagent to the resultant color. The preliminary

importance in quantitative analysis of drugs involve the knowledge of the functional

groups either acidic or basic nature present in the drug molecules(Table: 1.01, P: 46-

48), chemical reactions such as redox, substitution, addition, elimination,


14

rearrangement and complexaction ect between an analyte (drug) or its converted form

with preliminary treatment (cationic, anionic, oxidized, reduced ect.) and a reagent or

its converted form (cationic, anionic, electrophlic, nucleophlic, oxidized and reduced

ect.) with preliminary treatment to produce color. An electrophlic reagent i.e cation,

dipolar molecule, or molecule that has atoms with incomplete octet, is a species

having electron deficient atom or center. The nucleophlic reagent is electron rich.

Various reactions may involve the formation of three main intermediates namely free

radicals, carbonium ions (C+ )and carbanion (C

-)which then react with the reagents to

form products. The analytical application of each reagent has been discussed in detail

separately. Under proposed experimental conditions, the methods M1 to M19 refer to

the serial number. The alphabets „a‟ and „b‟ refer to the different dyes/oxidants used

in the present investigation. The selectivity and sensitivity of the visible spectroscopic

methods depends only on the nature of chemical reactions based on functional groups

present in the drug with suitable chromogenic agent involved in color development.

I. Dyes in ion association complex formation: Methods M1(a), M1(b), M1(c) , M1(d)

M2(a) , M2(b)) and M17.

Ion-Ion association complex or adduct is a special form of molecular complex

resulting from two oppositely charged components extractable into organic solvents

from aqueous phase at suitable pH. One component is a chromogen (dye or metal

complex) possessing charge (cationic or anionic nature) and so insoluble in organic

solvents and the other is colorless, possessing opposite charge (anionic or cationic) to

that of chromogen. The ion-ion association complex extraction has been applied to the

estimation of numerous compounds; possessing basic moieties (secondary or tertiary

aliphatic amino groups) by using an acid dye as a reagent and a chlorinated solvent as


15

an extractant. The structure of the species formed may depend upon the experimental

conditions (concentration of the components, pH of the aqueous phase). The color can

be altered or intensified upon acidification or re-extracted into a buffer. The presence

of hydrophilic substituents such as -OH or -COOH often prevents extraction of the

complex into organic phase. The selectivity of the reaction may increase by using

appropriate organic solvent (extractant) which depends upon the polarities of the

amine and of the dye. Chemical features of some acidic and basic dyes used in ion

association complex formation are listed in Table: 1.02, P: 49-51.

Simple extractive spectrophotometric methods have been developed for the

estimation of several drugs having basic /acidic moiety either in pure and

pharmaceutical dosage forms [50-60]. These methods are based on the formation of

ion-pair complex of the drug with acidic / basic dye. Alfuzosin [50] is estimated in

both pure and pharmaceutical dosage forms with acidic dye bromocresol green (BCG)

in acidic condition, followed by its extraction in organic solvent (chloroform). R.

Kalaichelvi et al [51] have developed an extractive spectrophometric method for the

assay of pantoprazole sodium either in pure form or in pharmaceutical solid dosage

form using bromothymol blue in aqueous acidic medium. The extracted complexes

showed absorbance maxima at 428 nm. Ashour [52] et al reported a method to

determine alfuzosin hydrochloride either in pure form or in pharmaceutical

formulations. S V Muralimohan Rao et al [53] have reported an extractive

spectrophometric method for the assay of bromhexine HCl both in pure form and in

pharmaceutical solid dosage form using acidic dyes such as TPooo, Napthalene Blue

and Azocarmine. A simple and sensitive extractive spectrophotometric method is

described [54] for determination of buspirone. Two spectrophotometric methods have


16

been developed [55] for the determination of quetiapine fumarate (QTF) in pure form

and in its dosage forms. These methods are based on the formation of ion-pair

complex between the drug and two sulphonthalein acidic dyes, namely, bromophenol

blue and thymol blue, followed by the measurement of absorbance at 410 and 380nm,

respectively. Extractive spectrophotometric determination of omeprazole is developed

[56] using acidic dyes- bromophenol blue and orange G - as ion-pairing agents in

aqueous medium (pH 7.0 and 6.0, respectively). A spectrophotometric method has

been developed by H. Abdine et al [57] for the determination of cinnarizine in

pharmaceutical preparations. Abdel-Aziz M [58] developed a spectrophotometric

method for the determination of guanethidine sulphate, guanfacine hydrochloride,

guanoclor sulphate, guanoxan sulphate and debrisoquine sulphate which involves ion-

pair formation of the selected compounds with bromocresol purple at pH 3.8. Two

acid dye reagents have been utilized for spectrophotometric determination of

triamterene [59] in pure form and in pharmaceutical preparations. The dyes used are

bromophenol blue (BPB), and bromothymol blue (BTS). They form a chloroform-

soluble, coloured ion association complex with triamterene, at pH 3.4 and 3.2 using

bromo phenyl blue and bromo thymole blue respectively. Krishna and Sankar [60]

have reported four simple and sensitive ion-pairing spectrophotometric methods have

been described for the assay of gemifloxacin mesylate (GFX) either in pure form or in

pharmaceutical formulations. In the present investigations, ARS, BTB, MO and

TPooo (Method M1(a), M1(b), M1(c) M1(d)) have been used as acidic dyes for the

formation of ion –ion association complex with the selected drugs NTT, LEF, OND

and TDP. TDP and LEF form ion association complexes with basic dyes MB and

SAF-O, which are extractable into chloroform from the aqueous phase and the max


17

and max values obtained with the two basic dyes with the responded drug LEF and

TDP are compiled in Chapter - III and Chapter - V.

II. Fe (III)/o-Phenanthroline: Method M3

Ferric salt, Fe (III) converts into a ferrous salt, Fe (II) by reduction process and

the reduced for of iron can be easily detected by the usual reagent o-phenanthroline

[61], bipyridyl or triazine [62].

3Fe+2

+ 2 [Fe (CN)6]-3

Fe3 [Fe(CN)6]2

The reaction between o-Phen and Fe (II) forms a red complex. Each of the

nitrogen atom in 1, 10-phenanthroline has an unshared air of electrons that can be

shared with iron (II). Three such molecules of the organic compound attach

themselves to the metallic ion to form a blood-red complex ion. The iron (II) can be

oxidized to iron (III), and the later ion also forms a complex with three molecules o-

phen. Based on its complexing tendency and oxidizing properties, ferric salt is

suggested in the estimation of several drugs. P.Nagaraju et.al [63] have developed

three spectrophotometric methods for the determination of Atorvastatin calcium in

pure and its pharmaceutical formulations. These methods are based on the oxidation

of Atorvastatin calcium by ferric chloride in presence of o-phenanthroline or 2,

2'bipyridyl or potassium ferricyanide. Kanakapura [64] has described two

spectrophotometric methods for the determination of etamsylate in bulk and in

capsule formulations. These methods are based on the oxidation of the drug with

ferric chloride in neutral medium and subsequent chelation of the resulting iron(II)

with 1,10-phenanthroline and with 2,2‟-bipyridyl. A simple, sensitive and accurate


18

spectrophotometric method [65] for the determination of Levodopa , Carbidopa and

-Methyldopa at the ppb level has been developed. This method is based on the

oxidation of the drugs by Fe(III), using a Fe(III)-o-Phenanthroline mixture and is

followed by the formation of a highly stable orange-red coloured tris-complex [Fe(II)-

(o-Phenanthroline)3]2+

in a moderate acidic medium (pH=5.0±0.2) which exhibits an

absorption maximum at =510 nm. Alaa S. Amin [66] et.al have reported two

methods for assaying domperidone (I) and metoclopramide (II) in a bulk sample and

in dosage forms are investigated. These methods are based on the oxidation of I

and/or II by Fe3+

in the presence of o-phenanthroline or bipyridyl. The formation of

tris-complex upon reactions with Fe3+

-o-phen and/or Fe3+

-bipy mixture in an acetate

buffer solution of the optimum pH-values is demonstrated. Ayman A Gouda and

Wafaa S Hassan [67] have described three simple, sensitive and reproducible

spectrophotometric assay methods for the determination of etodolac in pure form and

in pharmaceutical formulations. Two of the methods are based on the oxidation of

etodolac by Fe3+

in the presence of o-phenanthroline (o-phen) or bipyridyl (bipy). The

formation of the tris-complex on reaction with Fe3+

-o-phen and/or Fe3+

-bipy mixtures

in acetate buffer solution at optimum pH is demonstrated at 510 and 520 nm with o-

phen and bipy. Third method is based on the oxidation of etodolac by Fe3+

in acidic

medium, and the subsequent interaction of iron(II) with ferricyanide to form Prussian

blue, with the product exhibiting an absorption maximum at 726 nm. Ragaa El-Shiekh

[68] have reported three spectrophotometric methods for the determination of

pipazethate hydrochloride (PiCl) in pure form and in pharmaceutical formulations are

described. The first and second methods are based on the oxidation of the drug by

Fe3+

in the presence of o-phenanthroline (o-phen) or bipyridyl (bipy). The formation

of tris-complex upon reactions with Fe3+

-o-phen and/or Fe3+

-bipy mixture in an


19

acetate buffer solution of the optimum pH values is demonstrated at 510 and 522 nm,

respectively, with o-phen and bipy. The third method is based on the reduction of

Fe(III) in acid medium and subsequent interaction of Fe(II) with ferricyanide to form

Prussian blue, which exhibits an absorption maximum at 750 nm. In the present

investigations, (Method M3), the selected drug LEF is treated with access of Fe (III)

salt under specified experimental conditions. Acting as oxidant Fe (III) undergoes

reduction to Fe (II) in oxidizing (LEF) which corresponds to the drug concentration.

Fe (II) is estimated by the usual reagent for divalent iron, o-Phenanthroline. The

details of the investigations, scheme of reactions are compiled in chapter III.

III. Brucine – Periodate: Method M4

Brucine (2, 3 – dimethoxystrychnine) under acidic conditions has been sued as

an effective reagent for spectrophotometric determination of nitrates and nitrites,

cerium, manganese, cadmium and platinum. Several modifications have been

introduced for the spectrophotometric determination of nitrites and nitrates using this

reagent. Brucine can also been used for the spectrophotometric determination of

halides and cysteine and as an indicator in redox titration. Brucine forms a 1:1 colored

complex with p–dimethylamino cinnamaldehyde under acidic conditions

Sodium metaperiodate is an effective oxidant for converting methyl

substituted p-dihydroxy phenols to o-quinones and is also color stabilizer. brucine-

periodate reagent for spectrophotometric determination of tryptophan and some

sulphur compounds and for tetracyclines, chlorophenicol and streptomycin

.According to them, periodate converts most electron rich portion of the coupler

(tryptophan and other mentioned compounds) to yield 1-mono substituted


20

bruciquinone derivatives with an absorption maximum at 500-510 nm as the colored

species. Brucine – periodate reagent gave colored species with the compounds

containing either primary or secondary aliphatic amino and aromatic primary amine

groups. A simple, accurate and reproducible UV-Visible spectrophotometric method

[69] established for the assay of ceftiofur (CEFT) based on oxidative coupling of

CEFT with brucine/IO4. Determination of CEFT in bulk form and in pharmaceutical

formulations has also been incorporated. A spectrophotometric method has been

developed for the determination of nicorandil [70] in drug formulations and biological

fluids. This method is based on the reaction of the drug with brucine–sulphanilic acid

reagent in sulphuric acid medium producing a yellow-coloured product, which

absorbs maximally at 410 nm. On the basis of this observation, the author has

developed a specific method for the assay of NTT (Method M4) in bulk samples and

dosage forms. The details of the spectrophotometric investigations of the

corresponding drug are incorporated in chapter II.

IV. MBTH – Oxidant: Methods M5(a), M5(b), M5(c) and M5(d)

3-Methyl-2-benzothiazolinone hydrazone Hydrochloride (MBTH) is

synthesized by Besthorn [71]. The first procedure described by for the determination

of aldehydes, with which MBTH condenses to give a blue cation. This technique is

later improved, allowing more sensitive determinations. The reaction is applied to the

analysis of aliphatic aldehydes and the detection of the aldehyde groups in tissue and

collagen. Under reaction conditions, MBTH loses two electrons and one proton on

oxidation, forming the electrophilic intermediate, which has been postulated to be the

active coupling species. The intermediate reacts with amine or phenol by electrophilic

attack on the most nucleophilic site on the aromatic ring of amine or phenol (i.e., para


21

or ortho position) and the intermediate is spontaneously oxidized in the presence of

oxidant to form the colored species. MBTH also forms a strongly electrophilic

diazonium salt when acted upon by an oxidizing agent. These properties led the way

to colorimetric determinations based on the formation of formazans. Glyoxal reacts

with MBTH in the presence of acetic acid giving yellow diazine, which allows its

determination in the presence of unsubstituted monoaldehydes, when oxidant is

present. Phenol is so determined using the oxidant cerium (IV) ammonium sulphate.

This reaction is extended to miscellaneous other phenols, using various oxidants [72]

and an automated method MBTH can be used for the determination of polyhydroxy

compounds aromatic amines [73], aliphatic and alicyclic amines. Azodyes, stilbenes

and Schiff bases as well as pyrrole derivatives also react with MBTH under oxidative

conditions. This reaction is extended to the determination of bilirubin and its

oxidation products such as urobilin and biliverdin. Ferric chloride has been mostly

used as an oxidant for the determination of aromatic and heterocyclic amines by (in

neutral conditions) and (in acidic conditions). Other oxidants such as periodate (acidic

conditions), ammonium persulphate (alkaline conditions) and potassium dichromate

(acidic conditions) are employed for the determination of ethylenic compounds and

primary alcohols (after oxidation with Ruthenium tetraoxide. Sastry et al [74]

reviewed various aspects of MBTH chemistry in pharmaceutical analysis. Malipatil

et. al [75] have developed a spectrophotometric method in which oseltamivir

phosphate formed a green coloured chromogen when treated with MBTH in the

presence of oxidant ferric chloride. Vijaya Raja [76] reported a method which is based

on the oxidation of 3-methylbenzothiazolin-2-one hydrazone (MBTH) by ferric

chloride followed by its coupling with the drug producing colored complex measured

at 670nm in acidic medium. Adefovir dipivoxil [77] is subjected to acid hydrolysis


22

and the hydrolysed product used for the estimation. A simple and sensitive kinetic

method is described for the determination of ketoprofen [78] in pure form,

pharmaceuticals and biological fluids. Rekha Rajeevkumar et.al [79] have reported a

visible spectrophotometeric method for the quantitative estimation of Moprolol in

bulk drug and pharmaceutical preparations which is based on the reaction of Moprolol

with 1%w/v 3-methyl-2- benzthiazolinone hydrazone hydrochloride (MBTH) reagent

in presence of 2%w/v of ferric chloride salt to give a green colored chromogen with

absorption maximum at 629nm. Prakash et.al [80] have developed a method which is

based on oxidation followed by coupling of 3 methyl-2-benzothiazolinone hydrazone

(MBTH) with Ganciclovir in presence of ferric chloride to form bluish green color

chromogen and exhibiting absorption maximum at 611.8 nm and obeying beers law

in the concentration range of 50 – 250 μg/ml respectively. Vijaya Raja et.al [81] have

described a spectrophotometric method for the determination of bromhexine

hydrochloride in bulk and formulations which is based on oxidation of 3-

methylbenzothiazolinone-2- hydrazone by ferric chloride followed by its coupling

with the drug in acidic medium forming an intense green colored chromogen with

absorbance maxima at 630nm. Malipatil et al [82] have developed a

spectrophotometric method for the determination of Citicoline with MBTH. Lakshmi

et al [83] reported a spectrophotometric method utilizing the reaction of manidipine

with 3-methyl-2-benzothiazoniumhydrazone hydrochloride (MBTH) in the presence

of ferric chloride. A novel-coupling reagent is used for the simple and sensitive

spectrophotometric determination of caffeine (CF) and theophylline (TP) [84] in pure

or pharmaceutical formulations. In the present investigation the selected drugs NTT,

LEF, OND and TDP which possesses amino group, produced an oxidative coupling

product with MBTH in the presence of oxidants such as Ce (IV), NaIO4 and IBDA.


23

The probable sequence of reactions based on analogy is presented in the respective

chapters of the corresponding drugs.

V. Redox reactions- Folin Ciocalteu [FC] reagent : Method M6

Heteropolyacid complexes are formed by the combination of orthophosphoric

acid and periodic, molybdic, vanadic, tungstic and molybdovanadic acids. Treatment

of complexes with reducing agents result in the formation of the corresponding

reduction products, which are blue in color (eg: molybdenum blue from

phosphomolybdate, tungsten blue from phosphotungstate). This reaction is the basis

of several methods suggested for the determination of phosphate. Various reducing

agents have been used for the reduction of heteropolyacids. Stannous chloride is most

widely used one among several reducing agents (1,2, 4-aminonaphthol sulphonic acid

, ascorbic acid , hydrazine, ferrous sulphate ], p-amino phenol hydrochloride

,thiosulphate and sulphite ,thiourea, pyrogallol and metol. Among the various

heteropolyacids, phosphomolybdo tungstic acid, the well-known Folin-Ciocalteu

reagent (F.C reagent) is preferred by a number of workers for the determination of

drugs [85-87]. The wavelength of maximum absorption and stability of the blue

colored reduction product and the sensitivity and reproducibility of the reaction are

dependent upon pH, composition of the heteropolyacid complex, nature and

concentration of the reducing agent, temperature and time. Allopurinol [88], caffeine ,

pentazocine [89], oxymetazoline, isoxsuprine, orciprenaline, pholedrin, vitamin–K

and rutin [90] are some typical examples of drugs estimates in this manner. A

spectrophotometric method for the estimation of hydralazine hydrochloride [91] in

bulk drug and in their tablet formulations is described. The described method is based

on the formation of blue colored chromogen due to the reaction of hydralazine


24

hydrochloride with Folin Ciocalteu reagent in presence of alkali, which exhibits

maximum wavelength at 640 nm. A reproducible colorimetric method has been

developed for the estimation of Memantine by Jagathi [92] in bulk and in

pharmaceutical formulations. This method is based on the reduction of

Folin‐Ciocalteau reagent by the drug and the reduced species posses a characteristic

intense blue color (λmax 760 nm). Singh et.al [93] have reported a method for the

determination of ajmaline and brucine based on the development of blue coloured

product due to reduction of tungstate and/or molybdate in Folin Ciocalteu‟s reagent

by ajmaline and brucine in alkaline medium. Mohamed Abd El-Ghaffar [94] has

reported a spectrophotometric method for the determination of ritodrine hydrochloride

(RTH) either in pure form or dosage forms using Folin-Ciocalteu reagent producing

blue chromogen which is measured at 760 nm. Prakash S Sarsambi [95] et.al have

described a spectrophotometric method for the determination of which is based on

reduction of Ganciclovir, an acyclic guanosine analog used in the treatment of AIDS

using Folin-Ciocalteu reagent in presence of alkali to form intense blue color

chromogen exhibiting absorption maximum at 764.7nm. A spectrophotometric

method [96] has been developed for the estimation of diacerein in Pharmaceutical

dosage forms. A spectrophotometric method for the determination of penicillins [97]

(ampicillin, amoxycillin and carbenicillin) using Folin-Ciocalteu reagent (FC reagent)

is described. Basavaiah K et.al [98] have described a spectrophotometric method for

the determination of acyclovir in bulk drug and in formulations. A spectrophotometric

method is described for the determination of amoxicillin and ampicillin by Dhruv et.al

[99] using Folin-Ciocalteu reagent. The above method has been used in the

determination of NTT in the present investigations. The details of the investigation

have been incorporated in Chapters II.


25

VI. N-Bromosuccinimide (NBS) as an oxidant: Method M7

N-Bromosuccinimide contains weakly bound bromine and is used for

brominations and dehydrogenation in organic chemistry. The reagent behaves as a

mild oxidising agent and converts primary and secondary alcohols to the

corresponding aldehydes and ketones. It has been used as quantitative oxidizing agent

for hydrazines for thiourea and some of its derivatives for isoniazid, and for the ene-

diol group in ascorbic acid. The reagent is highly selective oxidant and after all the

compound being titrated has been oxidized a slight excess gives a blue color with

potassium iodide - starch or decolorises methyl red either of which can be used as the

end point detector. By the titrimetric procedure analyte can be estimated only at

milligram level. After completion of the reaction with analyte the unreacted NBS can

be determined using visible spectrophotometric method. The reacted NBS (NBS

originally added - NBS unreacted) corresponds to the analyte present in the

microgram level. Two reagents (CB) [100], PMAP-SA[101] have been used in the

literature for the colorimetric determination of NBS. A spectrophotometric method is

described for the determination of the commonly used antimycobacterial drugs such

as Isoniazid and Rifampicin[102] in their pure forms, pharmaceutical preparations and

biological fluids based on the reaction of drugs with N-bromosuccinimide (NBS),

then the excess of NBS is reacted with KI, the liberating iodine is determined at wave

length 572 nm. Ibrahim et.al [103] have reported a method which is based on the

reaction with N-bromosuccinimide (NBS) and subsequent reaction of the remaining

NBS with fluorescein (FLC) to give a pink colored product that is measured at 518

nm. A sensitive spectrophotometric method is presented [104] for the assay of

pantoprazole sodium sesqui hydrate (PNT) in bulk drug and in formulations using N-


26

bromosuccinimide (NBS) and two dyes, methyl orange and indigo carmine, as

reagents. This method involves the addition of a known excess of NBS to PNT in acid

medium, followed by determination of unreacted oxidant by reacting with a fixed

amount of either methyl orange and measuring the absorbance at 520 nm (method A)

or indigo carmine and measuring the absorbance at 610 nm. In the present

investigations the author proposed a simple selective and sensitive indirect

spectrophotometric method using NBS/CB for the assay of OND (Method M7). This

method involves two steps. First step involves the oxidation of OND with NBS. The

second step in the procedure is the quantitative decolorisation of CB by the unreacted

NBS. The probable sequence of reactions in two steps (drug – NBS, NBS - CB) based

on analogy are present in the schemes of the corresponding chapters. The details of

these investigations are incorporated in chapter IV of the individual drugs.

VIII. Condensation reaction - (a)Isatin - Sulphuric acid : Method M8

Isatin (1H – indole – 2, 3 – dione) is first obtained by Erdman and Laurnet as a

product from the oxidation of indigo by nitric and chromic acids. It usually exists in

two tautomeric forms (lactam and lactim). Isatin under alkaline conditions hydrolyze

giving o-amino benzene keto acid, which condenses with acetone giving 2 – methyl

cinchonic acid. The blue color of indophenin dye formation (pfitzinger reaction)

involving isatin and thiophene in presence of sulphuric acid is due to the formation of

compounds related to indigo [105-106]. Under acidic conditions isatin reacts with

proline or pyrrole to give colored condensation product. Isatin also produce a

fluorogenic derivative when reacted with tryptophan, which has been used for its

detection by TLC [107-108]. In the present investigation, a visible spectrophotometric

method has been developed for NTT, which possesses carbazone moiety by using


27

isatin and sulphuric acid in acetic acid medium. The details of these investigations

are presented in chapter II.

b) Vanillin: Method M9

It is well known that aromatic aldehydes form colored condensation product

(Schiff base) with aromatic primary amines in particular. It has been observed by

suitable alteration of experimental conditions, others such as hydrazine and its mono

substituted derivatives, primary alkyl amines, amino acids [109] converted to pyrrole

derivatives [110,111]. Primary heterocyclic amines and m-diphenol[112] also develop

color with aromatic aldehydes. Enoche Florence Oga [113] has described a method

for the determination of isoniazid in pure form and in pharmaceutical formulations.

Two simple and sensitive spectrophotometric methods have been developed for the

estimation of Famciclovir [114] in bulk and tablet dosage form which are based on the

condensation reaction of Famciclovir with carbonyl reagents such as p-

dimethylaminobenzaldehyde (PDAB) and vanillin in acidic condition to form orange

yellow colored chromogen with absorption maxima at 480 nm and 470 nm

respectively. Manikya Sastry et. al [115] developed a spectrophotomeric method for

the determination of Lerccnidipine HCl using vanillin as a chromogenic reagent

having maximum absorbance at 600nm. A new spectrophotometric method [116] has

been examined for the determination of the tranexamic acid by derivatization with

vanillin. A colorimetric method is developed for the quantitative determination of

hydralazine hydrochloride in dosage forms by Bala et.al [117]. Three

spectrophotometric methods have been developed for the quantitative estimation of

nitazoxanide [118] in bulk drug and pharmaceutical formulations. These methods are

based on the reaction of reduced nitazoxanide with p-dimethylaminobenzaldehyde, p-


28

dimethyl amino cinnamaldehyde and vanillin in acidic conditions to form pink,

orange red, and orange yellow coloured chromogens with absorption maxima at 559

nm, 534.5 nm, and 475 nm respectively. These observations have led to application of

aromatic aldehyde, Vanillin (p-hydroxy-m-methoxy benzaldehyde) as analytical

reagent for the analysis of pharmaceutical dosage forms. In the present investigation

the selected drugs NTT, it is observed that Vanillin under certain established

experimental conditions produce color of maximum intensity in methanol with the

selected drug NTT and the results of these investigations are presented in chapters II.

IX. Ortho nitro benzaldehyde: Method M10

It is well known that aromatic aldehydes form colored product due to

condensation with aromatic primary amines in particular. It has been observed by

suitable alternation of experimental conditions, others such as hydrazine and its mono

substituted derivative, primary alkyl amines, amino acids converted to pyrrole

derivatives, indole derivatives primary hetero cyclic amines and m-diphenol develop

color with aromatic aldehydes. These observations have led to numerous applications

of aromatic aldehydes such as p-dimethyl amino benzaldehyde (PDAB), p-dimethyl

amino cinnamaldehyde (PDAC), ortho notro benzaldehyde (ONB) as analytical

reagents. In the present investigation, NTT responded to condensation reaction with

ONB in the presence of Conc. H2SO4. The details of the investigation of the

corresponding drug (NTT) are incorporated in chapter II.

X. Poly acid complexes - AMV /H2SO4 : Method M11

The orthovanadate ion [VO4]-3

occurs only at very high pH. It is such a strong

base that the first step in its protonation, forming [HVO4]-2

, is already complete at pH


29

12. When the pH is gradually lowered to one successive protonation takes place that

ultimately leads to the formation of the pale yellow cationic species, usually

formulated as VO-1

2 Due to the great tendency of vanadate to oligomerize, the

protonated monomers [HVO4]-2

, [H2VO4]-1

, and VO+

2 (except at very low pH) are

predominant only in highly diluted solution. The chemistry of vanadium is

complicated. It forms compounds corresponding to oxidation numbers +2 to +5. The

most stable and commonly encountered compounds of Molybdenum are derived from

its oxide [VO4]-3

.The vanadium compounds corresponding to the oxidation states

ranging from +2 to +5 are mostly complexes species. The isopolyanionic or hetero

polyanionic species of vanadium undergo reduction to coloured vanadium species

with certain bioactive compounds. The max values of reduction products vary from

600nm – 850 nm depending upon the reaction conditions (nature and strength of acid

or base medium, temperature, time) nature of poly acid (very efficient if the

composition of hetero acids are more) and nature of reducing agent (analyte).

“Vanadium greenish Blue” is the result of mild reduction of an acidified solution,

which contains V(VI), either as an iso-or a hetero polymolybdate anion (or even

alkaline conditions). Wieslawa Misiuk and Ewa Kleszezewska [119] have reported a

spectrophotometric method for the determination of Prothipendyl HCl on reacting

with ammonium metavanadate forming a colored oxidation product exhibiting

maximum absorbance at maximum wavelength 374nm. In the present investigation,

TDP responded to condensation reaction with AMV in the presence of Conc. H2SO4.

The details of the investigation of the corresponding drug (TDP) are incorporated in

Chapter V.


30

XI. NaIO4 / Phenyl hydrazine hydrochloride (PHH)/[Fe(CN)6]-3

:Method M12

Periodic acid oxidation [120] is applicable to compounds having two hydroxyl

groups or a hydroxyl and an amino group attached to adjacent carbon atoms and are

characterized by the cleavage of the carbon-carbon bond. If the hydroxyl groups or a

hydroxyl and an amino group are not vicinal, no oxidation takes place. This selectivity,

which is the outstanding characteristic of periodic acid oxidation, adopts this reaction for

the presence of vicinal hydroxyl groups and hydroxyl and amino groups. Carbonyl

compounds in which the carbonyl group is adjacent to a second carbonyl (α-diketone) or

hydroxyl (α-ketol) group are also oxidized.

Sodium metaperiodate (IO4-) is considerably soluble in water (12.62g/100mL,

25oC). The solubility of sodium metaperiodate is greatly reduced in alkaline solution

because of the formation of disodium metaperodate (Na2H3IO6). This effect occurs at

pH>5.0.Aqueous solution of sodium metaperiodate at pH 4.0 or below is the most suitable

one as the oxidant. The rapid and generally quantitative nature of the reaction recommends

for a very wide variety of analytical applications. Certain analytical procedures have been

developed for the determination of aldehydes utilizing periodate oxidation. Different

reagents are used in developing the spectrophotometric methods for their determination.

Amoung several reagents used for the determination of aldehydes in particular

formaldehyde (existing or formed through some preliminary treatment such as periodate

oxidation of compounds possessing vicinal aminol, diol or ketol), the reagent like

schryver‟s appear to yield highly sensitive and stable chromogen with formaldehyde

especially. This method avoid the distillation or diffusion step and permit the determination

of the liberated formaldehyde directly in the reaction medium colorimetrically by oxidative

coupling reaction with schryver reaction with PHH and hexacyanoferrate (III) and this


31

method has been applied for the determination of doxorubicin [121]. In the present

investigation, LEF responded to oxidative coupling reaction with PHH in the presence of

hexacyanoferrate (III) giving formazan dye. The details of the investigation of the

corresponding drug (LEF) are incorporated in chapter III.

XII. Ce (IV) / 2, 4 DNP: Method M13

Condensation reaction is one in which two molecules join together with the

loss of a water molecule in the process. In this case, that small molecule is water. In

terms of mechanisms, this is a nucleophilic addition-elimination reaction. 2, 4-

dinitrophenylhydrazine is often abbreviated to 2,4-DNP or 2,4-DNPH. A solution of

2,4-dinitrophenylhydrazine in a mixture of methanol and sulphuric acid is known as

Brady's reagent. The 2,4-dinitrophenylhydrazine first adds across the carbon-oxygen

double bond to give an intermediate compound which then loses a molecule of water.

The ketone functional group can also take part in auto condensation reactions, which

eliminate water. A spectrophotometric method has been proposed by Nagaraja and

Aswinee Kumar [122] for the determination of four phenolic drugs; salbutamol,

ritodrine, amoxicillin and isoxsuprine. The method is based on the oxidation of 2, 4-

dinitrophenylhydrazine and coupling of the oxidized product with drugs to give

intensely colored chromogen. A simple spectrophotometric method is developed and

validated for the determination of Tolperisone [123] in bulk and its dosage forms. The

proposed method is based on the interaction of the drug with 2,4-

dinitrophenylhydrazine in the presence of an acid catalyst, followed by treatment with

a methanolic solution of potassium hydroxide; an intensely colored chromogen is

formed that is measured in dimethyl formamide as the diluting solvent at 520 nm.

Osama [124] developed a spectrophotometric method for the determination of


32

azithromycin (AZ), clarithromycin (CLA), and roxithromycin (ROX) in bulk powders

and their dosage forms.

XIII. Coordination complex formation with Cobalt thiocyanate: Method M14

Cobalt thiocyanate (CTC) (formed by combination of ammonium thiocyanate

and cobalt nitrate) has been proved to be a valuable chromogenic reagent for the

detection and determination of amino compounds. The colored species formed is the

coordination complex of the drug (electron donor) and the central metal atom of

cobalt thiocyanate which is extractable into nitrobenzene from aqueous solution. The

basis in the present investigation is the formation of the blue colored complexes by

LEF (the presence of cyclic tertiary amine group) when treated with CTC. The

probable sequences of reactions based on analogy are presented in the individual

chapters III.

XIV. p-CA + CHCl3 -Charge-transfer Complex formation reactions: Method M15

It is well known that electron donors and electron acceptors can interact in

solution to form intensively colored charge-transfer complexes. These complexes are

usually characterized by absorption bands, not present in either reagent, assigned to an

intermolecular charge-transfer transition [125-126]. Some substituted quinones such

as p-benzoquinone chlorimide [127], 2,5-dichloro benzoquinone[128] and p-N-

methylbenzoquinone monoamine[129], 2,3-dichloro-5,6-dicyano-p-benzoquinone

DDQ[130-131], chloranilic acid[132] and chloranil have been used in the studies of

amines. Two spectrophotometric methods have been proposed [133] for the

determination of milrinone in pharmaceutical formulations based on the charge

transfer complexation reaction of milrinone with 2, 3-Dichloro-5, 6-


33

Dicyanobenzoquinone, DDQ and p-Chloranilic Acid (pCA). Two Spectrophotometric

procedures are presented by M. Walash et. al. [134] for the determination of two

commonly used H2-receptor antagonists nizatidine (I) and ranitidine hydrochloride

(II). The methods are based mainly on charge transfer complexation reaction of these

drugs with p-chloranilic acid or 2, 3 dichloro-5, 6-dicyanoquinone (DDQ).

Kanakapura Basavaiah et.al. [135] reported two visible spectrophotometric methods

for the determination of bupropion hydrochloride in pharmaceuticals and spiked

human urine. These methods are based on charge transfer complexation reaction of

bupropion base (BUP) as n-electron donor with either p-chloranilic acid or 2, 3-

dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ) as π-acceptors to give highly colored

radical anion species.

Rahman and Haque [136] have developed two spectrophotometric methods

are based on the charge transfer complexation reaction of the drug with p-chloranilic

acid (pCA) in 1, 4-dioxan-methanol medium, 2, 3-dichloro 5, 6-dicyano 1, 4-

benzoquinone (DDQ) in acetonitrile-1,4 dioxane medium. Two spectrophotometric

methods have been proposed [137] for the determination of lisinopril in pure form and

pharmaceutical formulations. The methods are based on the charge transfer

complexation reaction of the drug with 7, 7, 8, 8, tetracyanoquinodimethane (TCNQ)

and p-chloranilic acid (pCA) in polar media.

Hisham E. Abdellate [138] have developed two spectrophotometric methods

for the determination of perindopril which are based on the reaction of this drug as n-

electron donor with 2,3-dichloro-5,6-dicyano-p-benzoquinone(DDQ)-7,7,8,8

tetracyanoquinodimethane (TCNQ), tetracyanoethylene (TCNE), chloranil (CL) and

p-chloranilic acid (p-CA) as π-acceptors to give highly coloured complex species.


34

Chloranil, a strong electron acceptor can form charge-transfer complex with a variety

of electron donors, including the above drugs, containing amino-group. Studies

showed that tetrachloro-p-benzoquinone (p-chloranil), acting as an electron acceptor,

could be used for determination of aliphatic and aromatic amines as well as some

amino acids. Taking these considerations into account, NTT, LEF, OND and TDP

drug molecules, which have amino groups, can act as an electron donor and react with

p-chloranil. The proposed mechanism for the reaction between these drugs and p-

chloranil to produce the charge transfer complex are showed in respective chapters

XV. H2O2 / per acids +MO: Method M16

Tertiary amines can be convertedto amine by oxidation. Hydrogen peroxide is

often used, but peracids are also important reagents for this purpose.In the attack by

hydrogen peroxide there is first formed a trialkyl ammonium peroxide,the

decomposition of this complex probably involves an attack by the OH +

moiety of

H2O2.

R3N + H2O2 R3N.H2O2 R3N+

- OH

The positive ion of the oxidisedform of the drug is associated with negative ion of the

methyl orange molecule to form ion-ion associated complex which is extracted in to

chloroform solvent.

XVII. Condensation reactions with 4-AP, INH: Method M18 and Method M19

The ketone functional group can also take part in auto condensation reactions

with elimination of water molecule. These reactions will produce an aromatic

molecule from a starting point of an aliphatic, straight chain molecule. Polymerization


35

across the carbonyl group is a very uncommon reaction, particularly compared to the

similar group in aldehydes. Carbonyl compounds, ketosteroids can be determined

through the formation of hydrazones or schiff bases with Isoniazid (INH) and 4-

aminoantipyrine (4-AP) [139]. The reaction with hydrazines is rather general,

condensation with hydrazides and amines may lead to more selective colorimetric or

fluorimetric determinations. In the present investigation 4-AP and INH have been

used for the determination of LEF and OND which possess keto group and the details

concerned are presented individually Chapter-III and Chapter – IV. Statement

showing functional groups/moieties in the selected drugs, methods proposed,

reagents, types of reactions involved are presented in Table: 1.03, P: 52-55.

1.08 Method Development and Optimization

In developing a quantitative method for determining an unknown

concentration of a given substance by absorption spectrophotometry, the first step will

be the selection of analytical wavelength at which absorption measurements are made.

The analytical wavelength can be chosen either from literature or experimentally by

means of scanning with a spectrophotometer. In order to enhance the sensitivity of the

method and signal to noise ratio, the wavelength of maximum absorbance is chosen as

analytical wavelength. Absorption spectrum is a graphical representation of the

amount of light absorbed by a substance at definite wavelengths. To plot a curve, the

values of wavelength in the visible region are laid off along the x- axis and the

absorbance values on y-axis. A characteristic of an absorption spectrum is a position

of the peaks of light absorption by the substance and also the intensity of absorption,

which is determined by the absorptivity at definite wavelength. After selection of the

analytical wavelength, the chromogenic reagent and the absorbing product must be


36

stable for a considerable period of time. Now a series of standard solutions are

prepared under the optimum conditions, and the absorbance values are measured at

λmax, these values are plotted against the concentration in μg/ml.

In each type of basic reaction, the colored species is formed or the final color

of the reaction mixture whose absorbance is measured and thus the sensitivity of the

method, rate of color formation and stability is affected by the concentration of the

reagent in the solution, nature of solvent, temperature, pH of the medium, order of

addition of reactants and intervals between additions. For simple systems having no

interaction between variables, the one variable at a time strategy appears to be simple,

efficient and effective to establish the optimum conditions [140]. The one variable at a

time approach requires all variables but one to be held constant while a univariate

search is carried out on the variable of interest. The details of fixing optimum

conditions used in different procedures of present investigations are furnished in

subsequent chapters.

1.09 Statistical Treatment of Analytical Data

1.09 (i) Calibration: Calibration is one of the most important steps in bioactive

compound analysis. A good precision and accuracy can only be obtained when a good

calibration procedure is used. In the spectrophotometric methods, the concentration of

a sample cannot be measured directly, but is determined using another physical

measuring quantity 'y (absorbance of a solution). An unambiguous empirical or

theoretical relationship can be shown between this quantity and the concentration of

analyte.


37

The calibration between y = g (x) is directly useful and yields by inversion of

the analytical calculation function. The calibration function can be obtained by fitting

an adequate mathematical model through the experimental data. The most convenient

calibration function is linear, goes through the origin and is applicable over a wide

dynamic range. In practice, however, many deviations from this ideal calibration line

may occur. For the majority of analytical techniques the analyst uses the calibration

equation.

Y = a + bx

In calibration univariate regression is applied, which means that all observations are

dependent upon a single variable x.

1.09(ii) Linear Regression- Method of least squares

Least-squares regression analysis [141,142] can be used to describe the

relationship between response (y) and concentration (x). The relationship can be

represented by the general function Y = f (x, a, b1, ……. bm ) Where a, b1......, bm

are the parameters of the function.

We adopt the convention that the x values relate to the controlled on

independent variable (e.g. the concentration of a standard) and the y values to the

dependent variable (the response measurements). This means that the x values have

no error. On the condition that the errors made in preparing the standards are

significantly smaller than the measuring error (which is usually the case in analytical

problems). The values of the unknown parameters a, b1, ... bm must be estimated in

such a way that the model fits the experimental data points (xi, yi) as well as possible.


38

The true relationship between x and y is considered to be given by a straight line. The

relation between each observation pair (Xi, Yi) can be represented as

Yi = + Xi + ei

The signal yi is composed of deterministic component predicted by linear

model and a random component ei. One must now find the estimates of a and b of the

two values and . This can be done by calculating the values a and b for which ei2 is

minimal. The component ei represent the differences between the observed yi values

and the predicted yi values by the model. The ei are called the residuals, a and b are

the intercept and slope respectively.

2

1 1

2

1 11

n

i

n

i

ii

n

i

n

i

ii

n

i

ii

xxn

yxyxn

b 2

1 1

2

1 11

2

1

n

i

n

i

ii

n

i

n

i

ii

n

i

ii

n

i

i

xxn

yxxxy

a

1.09(iii) Standard error on estimation ( Se)

The standard error on estimation is a measure of the difference between

experimental and computed values of the dependent variable. It can be represented by

the following equation,

)2/()(1

2

nyySn

i

iie

Yi, and yi, are the observed and predicted values, respectively. Standard deviations on

slopes (Sb) and intercepts (Sa) are quoted less frequently, even though they are used to


39

evaluate proportional differences between or among methods as well as to compute

the independent variables such as concentration etc. It is important to understand how

uncertainties in the slope are influenced by the controllable properties of the data set

such as the number and range of data points and also how properties of data sets can

be designed to optimize the confidence in such data.

1.09(iv) Standard deviation on slope ( Sb)

The standard deviation on slope is proportional to standard error of estimate

and inversely proportional to the range and square root of the number of data points.

)2(

)(1

2

n

yy

S

n

i

ii

b

Where Xi is the arithmetic mean of xi values

1.09(v) Standard deviation on intercept, Sa

Intercept values of least squares fits of data are often to evaluate additive

errors between or among different methods

)2(

)(1

2

n

yy

S

n

i

ii

a

1.09(vi) Correlation coefficient (r)

To establish whether there is a linear relationship between two variables xi and

yi, use Pearson‟s correlation coefficient r. The value of r must lie between +1 and -1;

n

i

ii xx1

2)(

1

n

i

ii xx1

2)(

1

n

i

ii xx1

2)(

1

n

xn

i

i1

2


40

the nearer it to ±1, the greater the probability that a definite linear relationship exists

between the variables x and y, values close to +1 indicate positive correlation and

values close to -1 indicate negative correlation.Valuer of r that towards zero indicate

that x and y are not lineary related.

222 )1/()()(

)1/())((

nyyxx

nyyxx

rn

ii

n

ii

1.09(vii) Selectivity of the method

Matrix and interference effects may disturb the determination of an analyte.

Some of the excipients, incipients and additives present in pharmaceutical

formulations may sometimes interfere in the assay of drug and in such instances

appropriate separation procedure is to be adopted initially. The selectivity of the

method is ascertained by studying the effect of a wide range of excipients and other

additives usually present in the pharmaceutical formulations to be determined under

optimum conditions. Initially, interference studies are carried out by the determination

of fixed concentration of the drug several times by the optimum procedure in the

presence of a suitable (1-100 fold) molar excess of the foreign compound under

investigation and its effect on the absorbance of the solution is noticed. The foreign

compound is considered to be interfering at these concentrations if it constantly

produces an error of less than 3.0% in the absorbance produced in pure solution.


41

1.09(viii) Linearity and Sensitivity of the method

Knowledge of the sensitivity of the color is important and the following terms

are commonly employed for expressing sensitivity. According to Beer's law

A= c t

The absorbance (A) is proportional to the concentration (c) of the absorbing

species, if absorptivity () and thickness of the medium (t) are constant. When c is in

moles per litre, the constant is called Molar absorptivity. Beer's law limits and max

values are expressed as g.ml-1

and l mole-1

.cm-1

, respectively. Sandell's sensitivity

[143] refers to the number of g of the drug to be determined, converted to the colored

product, which in a column solution of cross section 1 cm2 shows an absorbance of

0.001 (expressed as g.cm-2

).

1.09(ix) Limit of Detection and Limit of Quantification

The limit of detection (LOD) [144] of an analytical method may be defined as

the concentration, which gives rise to an instrument signal that is significantly

different from the blank. For spectroscopic techniques or other methods that rely upon

a calibration curve for quantitative measurements, the IUPAC approach employs the

standard deviation of the intercept (Sa), which may be related to LOD and the slope of

calibration curve, b, by

LOD = 3Sa / b LOQ=10Sa / b


42

1.09(x) Ringbom's Plots

The relative concentration error depends inversely upon the product

absorbance and transmittance. The relative error increases at the extremes of the

transmittance scale. The slope of plot 'C‟ versus T, i.e. Ringbom plot [145,146] gives

relative error coefficient (i.e. plot of log C T) The main limitations of Ringbom plot

is that it provides no concerning the concentration range of good precision unless it is

combined with T versus T relation. The above expression is valid whether Beer's

law is followed or not.

1.09(xi) Precision and accuracy

The purpose of carrying out a determination is to obtain a valid estimate of a

'true' value. When one considers the criteria according to which an analytical

procedure is selected, precision and accuracy [147] are usually the first time to come

to mind. Precision and accuracy together determine the error of an individual

determination. They are among the most important critical for judging analytical

procedures by their results.

Precision

Precision refers to the reproducibility of measurement within a set, i.e., to the

scatter of dispersion of a set about its central value. One of the most common

statistical terms employed is the standard deviation of a population of observation.

Standard deviation is the square root of, the sum of squares of deviations of individual

results for the mean, divided by one less than the number of results in the set. The

standard deviation S, is given by


43

n

i

i xxn

S1

2)(1

1

Standard deviation has the same units as the property being measured. The

square of standard deviation is called Variance (S2). Relative standard deviation is the

standard deviation expressed as a fraction of the, mean, i.e. S/x. It is sometimes

multiplied by 100 and expressed as a percent relative standard deviation. It becomes a

more reliable expression of precision.

% Relative standard deviation = S X 100/x

Accuracy

Accuracy normally refers to the difference between the mean x, of the set of

results and the true or correct value for the quantity measured. According to IUPAC

accuracy relates to the difference between results (or mean) and the true value. For

analytical methods, there are two possible ways of determining the accuracy, absolute

method and comparative method.

a) Absolute method

The test for accuracy of the method is carried out by taking varying amounts

of the constituents and proceeding according to specified instructions. The difference

between the means of an adequate number of results and amount of constituent

actually present, usually expressed as parts hundred (%) is termed as % error.

The constituent in question will be determined in the presence of other

substances, and it will therefore be necessary to know the effect of these upon the


44

determination. This will require testing the influence of a large number of probable

compounds in the chosen samples, each varying amounts. In a few instances, the

accuracy of the method controlled by separations (usually solvent extraction or

chromatography technique) involved.

b) Comparative method

In the analysis of pharmaceutical formulations (or solid laboratory prepared

samples of desired composition), the content of the constituent sought (expressed as

percent recovery) has been determined by two or more (proposed and official or

reference) supposedly "accurate" methods of essentially different character can

usually be accepted as indicating the absence of an appreciable determinate error.

1.10 Comparison of Results

To evaluate the accuracy of the method, one often compares [148] the method

being investigated of 'test method' with an existing method called the 'reference

method.

a) Student t-test

Student t-test is used to compare the means of two related (paired) samples

analysed by reference and test methods. It gives answer to the correctness of the null

hypothesis with a certain confidence such as 95% or 99%. If the number of pairs (n)

are small than 30, the condition of normality of x is required or at least the normality

of the difference (di). If this is the case the quantity

ns

dt

d

i

/


45

If this is the case the quantity has a student t-distribution with (n-1) degrees of

freedom, where di =XR (Reference method) – XT. (Test method) and Sd is the

standard deviation (S).

b) F- test

By the F-test we can test the significance of the difference in variances of

reference and test methods. Let us suppose that one carried out n1 replicate

measurements by test methods and n2 replicate measurements by using reference

method. If the null hypothesis is true, then the estimates ST2 (variance of the test

method) and SR2 (variance of reference method) do not differ very much and their

ratio should not differ much from unity. Infact, one uses the ratio of variances.

F = ST2

/ SR2

It is conventional to calculate the F - ratio by dividing the larger variance by the

smallest variance in order to obtain a value equal or larger than unity. If the

calculated F - value is smaller than F - value from the table, one can conclude that the

procedures are not significantly different in precision at given confidence level.

46

Table 1.01

Functional groups imparting acidic nature

1.Functional groups imparting acidic nature to a substance

i Carboxylic acid - COOH v

Enediol

- C (OH) = C (OH)

I

ii

Imide

- CO

- CO

N - H

vi

Phenolic Hydroxyl

OH

iii

Thiol

- SH

vii

Sulphonic acid

- SO3H

iv Enol

C = C (OH)

Cond…..

47

Table 1.01

Functional groups imparting basic nature

2. Functional groups imparting basic nature to a substance

i Primary amino group (R1 = R

2 = H)

ii Secondary amino group (R1 = H, R

2= alkyl)

iii Tertiary amino group (R1 = R

2 = alkyl)

The tertiary nitrogen is the necessary element in the molecules of alkaloids, heterocyclic compounds and mono & di substituted

hydrazine derivatives R2 – HN – NHR

1

ON

R1

R2

N

R1

R2

N

R1

R2 - (CH2)n - N

R1

R2

R,

,

and

Cond…..

48

Table 1.01

Functional groups imparting neither acidic nor basic nature

3.Functional groups which impart neither acidic nor basic nature to a substance

i Aldehyde

R-CHO vii

Ester

- COOR

ii Keto R-CO-R

1

viii

Lactam

NH

O

iii

Hydroxy methyl

R-CH2-OH

ix

Olefinic

C = C

iv

Nitroso

- N = O x Acetylenic - C C -

v

Methoxy

-O-CH3

vi

Ether

R-O-R1

Cond…..

49

Table 1.02

Chemical features of some acidic dyes used in ion association complex formation

S.No Dye name / CI No. Chemical category Structure Chemical name

1

Alizarine Red S

(ARS) / 58005

Anthraquinone dye

NaO3S

HO

O

O

2-Anthrcene sulphonic acid

- 9,10-dihydro-3,4-

dihydroxy

-9,10-dioxo, mono sodium

salt

2 BTB Triphenyl methane BrH3C

CHMe2

H3C

Br

Me2HC

HO OH

HC

O

SO2

Dibromo thymol sulphone

phthalein

Cond…

50

Table 1.02

Chemical features of some acidic dyes used in ion association complex formation

S.No Dye name / CI No. Chemical category Structure Chemical name

3 MO Azodye

N NNaO3S N(CH3)2

4

Tropaeoline ooo

(Tpooo) / 14600

Azodye

NNNaO3S OH

Benzenesulphonic acid,4[

(4-hydroxy-1-naphthalenyl)

azo]-, mono sodium salt

Cond…

51

Table 1.02

Chemical features of some basic dyes used in ion association complex formation

Sl.No Dye name / CI No. Chemical category Structure Chemical name

3

Safranine-O

(SFNO)/ 50240

Azines

N

NH3C

H2N

CH3

NH2Cl-

Dimethyl pheno Safranin

4

Methylene Blue

(MB)/ 52015

Thaizines

N

SCH3

N N+

CH3

CH3

Cl-

CH3

Tetra methyl thionin

52

Table 1.03

STATEMENT SHOWING FUNCTIONAL GROUPS /MOIETIES EXPLOITED IN THE SELECTED DRUGS, METHODS PROPOSED,

REAGENTS, TYPES OF REACTIONS INVOLVED –DRUG WISE

Drug Estimated/ Chapter

No.

Proposed

Method

Reagents used for the

exploitation of functional

group/ moiety

Type of reaction

involved

Functional group/moiety

in the drug exploited

Naratriptan HCl/Chapter –

II

M1(a) ARS+0.1M HCl+CHCl3 Ion-Ion Association Tertiary Nitrogen

M1(b) BTB+0.1M HCl+CHCl3 -----------do---------- -----------do----------

M1(c) MO+0.1M HCl+CHCl3 -----------do---------- -----------do----------

M1(d) TPooo+0.1M HCl+CHCl3 -----------do---------- -----------do----------

M3 Brucine+NaIO4 Oxidative Coupling Secondary nitrogen

M5(a) MBTH+Ce(IV) Oxidative Coupling -----------do----------

M5(b) MBTH+ NaIO4 -----------do---------- -----------do----------

M5(c) MBTH+ NaIO4 +AcOH -----------do---------- -----------do----------

M5(d) MBTH+IBDA -----------do---------- -----------do----------

M6 FC-Reg.+Na2CO3 Redox Reaction -----------do----------

M8 Isatin+H2SO4 Condensation -----------do----------

M9 Vanillin+H2SO4 -----------do---------- -----------do----------

M10 ONB + H2SO4 -----------do---------- -----------do----------

M15 p-CA+ CHCl3 Charge Transfer Tertiary Nitrogen

M16 MO+Ce(IV)+ H2SO4 Redox/Association -----------do----------

M17 PA+ CHCl3 Ion-Ion Association -----------do----------

Cond…

53

Table 1.03


REAGENTS, TYPES OF REACTIONS INVOLVED –DRUG WISE


No.

Proposed

Method



group/ moiety

Type of reaction

involved



Levofloxacin

/Chapter – III

M1(a) ARS+0.1M HCl+CHCl3 Ion-Ion Association Tertiary Nitrogen

M1(d) TPooo+0.1M HCl+ CHCl3 -----------do---------- -----------do----------

M2(a) MB+ Buffer + CHCl3 Ion-Ion Association -----------do----------

M3 Fe(III)+O-PHEN Redox Reaction Aromaticring

M5(c) MBTH+NaIO4 + AcOH -----------do---------- -----------do----------

M12 PHH+NaIO4 +[Fe(CN)6]-3

Condensation Keto

M14 CTC+Nitrobebzene Complex formation Secondary nitrogen

M17 PA+ CHCl3 Ion-Ion Association Secondary nitrogen

M18 4-AP+ H2SO4 Condensation Tertiary Nitrogen

Cond…

54

Table 1.03


REAGENTS, TYPE OF REACTIONS INVOLVED –DRUG WISE


No.

Proposed

Method



group/ moiety

Type of reaction

involved



Ondansetron

HCl/Chapter – IV

M1a ARS+0.1M HCl+CHCl3 Ion-Ion Association Tertiary Nitrogen

M1b BTB+0.1M HCl+ CHCl3 -----------do---------- -----------do----------

M5a MBTH+ Ce(IV) Oxidative Coupling Nitrogen in Imidazol

M5c MBTH+ NaIO4+AcOH Oxidative Coupling -----------do----------

M7 NBS+CA Redox Reaction

M15 p-CA+ CHCl3 Charge Transfer Tertiary Nitrogen

M16 H2O2+MO Redox/Association -----------do----------

M17 PA+Buffer+ CHCl3 Ion-Ion Association -----------do----------

M18 4-AP+MeOH Condensation Keto Group

M19 INH+MeOH -----------do---------- Keto Group

Cond…

55

Table 1.03


REAGENTS, TYPE OF REACTIONS INVOLVED –DRUG WISE


No.

Proposed

Method



group/ moiety

Type of reaction

involved



Trandolapril

/Chapter – IV

M1a ARS+0.1M HCl+CHCl3 Ion-Ion Association Tertiary Nitrogen

M1b BTB+0.1M HCl+ CHCl3 -----------do---------- -----------do----------

M1c MO+0.1M HCl+ CHCl3 -----------do---------- -----------do----------

M1d TPooo+0.1M HCl+ CHCl3 -----------do---------- -----------do----------

M2a MB+ Buffer + CHCl3 Ion-Ion Association Carboxylic Acid

M2b SFN-O+ Buffer + CHCl3 -----------do---------- -----------do----------

M5a MBTH+Ce(IV) Oxidative Coupling Basic Nitrogen

M11 AMV+H2SO4 Redox Reaction -----------do----------

M15 p-CA+CHCl3 Charge Transfer Tertiary Nitrogen

M17 PA+ CHCl3 Ion-Ion Association Tertiary Nitrogen

Chapter – I Part- C Derivative Spectrophotometriy

56

DERIVATIVE SPECTROPHOTOMETRY

1.11 Introduction to Derivative Spectrophotometry

The quantitative investigations of broad spectra are frequently difficult, especially

where the measurement of small absorbance is concerned. But this difficulty is overcome

by derivative spectrophotometry (DS), due to the increased resolution of spectral bands,

allowing the detection and location of the wavelengths of poorly resolved components of

complex spectra and reducing the effect of spectral background interferences [149].

Derivative spectrophotometry is widely applied in inorganic and organic analysis,

toxicology and clinical analysis, analysis of pharmaceutical products, amino acids and

proteins, in analysis of food and in environmental chemistry. General analytical

applications of UV-visible derivative spectrophotometry have been reviewed [150-151].

The main characteristic of derivative spectrophotometry, the enhancement of the

resolution of overlapping spectral bands, is the consequence of differentiation which

discriminates against broad bands in favour of a sharp peak to an extent which increases

parallel to the derivative order [152].

1.12Pharmaceutical analysis by Derivative spectrophotometry

Due to its increased selectivity and sensitivity compared to classical

spectrophotometry, DS (Derivative spectrophotometry) is especially widely applied in

analytical chemistry for the determination of pharmaceutical compounds and trace

elements of similar chemical properties present in mixtures at different concentration

levels. For the purpose, the first and the second order derivative are usually used,

although in some cases higher-order derivatives provide more reliable results [153-


57

154]. Methods for the determination of organic substances by the DS technique have

been developed mainly for application in the analysis of pharmaceuticals and/or

clinically and biochemically interesting systems. The interference of the formulation

excipients or other UV-absorbing components, such as co-formulated drugs and

degradation products, usual in conventional UV- spectrophotometry can be

successfully eliminated by the DS technique.

A variety of procedures that render the DS determination of drugs more

specific and sensitive, regardless of whether they are determined as single compounds

or in mixtures, have been published. First-and second-order DS methods have been

proposed for the assay of the anti inflammatory drugs such as fentiazac, flufenamic

acid, tiaprofenic acid and proquaone [155], matronidazole in tablets [156],

Carboplaton [157], anthralin in ointments [158] and paracetamol in blood sera [159].

Aspirin, phenacetin and caffeine in analgesic tablets have been determined by zero

crossing derivative spectrophotometry [160]. Chlorpheniraminemaleate, codeine

phosphate and ephedrine hydrochloride [161] have been estimated without separation

using second-order DS. For quality control of pharmaceutical preparations containing

clozapine derivative spectrophotometric method is suitable for different levels of the

drug [162]. Simultaneous determination of atropine sulfate and morphine

hydrochloride in their binary mixture [163] using spectrophotomtric methods is

proposed by Dinc et al. A method for the determination of cetrizine

dihydrochloride[164] in pharmaceuticals by first, second, third and fourth-order

derivation spectrophotometry is described using “Peak-Peak” (P-P), and “Peak-Zero”

(P-O) measurements. First and second derivative spectrophotometry is applied for the

simultaneous determination of amoxycillin and clavulanic acid[165] in


58

pharmaceutical preparations. A simple and rapid derivative spectrophotometric assay

procedure is described for the analysis of casteine, acetaminophen and

propyphenazone [166] in tablet formulations. Caffeine [167] content is determined in

cola, coffee and tea by second and third order derivative spectrophotometry without

using any separation or background correction technique and reagent.

Second derivative spectrophotometric determination of trimethoprime (TMP)

and sulfamethoxazole (SM) in the presence of hydroxyl propyl-β-cyclodextrin (HP-β-

CD) has been reported [168]. A fast and accurate method for the determination of

dropenidol in the presence of methylparaben and propylparaben is developed using

derivative spectrophotometry [169]. Derivative spectrophotometry in the

determination of phenyl-β-naphthlaine (PBN) used as an antioxidant in rubber

mixtures have been described [170]. Simple, fast and reliable derivative

spectrophotometric methods are developed for determination of indapamide [171] in

bulk and pharmaceutical dosage forms. A derivative spectrophotometric method is

developed for the three binary mixtures of pseudophedrine with fexofenadine (mix.I),

cetrizine (mix.II) and loratidine (mix.III) [172]. Derivative procedures reported for

vitamin mixtures are concerned with pyridoxine hydrochloride and thiamine

hydrochloride in tablets (first and third order) [173], vitamins B6 B1 and B12 uridine 5-

triphosphate in injections (second order) [174], and sodium salicylate, thiamine

hydrochloride and ascorbic acid in visalicyl tablets (first and second order). First-

derivative measurements have been used to determine benzimidazole and cinnamate,

as well as benzophenone derivatives in order to characterize sun-screens in cosmetic

formulations [175]. First- and second-order DS have been described for evaluating

bilirubin, albumin and oxyhemoglobin in amnionic fluid [176]. First-order DS is used


59

for the determination of intact ceftazidime cefuroxime sodium and cefotaxime sodium

in the presence of their degradation products [177]. For a simultaneous determination

of acetaminophen and Phenobarbital after their extraction from the corresponding

suppositories with borate buffer, pH 10, a first-order DS method is developed [178].

Simultaneous analysis of a ternary mixture containing etamizole, paracetamol and

caffeine [179] is carried out by derivative spectrophotometry. First DS methods for

determination of triamterene and hydrochlorothiazide respectively in combined tablets

have also been described [180]. A first order DS method has been developed for the

simultaneous determination of rifamycin SV sodium and lidocaine hydrochloride in

injection solutions [181]. The simultaneous determination of ethinyl estradiol and

norgestrel in tablets utilizing first-order DS has been reported [182]. A method for the

simultaneous determination of melatoninpyridoxine combination in tablets by the

zero-crossing technique of the first and second-order DS has been reported [183].

This method is successfully applied for the determination of both drugs present in

laboratory prepared mixtures and in tablets [184]. For the evaluation of diclofenac and

benzyl alcohol as an excipient in injectable formulations, the first- and second-order

DS method using the zero-crossing technique has been described [185]. Three new

methods (first-order DS, ratio spectra DS and Vierordt’s method) for the quantitative

analysis of tablet formulations containing pseudoephedrine hydrochloride and

triprolidine hydrochloride are developed and compared [186]. A rapid, simple and

direct assay procedure based on first-order DS using zero-crossing and peak-to-base

measurements for the determination of dextromethorphan HBr and bromhexine HCl

has also been developed[187]. A simple and economical DS procedure is developed

for the simultaneous determination of indomethacin and paracepamol in combined

dosage forms [188]. Applying the zero-crossing technique of the second-order DS a


60

method for the determination of 1,4-benzodiazepin, midazolam[189] and

lorazepam[190] in tablets is developed. A second-order derivative UV

spectrophotometric method for determination of vitamin C [191] content in a variety

of natural samples is described. A second-order derivative spectrophotometric

method for the determination of bifonazole in the presence of methyl-and propyl p-

hydroxybenzoate as preservatives has been developed [192].

UV derivative spectrophotometric method to quantify Losartan potassium

[193] in pharmaceutical formulations is developed. A procedure has been developed

for the fourth derivative spectrophotometric determination of

tetramethyldithiocarbamate [194-195]. Paracetamol and phenoprobamate are

determined by first-order derivative spectrum [196], mixtures of cocaine, procaine

and lidocaine in pulver samples by second-order derivative spectrum [197],

paracetamol and caffeine in tablets by first- and second-order derivative spectrum

[198], acetylsalicylic acid and free salicylic acid in sustained release tablets [199],

cetrimide and chlorhexidine glyconate in antiseptic solutions by first-order derivative

spectrum [200] have been described. Fourth-order derivative spectrum procedures

have been used for the determination of clopramide and pindolol in tablets [201],

lidocaine hydrochloride and 5-nitrox in liquid formulations [202]. Derivative

spectrophotometric methods have been described for the assay of phenobarbitone in

mixtures with oxyphenonium bromide and meprobramate, paracetamol, or acetyl

salicylic acid [203], procaine hydrochloride with benzoic acid, pyridoxine

hydrochloride, 4-aminobenzoic acid [204], sulfanilamide and sulfadiazine [205] and

sulfamethoxazole and trimethoprim[206].

general introduction - information and library...

Documents