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STUDIES IN APPLICATION OF ORGANIC REAGENTS IN
ANALYTICAL CHEMISTRY

DR. SUDHIR S. KAMAT

PH.D., M.Sc., F.I.C.

(Professor Emeritus)

Mumbai University.

1
 ACKNOWLEDGEMENT

I am very much grateful to late Dr. R. T. Sane, M.Sc, Ph.D (Ireland) my research guide, for his
valuable and purposeful guidance. I thank all my colleagues, friends and near once whose constant
encouragement and help kept my spirit above level, all the time, throughout the studies.

My special thanks to Mr. Raju P. Suryawanshi, for encouraging me for this publication
project and helping me through the manuscript preparation.

2
 INTRODUCTION

The drug abuse problem is a serious threat to mankind in general and young generation in
particular. This has wide ramifications in increasing crime rate and depleting family and social
moral values. There are different types of chemicals hazardous substances like insecticides /
pesticides, narcotics and psychotropic drugs. They are misused for suicidal/ homicidal purposes. It
is therefore constant tussle for Analytical Chemist to make available simple, quick and accurate
laboratory methods for the identification and determination of such compounds which will be useful
to medical personnel and crime detection team / judiciary.

The problem of “ Laboratory accuracy relates to the age old conflict of high sensitivity versus test
specificity the present work is an humble attempt to provide the analytical methods for various
compounds which are involved in number of fatal / nonfatal cases.

In this era of most sensitive instruments of chemical analysis like High performance liquid
chromatography (HPLC), Fourier Transform infrared spectrophotometry (FTIR), X-ray powder
Diffraction, Atomic Absorption spectrophotometry (AAS), Chemists in general and Analytical
Chemists in particular have forgotten the beauty of colour and chemical constituents. All those
instruments will not be useful to identify any unknown compound individually or in a mixture.
However, the chemical reagents will give specific colour with particular compound for the
identification as well as quantitation of the compound. The colour and chemical constitution theory
has always excited me during my career. The beauty of such chemical reaction increases, when a
selective compounds gives colour and another compound with same functional group do not give
any colour. For example acetaldehyde CH3CHO gives colour with 2-Thiobarbituric Acid whereas
formaldehyde with aldehyde group do not give any colour with 2-Thiobarbituric Acid.
Organothiophosphates having -P=S develops brilliant blue colour with Benzophenone reagent but
other sulphur containing organic compounds like sulphonamides or organophosphates do not form
any color with benzophenone. The methods describe in this book are published during the course of
studies, in International peer reviewed Journals / books (1- Clark’s Isolation and Identification of
Drug substances – By E. G. C. Clerke- Edited by Moffat, A. C et. Al pp 584, 1986. Pharmaceutical
press, London, 2- The Agrochemical Handbook of Royal Society of Chemistry Information Service,
University of Nottingham, England, Second edition p-177).

3
 INDEX

Chapter No. CO N T E N T S Page

Number

1 General introduction to colour formation and measurement of 05-18

colour.

2 Colorimetric determination of Organothiophisphate insecticides. 19-47

3 Colorimetric determination of Paracitamol. 48-65

4 Colorimetric determinations of acetaldehyde in presene of 66-89

formaldehyde and ethyl alcohol in presene of methyl alcohol.

5 Colorimetric determinations of organochloro insecticide 90-108

Endrine.

6 Bibliography 109-120

7 List of Publication 121

4
 CHAPTER I

INTRODUCTION 

5
 A@ General Analytical methods.

B@Color formation and Measurement.

C@Advantages of colorimetry over methods.

D@Limitations of colorimetric methods.

E@Necessity of this type of study.

6
 Out of the curiosity to find out the secrets and facts of nature, man has started analyzing
everything around him.During the process, he has learned that everything in the nature is made up
of some definite factorswhich he named Chemical Elements. This in fact is the starting point of the
Analytical ChemistryAs such the Analytical chemistry is as old as human race and it is the most

fundamental branch of science Today we can establish the identity of everything around us by
comparing the physical or chemical properties, which shows that analytical chemistry is not only
the fundamental branch, but the most essential branch of scienceAccording to the availability of
resources and individual intelligence of analyst, the analytical methods were developed.During the
course of time new methods have taken place of the old ones rendering the latter less effective or
sensitive with the ultimate desire to reach “Vanishing zero”.Man has learnt that mere identification
will not solve all the problems and quantitative determination is very much essential This made
man to develop mathematics as means to measure and calculate, and then started the quantitative
chemical analysis It is rather impossible to find the exact period of start of alchemie. Evidences

were found that skillful metal workers of Mohenjodaro C 2000 BC used copper arsenic alloy, for

its hardness, for making toolsThis clearly shows that alchemie must have taken shape much earlier

to this, as the alloy making essentially requires knowledge of mixing metals quantitatively The

rapid growth of alchemie is attributed mainly to the medicinal uses of plants During this long
course of development of alchemie various methods for the quantitative determination have been
found out such as

1@Gravimetric a Precipitation

b Electrodeposition

 c Volatilization

2@Volumetric-

Gasometric: Oxygen / CO2 contents of blood (Van Slyke instrument).

7
 Titrimetric  a Neutralization.

b Oxidationreduction

 c Precipitation

d Complexation

e Nonaqueous.

3@Biological a Animal assay.

 b Microbial assay.

c) Immunologiacal.

4@Resonancea Electron spin resonance ESR 

b Nuclear magnetic resonance NMR 

5@Physicochemical Electrical Methods a Photometric

b Conductometric

c Polarographic

6@Optical  a Nephlometric

 b Turbidometric

 c Colorimetric

d Fluorometric

e UV spectrophotometric

 f IR spectrophotometric

7@Chromatographic a Column chromatography

b Paper chromatography

8
c Thin layer chromatograph

 d Gasliquid  Gassolid chromatography

e High pressure liquid chromatography

8@Radioimmuno assay

9@IonizationMassa Chemical ionization

b Electron ionization

10@Differential thermal analysis

Every method has its own merits or advantages and disadvantages Therefore, even today

conventional methods like volumetric and gravimetric go handinhand with modern instrumental
methods of analysis such as Gas Chromatography, differential thermal analysis etc. The modern
instrumental methods will not identify an unknown compound. They will confirme the
configuration of the compound. e.g. Carbonyl group in an organic compound will show IR peak
around 1680 nm whereas, thiocaronyl group will show IR peak around 1180 nm.

But it is the color more often, responsible for man’s enhanced interest in the chemistry, and this
very visual aspect of science makes for much of its appeal to mankind in general and scientist in
particular One can only imagine the man’s urge, enthusiasm and restlessness to understand the

spectrum of rainbow when he must have seen it for the first timeToday color plays most vital role
in life.Color has long been exploited by research scientists and industrialists in almost every field
of life and in different arena such as from consumer products to staining of biological cellsVisible

spectroscopy is a complex phenomenon than the comparatively simple UV spectroscopy The

complexity of the most chromophoric colored systems and diversity of chemical types exhibiting
colors makethe systematic classification o structure color relationship very difficultIt is generally
true that color making and color using industries contribute greatly to the economy of any
industrialized country In view of the commercial importance of coloring matters, considerable
interest has been shown in the theoretical and empirical evaluation of relationship between colors
and molecular structuresThis interest has been accentuated by widening the areas in to which color
and colorant now introduced, and color structures relationship are now of vital importance in
various analytical disciplines in generalColorants envade our total environmentClothings, carpets,

9
painted walls, plastic veneers, foods, cosmetics, household items and even medicinal preparations,
all contain coloring matters Even so called white object such as paper are normally rendered

“whiter than white” by the incorporation of special fluorescent brightening agents In the board

scientific field of analysis, color is of paramount importance Thus, dye and chemicals become
more than pretty chemical curiosities to the analytical chemists, physicians in diagnostic medicines
and to the biologists involved in histophayhological studies

Formation of color

A certain amount of unsaturation in the molecule, with a part of it at least in the form of aromatic
rings, combined with structure of a minimum complexity usually lays the foundation of the
molecules that we recognize as colored dyesMuch has been correlated between the structure and

colour1

The general equation

Chromogen Auxochrom Colored complex or dye is widely accepted

The chromogen is an aromatic body containing a group called a chromophore By derivation

chromophore means colorgiver and generally represented by chemical radicals such as: 

a) The nitroso group NO  NOH

b) The nitro group NO2  NOOH

c) The azo group N N

d) The ethylene group>C C<

e) The carbonyl group>C 0

f) The carbonnitrogen groups>C NH & CH N

g) The sulfur groups >C S & >CSSC<

Such groups add color to the simpler aromatic bodies by causing the displacement of, or
appearance of, absorbance band in the visible spectrum These chromophores are so

important that many dyes are chemically classified by the chief chromophore they contain
These chromophore groups on reduction frequently loose the color, probably owing to the
removal of electron resonance Close packing of unsaturation, as conjugation, also tends

towards colorThus, even the hydrocarbon dimethyl fulvene has an orange color.

10
 The color depends upon the wavelengthThe wavenumber scale is directly proportional to
the frequency of the wave and according to the Plank equation
E hv

E photon energy , v frequency of wave and h Plank’sconstant 6625 x 1027 erg/sec.)

The wave frequency is proportional to photon energy or the electronic excitation energy of
the absorption band
Eexited state -Eground state hv

Accordingly, the lowest energy function is the ground state of molecule, and the change in the
energy function will eventually decide the color of the substance

It is often found that unsaturated molecules containing nitrogen, oxygen or sulfur atoms show long
wavelength absorption bands of low intensity, that are not present in the corresponding hydrocarbon
systemsMulliken2 was the first to interpret these bands as transition of lone pair electrons peculiar

to the heteroatomKasha3 in 1950 suggested the designation of these bands as

ղ>  ∗ transitionsThese transitions thus correspond to the excitation of an electron from the

lone pair nonbonding orbital of the heteroatom in to one of the vacant usually lowest   ∗

orbitals of the moleculeThe more negative the heteroatom the more firmly will the lone pair

electrons be held The nonbonding orbitals of a sulfur atom will be higher in energy less stable)

then those of the more negative oxygen atomThis is reflected in the ionization potential of oxygen

136eV  and sulfur 104 eV , where 1 eV  160210 x 1012 erg per particle Since an increase
inelectronegativity of an atom lowers the energy of the orbital, it follows that the more
electronegative heteroatoms show ղ >  ∗ band at shorter wavelengths than the less

electronegative heteroatomsWhen ղ>  ∗ excitation occurs, one of the n electrons is removed

from the nonbonding orbital and promoted to   ∗ orbital effectively, destroying the hydrogen

11
bond The excitation energy is thus raised by an amount roughly equal to the strength of the

hydrogen bond and a hypochromic shift is observed 4 Thio-ketones are brightly colored ranging
from red in the case of alkyl and cycloalkyl compounds to deep blue in the case of aromatic
derivativesThe colors is due to the ղ>  ∗band of the thiocarbonyl group and generally lies in
5,6
the range 500700 nm The ionization potential of the sulfur is much lower than that of oxygen,

which shows that lone pair electrons on sulfur are much less firmly held than that on oxygenAryl

conjugation gives a large bathochromic shift of ղ>  ∗ band and in thiobenzophenone derivatives

the band generally occurs at about 600 nm in nonpolar solvent It has been shown that ղ>  ∗

transition of thiobenzoophonone is polarized along the C S b and which is to be expected if the

transition has some >  ∗ character7 Aryl substitution in the thiobenzophenones exert a small

effect on the portion of ղ>  ∗ band, λ max generally increasing with the electron withdrawing

power of the substituents The bathochromic shift during conversion of the phenols to phenolates
can be extended to visible region if quinones are formed.

Measurement of Color formed

The intensity of the color formed can be measured by photoelectric colorimeter, where rays of
definite wave length pass through the solution of certain thicknessThe absorption of light radiation

passing through the colored solution depends upon two factors i  concentration of solute and ii 

path length of cell through which light beam passesThe law governing this relationship is known

as BouguerlambertBeer law of light absorption and is expressed as

follows

It Io.10∈C L

Whence log IoIt ∈ CL

Where IoIntensity of incident radiation

ItIntensity of transmitted radiation

Cconcentration of solution, mollit

L -Path length of cell, cm

12
∈Molar extinction coefficient, lit. mol-1. Cm-1

The common logarithm of the ration of the intensity of incident light to intensity of transmitted light
is known as absorbance of solution and is expressed as

A log IoIt

Or A ∈C L

It follows, therefore, that absorbance or optical density is directly proportional to the concentration
of the solute in the solution and the thickness of absorbing layer

Molar extinction coefficient

It is the coefficient of proportionality and it is equal to the optical density of solution having
concentration of colored substance 1 mole per liter when the thickness of absorbing layer is 1cmIt
can be calculated as

∈=ACL

Or molar extinction coefficient Absorbance cell path length x molar concentration

It can also calculated as

Molar absorptivity S x molwtx 103

Where, S slope of graph obeying Beer’s law

Maximum sensitivity in a colorimetric procedure is obtained at a wavelength showing maximum

absorbance (λ max) though it is not the only criterion for the selection of proper wavelength for
photometric measurement.From analytical point of view, the most satisfactory wave length is the
one which , at a given depth of solution , obeys Beer’s law over as wide a range as possible ,
facilitating this range to be read with in the most accurate region of photometric scale

The colorimetric methods, though not as sensitive as methods like GLC, HPLC, etc, are very
simple and do not involve high cost.Colorimetric methods are less time consuming in comparison
to the time required for cleanup of the GC columnsColorimetric methods are easy to carry out as

no specially trained operators are necessary as in the case of other instrumental methods of analysis

These factors are very important in a country like India

13
Reason for selecting particular compounds for the present study

The misuse of chemicals and drugs is as old as history of mankind The deliberate misuse of
chemicals is the silent weapons capable of destroying life mysteriously, secretly and without
violence.They have played very large part in the history at various periods, in romance as well as in
crime. Poisoning is the cruelest act and it is equally difficult to prove without proper analytical
techniques

Apart from such misuse of drugs and poisonous chemicals, pollutions and public health are also
important factors. During 1970, nearly 40,000 tons of pesticides were used, both in the field of
agriculture and public health in India and India has the lowest consumption of pesticides of 180 g
per hectare as compared to 1490 g in USA, 1870 g in Europe and 10790 g in Japan.

Organothiophosphate insecticides like Malathion, Parathion etcshow the maximum percentage of

50 to 70 followed by alcohol 25 to 30 and organochloro insecticides like Endrin 12 to 258.
Paracetamol, chemically acetaminophen, a compound widely used in antipyretics and analgesic
formulations world over, is the third rank as a killer drug as per Registrar General of England and
Wales9.

It was, therefore, thought worthwhile to find out easy methods for their quantitative determinations
which can be carried out in any laboratory, without the use of any high cost instrumentThis work

is a humble attempt to provide such methods having precession, specificity and sensitivity

Statistical parameters

Any measurements, whether, it be the determination of weight, volume, time, density or absorbance
etc, is only of limited accuracyThe accuracy is best define as the closeness of agreement between
an experimental value and the true value, while the precision is the closeness of agreement between
replicates experimental valuesThe closer these values the more precise are the method; the values

may not however agree with true value It is, therefore, necessary that a method must have both,

accuracy and precision The result will differ slightly from real figures and will, in all probability,

differ from replicates measurements made under apparently identical condition It is necessary to

have an index of its reliability which can be obtain by statistical parameters

A set of values will have variability giving rise to the deviation from arithmetical mean valueThe

average deviation or mean deviation is usually measured in relation to arithmetic meanThe mean

14
deviation is obtain by taking the sum of deviations of items from arithmetic means, without regard
to signs, and dividing by number of observations of items, thus

Accuracy of result

The standard error of the mean,

15
Where the standard deviation of the mean is defined as .

The value of t, can be obtained from standard table, and it depends upon the number of degrees of
freedom n1 and level of probabilityThe probability limits are generally listed as 05, 01, 005,

002 and 001It is customary to use the 005 level to determine the limitsThis means that 95of

readings will be within the calcu lted limits

Where,  t value from the table for 005 probability for high levels of significance,

the 001 level is used, which means that 99of the readings will be within the limit ,

 Where (‘t’ value from the table for 001 probability The variance

is measured of dispersion of observations around the mean values and is given by SD 2 The
coefficient of variation is calculate by the formula

Therefore, throughout this work, the statistical parameters are calculated using following
formule10,11

16
Accuracy of estimation for 95and 99probability limit

A typical set of calculation is shown below

Following is the set of concentrations determined in six experiments for a formulation

ExptNo Concentration determined Mean deviations

1 198 1981992
2 200 2001992
3 198 1981992
 4 199 1991992

 5 200 2001992

6 200 2001992

n 6 Mean 1992 SD 09831

n1 5

Meanconcentration 1992

Standard deviation 09831

17
t for 95probability is 2571

Therefore 95confidence limit 1992 ± 103

And 99confidence limit 1992±162

References

1 Watson, ER¯Color and its relation to chemical constitution, Longmans green and

Co, Inc, New York, p57,1918

2 Mulliken, RSJChemPhys3, 564,1935
3 Kasha, MDiscussions Faraday Soc2, 14, 1950
4 Griffith, J ¯color and constitution of organic molecule°, Academic Express,

London , p77 , 1976

5 Ibid ¦p136
6 Korver, O, Veenland, JUand Boer, TJde,RecTravChim84, 289, 1965 cf

CA6214459d ,1967
7 Gupta, SD, Choudhari,Mand Bera, SCJChemPhys53, 1293, 1970
8 Bami, HLJIndAcadForensSCI11,164167 , 1972
9 Registrar General¬s Statistics for deaths by poisoning ,1972cfCIBA Foundation

Symposium 26 New Series , Associated Scientific Publisher, New York &

London, p246,1974
10 James, A M ¯Practical Physical Chemistry°, J&A Churchill Ltd, London, p8,

1961
11 Croxton, FE,Cowden, DJand Klein, S¯Applied General Statistics3rd Ed

Appendix I, Prentice ¦Hall of India PvtLtd, New Delhi, p670671, 1973

18
 CHAPTERII

COLORIMETRIC METHOD FOR THE DETERMINATION OF

ORGANOTHIOPHOSPHATE INSECTICIDES

 Determination of Organothiophosphate Insecticides

19
PART - I

a) Development of organothiophosphate insecticides

b) Chemistry and nomenclature of organothiophosphates

c) Metabolism and Toxicity of the compounds

d) Review of the reported analytical methods

PART - II

a) Chemistry of benzophenone and thiobenzophenone.

b) Reaction of the proposed method

c) Proposed Methodi Standard Curve

ii Recovery

iii Determination from marketed formulations

iv Determination from biological materials

d) Results and Discussions

e) References

LIST OF TABLES

1) Table IUV and IR absorption peaks of benzophenone and thiobenzophenone

2) Table IISpectral Shift in thiobenzophenone due to substitution

3) Table IIIOrganothiophosphates giving the reaction

4) Table IVOrganic -S- containing compounds not responding to the reaction

5) Table V: Determination of Malathion by A. O. A. C. method and proposed method.

FIGURES

1) Nomenclture of thiophosphates
2) Absorption maxima
3) Standard curves for Malathion, parathion, sumithion, feanthion, dinethoate, Phorate
4) Recovery from standards
5) Effect of the heating time and stability of the color formed
6) GCMass of the reaction product

20
PART I

DEVELOPMENT OF ORGANOTHIOPHOSPHATE INSECTICIDE

Prior to 1940’s, farmers around the world sprayed their crops with insecticides such as
lead, arsenic, fluoride and exotic natural products like nicotine, pyrethrin, retenone etc
Occasionally Food and Drugs Administrations of various Governments used to analyze
the fruits, vegetables, dairy products etc for residual pesticides, but there were no

regulations for the minimum permissible limitsFirst such regulations were instituted in
1927 allowing a permissible limit for Paris Green and lead and latter for arsenic and
fluoride in raw agricultural crops to be set at 35 to 70 ppm1, for the determination of

which Hoskins2 and Cassil3 developed quantitative analytical methods

During World War II, the organic derivatives of phosphoric acid were developed in
Germany, as war gases, the so called “nerve gases” Since the war, they have been

intensively studied as nicotine substitute for the pest control German Chemists

discovered a new class of powerful insecticides, the organophosphates 4 During the

mid40’s an agricultural revolution took place in most of the advanced countries, as a

result, the production and use of such chemicals during the past decade and a half has
increased tremendously
Though there are virtually non toxic an organophosphate esters has ethyl phosphate,
certain aryl phosphates having technical importance in plastics, rubber and petroleum
industries, most of the organophosphate esters, possess an insidious neurotoxic action
Many of such organophosphorous insecticides are highly toxic in man and domestic
animals This necessitated the passing of regulation that would accommodate the
synthetic pesticides under the food and drugslegislation providing the safe use of these
compoundsThe concept of such lowpertaining to pesticides was that the presence of
any poisonous or deleterious pesticides chemicals in the daily diet of the population was
undesirable unless a “safe”tolerance limit for these chemicals had been established by
the government With the lapse of time, many of them have been found to be

carcinogenic
When parathion was first introduced in 1944 as pesticides, there were many
fatalities In countries like India, Japan, Egypt where pesticides are extensively used
under somewhat primitive condition and are easily accessible as bug killers or

21
cockroach repellents etc, death due to such chemical muster a very large number In
recent year organophoshates have become a very handy and effective means of
committing suicides or homicides Chemically finite tolerance can now be established

which are based on sensitive method of analysis

CHEMISTRY AND NOMENCLATURE OF ORGANOPHOSPATES

The first esterificationof alcohol and phosphoric acid is attributed to Lassaign5 in
1820. In 1903 and 1915, Michaelis published long paper dealing with the synthesis of
compounds containing phosphorous and nitrogen, particularly amidated of phosphinic,
phosphoric and phosphorothionic acid6, 7 In 1906 the synthesis of a number of
phosphastes and phosphonates were reported, which were forerunners of the compounds
used as anticholinestrase todayEster of phosphoric acid was made in 1913148, 9

In 1932, Lange and Won Krueger prepared some dialkyl phsphorofluorides10 In all

this early work, one finds no mention of poisonous nature of these compounds It is
only during the war their strength as potential chemical war fair agents, was recognized
by British and German scientistsThe insecdicidal importance of thes comppounds was
realisedc by the end of the war and many of the insecticidal phosphates which are being
used currently thesnthesiesed
The term ‘organophosphates’is a lose one representing all compounds which contain
carbon, oxygen and are derivatives of phosphoric acid Therefore, the terms

‘phosphates’ ‘Phosphorohionates’ ‘phosphorothiolates’ etc, hav been suggested. The

nomenclature of the organophosphates has been in a confused state, but in 1952 and
agreement was reached between Britin and USA upon naming of the compounds
11, 12
containing one phosphorous atom   The pattern of nomenclature is shown in the

fig1 Unfortunately, these ‘transatlantic’ rules of nomenclature are not accepted

internationally Subsequently there are five different systems of nomenclature13, two

American, One British, One German and the new Anglo - AmericanThere is a six one

recommended by Sweden

22
23
METABOLISUM AND TOXICITY OF ORGANOTHIOPHOSPHATES

Organisms of any kinds may be killed mechanically, physically or chemically

Mechanical killing means gross destruction by flyswatter, by fire, by squashing or
entangling materials, mechanical abrasives By physical, it means the action of agents

which kill by interacting with body components eg electric shock The chemical
means of killing is the term used for action where an agent kills by reacting usually
specifically with a body ingredient without disturbing physically This class includes

most of the insecticides, drugs, fumigants and chemicals In some cases, clearcut

chemical reaction involving covalent bonding occur eg hydrazides react with

pyridoxal phosphates vitamin B6  In other cases, weaker bonding may be involved,

24
such as ionic, van der Waals or hydrogen bondin in both the cases however; there is
molecular specificity of the reactionProtonated and unprotonated forms differ radically
in polarity and hence in permeability and partitioning properties. This is a very
important factor while studying the metabolic fate of any compounds.
It is widely accepted that organophosphates kill animals, both vertebrates and
Invertebrates, by inhibiting the enzymes cholinesterase space with the consequent
disruption of nervous activity caused by accumulation of acetylcholine at the nerve
ending14, 15. Cholinestrase is a vital enzyme and its severe inhibitionis associated with
death In comparison with chlorinestrase the inhibition of other enzymes by

organothiophosphates is very poor

The clear visible symptoms of organothiophosphates insecticides poisoning are
parasympthatic stimulation, specially defection, urination, lachrymation, contraction of
pupil, slowing of the heart and consequent drop in blood pressure16

These effects are called muscarinic effects In addition, nicotinic effects involving the
neuromuscular junction are seen, at first excitatory, leading to muscle Tewitching, then
inhibitory leading to paralysis Convulsions are usual in severe poisoning and are

primarily clonic that is with rapid repeatative movementsDeath is due to asphyxiation,

ielack of oxygen, due to respiratory failure17, 18, 19, 20

Chemical degradation of all the thiophosphate in the body takes place through various
stages finally leading to oxidative form This activation forms the oxidative form

increases their potency as cholinesterase inhibitore21 The commonest activation is the

conversion of phosphorothionates or P S compounds to phosphtes or P O compounds

Thus, parathion is converted to paraoxane and Malathion to malaoxan Though this

conversion is often called as oxidation, in fact no change of valence state occure and
therefore does not require such activationThe reason for effctiveness of such activation

is found in electronic factor. S is less electrophillic than O and consequently the P in

most P S compound is inadiquently positive to undergo rapid reaction with

cholinesteraseThis very electronic affinity theory is responsible in the present reaction

of phosphorothionates with benzophenone, S from phosphorothio compound is being

replaced by strong electrophilic O from benzophenone converting colorless

benzophenone to blue thiobenzophenone

25
REVIEW OF THE ANALYTICAL METHODS

Various methods are reported for the determination of organothiophosphates
insecticidesMost of them are based on the phosphoric acid moiety in the compounds22
23
Getz and Watts reported a rapid colorimetric method for the determination of
organophosphates pesticides, by using the reaction with 4 pnitrobenzyl pyridine in a

slightly alkaline solution at 1750180 0cTurner 24 has further modified the method and

carried out the reaction at slightly lower temperature of 1000 c The color is very
unstable and stability varies according to pesticides occasionally giving erratic result
due to rapid evaporation of the solvent Ott and Gumther25 have reported a method of
determination of organophosphates by wet digestion oxidation or alkali hydrolysis
followed by wityh ammonium molybdate and 1amino2 napthol4 sulphonate

forming molybdium blue complex There are certain methods useful for individual

organophosphorus pesticides A method involving formation of copper complex has

26,27,28,29
been reported for the determination of Malathion  A method for the
30
detrermination of feni trithion residue from milk is reported where alkaline hydrolysis
of fenitrithion gives the yellow 3methyl 40 nitrophenateSimilarly, hydrolysis to the
corresponding phenols was applicable to the residue of fenchlophos and
dichlorofenthion using 4aminophenazone as a reagent31Enos and frear 32
reported a

method for dimehtoate based on the formation of methylene blue Formation of a

complex betweendimethoate and 1chloro24dinitrobenzene is claimed by George et

al33. Recently a method based on the reaction mercurous nitrate with pphenyl

organophosphate insecticides has been reported34 Several workers have used an

enzymatic method of cholinesterase inhibition35,36,37,38 The method is based on the

determination of unused acetylcholine left at the end of the enzyme substrate reaction
Such esterase activity measurement is used only as screening test provided negative
results are obtained For this enzyme inhibition method, the previous identification of
insecticides is essential as the enzyme cholinesterase show inhibition towards many
compounds like organochloro39, barbiturates14, carbamates40, cholral hydrates14 etc.
Fisk etal41 has reported an ultraviolate scanning method for the   Determination of

organothiphosphates from dermal residue gastic lavage fluidThe color change procede
by the acid involved in the feebly buffered phenol red solution has been used by
26
 Gregoire42The turbidity caused by production of acid in a casein solution was used to
43
follow acetylcholine hydrolysis, Avrell and Norris reported a method were patathion
ios reduced with zinc and hydrochloric acid to the corresponding amino compound
which is further diazotized and coupled with N1 napthylethylene diamino to yield a

magenta dye A method based on methylene blue dye formation by reacting

organothiophosphate with paminodimethylaniline and ferric chloride is reported by

Suter et al44
Apart from colorimetric methods, there are a number of other methods based on
45,
the techniques such as the paper chromatography thin layer chromatography46, gas
liquid chromatography47, high pressure liquid chromatography 48
and GCMass 49,50

which are used for quantitative determination of such insecticides

PART II

CHEMISTRY OF BENZOPHENONE AND THIOBENZOPHENONE

Benzophenone is a diphenyl ketone with molecular formula C13H10OIts molecular weight is

18221 with the elemental analysis C8569; H553and O878It is orthorhombic colorless

prism having melting point 485o Cthe UV51and IR52,53 absorption peaks of benzophenone are

shown in table I

It boils at 360OCIt is generally used as a good fixative for heavy perfumes such as geranium, new

mown hay etc, used in manufacture of antihistamines and hypnotic preparations and insecticides
Its structure is as follows.

27
 TABLE I

Compound UVVisible absorption in ethyl IR principal peaks in KBr

alcohol medium

Benzophenone 250, 335 nm 1651, 1671 cm 1

Thiobenzophenone 235, 260nm 1180, 1220cm1
3165, 599 nm

Thiobenzophenone is S analog of benozophenoneIt is dipheyl thioketone, having molecular

formula C13H10S and molecular weight 19821In the natural form, it is blue needle shaped crystals

having melting point 53O54OCBrocklehurst and Burawoy54 studied UV and visible spectra of

thiobenzophenone extensivelyUVvisible and infrared peaks of thiobenophenone are shown in

Table IIts structural formula is

Thiobenzophenone in ethanol shows i a Kband of fairly high intensity λ max 3165 which

originates in a transition involving and electronic migration along the conjugated system, ii a R

band of fairly low intensity max 599 , a characteristic of thiocarbonyl group and iii an

absorption band at much shorter wavelength λmax 235 which may be due to the solvent used

There is shift in peaks due to different solvents as well as various substituents in benzene rings of
thiobenzophenone54Substituents of the chromophoric double bonds have the same qualitative

effects on Rbands as substituents in benzene ring, of an absorbing system on KbandsRbands are

displaced to shorter wavelength influence such as solvent effect, proton addition, substituents,
hydrogen bond formation etcwhich increase the polarity of double bonds55,56 Effects of

substituents on Rband and Kband of thiobenzophenone in ethanol are shown in table II

28
 TABLEII

Spectral shift in thiobenzophenone due to substitution at X & X’

Substituents RBand D Kband D

o
Px Px¬  max A o
∈ A  max A o
∈ Ao

H H 5990 1858  3165 15600 

H OH 5800 254 190 3685 16000 520

OH OH 5662 370 128 3595 25000 430

CH3O H 5800 175 190 3595 12100 430

C6H5CH2O H 5875 275 115 3610 17000 445

CH3O CH3O 5770 288 120 3580 20000 415

CH3 2N H 5475 452 515 4390 23600 1225

CH3 2N (CH3)2N Inflexion  4330 37000 1165

C6H5 H 6005 246 15 3545 17800 380

C6H5 C6H5 6025 318 35 3568 25000 403

REACTION OF THE PROPOSED ANALYTICAL METHOD:

The synthesis of thiobenzophenone from benzophenone was first reported by Staudinger and
Freudenberger57 in 1928In their first method H2S and HCl gases were passed simultaneously in a
solution of benzophenone in ethyl alcohol for three hours.H2S was continued to pass for another 20
hours with constant icecoolingThe method is time consuming and the reaction conditions are very

critical

29
In 1943 another method was reported58, where benzophenone dichloride was made to react with
sodium hydrogen sulfide under reduced pressure and high temperatureThis method also requires
critical reaction conditions

In the proposed reaction benzophenone was directly heated with organophosphorothionates over a
low flame Since  0 is having more affinity towards P than  S, ‘desulfuration’took placeThe

O in benzophenone was replaced by S forming blue thiobenzophenone The blue mixture

contained thiobenzophenone III), excess benzophenone (I)and oxidised or desulphurated product

of organothiosulphate IV) This reactionwas confirmed by GCMass and UV visible spectral

studies 59, 60 represented in fig2 and fig6The reaction is as follows

30
Thiobenzophenome colour after reaction (III)

The reaction mixture was dissolved in isooctane and the solution was washed several times with

20 aqueous methanol till the washing become clear and colorless The brilliant blue isooctane

layer was washed five times with distilled water, dried and solvent removed under vacuum The

product was chrmatographed on a column of which 225 cm was packed with silica gel 60120mμ)

and 75 cm with TLC grade silica gel GThe column was eluted with petroleum ether (40-60 oC):

iso octane mixture 5050vv The solvent from the collected eluate was removed under nitrogern

atmosphere The blue needles thus obtained gave melting point 5353oc various
organothiophosphate compound (shown in table III) being used in insecticidal formulations, were
subjected to this reactionThe thin layer chromatographic studies of the reaction products of these

compound showed bluespot having the same Rf (069) for the same solvent system iso octane
31
benzene 64  UV and visible spectra of the blue compound in ethanol showed a kband of high

intensity at 3165nm and Rband of fairly low intensity at 5988 nm fig2 This is in confermation

with the earlier work of Broklehurst and Burawoy54

Some of the benzophenone derivatives like 44’dimethylaminobenzophenone were treated with

organothiophosphate insecticide No appreciable change in sensitivity was observed Since

benzophenone was easily available than its derivatives, it was found to be the best choice for the
routine analytical method based on this reaction

32
Table- IIIA-Above comppouds responds to roposed radtion due to P=S in the structure

33
Table IIIB- Above compounds do not give thiobenzophenone reaction.

34
COLORIMETRIC METHOD:EXPERIMENTAL

Material 1@Benzophenone

2@Methyl alcohol

3@Acetone

4@Malathion (Analab IncUSA)

All the chemicals used were of analytical grade except specified otherwise, and were absolutely free
from moisture

Preparation of the sample

 10 g of the material under test was accurately weighed, extracted five times with solvent
ether using 100ml ether each time Ether layers were together and evaporated to dryness under

vaccumThe residue was dissolved in about 70 ml acetone and made up to 100ml in a volumetric

flask using solvent acetoneThis was the stock standard solutionThis stock standard solution was

diluted appropriately using solvent acetone to get the working standard of concentration 1mg ml1

This dilution depended upon the original concentration of formulations, eg in case of CythionR

Cynamid India , which was malathion 50ww, stock solution of 10g extract should be diluted 1

volume to 50 volumes to get a sample of concentration 1000μg ml1 whereas DalfR Bayer India 

which contained fenthion 2 ww, stock solution of 10g extract should be further diluted to 1

volume to 2 volumesto get sample of concentration 1000μg ml1

GENERAL PROCEDURE

The 10g accurately weighed benzophenone was taken in a test tube with 3ml graduation markThe

tube was heated on a microflame till the benzophenone was melted completely1ml of the sample

under test was added to molten benzophenone The tube was then heated till the contents started
boiling and the boiling was continued for further 3 minutes, making the total heating time 4
minutesThe tube was cooled to room temperatureThe final volume was made up to 3ml mark on

the test tube using methyl alcohol as solvent for dilution The optical density of the blue solution

was measured at 600nm using 1 cm matched cell, against the corresponding reagent blank The

35
reagent blank was obtained by treating 1ml solvent acetone in place of sample solution, under the
identical experimental conditions

CALIBRATION GRAPH

A series of Malathion standards were prepared by diluting stock standard solution 1mg ml1 
appropriately to give concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900 and1000μg
ml11ml of each standard solution was pipetted in separate test tubesThe color was developed

for each standard solution using the procedure described earlier The optical densities of the

color produced by each standard were measured at 600nm using 1cm matched cellThe graph
obtained by plotting concentrations against optical densities gave a straight line passing through
the originBeer’s law was obeyed for the concentration range 35μg ml1to 350μg ml1 Fig3 

Recovery experiments
The usual method of recovery experiment to check the accuracy and precision of the method
was followedDifferent concentration of standard organothiosulphate compound was added to a

fixed initial concentration of a formulationThe concentration of each mixture was determining

by using the procedure describe earlierThe percentage recovery was calculatedThe graph of

concentration against optical densities for the recovery experiment is shown in Fig4 The
intercept on Y axis represents the optical density of fixed initial concentration of the
organothiophosphate in the sample aliquot

DETERMINATION FROM MARKETED FORMULATIONS

Five marketed formulations A, B, C, D and E having concentration 50g, 50g, 50g, 50g and 20g
malathion per 100g respectively, were subjected for the determination of malathion by the
proposed method1g of each of the formulations was weighed accurately and dissolved in 50ml

acetoneThis was transferred to 100ml volumetric flask and the volume was made up to 100 ml

with acetoneThe concentration of first four formulations A, B, C and D these solutions were 5

mg mL1whereas concentration in the fifth solution E was 2 mg ML101 mL solution of each

of the five formulations A, B, C, D and E was added to 1 g molted benzophenone in five
different test tubes Each test tube was boiled for four minutes as described in the procedure

earlier The concentrations of the individual sample were calculated from optical density

readings extrapolating the standard curve The amount of Malathion present per 100g of the

formulation was calculated as follows

36
 a amount obtained by extrapolating standard curve

i e μg per 01 mL of solution

Therefore, a x 1000 b amount per 1 g of formulation

bx100 c amount per 100g of formulation

Sample Amount calculated Amount per g Amount per 100g

from graph μg formulation mg, formulation g100
 a b c

A 4980 4980 498

B 4970 4970 497

C 4983 4983 498

 D 4982 4982 498

E 1992 1992 199

The concentration of the formulation determine by the proposed method were

compared with those determined by AOACmethod of copper complex formulation61

Table- Comparision of A. O. A. C. method and Propos method for Malathion determination is

shown in following table.

Sr. No Product Declared Number of Malathion Malathion determined

concentration g% observation determined A.O.A. by proposed
w/w C. method method% of declared
% of declared
1 A 50 8 98.3 ± 1.2 99.3 ± 1.7
2 B 50 8 98.5 ± 1.7 99.2 ± 1.2
3 C 50 8 98.3 ± 1.1 99.3 ± 0.5
4 D 50 8 98.7 ± 2.2 97.5 ± 2.6

APPLICATION OF THE PROPOSED METHOD FOR THE DEtERMINATION FROM

BIOLOGICAL MATERIALS

Preparation of sampleBloodUrinePlasmaSerum

The 5 ML of the sample was taken in 100 mL separating funnel The sample was extracted

repeatedly for five times with 25 mL of solvent mixture isooctane ethyl ether 5050 vv  each

37
time. All the extracts were collected togetherThe solvent was evaporated to drynessThe residue

was dissolved in 1 mL of acetone added by pippeteThis 1 mL solution was further processed by

adding to 1g benzophenone as described in the procedure earlier

Viscera

50 g homogenized viscera were extracted with isooctane ethyl ether 5050 vv  mixture five

times, using 100ml solvent mixture each time All the extracts were collected together The

insecticides being lipophilic will be extracted along with fatty material of visceraTo separate the
insecticide from fat the extract was further pushed through the celite column62.A glass column of
40 cm in high and 15 cm in dimeter was packed with celiteTo 100 g celite EMerck 60ml of

methanol, 23 saturated with isooctane 20ml methanol 40ml isooctane  was added, mixed and

slurried with 500 ml isooctane

This slurry was then packed in the column The extracted compound was lloaded on the column

using isooctanemethanol solvent mixture 20ml meyhanol 40ml isooctane for elutionThe flow

of the eluting fluid was adjusted 10ml per minuteThe eluate of 5th and 6th minute was collected in

one tubeThe tube was then placed in hot water bath to reduce the volume of the solvent from 2ml

to less than 05ml This was further treated with benzophenone as described in general procedure

earlier The experiment showed average % recovery 99.2 % with standard deviation 0.32
confidence limit for 95% is 99.2±0.3 and confidence limit for 99% was 99.2±0.4 the athoe
experimental details n=8, n-1=7, t for 95% confidence limit was 2.365, and t for 99% confidence
limit was 3.499 (n= number of observations).

RESULT AND DISCUSSION

The effect of time of heating on the rate of reaction of organothiophosphate insecticide and
benzophenone was studied It was observed that the reaction was accelerated at high temperature

and was complete after 4 minutes of heating The color developed was stable for 30 minutes at

room temperature 29o30o C The effect of heating time and stability of the color formed is shown

in fig5

Malathion has been chosen as a representative sample for the work of the recovery
experiments etc All the other organothiosulphate insecticides like parathion, sumithion, fenthion,

38
 59, 63
dimethoate and phorate etc gave similar linearity response for the reaction  The percentage
yield of reaction of these insecticides with benzophenone, € the extinction coefficients and
concentration range obeying Beer’s law is shown in the following table. This is the first
colorimetric method based on the reaction with P S moiety of the thiophosphate insecticidesOther
sulfur containing compounds such as sulphanilamide, sulphadiazine, sulphaguanidine,
thiobarbituric acid, thiourea, carbon disulfide and thiocarbamate etcdo not interfere in the reaction

The sulfur containing compounds not forming blue color with benzophenone are listed in table III

B

The proposed method may be more useful in studying the “shelf life” or the stability of
organothiophosphate insecticides since, it is based on the reaction of P S moiety Most of the

insecticides belonging to this group undergo autooxidation resulting in desulfuration there by

losing its potentiality as an insecticideTo study of the shelflife of these insecticides hitherto was
very difficult as most of the methods reported were based either on determination of phosphorous
moiety or phenol moietyThese methods are, therefore, not specific egbarbiturates, interfeare in

coppercomplex method, sulphadrugs interfere in method based on diazo reaction and phenolic

derivatives interfere in method based on the reaction with phenolic moietyThe proposed method is
move specific as it does not suffer interference of most of the drugs and insecticides including
sulfur containing compoundsIt is also useful in studying photochemistry of organothiophosphates

besides shelflife study

39
40


41
42
43
44
References

1 Agricultural chemicals, Livestock remedies and commercial feeds produce carrying spray
residueCalifAgrCode Div7, chapter1, 1975
2 Hoskins, WMand Ferris, CAIndEngChemAnalEd8, 69,1936
3 Cassil, C. C. and Wichmann, HJJAssoOffAgiChemist,22, 436445,1939
4 Schrader, G: “Die Entwicklung neur insekyizider phosphorsaureEster” P 444, 3rd Ed
Verlag chemie, Winheim,1963
5 Kosolapoff, G.M“organophosphorous compounds”, p1, Wiely, New York, 1950
6 Michaelis, A:Ann,326,1291903
7 Ibid,:Ann, 407, 290, 1915
8 Balareffm D:ZanorgChem, 88, 133, 1914
9 Nylen, P Studien uber organische phosphorver bindungen PhD Thesis, University of

Uppsala, Sweden, 1930 off o’ Brien, R D “Toxic phosphorous Ester, Chemistry

metabolism and bilological effectd”p178, Scademic press Inc Lomdon ltd, LONDON,
1960
10 Lange, W and Van Krugel, BBer65, 1598, 1932
11 Committee on nomenclature, spelling and pronunciation ACS offi Reports chem Eng

News, 30, 4515, 1952

12 Editorial report on nomenclature, 1952LarJchemsoc, p5122, 1952
13 Larsson, Land Holmsted, Band Tjus, EActaChimScand, 8, 1593,1954
14 Schutz, fPhysiol, 102, 25968, 1943
15 Nachmansohn, Dand fled, EAJBiolChem, 171, 715,1947
16 O,Brien, RDInsecticides, Action and metabolismP 56, Academic Press inc London 

ltd, London, 1967

17 Barnes, JM:BritJPharmacology, 8, 208, 1953
18 Barnes, JM:Chem& Ind London , p478, 1954
19 De Candole, C. A Douglas, W W, Evans, C L, Holmes, R, Spencer, K E V,

Torrance, RW and Wilson, KMBritJpharmacology, 8, 466, 1953

45
20 Douglas, WWand Matthews, PBCJPhysiol, 116, 202, 1952
21 O,Brien, RDInsecticides, Action and metabolismP 44, Academic Press inc London 

ltd, London, 1967

22 Purvis, JEand McCleland, NPJChemSoc, 101, 1516, 1912
23 Getrz, MEand Watts, RRJAssoOPffiAgricChem, 47, 1094, 1964
24 Turner, CRAnalyst London , 99, 431434, 1974
25 Ott, DEand Gunther, FAJAssoOffiAnalChem, 51, 1215, 1968
26 Norris, MV, Vail, WA and Averell, PRJAgriFdChem,2, 570, 1954
27 Upham, SDJAssoOffiAgricChem, 43, 360, 1960
28 Analytical Methods Committee Report, “The Detrermination of Malathion residue in
Cereals and oil seeds”Analyst London , 85, 915, 1960
29 Weiserberg, E, Gartner, Sand Scholonberg, jAnalyst London , 93, 443, 1968
30 Franz, Jand Kovac,JZAnalChem, 210, 354, 1965
31 Claborn, HVand IveyNCAssoOffiAgricChem, 47, 871, 1964
32 Enos, HFand Frear, DEHJAgriFDChem,12, 175, 1964
33 George, DA, Walker, KCMurphy, RTand Giang, PAJAgricFdChem, 14,

371, 1966
34 Prasad, BN, Kawale, GBand Joglekar, VDCurrSci, 47 3 , 77, 1978
35 Mekinley, WPand Red, SIJAssoOffiAgriChem, 45, 467, 1962
36 Hestrin, SJBiolChem, 180, 249, 1949
37 Robbins, WE, Hopkins, TLand Roth, SRJ EconEntamol, 51, 3265, 1958CfC

A 55, 224521, 1961

38 Fleisher, JH, Pope, EJand and Spear, DSFArchIndHealth, 11,332, 1955
39 Vibncent, Tand SegonzacGAnnBiolClin Paris , 165, 227, 1958CFCheAbstr,

52, 13850, 1958

40 O, Brien, RDInsecticides, Action and metabolism P 88, Academic Press inc London 

ltd, London, 1967

41 Fisk, AJ, Czerwinski, GRand Kenhart, JHJForenSci, 10 4 , 473, 1965
42 Gregoire, J and Cotte, M Compt Rend Soc Boil, 146, 741, 1952 Cf Chem Abstr,

47, 3912, 1953

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43 Averell, PRand Norris, MVAnalChem, 20, 753, 1948
44 Suter, R, Dalby, Rand MeyerRZAnalChem, 147, 173, 1955
45 Zwing, Gand Sherma, JAnalChem, 48 50, 66R83R, 1976
46 MacNeil, JDand Frei, RDJChromatogrSci, 13, 27982851975
47 Hall, RCJChromatogrSci, 12,152160, 1974
48 Koen, J G Huber, J F K, Poppe, H and Den Boei, G J J Chromatogr Sci 8, 192,

1970
49 Ryan, J F Analytical Methods for pesticides and Plant growth Regulatirs P1 vol9,

Zweing, Gand Sherma, J Ed , Academic press Inc, London, 1977

50 Vander velde, Gand Ryan, JFjChromatogr, Sci13, 322329, 1975
51 Organic Electronic Spectral Data, VolII, Ungnade, H. E. (Ed), Interscience publisher, Inc,

New York, 1960

52 Kauffman, FLJAmIol Chemist Siciety, 41, 8, 1964
53 Wallace, J, Biggs, Jand Dahi,EVAnalChem37, 412, 1965
54 Brocklehurst, Pand Burawoy, ATetrahedron, 10, 118, 1960
55 Burawoy, ABerDtschChemGes, 63, 3164, 1930
56 Burawoy, ATetrahedron, 2, 122, 1958
57 Staudinger, Hand Freudenberger, HBerDtschChemGes, 61, 1577, 1928
58 Blatt, AHOrganic synthesis, p573, Colletive VolumeII, John Wiley, New York, 1943
59
Sane RTand Kamat, SSCurrSci, 47 20 , 765766, 1978
60 Sane RTandKamatSSIndJChem, 17B 4 , 398399, 1979
61 Official Methods of Analysis of A O A C William Horwitz Ed , p 121, Twelfth

Edition, AOAC, Washington DC, 1975

62 Ahmed, MK, Casida, JEand Nichols, REJAgriFdChem,6740, 1958
63 Sane, RTand Kamat, SSAnalyst London , in press 

47
 CHAPTER III

COLORIMETRIC METHOD FOR THE DETERMINATION OF

PARACETAMOL

48
DETERMINATION O F PARACETAMOL

i Development of paracetamol as an effective therapeutic compound

ii Chemistry of paracetamolStructure activity relationship

iii Metabolism and toxicity of the compound

iv Review of the analytical method

v Proposed method

LIST OF TABLES

Table ITable depicting structure relationship and biotransformation

Table IIPercentage recovery from the standard acetaminophen

FIGURES

Figure 1Absorption maxima of the color formed

Figure 2Standard curve

Figure 3Recovery experiment graph

Figure 4Stability of the color formed

The scarcity and consequent high prices of drugs like quinine during the last
quarter of the nineteenth century motivated the search for synthetic antipyretics As a

result, a large number of compounds were introduced in the medicine After

introduction of sodium salicylate as an antipyretic by Buss in 1875, synthetic salicylates
completely displaced the more expensive compounds obtained from natural sourcesIt
was observed that some of the patients are allergic to aspirin or the aspirin is
contraindicated in patients with gout or peptic ulcer A search for the suitable substitute

form as aspirin for such patients was startedIt was in 1886, Cahn and Hepp introduced
acetanilide in medicines under the name antifebrin, who had accidently discovered its
antipyretic action Although acetanilide is not the paminophenol derivative, it is the

parent member of this group of drugsAcetanilide was found extremely toxic and was

discarded as a therapeutic compound This had prompted a search for the less toxic

compound It was thought that acetanilide is oxidized in the body to paminophenol,

therefore, paminophenol was given a first try as an alternative to acetanilideToxicity

was not lessened; however, and hence a number of paminophenol derivatives were then

tested One of the more satisfactory compounds was phenacetin, chemically

49
acetophenetidin, introduced in 1887 Since phenacetine was found to cause
methemoglobinemia, and hemolytic anemia as a form of acute toxicity and more
commonly as a consequence of chronic over dosage, the search for the less toxic
compounds was continued Paracetamol, chemically acetaminophen or Nacetylpara

aminophenol was first used in medicines by Von Mering in 1893 However, it has
gained popularity only since 1949, after it was recognized as a major active metabolite
of acetalanide and phenacetineIt was soon very popular and is now a commonly used

as an antipyretic and analgesic, which is readily available to public without prescription

CHEMISTRY AND STRUCTURE ACTIVITY RELATIONSHIP

Paracetamol, chemically acetamidophenol, is a white crystalline compound with
melting point 1690 1720 C Its molecular formula is C8H9NO2 and molecular weight

1512 It is soluble 1 in 70 parts of water, 1 in 20 of boiling water, 1in 7 of ethyl

alcohol, 1 in 50 of chloroform and insoluble in etherParacetamol is official in NF1 and

shows UV absorption at 249 nm in methanol It shows principal peaks in infrared

spectra2 KBr disc at A1650, B1563, C1443 cm1Paracetamol BPAcetaminophen

USP is marketed in India under many trade names as Crocin, Elcin, Calpol, Metacin
etcin tablet form It is also marketed in tablet form in combination with other
therapeutic compounds like dexamethasone, phenylbutazone, caffeine, analgin,
dextropropoxyphene hydrochloride, diazepam etc under the trade names such as
Dexarin, Aarelgin, Proxyvon, Canapar etcIt is also marketed in the form of elexir and

syrup under trade name Akneil Most of the tablets contain 500 mg paracetamol per

tablet, although there are some containing 250 mg per tablet

Acetaminophen and phenacetine have analgesic and antipyretic effects similar those of
aspirin The antipyretic activity resides in aminobenzene structure Acetaminophen
reduces fever by inhibiting the action of endogeneous pyrogen on the hypothalamic heat
regulatory centres3 Acetaminophen is more active than aspirine as an inhibitor of
prostaglandin synthetase of brain but it is weak inhibitor of postagalndin synthetase by a
preparation of spleen4 The structure relationship of paracetamol and its metabolide is

depicted in Table IThough antipyretic activity residue in the aminobenzene moiety of

the compound, aniline itself is very toxicIntroduction of other radical in to the OH of
para amino phenol and into the free amino group of aniline residues toxicity without

50
reducing antipyretic effect Best results were obtained when an alkyl group is

substituted in the OH eg ethyl in phenacetin and or an acid group is introduced in

the NH2of aniline, egphenacetin and acetaminophen

METABOLISM, PHARMACOLOGICAL EFFECTS OF TOXICITY

Acetaminophen is metabolized primarily by the hepatic microsomal enzymes

5, 6, 7
TableI also shows, the pathway of biotransformation Acetaminophen is rapidly
absorbed through the gastrointestinal tract, reaching the plasma concentration at the
peak within ½ to 1 hourIt is distributed uniformly throughout the body fluid75to

80of the phenacetin gets metabolized to paracetamol in normal individualsThe fate

of the paracetamol metabolized from phenacetin or administered is the sameAbout 3

of paracetamol is excreted unchanged in urine, where, as 80 gets excreted in the

conjugated form either as glucuronide or Sulphate The conjugation takes place in the

liver The hydroxylation is responsible for the heamolysis of the red blood cells,

methemoglobin formation and hepatotoxicity8 Acetaminophen relieves pain of

moderate intensity such as in headache and in muscle jointsIntense pain or that arising

from smooth muscle spam in hallow viscera is not alleviated The pharmacological

properties are reviewed by Smith9, Rasndall10, and Beaver11, 12 Skin rash and other

allergic reactions occur occasionallyThe most serious adverse effect of acute overdose

is the hepatic necrosisRenal tubular necrosis and hypoglycemic coma may also occur

A dose of 25g or more is potentially fetal Nausea, vomiting, anorexia and abdominal

pain occur during the 24 hours of poisoningThe hepatotoxicity may progress to coma

and death13Due to the wider interest in its medicinal uses, considerable interest in the
measurement of plasma concentration led to the development of methodology over the
last few years

51
Reaction product with parcetamol λ 463 nm

52


53
REVIEW OF THE ANALYTICAL METHODS

The methods currently available can be classified in to four main groups

i The first group involvesmeasurment by UV Spectrophotometry

ii The second group includes colorimetric methods of determination based on various

types of color reactions

iii The third group deals with gasliquid chromatography and

iv The forth group include high pressure liquid chromatography HPTLC 

In the UV spectrophotometric methods some workers used simple extinction

measurements at 250 nm14,15 while some other workers in the field used differential
extinction method, in order to remove the interference due to other drugs in
combination16, 17, 18
Various colorimetric methods for the determination of paracetamol are described,
involving reaction with 2, 2diphenyl1phenylpycryl hydrazyl dye16, different

aldehydes19, 21, phenol in combination with ferricyanide and dichromaste22Nitration 23,

24
nitrosation25 and diazotization followed by subsequent coupoling26 are some of the
other reaction used for colorimetric determination of paracetamol Several

modifications are reported for the method based on blue indophenol dye formation2729
All these methods are based on conversion of paracetamol to par aminophenol and
subsequent chemical reactionAn excellent review of all these methods is given by K

Wiener30 These fluorometric methods through oxidation of acetaminophen with

ferricynide31are or through hydrolysis and subsequent reaction with benzylamine32 are
also reported There are number of GLC3338 and HPLC3944 methods put forth by

several researchers
All these colorimetric methods lack simplicity and specificity of routine method, as
drugs containing aliphatic and aromatic nitro, nitroso, phenolicOH and or aromatic

amino groups interfere in one or the other methods45,46

PROPSED METHOD
 Acetaminophen reacts with sodium hydroxide reagent at elevated temperature
forming a crimson color reaction product The reaction product has absorption

maximum at 4630 nm fig1

54
REAGENTS

Standard acetaminophen solution 100mg accurately weighed acetaminophen was

dissolved in about 70mL methyl alcohol in 100mL volumetric flask After dissolving

complete by shaking, the solution was made up to 100ml volume with methyl alcohol

This gave the standard acetaminophen solution of the concentration1 mg.mL1

Sodium hydroxide reagent10g sodium hydroxide was dissolved in about 70ml distilled

waterThe solution was cooled to room temperatureThe final volume of the solution

was made upto 100 ml in volumetric flask using distilled water All the chemicals used

were analytical grade 

GENERAL ASSAY PROCEDURE

1 ml of the sample under test was pipetted out in a 10mL graduated centrifuge tubeTo

this 1mL of the 10sodium hydroxide reagent was addedThe tube was then kept in a

boiling water bath for 15 minutes After removing the tube from water bath, 15ml of
distilled water was added immediately and the content of the tube were mixed with
gentle shakingThis was then allowed to stand for 100 minutesThe final volume of the
solution was made to 4 mL by adding appropriate quantity of distilled water and the
color intensity was measured at 4630 nm against the reagent blank For the reagent

blank, 1 mL of methyl alcohol instead of the sample was treated simultaneously The

concentration of the test solution was then calculated from calibration curve

CALIBRATION CURVE
A series of acetaminophen standard were prepared by diluting the standard solution
to give concentration of 100,200,300,400,500,600,700,800,900,1000 micro gram
mL1By using the general procedure describe the above, the optical density of the color

produced with each standard concentration was measured The graph obtained by the
plotting optical densities against concentration of acetaminophen gave a straight line
passing through origin and obeyed Beer’s law for the concentration range 25 μgml1 to

250μg ml1The calibration graph is represented in fig2

RECOBVERY EXPERIMENT IN TABLET

To study the accuracy and precision of the proposed method recovery study was done
A fixed volume of the sample solution, equivalent to about 500 μg of paracetamol was
taken and five different known concentration of paracetamol were addedEach level of
55
added paracetamol was repeated eight times The total amount of paracetamol was

determined by the proposed method A graph of the total paracetamol determined

against the amount of paracetamol added is shown in fig3 The intercept on Y axis

represent the initial fixed concentration of paracetamol taken

DETERMINATION O F ACETAMINOPHEN FROM TABLETS

The tablets were powdered The powdered was weighed to have a quantity

equivalent to 50mg acetaminophenThe weighed powder was dissolved in about 70ml

methyl alcoholThe solution was filtered and the filtrate was transferred along with the

repeated washing in to 100ml volumetric flask The volume was up to 100 ml using

methyl alcohol The solution gave 500 μg mL1 concentration of acetaminophen The
exact amount of acetaminophen per tablet was determined by using the general
procedure describe earlier and the calibration curve The amount determined four
marketed formulation A, B, C and D with the declared paracetamole concentration 500
mg each where analysed by proposed method over 8 experiment of each product
showed average percentage recovery 98.8, standard deviation 1.3, 95% confidence limit
(t=2.365) 98.8±2.9 and confidence limit is 99% (t=3.499) is 98.8±3.6 where n-1=7,
n= number of experiments.
DETREMINATION OF ACETAMINOPHENE FROM BIOLOGICAL MATERIALS

From BloodUrineSerumPlasmaStomach contain etc

To 5mL of the sample diluted acetic acid was added till the medium become acidic

The sample was then extracted five times with 50 mL chloroform each time The

chloroform extracts were collected together and solvent was evaporated to drynessThe

residue was dissolved in 2 mL methyl alcohol 1 mL of this methanol solution was

processed according to the procedure described earlier for the determination of
acetaminophen in the sample

From Viscera

50g homogenized viscera was extracted five times with chloroform 100 ml each time 

in acidic medium using dilute acetic acid The extract was collected together and

solvent was evaporated The residue was dissolved in 5 mL methyl alcohol 1 mL of

methyl alcohol solution was treated by the procedure to determine acetaminophen
amount present in sampleAmount of standard acetaminophen added to the biological

56
sample and amount recovered showed avaragr percentage recovey of 98.2 over 8
experiments per specimens the confidence limit for 95% isequal to 98.2±1.7 ( t=2.365).
Confidence limit for 99% is equal to 98.2 ±2.9 (t=3.499) where n=8, n-1=7.
RESULT AND DISCUSSION
 The color of the reaction of acetaminophen and sodium hydroxide is very stable
figIV  Compounds such as phenacetin, acetanilide, aspirin, pacetanisidine etc
having structures very closed to that of acetaminophen do not give any color with
sodium hydroxide reagent TableV  pnitrophenol gives a faint yellow color with

absorption maxima at 405nmPaminophenol, a metabolite of paracetamol gives similar

color like the one with paracetamol This interference was removed by chloroform

extraction in acidic mediumThe other compound listed in Table VI which is used for
their therapeutic properties in combination with acetaminophen in formulation; do not
interfere in the proposed method Amount of acetaminophen determine per tablet and
the percentage of the declared amount in such combinations was worked out and is
presented in TableVII. The compounds such as barbiturates, hydantoins etcwhich are
being marketed in combination with acetaminophen, therefore, the pure standard
compounds were checked with sodium hydroxide reagent and were found not to give
any color with the reagent The absorption maximum in acetaminophen spectrum

undergoes bathochromic shift on alkalinization This displacement is due to the

ionization of phenolic hydroxyl group The phenate anion thereby produced has extra

electrons which interact with the electrons of the aromatic ringThis bathochromic shift

observed in official method1, 47 is from 249 nm acetaminophen  to 257nm in alkaline

medium By changing the reaction conditions, the bathochromic shift was effected to

463 nm in the visible region This helped in eleminating interference of many

compoundsAccording to Spooner et. al15 the method involving simple extraction into
ether is susceptible to slight interference from the salicylates, barbiturates and
methaqualone while the interference by phenylbutazone is quite considerable Though
the differential extinction technique has eliminated salicylates interference, the problem
of oxyphenbutazone and phenylbutazone is still encountered48 Barbiturates, salicylic

acid chlordiazepoxide all gave differential spectra in the region of 225nm to 350 nm

Amongst the methods involving paminophenol formation, methods based on

57
diazotization, indophenol reaction etcwill have serious interference due to compounds

with aromatic secondary and tertiary amino and phenolic groupsThe method based on

diphenyl picrylhydrazyl dye was found free from the interference of salicylates etc, but
potentially, secondary and tertiary amines and naturally occurring amine in plasma
contributed towards the interference Even the instrumental methods of analysis like

GLC, and HPTLC have interference due to barebiturates36 and sulpha drugs44 The
proposed method is found to be free from the interference from all such compounds
have specificity and precesion49

58


59
60
61
62
References

1 National formulary, NF XIII p18, 1970, American pharmaceutical

Association, Washington DC

2 Clarke, E G C, Isolation and Identification of Drugs, p 461&751, 1969,

The Pharmaceutical press, London

3 Clark, WGand Moyer, SGJPharmacexp Ther181, 183191, 1972
4 Farreira, SH& Vane, JRA Review Pharmac14, 5773, 1974
5 Brodie, BBand Axelrod, JJPharmacExpTher97, 5867, 1949
6 Prescott, LFClinPharmacTher10, 383394, 1969
7 Abel, JAClin PharmacTher12, 583598, 1971
8 ¯The pharmacological Basis of Therapeutics°, Goodman, L S, Gilman,

A Eds , p345, Vth Edn, 1975Macmillan publishing Co, New York

9 Smith, P KAcetophenetidin A critical Vibliiographic Revie, Interscience

Publisher, Inc, New York, 1958

10 Randoll, LO¯Physiological Pharmacology°, volI, p313416Academic

press, New York,1963

11 Beaver, WTAmJMed, Sci, 250, 577604, 1965
12 Beaver, WTAmJMed, Sci, 251, 576599, 1966
13 ¯The pharmacological Basis of Therapeutics°, Goodman, L S, Gilman,

A Eds , p346, Vth Edn, 1975Macmillan publishing Co, New York

14 Dordoni, B, Willson, RA, Thomson, RPH and Williams, R Brit Med

Jour,38687, 1973
15 Spooner, RJ, ReaveyPU, McIntos, LJournal of clinical Pathology, 29,

663, 1976
16 Routh JL, Shane, N A; Arrendondo, EG and paul, W D clinical

Chemistry 14, 883889, 1968

17 Varley, H, Gowenlock, AH& bell, MPractical Clinical Biochemistry5

63
 th Edition, vol2, 1976
18 Gibson, PF¦Lancent, 2 607, 1972
19 Sanghavi, NM& Vishwasrao, DRIndJPharma55p172, 1973
20 Inoue, T, Tatsuzawa, M, Lee, SC, Ishil, TJHygChem21, 313, 1975

CfAnalAbstr, 1E29, 1976

21 Vaughan, JBJPharmaSci,58, 4691969
22 Deodhar, RD, ShastriMR& Mehta, RCindJPharm,35,120, 1930
23 Glynn, JP and Kendal, SELancet, 1,1147, 1975
24 Chafetz, L Daly, RE, Schriftman, H & Lomner, J J J Pharm Sci,
60,463, 1971
25 Inamdar, MCGore, MA, and Kaji,NNIndJPharm31, 79,1969
26 Inamdar, MCGore, MA, and Phide, R,VIndJPharm36, 7,1974
27 WelchRMConney,AHClinical chem, 11106467, 1965
28 Love, EBLancet, 1,195, 1977
29 Gwit, JRRobertson, A, Mc Chesney, EWJPharmPharmacol15, 440,
1963
30 Wiener, KAnnal of Clinical bIochemistry, 15, 187196, 1978
31 Kaito, T Sagara, K, Yoshda, T and Ito, Y J, Pharm So Japan94, 633,

1974
32 Kaito, TSagara, KJ, PharmSoJapan94, 639, 1974
33 Prescott, LFJ Pharmacy and Pharmacology23, 111115, 1971
34 Thomas, B H, Coldwell BB J Pharmacy and Pharmacology 24, 243,
1972
35 Prescott, LFJ Pharmacy and Pharmacology 23, 8078, 1971
36 Street, HV¦jOurnal of Chromatography, 109, 2936, 1975
37 Dechtiruk, N A, Johnson, G F, Soloman, H, M 0 Clin; Chemistry 22,

879883, 1976
38 Garland, WAPharmSci, 66, 34044, 1977
39 Mrochek, JE, Katz, S, Christie, W H and Dinsmore, S R clin,

chemistry20, 10861096, 1976

64
40 Riggin, RMSchidt, ML& KissigenPTJPharmSci, 64, 68083,

1975
41 Wong, LT, Solomonra JG &Thomos, BM, Pharm Sci,65, 1064
66,1976
42 Gotelli, CR, KabraPM, MortonL,JClinChem23957591977
43 Blair, DRumack, BHClinChrm,23, 743745, 1977
44 Horvitz, R,AY& Jattyow, PJClin, Chem23, 159698,1977
45 Feigl F Organic Spot Tests Elsevier Publication co, Amsterdam, Vth

English Edition, P154 & 182, 1954

46 Davis, DR Fogg, A G, Thornburn Burns D, & Wragg, J S Analyst

99 1174 , 12, 1974

47 British Pharmacopia, Brit, Pharmaceutical Association, London, p341, Her

Majesty¬s Stationary centre, 1973

48 Wiener, K Loglands, M G and Tan B H A AnnClin Biochem 13,

45253, 1976
49 Sane RT& Kamat, SSJAssoOffiAnalChem in press 

65
 CHAPTER IV

DETERMINATION OF ACETALDEHYDE IN PRESENCE OF

FORMALDEHYDE
AND
ETHYL ALCOHOL IN PRESENCE OF METHYL ALCOHOL

66
PartI

i Role of ethyl alcohol in human society

ii Chemistry of ethyl alcohol

iii Metabolism and toxicity of ethyl alcohol, methyl alcohol, and acetaldehyde

iv Review of the analytical methods reported

PartII

i Proposed method for the detrermination of acetaldehyde in presence of formaldehyde

a standard curve

b Recovery in standard

c Recovery in biological materials

ii Proposed method for the detrrmination of ethyl alcohol in presence of methyl

alcohol

 a standard curve

b Recovery in standard

C Determibnation from marketed drinks

d Determination from biological materials

iii Result and discussion

iv References

List of figures

Figure1Absorption maximum of the color formed due to acetaldehyde and 2

thiobarbituric acid reagent

Figure2Standasrd curve of acetaldehyde in presence of formaldehyde

Figure3Percent recovery of acetaldehyde in standard

Figure4Standard curve of ethyl alcohol in presence of methyl alcohol

Figure5Percent recovery of ethyl alcohol in standard

Alciohol is an enjoyable pastime of the mankind eversince the dawn of
human civilization throughtout the worldOne can produce convincing data to support

the thesis that, of all poisons, grain alcohol ethyl alcohol  tops that the list in the

seriousness of its consequences Alcohol takes a far greater toll in death, permanently
disabled and seriously injured, either directly or indirectly due to vehicle accidents

67
under drunken conditions Statistically about 30 to 40  of all accidental fatalities

involve persons under drunken conditions In addition to this, underground traffic in 

illicit liquor gives rise to variety of dangerous social problems like gangsterism, rape
etc, disrupting the moral of society in general and lawenforcement agency in

particular
Counterbalacing the damaging effect of alcohol, there are its somewhat
beneficial effects Alcoholic liquir has always served as a means of escape from or

amelioration of, wordly cares and tensions If this were true in sixteenth and
seventeenth centuries as indicated by Shakespear’s or Ghalib’s references to the utility
of alcoholic beverages, it must be much more true in the present era The human
nervous system is reasonably static, but the tempo of living, the tensions, tortures and
turmoils of our modern civilization continue to mountIn the metapolitan centres man is

pressed between two grinding stones, a nontooredily adaptable static nervous system
and faster way of life through production, transpoprtation and intensive living in all
phasesUnderstandably to ease his frayed nerve such a man offen turns to cushioning

action of alcoholic beverages They serve as a sedative, in easing his worries, tension

and frustrationmaking life more tolerable for the momentBut with habit, his tolerance
for the sedative action goes on increasing, needing hom to go on increasing the dose day
by day ultimately pushinfg him beyond th e “deadline”.Due to thevincresesd tolerance
limit, one has to consume more and more quantity of liquir This naturally becomes

costly affair to the drinkerHe then turn to cheaper substitutes like illicitvliquir obtained

from “French polish”or denatured spirit, which gives similar sedative effectThis liquir

obtained from denatured spirit or “French polish”contain methyl alcoholThe presence

of methyl alcohol always gives mislesding figures of ethyl alcohol quantitativelyIt is,
therefore necessary to have a method for the determination of ethyl alcohol in presence
of methyl alcohol, in a shortest span of time.
Formaldehyde and acetaldehyde are well known products of in vivo
and in vitro drug metabolism, particularly in carcinogenic compounds and
oreganophosphorous esters When both foemaldehyde and acetaldehyde are present in
the same system, difficulties arise in individual quantitative analysis due to the
similarity of their chemical properties and reactvitiesThis nescessiciated for a search of

a reagent which will from color selectively with ione of the member from the system
68
ETHYL ALCOHOLAND ALCOHOLIC BEVERAGES

Ethyl alcohol is chemically hydroxy etahne with molecular formjula C2H5OH It is an

boils at 783oC at standard barometric pressure and has specific gravity of 079at 20 oC

Its molecular weight is 46It is completely misciblewithwater and has pleasant odour
It reduces certain oxidizing agent like portassium dichromate, potassium permagneate
etc

The alcohol is various beverages results from the fermentation of sugar or malt All
alcoholic beverages are chiefly water and alcohol, plus very small amount of other
substances called “congener”, which signifies “simultaneous formation”.The congeners
are chiefly are organic acid, esters and acetaldehyde Win are brandy contain methyl

alcohol up to1  from the brakedoun of methoxy group in the pectin of the grape1

The percent of alcohol by volume in some common alcoholic beverages is

Beer4 to 5, Ale5 to 8, Toddybelow 5, Wine10 to 22, Wisky, Gin, Vodka

40to 55
With distillezd beverages, the alcohol contents are expressed by thye term “proof”. In
USA 100Proof means 50alcohol by volume, whereas British 100 proof means 57
alcohol by volume, In India “Proof spirit” is defined by the act of parlliment as “Being
such as, shall, at tempersature of 51oF, weigh exactly 1213 part of an equal measure of

distilled water” 100 proof 571 vv ethyl alcohol  The term on the marketed

beverages are printed as under proof weaker spirit or over proof stronger spirut 

ACTION OF ALCOHOL ON HUMAN BODY

Although alcohol may be introducesd in to the body in other way, such as by
intravenous injection of 25 alcohol solutions, inhalation of alcohol vapours2 or by

rectal absorption from an enema2, the most common rout is oral

ABSORPTION
In contrast with ordinary foods and many drugs, at least half of the alcohol is quite
rapidly absorbed from the stomach3Hager et at4 found that fasting dogs which received

24alcohol by stomach showed an average absorption og 52in 15 minutes, 5194

in onehalf hour and 9094in one hour, with practically complete absorption in 23

hour The absotrption will be complete when intire gastrointestinal tract reaches

equilibrium with the remainder of the bodyComplete absorption may be delayed by an

69
hour or more if stomach contains a large amount of semifluid, aqueous material5The
rate of absorptioin of alcohol from stomach and small intestine in to the blood stream
depend upon a  concentration and total quantity of alcohol in stomach contents, b 

nature of the food alredy in the stomach fatty food and sugar apper to slowdown

absorption slightly , c the length of the time gastribc contents are held in stomach

prior to opening of the pylorus and d  permeability of the stomach and intestine

membranes

Distribution
Alcohol fromthe stomach and intestine is carried by the blood through the portar vein
to the liver, where blood from hepatic artery joins The mixture is carried away by

hepatic vein and merges with the general blood circulation in the vana cavaOrgans like
liver, brain, kidney, tissues etc, which have rich blood supply quickly attain akcohol
equilibrium with arterial blood On the other hand, voluntary muscle tissues, which
have much snaller blood flow per unit weight require at least an hour to reach the
equilibrium6, 7 Once the storage equilibrium is reached, the resulting concentration of

alcohol in each body tissues will be proportional to the water contents of the tissue

Obviously, blood, urine, spinal fluid content more alcohol than the other tissues

Relative concentration of alcohol in body tissue and fluids8

When Blood alcoholconcebtration 100 g.

SrNo
1 Urine 125
2 Cisternal Spinal fluid 110
3 Saliva 10 to 110
4 Blood Serum 110
5 Whole blood 100
6 Brain 085 to 090
7 Liver 085 to 090
8 Kidney 083

After equilibrium, the blood alcohol level in all parts of vascular system is the same

70
Elimination
The elimination of alcohol, distributed in the body, takesplace through two
mechanisms oxidation and elimination About 95 of the alcohol is completely

oxidized in the body to catbon dioxide and water Remaining 5 alcohol is excrted

unchanged in the breath, urine, stools and perspirationOxidation takes place entirely in

liver, where enzyme alcohol dehydrogenese ADH and coenzyme

diphosphopyridinenucleotide DPN  convert alcohol to acetaldehyde and further to

acetic

CH3CH2OH DPNADHCH3CHO DPNH2

The acetic acid formed reacts with body buffer system to form acetates This acetate
adds to the body acetate pool and is finally converted to CO2 and water, via Kreb’s
cycle The entire oxidation process is accompanied by generation of heat serving the

ingested alcohol as a redy source of thermal energy

Pharmacological and Toxicological effects

 Alcohol causes dilation of capillaries of the skin producing general hyperemia of
the body surface Since skin contains temperature sensitive nerve endings, the
hyperemia creates feeling of wrath through actually internal body temperature is
decreses Alcohol increses gastric acidity causes irritation and inflammation of gastric

mucas resulting in vomitingAlcohol in moderate doses causes vasodilation especially

of cutaneous vessels producing warmed and flushed skinHowever, chronic excessive

use of alcohol has a deliterious effect on heart9, 10 It is observed that liver cirrhosis is

frequent among alcoholics Alcohol produces diurestic action due to the inhibition of

certain antdiuretic hormony by the posterior pituitary gland Due to the depressant
effect on CNS that ethyl alcohol produces, the hearing and vision efficiency is lessened
11, 12
 And coordination of voluntary muscvles in impaired Judgment and selfcontrol

functions of the brain are impaired by lower concentration of body alcoholThis result

ion euphoria and loss of inhibitionEuphoria, originated from the Greek word, meaning
over optimistic state, is probably the chief reason for the popularity of alcoholic
beverages Loss of inhibition means, losssing the sence of caution or lowering the

normal restraintThis finally results in antisocial trends, crimes and violence

71
 In several acute alcoholic intioxication the skin is cold and clammyThe body
temperature is low, respirations are slow and noisy, the pupil may be normal or dilated
and heart rate is accelerated Diabatic coma may be mistaken for severe alcoholic
intoixication Drug intoxications, cardiovascular accidents; schizophrenia and fracture
skulls are also many times mistaken for drunken stateTherefore, determination of
alcohol concentration in body fluidsis very important from diagnostic point of view, as
well as medico legal points

METHYL ALCOHOL
Methyl alciohol also called methanol wood alcohol or Columbia spirit; it is the simplest
of alcohols It is the chemically hydroxy methane with boiling point 64oC at normal

temperature and barometric pressure Its molecular formula is CH3OH and molecular

weight is 32 Its specific gravity is 0793 It teduces certain oxidation agents like

potassium dichromate, potassium permagnete etc Thus, it has very close physicaland

properties to that of ethyl alcoholMethanol is widely used as an industrial solventIt is

also uised as a denaturant toorender ethyl alcohol unfit to drinkMethyl alcohol is very

hazardous and causes permanent blindness if cousumed even in nvery small quantity

The larger doses result in deathIts toxic limit is 100 ppmThe toxicity of methanol is

due to its cumulative effect The pharmacology, biochemistry and toxicology of

methanol has been extensiveviewed by many scientists 13, 14, 15

The denatured ethyl alcohol is less expensive than the conventional alcoholic
beverages and offer considerable temtation to derelict Poisoning result from the
consumption of such alcoholic preparation due to serious hazards of methyl alcohol. 6%
of all blindnesss in the unioted states Armed Forces during Worldwar II ws caused by
methyl alcohol16Though statistical figures of blindness due to methyl poisoning are not
17
available in India, there are a number of incedences reported in 1973 , where more
than 100 deaths were reported due to consumption of “Khopdi”an alcoholic preparation
contaminated with methyl alcohol, the survival suffered blindness
This toxicity of methanol is attributed to formaldehytde and formic acid
formed as a tresult of the enzymatic oxidative degradation of methanol18
It is obvious that determination of ethyl alcohol is bound to clash with that of
methyl alcohol and aldehyde determination with that of formaldehyde in cases where
liquir consumed is contaminated with methyl alcohol
72
REVIEW OF THE ANALYTICAL METHODS
There are several colorimetric methods reported for the determination of
formaldehyde19, 20, 21.
 The accuracy of some of the method is not affected by the
presence of acetaldehyde in the sampleBut so far, there is no satisfactory colorimetric
method fotr the determination of acetaldehyde in presence of formaldehyde without
removing any of them from the systemAt leastthree general methods have been used

for the determination of acetaldehyde In one of the oldest metyhods, the oxidation of
acetaldehyde to acetic by Nessler’s solution was utilized where the metalic mercury
liberated was combined with iodine and the excess iodine titrated22 Another general
principal has been, to titrate the liberated hydrochloric acid from hydroxylamine
hydrochloride coincident with the formatiom of acetaldoxime23The bisulphite binding
24
power either directly or in conjunction with silver nitrate treatment 25 has also been
utilized The widely used colorimetric method for the acetyaldehyde determination is

the pphenylphenol procedure of Berker and Summerson26, further modified by stotz27

This method can not be used when formaldehyde is present in the systemDagani and

Archer28 remove this formaldehyde interference by the treatment of acidic 2, 4

pentadione reagent This method is not useful for routine anlysis as the amount of

pentadione varies at higher concentration of formaldehydeIt is, therefore, necessary to

develop a method for the determination of acetaldehyde in presencr of formaldehyde

Such a method will be more simple if the determination if is possible without separating
any one of them from the system

The official AOACmethod for the quantitative determionation of alcohol

in marketed drink is based on distillation and measurement of specific gravity29Other

method reported, are based on measuring the volume of alcohol in pur form30, refractive
index31 etc There are number of methods reported such as oxidation of alcohol by
KMnO4 and noting the amount of oxidizing agent used, by disappearance of typical
32
purple color of the permagnate , oxidation of alcohol in vapour or gaseousstate by
passing it over hot iodine pentaoxide, at 155oC and measuring the amount of iodine
33
liberated , bromometric mesurment of ethelene formed by pyrolytic degradation of
alcohol vapours33, conversion of alcohol to ethylnitrite and measuring the amount of
nitrite formed33, oxidation of alcohol to acetaldehyde and subsequent determination of
acetaldehyde34Widely used chemical methods are based on the diffusion oxidation of
73
ethyl alcohol and either titrating the resulting acetic acid with standard alkali solution35
or detrmining residual potassium dichromate by adding potassium iodide and titrating
3640
liberated iodine against standard sodium tiosulphate , or determining reduced

trivalent chromium spectrophotometrically   Some other methods include enzymic42,

41

amperometric43, interferometric44 and spectrophotometric The methods described

above are either not specific as compound like methanol, ether, chloroform,
formaldehyde etc interfere or ther methods are not useful for the smaller amount of

sample as in xcase of biological specimen The colorimetric method for the

determination of ethyl alcohol in presence of methyl alcohol without preseparating any
one of them from the system will be an asset to the analytical chemists in general

PARTIITHE PROPOSED METHODS

The method is based on the condensation reaction of acetaldehyde and 2thiobarbituric

acidThe reaction can be written as

METHOD

Reagents

2thiobarbituric acid reagent 001M

144g accurately weighed 2thiobarbituric acid BDH was dissolved in 70mL glasial

acetic acid AR grade  and 30mL concentrtated sulphuric acid AR grade  in beaker
The contents were heated till the solution become clear, cooled to room temperature and
made up to 100mL in a volumetric flask, using distilled water as a solvent

ii Axcetaldehyde standard

74
065mL acetaldehyde BDH995vv, specific gravity 0779 in about 70mL distilled

water and made up to 100mL using distilled water 50mL of this stock standard
solution was diluted to 100mL using distilled water to get standard solution of
concentration 250μgmL

iii Standard formaldehyde solution

125 mL of formaldehyde 40wv  was dissolved in 100mL distilled water.

50mL of this stock standard solution was diluted to 100 mL using distilled water to get

a standard formaldehyde solution of the concentration 250μgmL

Preparation of biological specimen

BloodurineStomach wash10 mL of the sample was taken in a 25ml distillation flask

and 90mL of water was added 10mL of the distillation was collected by gently

heating the flaskViscera iestoachintenstinesliverspleenkidneybraineyc 10g

viscera were weighed in 250 mL distillation flask50mL distilled water was added and

10mL distillate was collected by gentle heating of the flask The distillatets thus

obtained were processed according to the analytical procedure described

Ansalytical Procedure

To 10mL of the distillate in a 10mL graduated centrifuge tube, 10mL of the 2

thiobarbituric acid reagent was added The tube was kept in boiling water bath for 30

minutesAfter half an hour, the tube was removed from water bath and cooled to room

temperatureThe volume was made up to 30mLContents were centrifuge and optical

density of the supernant solution was measured at 504nm against the reagent blankThe

reagent blank was prepared by trating 10mL distilled water under identical
experimental conditions

Calibration Curve
A series of standards with concentration as 25μg, 50μg, 75μg, 100μg, 125μg,
150μg, 175μg, 200μg, 225μg, and 250μg per mL acetaldehyde and equal amount of
formaldehyde were treated with 2thiobarbituric acidreagent under the conditions

described earlier The graph of concentration of acetaldehyde against optical density

was a straight line passing through origin and obeyed BeerLambert’s lawa for the

75
 concentratrion range 8 μgmL1 to 80 μgmL1  fig2  The color produced in the

reaction was stable for over 24 hours fig3 

Recovery of acetaldehyde in standards

Different concentration of acetaldehyde was added to a fixed concentration if
acetaldehyde containing equal quantity of formaldehyde The total amount of

acetaldehyde was determined from thestandard Beer’s Law ploteThe percent recovery
obtained with the 8 observations showed average percent recovery =98.1, standard
deviation =0.87, Confidence limit with 95% (t=2.365) recovery = 98.1±1.4 and
confidence limit with 99% (t=3.499) recovery =98.1±2.5 where n=8, n-1=7.A graph of
concentration of acetaldehyde added against concentration determined showed on
intercept on Y axis rerpresenting the original fixed concentration of acetaldehyde fig3)

Recovery of acetaldehyde from biological materials

Definite quantities of acetaldehyde with equal concentration of formaldehyde were
added to various biological specimens Threse are specimens were confirmed

previously for the absence of acetaldehyde, by quantitative tests The specimens to

which acetaldehyde was added, were processed according to the procedure “preparation
of Biological Specimens”described earlier10mL of t he distillates was further treated

with 2thiobarbituric acid reagent according to analytical procedure described earlier

The percent recovery showed for 8 observation average % recovery =98.9, stamdard
deviation 0.62, confidence limit with 95%=98.9±0.8 (t=2.365) and confidence limit
99%recovery =98.9±1.3 (t=3.499), where n=8 and n-1=7.
METHOD AS APPLIED FOR THE DETRMINATION OF ETHYL ALCOHOL
IN PRESENCE OF METHYL ALCOHOL

Alcohols were converted to aldehydes by treating with potassium dichromate solution

46
and dilute sulphuric acid  The mixture of aldehyde was then made to react with 2thiobarbituic
acid reagent in acidic condition to obtain the pink complex showing absorption maximum at 504
nm fig1 

Rreagents

i Saturated aqueoius solution of mercuric chloride contains 75g HgCl2 per 1 mL

H2O

76
ii Aqueous sodium hydroxide solution 20 200g sodium hydroxide dissolve in

about 70 mL distilled water and made up to 100 mL

iii Ethyl alcohol stock standard obtained by distillingethyl alcohol with unhydrous

calcium chlorideThe specific gravity of the distilled ethyl alcohol was 07783 at 300C

iv Ethyl alcohol working standard 1285 mL of the stock standard ethyl alcohol

pipetted out in a 100mL volumettic flask and diluted to 100 mL with distiolled water

The strength of this solution 100mgmL 01mL of this solution wsa again diluted to

100 mL in volumetric flask This working standard has concentration of ethyl alcohol

100μgmL

v Aqueous potassium dichromate solution N20  2452g K2Cr2O7 dissolved in

distilled water and made up to 1000mL with distilled water

vi Sulphuric acid 2N 1041g sulphuric acid specific gravity 184 was slowly added

to 500 mL water and made up to 1000ml, with distilled water

vii 2thiobarbituric acid reagent 001M  144g accurately weighed 2thiobarbituric

acid BDH  was dissolved in 70 mL glacial acetic acid AR grade  and 30mL

concentrated sulphuric acid AR grade  in abeaker The content were heated till the
solution become clear, cooled to room temperature and made up to 1000mL volume in a
volumetric flask, using distilled water as a solvent

PREPARATION OF THE BIOLOGICAL SPECIMENS

BloodUrine

10mL of blood urine was taken in a 50mL distillation flask and 10mL saturated

mercuric chlotride solution and 10mL sodium hydroxide 20 solutions were added to

the flaskThe contents of the flask were then heated to boiling and 10mL distillates was

collected The 10mL distillate was taken inanother distillation flask to which 10mL

potassium dichromate solution and10 mL dilute sulphuric acid were added 10mL of

distillate was collected by boiling the contents of the flaskThis distillate was used for

the determination of ethyl alcohol as per general analytical procedure described later

VisceraStomach contents

500g of the samplr was accurately weighed, macerated and transferred to distillastion

flask 10mL saturated mercuric chloride solution and 10mL sodium hydroxide 20 

77
 solution were added Contents were diluted by addind 100 mL distiolled water The

mixture was heated to boiling and 100mL of the distillate was collected
To the 10mL distillates out of 100mL, 10mL potassium dichromate
solution, 10mL dilute sulphuric acid solution and 80mL distilled water were addedThe

contents were distilled over gentle flame to collect 100mL distillate This distillate is
further used for the determination of ethyl alcohol as per the analytical procedure
described bellow

GENERAL ANALYTICAL PROCEDURE

10 mL of the final distillates, as described in preparation of the

sample, was pipetted out in a 10mL graduated centrifuge tube 10mL of the 2
thiobarbituric acid reagent was added the tube and the tube was kept in boiling
waterbath for 30 minutes The tube was then cooled to room temperature and the

contents were made up to 30mL with distilled water Ther solution was then

centrifuged and the supernant was read at 504 nm against the reagenr blank The

reagent blank was prepared by treating 10mL of the distilled water with 1 mL

thiobarbiturate acid reagent (instead of distilled under indetical conditions

STANDARD CALIBRATION CURVE

The calibration curve was obtained by trating a series of ethyl alcohol standard
concentration from 25μg, 50μg, 75 μg, 100 μg, 125 μg 150 μg, 175 μg, 200 μg,
225,and 250μgmL along with equal amount of methyl alcohol concentration per mL 

as described in analytical procedureA graph of concentration of ethyl alcohol against

optical density shown in fig4 is a straight line passing through origin and obeys Beer

Lambert’s Law for the concentration range of 25μg to 250μg/mL

Determination of ethyl alcohol from alcoholicpreparation

The alcoholic preparation bearing declared percentage of alcohol on the label, were
diluted approximately to about 2g100mL 1mL of this solution of mercuric chloride

and 10mL sodium hydroxide solutions were added The contents were boiled and

100mL of the distillate was collected

10mL of the distillate was further taken in a distillation flask and 10mL potassium

dichromate and 10mL dilute sulfuric acid was addedThis was then heated to boiling and 10mL of

78
the distillate was collected This final distillate was used for the determination of ethyl alcohol as

per the procedure determined earlier

The following five alcoholic drinks from market were tested for their ethyl alcohol contents by the
proposed method

Srno Type of drink Ethyl alcohol contents declared gwv Declared proof spirit

1 Rum 333 25 up

2 Brandy 333 25 up

3 Whisky 333 75 p

4 Golkonda Ruby wine 1463 33 p

5 White wine 843 19 p

6 Industrial methylated spirit 95 contained 5wood napthal

Where p proof and up underproof

In addition to these five samples, one sample of“Industrial methylated spirit”was also tested by the
proposed method All the six samples were first diluted using distilled water to bring their ethyl

alcohol concentration around 2g 100 ml1

Srno Type of preparation Declared ethanol Dilution vv Approximate

concentration gwv concentration in diluted
solution gwv

1 Rum 3330 1 to 20 166

2 Brandy 3330 1 to 20 166

3 Whisky 3330 1 to 20 166

4 Golkonda Ruby wine 1463 1 to 10 146

5 Golkonda White wine 843 1 to 5 168

6 Industrial methylated spirit 9500 1 to 50 190

79
These six samples were analysed by the proposed method using the analytical procedure described
earlierThe results are calculated using Beer’s law plot and are shown in table a and b The level

of ethyl alcohol in these samples were also determined by the AOACofficial method and shown
in the same tables.

80
81
Different concentrations of ethanol with equal methanol concentrations  were added to a fixed

ethanol of diluted preparation The ethanol was determined by proposed method as in in fig 5

shows initial fixed ethanol concentration on Y axisThe recovery of ethyl alcohol in thes
experiments with 8 observations showed average % recovery 99.8, standard deviation =0.57,
confidence limit with 95% =99.8±0.9 ( t=2.365) and confidence limit with 99%=99.8±1.7(t=3.499)

RESULT AND DISCUSSION

The alcoholic beverages are the fermentation products of suger or malt and, therefore, contain
organic acids, esters and acetaldehyde as by product The official AOAC method is based on

distillation and subsequent determination of specific gravityThe volatile acids as well as aldehyde

interfere in this method In pphenylphenol reaction26, 27

formaldehyde interferes Whereas
28
according to Dagani and Acher , in their method, removal of larger amounts of formaldehyde may
require somewhat longer reaction times, higher temperatures or higher concentrations of 2, 4

pentandione The unconsumed formaldehyde will give blue color  max 600 nm  with p

phenylphenol, thereby seriously interfering acetaldehyde determination  max 560 nm  This

means that one has to determine formaldehyde concentration in the mixture and then set the
reaction conditions accordingly This makes the already critical reaction conditions more

laboureous and time consuming In the proposed method 2thiobarituric acid selectively reacts,

under experimental conditions described, with acetaldehyde only and not with formaldehyde

Baker noted that copper lime treatment of blood or redcells liberated acetaldehyde It was found
that the amount of bound acetaldehyde liberated27 from human blood by this treatment depends
upon the dilution of blood, concentration of reagents and specially the time and temperature of Cu

lime treatmentValues as high as 90 mg percent were obtained by Culime treatment in water bath
Such a value may be too high and will be misleading in traffic offences due to drunken driving,
especially if the method is used for ethyl alcohol determinationNormal viscera, blood etcdid not

show any false value of acetaldehyde or ethyl alcohol by proposed method

Several workers have used 2thiobarbituric acid as a reagent for thin layer chromatographic analysis

of sorbic acid, polyhydric alcohols etcafter potassium dichromate treatmentChloral hydrate reacts

with 2thiobarbituric acid in alkaline mediumIn the proposed method, proper care has been taken

to eliminate the interfering compounds during first distillation with HgCl2 and NaOH All acids
82
such as lactic acid, sorbic acid, get neutralized while chloral hydrate is converted to chloroform by
NaOH Chloroform does not react with 2TBA Higher alcohols such as butyl alcohol, propyl

alcohol etcwhen treated under identical reaction conditions, did niot give any color with 2TBA,

whereas immiscible layer of aldehyde formed by amyl alcohol was slightly off whiteTherefore, the

proposed method of 2thiobarbituric acid was found more specific, accurate and sensitive for the
determination of acetaldehyde and ethyl alcohol even in presence of formaldehyde and methyl
alcohol respectively The graph of acetaldehyde concentration against optical density and

acetaldehyde plus formaldehyde against optical density was superimposable fig6 This confirmed

that there is no deviation due to the presence of formaldehyde in the mixture



83
84
85
86
References

1 Harger, R N Toxicology Mcchganism & Analytical methods, Vol II p87, 1961

EditorStewart, CP& Stoman, A ed , Academicc press, New York & London

2 MUchlberger, CW ¦ Alcohol Inloxication, Legal Medicine R B H Groundwohl 1954

The CVMosbey & coLondon

3 Berrgren, SM& Goldberg, LActa physiolScandVolI, 246, 1940

4 Harger, RN, Hulpieu, HR & Lamb, EBJBiolChem, 120, 689, 1937

5 Tuovinen, PJSkand ArchPhysiol60, 1, 1930cfCA25, 334, 1931

6 Harger, R N, Hulpieu, HR ¦ Alcoholism GN ThomsonEd  chapter 2, 1956,cc

Thomas, Springfield, Illinois

7 Harger, RN, Ferney, RB& Barnes, HBJLabClinMed36, 3061950

8 Gradwohl, RBHEdLegal Medicine, p756,1954The CVMosbey &coLondon

9 Myrson, RM Biological Basis og Alcoholis, John Miley & Sons IncNew York, p 183

208, 1971

10 Burch, GE& Giles, TD¦The Biology of Alcoholism, vol3p435460, 1974Clinical

Pathology, Plenum Press, Neew York

11 Newman, HW& Fletcher, EAmJMed Sci, vol202, 723, 1941

12 Goldberg, LActa PhysiolScand5 SupplNo16, 1943

13 Octtingen, W F von public Health Bulletin No281, U S Govt Printingofgice,
Washington DC, 1943

14 Kini MM & Copper, JR,Biochem, J,82, 16472,1962

15 Morgan R & Cagen EJ¦The Biology of Alcoholism, vol3 Clinical Pathology Kissin,

87
 B& Begleiter, HEd , p1630189, 1974Plenum Press, New York

16 Greear, J N The causes of blindness Blindness Modern Aproaches to the unseen

Environment Zahl, PAEd Princeton University press, 1950Princeton, New Jercy

17 Personal Communications

18 Haggard, HWand Greenberg, LAJPharm& ExprThera p66, 47996, 1939

19 Nash TBiochemJ55, 41621, 1953

20 Cochin, J& Axelord, JJPharma &ExprTherapVol125, 10511, 1959

21 Kaniewski, JRocznZakiHig, warsaw, 11 3 , 241246,1960 cfAnalAbstr82004,

1961

22 Supniewski, JVJBioChem70, 13, 1926

23 Sieber, RVhemZtg45, 349, 1921

24 Briggs, APBiolChem7167, 1926

25 Stepp, WJBiolchem146, 349, 1924

26 Barker, SB& Summerson, WH, JBioolChem138, 535554, 1941

27 Stotz, EJBiolChem148, 58591, 1943

28 Degani, Dand Archer, MCAnal, Biochem87,45559, 1978

29 Official methods of Anaslysis of AOAC¦ William Horwitz Ed  12th Ed, p193 with

specific gravity tables p96178, 1975 published by AOAC, Washington DC 20044

30 GetlerAO& SiegelGAmJClinpathology, vol7, 8593, 1937

31 Book, JCJBiolChem93, 645, 1931

32 FriedmanTE&Klass, RJBiolChem115, 4751, 1936

88
33 Gradwohl, RHBEdLegasl Medicine, p796797, 1954The Mosbey ^& co, London

34 Henry, RJJ LabClin, Med33, 24145, 1948

35 Harger, RNJLabClilnMed20, 74651, 1935

36 Widmark, EBiochemZtschr131, 47374, 1922

37 Cavett, JWJLabClinMed3543, 1938

38 Kozelka, FL& Hine, CHIndus and EnginChem analEd 13, 9057, 1941

39 Kozelka, FL& Hine, CHAnalyst London 67, 174, 1942

40 Mahal, HSAnalChem31, 1908, 1959

41 Gradwohl, RHBEdLegasl Medicine, p770, 1954The CVMosbey & co,St, Louis

42 Kirk, PL, Gibor, A, Patker, KPAnalCHem30, 1418, 1958

43 Smith, MD& Olson, CLAnalChem47 7 , 10, 1975

44 Kurhekar, MPand Matto, BNCurrSciSci43 2 ,4546, 1974

45 Berkar, SBJBiolChem, 137, 783, 1941

46 Feigl, FOrganic Spot Tests Elsevier publication Co, Amsterdam, Vth English Edition,

1954

89
 CHAPTER -V

COLORIMETRIC DETERMINATION OF ENDRIN

90
1) Introduction
2) Chemistry of Endrin
3) Metabolism of Endrin
4) Review of analytical methods
5) Proposed method
a) Standardization of experimental methods
b) General procedure
c) Standard curve
d) Application of the method to emulsifiable concentrates
e) Recovery of added endrin to formulation
f) Application of the method to biological specimen

6 Results and discussion

List of figures

1) Figure 1 -Absorption maximum of the color formed

2) Figure 2 -Effect of sodium perborate on reaction
3) Figure 3 -Effect of formaldehyde on reaction
4) Figure 4 -Effect of sulfuric acid on reaction
5) Figure 5 -Rate of reaction and stability of color formed
6) Figure 6 -Standard curveBeer’s law plot

7) Figure 7 Recovery of added endrin to formulations

Endrin is a widely used pesticide for agricultural porposes through out the world This naturally,

has its contribution towards population and health hazardsThe magnitude of pestcidal pollution
in India has primarily resulted fron the iondiscriminate and injudicious use of number of potent
insecticidal products which are inimical to ther human health and hazards The gretest hazards

has come from organo chloro pesticides which are notoriously persistent and capable of

accumuilation in the environment from which they are ultimately stored in the human body fat

Accumation of such pesticides in stored fat may lead to the serious hazards like carcinomaThe
hazards associated with the modern pesticides is mainly due to chronic poisoning and
envirinental pollution originating from pestcidal residue, industrial manufacture are normal
exposure during use The poisoning through pestcides including homicidal, suicidal and

91
accidental is also associated with the insecticidal hazards Although limited survey have been

conducted regarding contamination of vegatables Oil seeds, cereals, animal feeds, eggs, meat,

milk etsWith DDT, Malathion, it has been found that the problem is very seriousKeeping this
trend in view all over the world, the prob lem of fixing the safe limit for pesticide residues in
food, feed, water, air etchas been engaging the attention of number of national and international

bodiesAt the intrrnational level, codex Alimetarius Commission, Joint Committee of the World
Health Organisation and Food and Agricultural Organisation are setting up standards for
pesticides and commodities involved This work is also being considered by the committee
under the prevention of Food Adultration Actunder the Union Ministry of Health, and
tolerewnce limits for some pesticides residues have already been prescribed More recently,
Insecticide Act 16970 has been enforced under the Department of Agriculture, which would
regulate production, sale and use of pesticides from all aspects, keeping the health hazards of
pesticides in view, Indian Standards Institution is also actively collaborating with the above
agencies in the country to deverlop analytical method for the determination ofpesticideresidue
in various food materials

Chemistry of Endrin

Chemistry endrin is, 1, 2, 3, 4, 10, 10Hexachloro67epoxy1, 4, 4a, 5, 6, 7, 8, 8a

octahydro1, 4 exo  dimethonapthalene Its molecular formula is C12H8Cl6O with molecular

weight 380.9It is white crystline solid with melting point 200202ocIt is insoluble in waterIt

is soluble in acetyone, carbon disulphide and benzeneIts UV in ethanol is 224nmIt shows IR

peaks 1, 2 , 3 at 1176 μ 850cm1 It has the following strucral formula

92
Metabolism of Endrin

The metabolism of endrin has been extensively studied in rats4, 5The major metabolite is found

to be anti12hydroxy endrin IIb which is eliminated in bile in the form of its glucuronide

conjugate IId It is excerted deconjugated in facesEndrin inhibits enzymes, like succinic

dehydrodenase, cytochrome oxidase, Lipase, Chlinesterase etc, in the body

Substitution at

A B

A Endrin H H

B Ani12hydroxyendrin H Hydroxyl

C 12keto endrin Carbonyl

D glucuronide of anti12 H D glucuronic acid

hydroxyendrin

Review of Analytical methods

Quantitative analysis of endrin contents by determining total organic chloride and Infrared

spectrophotometric methods are recommended by Indian Standards Institute and AOAC1,2 in

case of formulations Colorimetric method based on dichlorination of endrin and subsequently

93
coupling with diazotized sukphanilic acid is reported by Bann et al6 There are some other

colorimetric methods reported in the leterature7,8,9  Two thin layer chromatography method
based on the use of zinc chloride in hydrochloric acid10 and the sulphuric acid catalyzed
isomerization11 are also being used for quantirative analysisTreatment of ethanolic silver nitrate

followed bu UV radiation12, silver nitrateformaldehydepotassium hydroxidenitric acid

hydrogen peroxide in succession13, alcoholic otoluidin13 or odianisidine14 are some other

chromogenic reagents used for quantitative thin layer chromatographic method Some of the
workers used bioassays for the determination of endrin as they are capable of detecting very
small amounts of toxicantsThe most commonly used species for bioassay were vinegar flies15

Drosophila melanogaster through other species such as water fleas Daphnia magna and gold

fish16 carassius auratus  were also used There are number of methods used on gas

chromatography10,17,18 and mass spectrophotometry17,18 Excellent reviews of all those methods

are reported by Beynnon et al19 and Wlliam et al20 White and McKinley3 had reported

advantages and disadvantages of various analytical methods while reporting an IR method

SrNo Method Adavntages Disadvantages

1 Total Chlorine Rapid, Inexpensive Nonspecific

2 Colorimetric Semispecific Slow, unstable reagent

3 Hydrobromic acid Semispecific Slow, unstable reagent

4 Infrared SpecificRapid Initial expenses

But later studieds shows tht there is a shift in IR peak at higher concentration of endrin1

Proposed Metyhod

In this proposed metyhod, endrin wss treated with formaldehyde followed by sulphuric acid in
presence of sodium perboraye The yellow color developed is measured at its absorption

maximum 405nm fig1  The method is sensitive and obeyed Beer¬s law for the concentration

range 4μgmL1 to 40μgmL1

94
Method

Materials

i Methyl alcohol

ii Acetone

iii Acetic acid glacisal

iv formaldehyde 40vv 

v Sulpohuric acid concentrated

vi Sodium perborate

vii Endrin standard shell Chemical corporation, USA

viii Carbon disulphate

ix Sodium hydroxide 10aqueouswv 

All the cjemoical used were ofanalytical grade unless specified otherwise

Preparation of the sodium perborate solution saturated 

150 mL methyl alcohol was mixed with 152mL glacial acetic acid and 100g sodium
perborate was dissolved in this mixtureThe solution was kept overnight in stoppered bottle to

ensure the saturation

Preparation of thestandad solution of Endrin

500 mg of pur recrystallized endrin was accurately weighed and dissolve in about 20 mL
acetone The final volume waa made up to 50 mL in a volumetriv flask using methynol as

solventThis gave the standard solution having concentration 10mg mL11 mL of the standard

solution was transfered to 10 mL volumetric flask and volume was made up by methanolThis

endrin working standard solution had concentration 1 mg mL1

95
 Standardisation of experimental conditions

Formaldehyde and siulphuric acid react with endrin in presence of sodium perborate forming a
yellow colored compound Sodium perborate gives out H2O2 in ptresenmce of acid, which

promotes the reaction of formaldehyde sulphuric acid with endrinThe Maximum absporption of

the color formed, amount of sodium perborate, sulphuric acid and formaldehyde required etc

were studied

(A) Absorption maximum

A standard endrin solution containing 600μg of endrin was tranfered to a 25

mL volumetric flask1 mL sodium perborate, 1 mL formaldehyde and 20 ml sulphuric acod

were adde in successionThe comntents were kepy for 30minutes, after which the voliume

was made upto 25 mL with methanol This colored solution was then scanned between

380nm to 650 nm against the reagent blank The maximum absorption peak was noted at

405nm fig1 

Reaction color λ 405nm

B Effect of sodium perborate

In a series of 25mL volumetric flask, 600μg endrin standard solution was transferred and
different volumes of sodiumperborate solution were added The color was developed by

adding 1 mL formaldehyde and 2 mL sulphuric acid as per the procerdure described earlier

The absorbance was measured at 405nm It was observed that absorbance increased with
increasing amount of sodium perborate and reached maximum at 1 ml sodium perborate,

96
after which the absorbance remained constant for higher concentration of sodium perborate

The graph of absorbance against milliliters of sodium perborate added is shown in fig2

Effect of Formaldehyde

In a series of 25 mL volumetric flasks, 600 μg endrin standard solutions were transferred

1ml of sodium perborate was added to each flaskDifferent volumes of formaldehyde were

added to a series of flasks20 mL sulfuric acid was then added to each of the flaskAfter 30

minutes, the contents were diluted to 25 mL mark using methanol The absorbance of the

color developed was measured at 405 nmIt was observed that the absorbance increased with
the increasing volume of formaldehyde till 1 mL, thereafter the absorbance remained
constantfor more quantity of formaldehydeThe graph of milliliters of formaldehyde added

against absorbance is shown in Figure 3

EFFECT OF SULFURIC ACID

In a series of 25 mL volumetric flasks, 600 μg of endrin standard solution was transferred

To each flask 1mL sodium perborate and 1 mL formaldehyde were addedDifferent volumes

of sulfuric acid were added to different flasksAfter 30 minutes, the contents were diluted to

25 ml mark with methanol and absorbance was measured at 405nmThe graph of volume of
sulfuric acid againt absorbance showed that the absorbance increased with the increase in
volume of sulfuric acid till 20 mLAfter 2 mL, the absorbance was same for further addition

of sulfuric acidThe graph iis shown in figure 4

RATE OF REACTION AND STABILITY OF THE COLOR

It was observed that the color formation was accelerated because of the heat formed due to
exothermic reaction of sulfuric acid with the remaning mixture In a series of 25 ml
volumetric flasks, 600 μg endrin standard solution were treated with 1 mL sodium perborate,
1 ml formaldehyde and 2 ml sulfuric acid The contents were diluted at different time

intervals and absorbance was measured at 405 nm A graph of absorbance against time in

minutes figure 5 showed that the maximum absorption was attained after 30 minutesThe

absorbance was constant over 100 minutesThat showed the stability of the color formed is

over 100 minutes

It was therefore, concluded that 1 ml sodium perborate, 1 ml formaldehyde and 2ml sulfuric
acid were the optimum volumes of reagents and 30 minutes was the optimum reaction time

ANALYTICAL PROCEDURE
97
1 ml sample under test was pipetted in a 25ml volumetric flask1 ml sodium perborate and 1

ml formaldehyde were added The contents were properly mixed mixed by gently shaking

the flask2 ml sulfuric acid was added very slowly, swirling the flaskThe flask was allowed

to stand for 30 minutes after which contents were diluted to 25 ml mark by methanol

Absorbance of the color developed was measured at 405 nm, against reagent blank The

reagent blank was prepared by treating 10 ml methyl alcohol in place of sample

STANDARD CURVE
A series of endrin standard solution having concentrations 100 μg , 200 μg , 300 μg , 400 μg,
500 μg , 600 μg , 700 μg , 800 μg , 900 μg , 1000 μg were taken in 10 different 25 ml
volumetric flasks, and treated as per the analytical procedure described earlierThe graph of
endrin concentrations against optical densitywas a straight line passing through originand
was found to obey Beer¬s law for the concentration range of 4 μg to 40 μg ml1 endrinThe

graph is shown in figure 6

APPLICATION OF THE METHOD TO EMULSIFIABLE CONCENTRATES

The marketed formulations of endrin are available in the form of emulsifiable concentrates of
different strengthsFive such concentrates were analysed by the proper method

PREPARATION OF THE SAMPLE

To accurately weighed 1 g emulsifiable endrin formulation 50 ml aqueous sodium hydroxide
10wv was addedThe mixture wa tranfered to separating funnelThis was extra cted five

five tinmes using ml carbondisulphide each timeAll the extracts were collected together and

solvent was evaporated completelyThe residue was dissolved in 10ml acetone and dioluted

to 100ml withyh methanol in a volumetric flask 1ml of this solution was pipetted in 10ml

volumetric flask and made up to volume using methanol 1ml of this solution was transferred

in to 25 ml marked volumetric flask and processed as per procedure desciged earlier The

concentration was determined by extrapolating the absorbance from the standard curve If

this amount determined in 1ml say x μg

In 10ml >X x 10@

Thios was in 1 ml of undiluted solution of 100ml
In 100ml >X x 10 x 100@
This was in 1gh of formulation
In 100 g formulation

98
X x 10x100 x 100 μg per 100g X x10x100x1001,000,000 gww

The amount determined from formulation and percentage recovery is show in Table I a &

b

Recovery of Standard added to formulation

To a fixed amount of 500 μg of endrin formulation, ifferent concentration such as
100μg, 200 μg, 300 μg, 400 μg, and 500 μg of standard endrin were added The total

amount of endtin in each of the sample was determined by procedure described earlierThe

graph of endrin added and totsal endrin determine is shown in fig7The interception on the

Y axis represented the original fixed concentration of endrin The amount determined and

percent recovered is shown in Table II a & b 

Application of the method to biological specimens in vitrio 

Preparation of the sample 6,21 visceraBloodStomach contentsUrin etc

50g macerated sample was taken in a Erlenmeyer flask 50ml of 50 aqueous potassium

hydfroxide solution and 150 ml of 95ethyl alcohol were addedThe flask was attached to

a reflux condenser and was gently refluxed on a steambath for one and half hours The

contents were cooled to room temperature and transferred to 500ml separating finnel This

was extracted five times with 100ml carboidisulphide each time Alll the extracts were

collected together and solvent was evaporated to dryness The residue was dissolved in

petroleum ether 40o600c  and was placed on the column prepared as follows 500g
magnesium oxide slurried with 500ml distlled water, heated on water bath for 30 minutes
and filtered The magnesium oxide was further dried over night at 1100c This activated

magnesium oxide was mixed with celite in the proportion 11 by weightAn eluting mixture
was prepared by mixming 500ml carbon disulfide, 5ml dioxane and 445 ml petroleum ether
40o600c  The slurry of magnesium oxide celitemikxture was prepared by using this
eluting solvent mixture and poured in a chromatographic glass column of 30cm height and
25 cm diameter, with glasswool pluged at the bottomThe slurry was fixed with tapping

down the column to 20cm column hightEndrin extracted residue  dissolved in petroleum

ether was placed on top of the column The column was eluted using eluting mixture The

eluting rate was maintained at 5 ml min1 300ml eluate was collected and solvent was

evaporated completelyThius fat free endrin extract was dissolved in 1 ml acetone and made

99
up to 10ml in volumetric flask This solution was further processed as per the analytical

procedure described earlier

Calculations

If «Y¬μg was the amount of endrin determined by extrapolating Beer¬s low plot in 1ml

soltution  then Yx 10  μg was endrin present in total ectract Therefore >Yx101000@ mg

was the amount in total 50g biological specimen taken for analysisThe amount of endrin in

mg shown inTableIII a, b were calculated accordingly

The recovery of endrin in vitro  from biological material was studied by adding definite

quantity of endrin to various biological specimen such ass viscera stomach, intenstine, liver,

spleen, kidney etc , blood, urine and stomach contents etcThe biological specimens were
extracted and extract were purified by column chromatography as per the procedure
described for the preparation of the sample The final residue thus obtained was dissolved

inn about 4ml acetone and made up to 10ml in volumetric flask using methanol This

solution was used for the determination of endrinThe percent recovery of the endrin added

is shown in Table III a & b  The statistical parameters such as standard deviation, co

efficient of variation and confidence intervals at 95and 99level were calculated

The molar absorptivity of the yellow color formed by endrion was calculaterd from
Beer¬s Law plot using formula

Molar absorptivity slope xmolWeight x103

 0375120 x3809x103

 119x 103moles litre1

This shows that ye method isd sensitive for tge routine analytical procedure

Result and Dioscussion

Very little work has been reported on endrin so far, inn comparison to the work on organo
phosphorous insecticides, DDTY, various drugs etdAt low concentration endrin shows IR

peak at 1176 microns 850cm1  but a shift was reported at higher concentration1 This

affects the specicity and accuracy of the methodColorimetric method reported by Bann et
al6 using diazotized sulophanilic acud gives stong background color as diazotized sulphanilic
22
reagent react with excess sulphanilic acid  Though this background color was reduced

using ammonium sulfamate by Fahey abnd Schechter22, it could not be eliminated totally

The reaction used on TLC were not found usuful for colorimetric methodIt was observed
100
that formaldehyde sulphuric acid reagent, known as Liberman¬s reagent is used foir the

identification of opium alkaloids, petroleum hydrocarbons etc, also form color with endrin

in presence of sodium perborate The freaction was also responded by other organochloro

insecticides like Aldrin, dieldrin, DDT, etcThe method proposed is sensitive and usdful for

the routine colorimetric deyermination of endrin



101
References

102
103
104
105
106
References;

Indian Standard specification for Endrin Emlsifiable concentratesIS 13101958

1
Indian Standard Institution, Mnak Bhavcan, 9, Mathura Rd, New Delhi, India,

1959

Official Method of Analysis of AOAC, William Horwitz ED , 12th Ed , 98,

2
1975

White TT& McKinley, JJAssoOffAgricherm,44 3 , 591, 1961

3
Baldwin, MK, Robinson, J& Parke, DVKJAgriFdChem18,1117, 1970
4
Baldwon, MK&Thuston, DHAnalyst London 105 1246 60, 1980
5
BannJM, Lau, SC, Potter, JC, Johnson, HW, O¬Donnell, AE& Weiss, F
6
TAgriFdChrem6,196, 1958J

O¬Donnell, AEJAgriFdChrem2,573, 1954

7
O¬Donnell, AEJAgriFdChrem3, 757, 1955
8
Beckman, HFAnalChem26, 922, 1954
9
Wienke, WW & Burke, JQJAssoOffAnalChem52, 1277,1969
10
Chou, ASY & CochraneWPJAssoOffAnalChem52,1220, 1969
11
Abbott, DC, EgonH & Thomson, JJChromat16, 481, 1961
12
Salo, T, Salminen, K& fiskari, Kz, Lebensmiottel untersuforch, 117, 369, 1962
13
CfChemAbstr57, 29571963

Kawashiro, I & Hosogai, Y Shokuhion Eisengaku Zasshi, 5, 54, 1964cf Chem
14
Abstr61, 6262, 1964

Phillips, WF, Bowman, M C & Schukltheisz, R JJ Agri Fd FdChem 10,
15
487, 1962

Davidow, B& Schwartzman, GJAssoOffiAgriChem38, 533, 1955

16
Zweig, G& Sherma JAnalChem48 5 , 66R83R, 1976
17

107
 Zweig, GJAnalChem61 2 , 229248, 1978
18
Beynon, KI& Elgr, KE¦Analyst London 91 1080 , 143, 1966
19
Willliams, S& Cook JW¦AnalChem39, 142R157R, 1967
20
Onley, JH¦JAssoOfficAgriChem, 47, 317, 1964
21
Fahey, JE & Schechter, MS– AgriFdChem9 3 , 192, 1961
22

108
109
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Publications;

1 Synthesis of thiobenzophenone, Curr. Sc. 47(20), 765-766, 1978.

2 Synthesis of thiobenzophenone, Ind. J. Chem. 17(8) 4,398-399, 1979.
3 A simple colorimetric method for the determination of paracetamol from
biological specimen, Curr. Sci. 49(17) 650-652, 198. Anal Abstr. 41(4),
4094,1981
4 A simple colorimetric method for the determination of acetaminophen, J.
Asso. Off. Anal. Chem. 63(6), 1189-1190, 1980. Anal .Abstr. 40(6), 6E53,
1981.
5 Colorimetric method for the determination of ethanol in presence of
methanol, J. Asso. Sci. Off. Anal. Chem. 64(5), 1145-1148, 1981.
6 Colorimetric determination of Endrin in biological preparations, Foren. Sci.
International (The Natherand) 18(1), 63-68, 1981.
7 Determination of organothiophasphate insecticides in technical material and
formulation, J. Asso. Off. Anal.Chem. 65(1), 40-42, 1982. Anal.Abstr.
43(6), 6G34,1982
8 Determination of organochloroinsecticides Endrin informulation J. Agric.
Food. Chem. 29(3)614-615, 1981.Anal. Abstr. 43(1), 1G59, 1981.
9 Determination of acetaldehyde in presence of formaldehyde, Ind. Grug.
19(3), 1118-120, 1981.Anal. Abstr. 43(1), 1D122, 1981.

121