MODULE -03

INTRODUCTION

1. Definition:

The word “Toxicology” is derived from the Greek word “Toxicon” which was used
as a poisonous substance to arrowheads. Traditionally, the toxicology is defined as the
science embodying the knowledge, source, character, fatal effect, lethal dose and analysis
of poisons and the remedial measures. A poison is defined as the substance, which is
capable of producing injury or death when absorbed. Appropriate dosages can differentiate
poison and also the remedial measures. All chemicals can produce injury or death under
certain conditions. Hence, a poison can be defined as a substance that is capable of
producing detrimental effects on a living organism. As a result, there may be a change in
the structure of the substance or functional processes, which may produce injury or even
death. The toxicologist is specially a trained expert to examine the role of such substances
and their adverse effects. The variety of potential adverse effects and the diversity of
chemicals present in our environment contribute to make toxicology a very broad field of
science. Therefore, toxicologists are usually specialized to handle various areas of
toxicology.

The professional activities of toxicologists fall into four main categories i.e.
Forensic, Industrial, Clinical and Environmental Toxicology. Forensic toxicology emerged
on the hybrid of analytical Chemistry and toxic principle effects. Forensic toxicologists
are also primarily concerned with the medico legal aspects of the harmful effects of
chemicals on human and animals. The expertise of forensic toxicologists is primarily
utilized in establishing the cause of death and elucidating its circumstances in post-mortem
investigation. The work of forensic toxicologist is therefore considered as highly
complicated as small quantities of poisons and their metabolites are to be isolated, purified
and quantified from a highly complex matrices.

1. ISOLATION AND PURIFICATION OF POISONS

Isolation and purification of poisons.

1. BASIC STEPS IN ANALYTICAL TOXICOLOGY:
 Extraction of active constituent i.e, poison in matrices of interest.
 Stripping or purification of active constituent thus separated.
 Rapid Screening and Identification
 Quantitation
  Interpretation / Conclusion.
GLOSSARY OF TERM RELATED TO EXTRACTION:
Matrix: Any material substance wherein the active constituent may be dispersed,
accumulated, left, absorbed or chemically bound.
Active Constituent: The toxic chemical of interest i.e. poison.
Stripping: Purification.
3. Classification of Matrices:
 Biological: Viscera, blood, urine, saliva, stomach contents, intestinal contents,
gastric lavage, vomit, brain matter, stool, faecal matter, bone, nails, hair, skin.

 Non Biological matrices: Water, remnants or traces of poison in small container,

food and food products, milk and milk products, fruits, vegetables, tea, coffee,
cooked materials, drinks, cereals, pulses, wines, etc.

DIFFERENT CLASSES OF POISON:

For the purpose of chemical analysis, poisons are grouped according to the methods used
for isolation of the substance from matrices. These are given below.

 Noxious gases
 Volatile poisons (organic and inorganic).
 Non-volatile poisons (organic and inorganic).
 Plant poisons
 Miscellaneous poisons.

4.DIFFERENT METHODS OF EXTRACTION:

There are various classical and modern methods of extraction. The selection of
proper method of extraction depends on various controlling factors viz. nature of poison,
matrix or matrices and also quantity of samples available or forwarded for analysis. The
active constituent should be extracted from sample in minimum steps to avoid loss during
processing. The extracted materials also require proper stripping or purification to avoid
interferences of matrices as far as possible.
The efficiency of extraction and stripping determines the lower limit of detection,
precision and accuracy in the determination. The analyst plays a significant role in the
selection of proper methodology on the basis of different parameters viz. case history,
amount physical state of matrices, analytical requirements and also infrastructural facilities
available. It will be befitting if the methods are presented Schematically depending on the
classification of poisons.
Class of poison Classical Method Modern Method
Gases Micro-diffusion, Adsorption- desorption Sensor Based Gas Analyzer,
Gas Chromatography.
Volatile Gutzeit Method, Massh-Berzelius Microwave oven technique for digestion
Inorganic Method, Micro-diffusion, Digestion followed by Ion Chromatography using
with specific reagents / under specific Ion exchange resins,. Spectroscopy etc
conditions of PH, Crystal test, Color
tests etc.
Volatile Distillation, Steam Distillation, Diffusion Gas Chromatography/mass spectrometry.
Organic
Non-Volatile Dry and wet ashing, Group analysis, Microwave oven technique for digestion
Inorganic Electro- dialysis, Digestion under followed by Ion Chromatography using
appropriate analytical conditions, Ion-exchange resins, Inductively coupled
Titrametric, Paper and Thin layer plasma atomic emission spectroscopy,
chromatography (ICPAES), ICP-MS.
Non-Volatile Solvent Extraction, Stas- Otto, HPLC, Paired ion extraction /
Organic Digestion with ammonium sulphate, Chromatography, HPTLC, Supercritical
Sodium tungstate or related method. fluid chromatography, Solid phase
extraction, Micellar extraction, Affinity
chromatography, , Microwave assisted
Reaction system, Accelerated solvent
Extractor, Sweep Co-Distillation Universal
Trace Residue Extractor.
Anion Dialysis, Chemical Digestion, Paper and Ion-Chromatography by Ion-Exchange
Thin Layer Chromatography resins.

Unit processes/operation in extraction methods: The various unit processes / operation related
to extraction are given below for convenience.
Solvent Extraction:
A system of two immiscible liquid is required for the separation of material by solvent
extraction. The active constituent should be unevenly soluble in the system thereby
facilitating extraction of the constituent from one phase to the other. The efficiency of
extraction is determined by distribution co-efficient (D).
 Total wt(gms.) of solute in the Organic Phase
D=
Total wt (gms.) of solute in the aqueous phase

If one of the two liquids contains a solute and the system is shaken and then allowed to
settle, some of the solute will be transferred to other liquid. Each of the liquid in a mixture
of two immiscible liquids of this kind is referred to as a phase. Thus, some of the solutes is
transferred from one phase to the other in the two phase system. The amount transferred
depends on the relative affinity of the solute for each of the two solvents (relative
solubility). It is determined by D. Greater the value of D, greater is the efficiency of
extraction.

The immiscible system may involve two organic solvents. The extraction for this
system may be impaired due to formation of emulsion. Solvent extraction is a common
technique in forensic toxicology related to biological matrices. Solvent extraction method
has know been upgraded and made automatic viz. accelerated solvent extraction. In case of
solid non-biological matrices, continuous extraction by a soxhlet may be employed i.e.
continuous extraction.

Distillation: The process involves heating a sample of liquid to convert it into vapour which is
then allowed to flow in another location, where it is cooled, considering it back into a liquid.
Vapour modification of the basic distillation process are used for specific Purpose viz.
distillation under reduced pressure, fractional distillation, sweep co-distillation.
Steam distillation: Volatile substances can be separated or isolated from blood, urine
homogenates or properly minced viscera by the operation of steam distillation. Steam is passed
into the solution and the aqueous distillate collected by condensation. Toxicants from acidic
distillation process include ethanol, methanol, phenol, halogenated hydrocarbons, cyanides etc.
On the other hand toxicants from basic distillation process include basic drugs amphetamine,
methadone and also aniline, pyridine, nicotine etc.

Fractional Distillation: This is a type of distillation which enable us to separate a mixture of

volatile liquid differing in boiling point. A mixture of kerosene oil of mineral turpentine oil in an
oil-water emulsion may be separated by steam distillation.

Distillation under vacuum: This is another type of distillation which provide separation of a
thermally labile volatile compound at a low temperature without decomposition.
Sweep co-distillation: It is a special case of distillation that is based on the preferential
volatalisation of organic compounds specially pesticides from oil, lipids or plant extracts using a
stream of inert gas and subsequent isolation of volatiles on cola traps or solid adsorbent. It is a
purge and trap technique involving dispersion of the sample in thin films on deactivated glass
beads or florisil or alumina or silica gel or tenax as trapping media at elevated temperature.
Universal Trace Residue Extractor (UNITREX) and Accelerated Solvent Extractor (ASE) i.e. the
highly automatic extraction systems for rapid extractions of multiple samples work on this
principle.

Micro diffusion: Micro – diffusion is a convenient and popular operation that facilitates
toxicants (gaseous and volatiles) in blood, urine and gastric, aspirates to be detected or
determined after isolation by various techniques. This is done by Conway Micro-diffusion dish
(elaborated separately for convenience).

Dialysis: It involves separation of a crystalloid from a colloid by filtering through a semi

permeable membrane. This separation method may be employed for the separation toxic cations
and anions in a colloidal solution or dispersion or colloidal matrices specially biological
materials including blood. The separation process may be accelerated by applying e.m.f. i.e.
electro-dialysis.

Sublimation: This is similar to distillation except the sample is a solid to begin with and is
converted directly into vapour and then back into solid. Sublimation is applicable to isolate a
toxicant in solid matrices viz. naphthalene, an thracene which sublimes.

Digestion or chemical treatment: Sometimes active constituents (Toxicant) are separated on

treatment with acid or alkali or digestion on a water bath or muffle furnace viz. biological
matrices are digested on a water bath for 1 hour or above or digested in muffle furnace with
acid / alkali / or Chemicals to isolate active constituent. Volatile inorganic poisons or
phosphine, arsine and hydrogen sulphide are isolated from their salts on treatment with
dilute acids.

Microwave Digestion: Matrices are digested with acids / alkalis in microwave oven to facilitate
isolation of inorganic poison in organic matrices under a specific analytical condition of
operation viz. operation of over at a specific microwave for sometime. The interaction of
microwave with matrices results in production of heat with rise of temperature for which
digestion occurs.
Absorption:It is a slow process (compared to adsorption at the surface) involving diffusion of
one substance into the interior of absorbent material. Toxic gases and volatiles in oil are
entrapped and enriched by the process using a tube containing diverse absorbing materials
specific for a particular toxicant and on line detection and determination is facilitated.

Chromatography:Chromatography involves the separation of substances based on their relative

affinity for two phases, one stationary and one mobile. Substances which have higher affinity for
the mobile phases are moved or carried along with it and are thus separated from those with
higher affinities for the stationary phase. Thus, the toxicants in molecular mixtures may be
separated convenient under different chromatographic methods and operating condition in a
particular Chromatography. There are different controlling parameters viz. nature of toxicant,
mobile and stationery phase and temperature. Salient aspects of different Chromatographic
methods are given below.

Column Chromatography:Separation of active constituent is achieved here by preferential

absorption of active constituents on the adsorbent or stationary phase and also adsorption by the
mobile phase. In this method a vertical glass tube is filled with a granular adsorbent. This
adsorbent acts as the stationary phase. The sample is then added to the top of the column in the
form of a solution in a suitable solvent or mixture of solvents. The system of solvent is then
added as a mobile phase, which is to selected beforehand. As the solvent system flows through
the column under the influence of gravity or pressure, various components of the sample mixture
will migrate at different rates and then arrive at the lower end of the column at different rates i.e.
time. Fractions collected at various intervals will thus contain the different component separated
at different intervals. The fractions thus separated can be subjected to further analysis by
different methods. The process is also known as elution.

The efficiency of separation in the column is dependent on the adsorbent material,

selection of solvent system, nature of active constituent as well as flow of mobile phase.
These controlling factors have been explored to develop different method of
chromatography viz. HPLC, HPTLC, affinity, gel permeation, ion-exchange
chromatography and ion chromatography, solid phase extraction and super critical fluid
chromatography. These have been discussed separately. The analytical condition required
for separation of toxicants are either available in standard texts or references or may be
developed conveniently by trial depending on nature of toxicant and matrices.
Paper Chromatography: The separation of active constituent occurs on cellulose paper as the
stationary phase. This is the primitive method in chromatography methods and used for
separation of organic dyes, pigments, inks, cations and anions.

Thin Layer Chromatography: The separation takes place kin a thin layer of adsorbent material
such as alumina, silica gel G or cellulose coated onto an inert backing material such as glass
plate, plastic sheet or alumina foil. The chromatography development is made by applying
samples as small spots. The plate is then dipped in a chamber containing the developing solvent.
The solvent is allowed to migrate for some distance and separation is achieved. There are
different methods that are popular in TLC viz. ascending, decending, two dimensional etc.
Quantitative separation is achieved by optimizing analytical conditions.

High Performance Thin Layer Chromatography:A type of thin layer chromatography

wherein the stationary phase is designed to offer enhanced separation and resolution properties.
The enhanced separation is due to special structural feature of adsorbent attained by special
processing for which optimum resolution of molecular mixtures (for separation on
chromatography) coated with specially prepared adsorbent. The system (HPTLC) is now fully
automatic and multiple samples may be handled quickly, precisely and conveniently. Diverse
literatures are available on different classes or organic sample for their separation in simple and
complex matrices.

High Performance Liquid Chromatography: It is based on the different attraction of non-

volatile analytes viz. drugs, pesticide, explosive, organics etc. between a liquid phase ( pumped
through a column) and a solid phase ( packed within the column-stationary phase ). The
operating parameter include composition of mobile phase, adsorbent, nature of analyte etc. for
optimizations of resolution.

Ion Chromatography: It works on the same principle as in HPTLC. The composition of

stationary phase and also the mobile phase is so selected that cations and anions can be separated
conveniently. The active constituents in the matrix material and chemically bonded to molecules
which have a fixed charge. In suitable form ion exchange resins can be packed into columns and
used for separation of mixture of molecules which have the opposite ( because unlike charges
attract ) viz. cations and anions. In some cases, the net charge on the column material or sample
molecules or both is dependent on pH giving rise to greater flexibility.

Gas Chromatography: It is a procedure whereby volatile components of a mixture may be

separated by partition between a solid or liquid stationary phase and a gaseous mobile phase. The
efficiency in the separation is achieved by controlling several factors by the analyst viz. column
type (capillary or wide bore), column length, column diameter, nature of liquid phase, carrier gas
flow rate and temperature. This technique has now been hyphenated with other technique viz.
SFC-GC, HPLC-GC-MS. The method is specially applicable for pesticide, drugs, volatile
organic, solvents etc.

Modern Methods of Extraction: The methods include various methods in Chromatography as

well as special extraction methodologies of the present generation viz. ion-pair formation, solid
phase extraction, solid phase micro extraction, micellar extraction. Special types of digestion as a
means of isolation of toxicant through chemical processing i.e. micro wave digestion and
digestion by plasma source for inorganic samples are also emerging for their consideration in
routine toxicological analysis. Thus, there are various methods of extraction under the head “
Modern Method” which are either underutilized or unexploited in the Indian perspective due to
lack of infrastructural facilities and analytical expertise. As it has become very difficult to cope
up with much inflow of exhibits, modern methods are replacing traditional methods of extraction
for speedy analysis. A few methods viz. HPLC, HPTLC, GLC, GC-Head Space, Micro Wave
Oven Technique have become the methods of choice. The other methods including hyphenated
techniques HPLC-GC, SPC-GC, GC-MS are under processing for standardizing in the Indian
perspective. The available references in literature are to be followed for standardization or
standard analytical conditions are to be arrived at after trials and comparison with other methods.
However, the principles of the methods and analyticals concerning a few methods have been
covered in the present monograph.

Head Space Technique:The headspace method is specially suitable for the very fast separation
of volatile components (alcohols, acetone, aldehydes) in complex biological matrices specially
blood in mass-liquor and prohibition low related cases. This methods has the advantage that the
risk of contamination of non-volatile component may be eliminated for on line analysis of
toxicant by gas chromatography. The principle underlying head space analysis is that in a sealed
vial at constant temperature, equilibrium is established between the volatile components of a
liquid sample in the vial and the gas phase above it ( the head space ). After allowing the time
for equilibrium (normally 15 minutes or so for 50 or more samples or more samples in a single
run) a portion of the head may be withdrawn one by one from vials using a gas-tight syringe and
injected to GC on line for further analysis of the separated components.
Dynamic Headspace, Purge and Trap Technique: These techniques are basically modified or
higher version of Headspace method to optimize the separation of volatiles for on-line analysis
by GC. In the purge and trap method, volatile compounds (toxicants) are liberated from the
sample by bubbling with an inert carrier gas and subsequently either condensed in a receiver
cooled usually with solid carbon-dioxide or liquid nitrogen adsorbed on a catridge filled with
solid dsorbent material such as Tenax. It is polymer based diphenyl- p-phenylene oxide and is
available in various forms. Tenax TA is a highly purified form of the polymer and stable
upto375oC and gives insignificant bleeds of organics. A material suitable for the recovery of low
molecular weight compounds is Tenax GR which contains 23% graphite. This adsorbent is
suitable for efficient trapping of compounds of low to medium polarity and recovering them
quantitatively by either solvent extraction or thermal desorption. After some time trapped
volatile are flash vaporized into a stream of carrier gas for GC analysis.
Alternatively, catridges filled with activated charcoal can be used to trap the volatiles
which are then extracted into a small volume of carbon disulphide prior to the analysis. This
technique is widely used in the analysis of volatile compounds in water samples till date mainly
because of the difficulty in interpreting the results at concentration below those which can be
measured using ordinary headspace method. Purge and trap technique are similar to dynamic
headspace sampling except that the gas is passed through the sample. Clearly any apparatus used
for dynamic headspace sampling can also be used for purge sampling by using on appropriate
sampling vessel. Both dynamic and purge methods are available in a variety of automatic
systems, to enable separation of constituent of concentration in the ppb – ppt range.

Solid Phase Extraction (SPE): The old observation in the extraction of drugs by adsorption on
solid materials viz. Florisil ( A synthetic magnesium silicate ) or activated charcoal and selective
elution by solvent thereafter has given with to solid phase extraction method. In this method,
siliceous materials with relatively close size distribution (15 to 100 Mm ) and various silica
bonded phases viz. n- octadecyl (C18, ODS), n-octyl (C8), n-hexyl (C6), ethyl (C2), methyl
(C1), cyanopropyl (nitrile, CN) aminopropyl (amino, APS etc.) have been employed as adsorbent
with much greater efficiency of separation. The method is based on the use of small tubes or
catridges filled with 100-500 mg.
There lies however a recently developed type of SPE catridge in which very small
particles of the adsorbent are enriched in a well of PTFE micro fibrils having similar efficiency
as in a packed column but require less pressure drop. The sample is applied to the SPE catridge
ether from a syringe or sampling manifold by applied pressure or suction at the lower end.
Components are subsequently recovered by solvent flashing. The method is attractive because of
the small amounts of the sample and materials necessary and also much greater speed of the
procedure compared to classical adsorption method.
Analyte concentration may often be achieved more easily with SPE than with
liquid-liquid extraction while use of SPE column to concentrate an analyte from solvent
extract may provide a quicker and possibly safer alternative to solvent evaporation. A
major advantage of SPE is that latch processing can be simplified. Further feature when
screening for unknown is that a range of analyte can be extracted simultaneously although
this may create problem if analyte of a single component is required. Moreover, SPE
columns are expensive and it may not be possible to retain very water soluble analyte. The
SPE protocol is now available.

Solid Phase Extraction (SPE) cartridges are used primarily to clean up samples for
analysis and/or concentrate samples to improve detection limits. The lack of sufficient
sample preparation will result in poor detection limits, identification and quantitation
errors, contamination problems and rapid deterioration of GC or HPLC column
performance. SPE techniques usually provide better sample cleanup and recoveries than
liquid-liquid extraction techniques. SPE uses small volumes of common solvents, requires
very simple laboratory skills, does not require the use of highly specialized laboratory
equipments and allows rapid sample throughput. A liquid sample or solid sample dissolved
in a solvent is poured into the conditioned SPE cartridge. Vacuum or pressure is used to
force the sample through the sorbent in the cartridge. In SPE, vacuum manifold is normally
used to simultaneously process multiple cartridges. Usually, SPE methods are designed to
retain the analytes of interest; other sample components similar to the analytes also will be
retained. The analytes of interest are then eluted from the sorbent using another solvent.
This solvent is collected for analysis for additional processing.

SPE Cartridges
An SPE cartridge is composed of three basic parts:

1) Cartridge or tube body

2) Frits
3) Phase or sorbent
Cartridge or Tube Body : The cartridge body usually is a syringe like barrel made of
serological grade polypropylene.
Frits: The frits are used to hold the sorbent in the barrel and to act as a particulate filter.
Phase or Sorbent : The most common SPE phases are bonded silica-based materials. Irregular
shaped, 40 µm silica particles with 60Å pores are used as the starting material. Various silanes
are used to attach functional groups to the accessible areas of the silica particle. In addition,
several non-silica based phases are commonly used. Solvent reservoirs can be used to increase
the volume of barrel above the phase. Large amounts of sample or solvent (up to 75 ml) can be
added directly to SPE cartridges in one volume instead of in small increments. Coupling fittings
are used to attach the reservoirs to the SPE cartridges.

Phases: There are three types of phases: normal, reverse and ion- exchange.
Normal Phase : Table 2 lists common normal phase sorbents. All of these phases are polar and
are used to retain (extract) polar analytes. For normal phase sorbents, solvent strength increases
as the solvent becomes more polar. For example, a retained analyte will completely elute from a
normal phase sorbent in a smaller volume of methanol than chloroform. All of the solvents in
Table 1 are commonly used with normal phase sorbents. Mixtures of two solvents often are used
to refine the solvent strength for optimal sample cleanup and analyte recovery.
Reverse Phase : All of these phases are non-polar and will be used to retain (extract) non-polar
analytes. For reverse phase sorbents, the solvent strength relationship is the opposite from
normal phase sorbents (Table 2). For reverse phase sorbents, solvent strength increases as the
solvent becomes more non-polar. For example, a retained analyte completely elutes from the
sorbent in a smaller volume of acetonitrile than water. In most cases, the solvents used with
reverse phase sorbents are limited to water, methanol, isopropanol and acetonitrile as mentioned
in Table 1 (Reverse Phase). On occasion, acetone or dichloromethane may be used as an elution
solvent for highly retained analytes.
Ion Exchange Phase : Ion exchange phases are more dependent on pH, ionic strength and
counter ion strength than solvent strength. Ion exchange phases depend on ionic interactions as
the primary retention mechanism. Ionic interactions occur between an analyte molecule carrying
a positive or negative charge and a sorbent carrying an opposite charge. There are two groups of
ion exchange phases. The cation exchange phases retain positively charged or “cationic”
compounds. Amines and carboxylic acid are not charged species. They can be charged by
varying pH. The anion exchange phases retain negatively charged or “anionic” compounds.
Table 3 lists the classifications and characteristics for several common ion exchange phases
charge).

Table 1: Solvent strengths

NORMAL PHASE Weak REVERSE PHASE
Hexane Water
Isooctane Methanol
Toluene Isopropylalcohol
Chloroform Acetonitrile
Methylene chloride Acetone
Tetrahydrofuran Ethyl acetate
Ethylether Ethylether
Ethyl acetate Tetrahydrofuran
Acetone Methylene chloride
Acetonitrile Chloroform
 Isopropylalcohol Toluene
Methanol STRONG Isooctane
Water Hexane

Table 2: Sorbents

NORMAL PHASE

Cyano (CN)* Diol (DIOL) Silica (SI)

Amino (NH3) +1** REVERSE PHASE

Octadecyl (C18 OR ODS) Octyl (C8)

Methyl (C 3) Phenyl (Ph)

*may be used as a reverse phase also

**may be used as an ion exchange also
Relative counter ion exchange.

Table 3: Ion Exchange phases

CATIONS ANIONS
-1 -1
Li +1 H +1 0.5 OH ,F ,Propionate 0.1

Na+1 1.5 Acetate,Formate 0.2

(NH4) +1 2.0 (HPO 2-) ,(HCO –1) 0.4
4 3
–1 -1
Mn+2,K+1,Mg+2,Fe+2,+ 2.5 CI , (NO2) 1.0
3
Zn+2,Co+2,Cu+1,Cd+2 3.0 (HSO3) -1 ,CN -1 1.5
Ca+2 4.5 (NO2) -1 4.0

Cu+2 6.0 (CIO3) –1 4.5

pb+1,Ag+1 8.5 (HSO4) -1 5.0
Ba+1 10.0 Citrate 9.5
Benzene sulfonate 10.0

Solid Phase Micro Extraction (SPME): Solid phase micro extraction (SPME) is an extraction
technique for organic compounds in aqueous samples in which analytes are adsorbed directly
from the sample onto a fused silica fiber that is coated with an appropriate stationary phase.
When the fiber is inverted in the sample, the analyte portion from the sample matrix enters the
stationary phase until equilibrium is reached. The fiber in then inserted into the injection port of
a gas chromatograph (GC) where it is heated and the analytes are rapidly thermally desorbed into
a capillary GC column for analysis.
The fiber holder can be used manually with any GC having appropriate straight
inlet liner. The holder is designed to be used with a reusable, replaceable fiber assembly.
Each fiber can be used for 50 to 100 analysis or more depending on the particular
application and the care that is given. The holder consists of a stainless steel barrel, a
bisack polymeric plunger on adjustable depth gauge with needle guide a stainless steel
relating nut.
Each disposable fiber assembly for manual injection has been outer septum piercing
needle with a flanged brass ferrule a gray sealing septum and an inner fiber attachment
needle. The fiber attachment needle has a coated fused silica extraction fiber secured at one
end and a colour- coded thread at the other. A tensioning spring is located between the nut
and the sealing septum. The nut color indicate the type of bonded phase coating on the
fiber.
Super Critical Fluid Extraction: Gases above their critical pressure and temperature are in a
supercritical state, intermediate between that of a gas and liquid. Supercritical fluids have
strong extraction properties because the solubility of compounds in fluid is close to that of a true
solvent and much lower viscosity allows it to percolate through packed bed of sample. Thus,
knot only therein an efficient contact between the extracting fluid and the sample but the fluid is
easily removed when it is released from its supercritical state. Carbon dioxide is nearly always
the chosen gas for SPE in view of its innocuous nature and mild critical condition namely
critical pressure of 75 bar and a critical temperature of 310C which are relatively easy to achieve
at present.
The sample holder is composed of a number of small stainless steel catridges which
are filled with the sample in a particular state. Solid sample such as soil or sediment are
packed into the cartridges without any pretreatment and aqueous samples can be flashed
through the cartridges filled with an appropriate adsorbent to concentrate all of the
contaminates. The catridges are subsequently fed into the extraction oven and the carrier
gas line which at this stage consists of supercritical carbon dioxide. The extracted
compounds are carried to the cold trap and condensed after the heated constriction which
restore carbon dioxide to its true gaseous state. After the appropriate extraction period, the
circulation of coolant cases and trap is rapidly heated to vaporize the components. At the
same time the column temperature is programmed according to the required condition. The
adjustable split partitions the sample size to avoid the possibility of overloading effect, SFC
is suitable for extraction of pesticide traces in solid and aqueous sample
Micellar Extraction: Micellar extraction is a special type of extraction procedure that appears to
be unique in the separation of drugs, plant poisons and pesticides in biological matrices (viscera).
In the extraction of active constituents as above, micellar environment of surfactant kef different
classes is employed. Surfactant or surface active agents at a particular concentration in solution
known as critical micellar concentration (CMC) form micelle or association colloid. At this
concentration or above marked changes in the properties viz. viscosity, conductance, electrical
conductance are exhibited. Surfactant in solution also acts at the interface of a two phases
system of oil and water or organic solvent and water resulting solubilisation of one phase into the
other.
An emulsion or micro-emulsion is formed by the process. The emulsion may also be
stabilized by increasing the ionic concentration of additives including surfactant. Biological
matrices (Viscera) in the Indian perspective contains fats, degraded protein and colouring matter
etc. resulting extraction of active constituent difficult. In the solvent extraction process if
surfactant is added to the extractant (organic solvent) deproteinization and also solubilisation
with the formation of emulsion occur due to micellar interaction. The emulsion thus formed is
due to solubilisation of fat in biological matrices in the added solvent (containing traces of water)
with the simultaneous formation of emulsion occurs due to micellar interactions. The emulsion
thus formed due to solubilisation is destabilized on increasing the concentration of surfactant in
the system. As a result, fats are separated and separated as semisolid material due to lowering of
zeta potential between the electrical double layers of the colloidal system.
As protein and fats are separated out, the supernant liquid containing active
constituent may be extracted for poison by organic solvents. The detailed analytical conditions
have also been presented at places in the manual.
Microwave Accelerated Reaction System: The method of extraction is used for isolating
pesticides in biological materials especially in liver and kidneys. In this process, the sample is
subjected to rapid heating with organic solvent by microwaves at elevated pressure resulting
isolation of active constituent. The biological material (1 – 2 gms.) is placed inside a microwave
transparent vessel with a polar solvent o ionic solution (usually an acid) and is subjected to rapid
heading by microwave in a Microwave accelerated reaction system (digester).
The analytical conditions (temp., time of digestion, pressure) may very depending on
active constituent and nature of sample viz. monocrotophos and phosphamidon are successfully
extracted within 15-20 minutes from viscera using dichloromethane as a solvent at 80-1000C and
100 Psi. However, optimization of analytical conditions to covers different classes of pesticides
are required for a rapid extraction by this method. The method finds application in the digestion
of biological materials for isolation of some toxic metals (Cu, As, Pb etc.) and determination by
AAS and ICP thereafter.
Universal Trace Residue Extraction: It’s a system that has been developed for the recovery of
pesticides and organic residue from a wide range of samples including biological materials. It is
based on the principle of sweep co- distillation that relies on preferential volatalisation of
pesticides or other organic chemicals from biological materials, lipids, plant extract using a
stream of inert gas and subsequent isolation of volatiles on cold traps of solid adsorbents. It is a
purge and trap technique involving dispersion of the sample in thin films on deactivated glass
beads at elevated temperatures.
The extractor system is specifically designed to recover volatile, thermally stable
organochloro and organophosphorous from lipids, meat, butter, viscera etc. At present the
distillation tube does not contain glass beads or glass wool as it renders less recovery.
Florisil in conjunction with sodium sulphate has been found satisfactory for trapping many
different classes of volatile organic compounds Alumina, Silica gel and Tenax are
materials that have potential for used as trapping media with advantages over Florisil in
specific applications. The method is expected to fail for thermally labile pesticides. The
consumption of solvent is minimum. The method requires optimization of analytical
conditions before its application to biological samples (viscera) in forensic cases. However
it is a 2 in 1 process i.e. extraction cum clean up in a very short time with a very
economical use of organic solvents unlike solvent extraction methods.
Accelerated Solvent Extraction: The name of method signifies multiple sample handling in
a very short time by a very updated extraction system which also works in the same as in the
case of Universal Trace Residue Extractor i.e. Sweep Co-distillation. In this method a
commonly used solvent is pumped into an extraction cell containing the sample which is
then brought to an elevated temperature and pressure. Minutes later, the extract is
transferred from the heated cell to a standard collection vial for clean up analysis.
The entire extraction process is fully automated and performed in minutes for fast
and easy extraction of multiple samples with a very minimum solvent consumption. The
standard or optimum analytical conditions are to be arrived for its application to biological
matrices in forensic toxicological work covering a broad spectrum of pesticides. However,
the method has been found to be effective for soil samples.

Size Exclusion Chromatography: The technique can be used to advantage as a preparation

technique for the prior fractionation of oils, fats, environmental samples etc. into discrete
molecular weight fractions and as a way of removing very high molecular weight material
from complex sample.

Ion-Pair Extraction: This method is applicable for the extraction of highly water soluble
organic compound. These compound can not extracted from samples by direct solvent extraction.
The difficulty has been overcome by forming ion pair with a suitable reagent viz. quarternary,
ammonium salts as ion- pairing agent. The ion-pair formation is an attraction between a positive
and a negative charge. This is not a reaction it is only an attraction. Once this pair is formed it
will act exactly like an organic molecule and not like as ionic compound at all. The extraction
may be carried out thereafter by direct solvent extraction preferable in presence of a buffer. The
method is also applicable for extraction of drugs in the form of quarternary salt in urine or blood.
The biological matrices are deproteinized and treated with a dye(selective) in presence of buffer
to form drug-dye complex which is extracted by organic solvent. The dye is destroyed by the
action of acid or alkali and the drug is isolated for analysis.

Hyphenated Techniques (HPLC-GC AND SFC-GC): The hyphenated techniques viz. HPLC-
GC and SFE-GC allow complex samples to be pre- fractionated rapidly according to molecular
weight or chemical classification on a suitable HPLC pre-column or SFC and appropriate
fractions then separated by on line capillary GC. The coupling of two technique has been made
by a bypass valve fitted with a sample loop of appropriate volume. Selected fractions are passed
to the capillary column via a large retention gap which utilizes the process of concurrent solvent
evaporation to retain the fraction in the retention gap thus avoiding any preliminary contact with
column must be adjusted suitably.

Extraction of Volatile Poisons by Distillation: The method is applicable for volatile organic or
inorganic poison under different conditions of PH acidic and alkaline.

Neutral and Acid Distillation:

Procedure : 50 gms. of viscera (properly minced), stomach contents, vomit or other materials to
be examined are knot to be mixed together but should be examined separately. They should be
brought to the consistency of a thin gruel by adding 3-5 times of distilled water and acidified
with tartaric or sulphuric acid and submitted to steam distillation. The condenser and the
receiving flask should be well cooled especially during the hot season with ice, the outlet of the
condenser being dipped in a little water or NaOH solution or any other reagent as necessary. A
few pieces of pumice store may be taken in the flask to prevent bumping. It is better to collect
the distillate in 4 or 5 fractions, of which the first one should not exceed 20 ml. and the
remaining fractions should be 50 ml. each. The flask containing the material should preferably be
heated on the water bath. If phosphorous is suspected, the distillation should be carried out in a
dark room and a black screen placed between the burner and condenser so that phosphorescence
may be seen clearly. The distillate as above contains alcohols, paraldehyde, other aldehydes,
acetone, carbolic acid and phenol, carbon disulphide, thymol, camphor, turpentine,
nitroglycerine, benzene and other volatile acids etc. For cyanides and carbolic acid, the distillate
is collected in 10 ml. of 0.1N sodium hydroxide solution.
Alkaline Distillation: After the completion of the acid distillation, the flask is allowed to cool
and its contents are rendered alkaline by adding NaOH solution. The alkaline mixture is then
distilled again in the same way as before and the distillate collected in two fractions – the first
fraction of about 20 ml. and the second fraction of about 50 ml. The distillate from the alkaline
mixture may contain aniline, pyridine, nicotine, conine, ammonia and volatile bases.

Extraction of Toxic Metals in Matrices:

Non-Biological Matrices: The non-biological matrices may be subjected to chemical analysis by

preparing solution of samples and their systematic group analysis.

Biological Matrices: The extraction of metals in biological matrices may be carried out by the
following methods.
 Dry Ashing Method;
 Wet Digestion or Acid Digestion
Method;
 Fresenius and Babo Method;
 Selective Chemical Treatment.
The organic matter which constitute the bulk portion are destroyed by chemical means to
get the active constituents (metal ions) free completely for qualitative and quantitative
analysis.
Dry Ashing Method: About 10 gm. Of tissue or other biological materials is taken in a
silica crucible and heated in a Bunsen burner for removing the moisture and partially
destroying the organic material. Then the crucible is kept in a muffle furnace. The
temperature of the furnace is raised up to 5500C and at this temperature the incineration of
the organic matter is performed by keeping the silica crucible for one hour. After
incineration in complete, the crucible is taken out. The colour of the residue is noted when
hot because in presence of zinc the residue assumes yellow colour while in presence of
copper the colour of the residue is somewhat bluish green. The residue in the silica basin is
boiled with 10 ml. of 4(N) hydrochloric acid and then filtered. The clear acidic solution is
tested for metallic poisons such as copper, bismuth, zinc, barium etc. by performing
general group analysis by suing micro methods, chromatographic and instrumental
techniques.

Wet Digestion Method: Procedure : 100 gms. of biological materials or 10 ml. of blood are
taken into a large Kjeldahl flask and 20 to 40 ml. of Conc. HNO3 are added to cover the material
and flask is gently heated in a small flame when the mass begins to liquefy. The heating is
continued until the liquefaction of the material is complete and that must be done in the presence
of copious brown fumes of nitrogen dioxide in the flask. At this stage about 20 –30 ml. of Conc.
H2SO4 are added and the flask is heated strongly over a wire gauge and Conc. HNO3 is added in
drops (by using dropping funnel) to the contents of the flask at the rate of about 10 drops per
minute so that the atmosphere in the flask must at no times be free from brown fumes. Heating is
continued until all organic matter is destroyed and the liquid becomes clear and colourless or
straw coloured.
To find out if the oxidation is complete, the flask is heated without adding any
HNO3. If there is any un-burnt organic matter, the liquid begins to darken and if the
digestion is complete no darkening takes place and the white fumes of SO3 are given off.
In the former case, the addition of HNO3 and heating are continued further till the organic
matter is completely oxidized. strong is continued for 15 minutes more to expel the nitric
acid completely. Then, after cooling 25 ml. of saturated ammonium, oxalate solution is
added. The liquid is boiled until SO3 fumes appear. This ensures complete removal of
HNO3. It is then cooled, diluted with an equal volume of water and carefully transferred to
a beaker.
The beaker is heated on a hot plate or sand bath to expel the excess H2SO4. The
solution is cooled and diluted with water in such a way that the strength of acid is in the
neighborhood of 10%. At this stage a precipitate may be formed which contains the
insoluble salts of lead, bismuth, tin, barium, strontium or silver etc. The precipitate is
filtered off and tested for the metals mentioned above. The filtrate will now contain all
other metals except mercury. It is subjected to systematic group analysis and quantitative
determination thereafter as and required.
Fresenium and Babo Method (for Mercury): The nitric-sulphuric acid method of destruction
of organic matter is not at all suitable for mercury, which is almost completely lost by
volatilsation. The method is considered most suitable for liberation of mercury although there
is a possibility of some loss of mercury by vaporisation.
Procedure: A definite amount of biological material viz. 20-25 gms. of viscera or 5-10 ml.
of blood is taken in a flask fitted with a reflux condenser. In the case of viscera or other
solid material sufficient water is added to make a gruel like consistency. One third of its
volume of chemically pure hydrochloric acid and a few gms. of solid KC1O3 are added.
The content are mixed by shaking. The mixture is heated over a wire gauge on a burner
flame or on a boiling water bath. Small amount of KC1O3 is added time to time and the
flask is shaken. Chlorine gas evolves. The heating is continued until the contents of the
flask becomes a uniform, straw coloured liquid free from organic matter except some fatty
substances in suspension which can not be oxidized. If heating for an hour after the last
addition of KC1O3 produces no darkening of the mixture, the oxidation of organic matter
may be taken as completed. It takes 4-6 hours to attain the stage. It is filtered and washed
with water. The filtrate and washings are collected. Sufficient sodium sulphite or bisulphate
is added to reduce the excess of chlorine into hydrochloric acid. The liquid is warmed on
water bath and a current of air is passed to expel the excess SO2. The solution is now ready
for analysis.
Selective Chemical Treatment: A few toxic metals viz. arsenic and antimony in their specific
oxidation state (+3) may be subjected to the reduction process by nascent hydrogen (by the
reaction between zinc and dil sulphuric acid) for isolation of metals in matrices viz. burnt bones,
nail, hair and non-biological matrices like food preparation, drinks, tea, coffee etc. in the form of
their volatile hydrides (ASH3 and SbH3). The process may be carried out by Gutzeit or Marsh
Berzelius method. This is actually two in one method for isolation and on line detection or
determination.
In case of presence of As and Sb in their higher oxidation state (+5), reduction to +3
state is to be carried out prior to chemical treatment. The method and analytical have been
presented separately. The analyst should not be biased by positive findings as there are
chances of interference due to the presence of Phosphorous, sulphide, As or Sb in the
matrix itself. A blank test should invariably be carried out as As or Sb may be present in
matrices or the reagent.
Extraction of Toxic Anions in Forensic Matrices: The extraction of toxic anions in non-
biological
matrices require very minor processing and clean up. In- convenicneces are felt in case of
biological
matrices viz. viscera, stomach and gastric contents, stomach wash, urine and blood. The
extraction
procedures include the following method.
 Protein Precipitation.
 Dialysis.
 Selective Chemical Treatment.
 Micro Diffusion.
 Ion Chromatography
Protein Precipitation: A slurry of the sample (minced viscera 10-20 gm. of stomach contents or
gastric lavage etc. is prepared. The protein in the sample is coagulated by adding 0.5 – 1.0 gm. of
ammonium sulphate. It was filtered through a 2 cm. layer of cotton wool held in the barrel of the
syringe. The excess of ammonium sulphate remaining in the filtrate is precipitated by methanol.
The supernant liquid is collected and evaporated to a small volume on a water bath. The
concentrated liquid is ready for analysis of anions.
Dialysis: 10 gms, of the tissue is cut into small pieces and placed in a cellophane membrane
made into the shape of a bag. The bag is then slowly rotated in a beaker containing 100 ml of
distilled water by means of an electrical motor or mechanical device. Dialysis occurs rapidly.
After one hour, the water in the beaker is replaced by fresh water and the bag is rotated for
further half an hour. The water is then taken out, mixed with the previous fraction and
evaporated on water bath to a small volume. This is filtered, if necessary and tested for toxic
anions.
Selective Chemical Treatment: Phosphides and sulphides present in biological matrices viz.
Viscera, stomach content and gastric lavage is subjected to treatment with dilute acid on hot plate
or water bath. The toxicants that are liberated in the form PH3 or H2S are subjected to chemical
analysis. This has been covered separately in the monograph.
Micro-Diffusion: The unit operation may be employed for biological matrices viz. blood, urine
and stomach wash and also non-biological matrices viz. water, drinks, tea, coffee containing
traces of toxic anions (cyanide, phosphide, sulphide,) by using selective liberating, sealing and
detection reagent in Conway Micro-Diffusion assembly. (described as and when required in case
of volatile poisons. Viz. ethanol, methanol, acetaldehyde, chloroform, toxic gases viz. carbon
monoxide, phosphine, ammonia, hydrogen sulphide), and toxic anions (cyanide, nitrate etc.).
Description of Conway Micro-Diffusion Assembly: The assembly consists of obrink type,
polypropylene cells with clear polystyrene covers and the cells have an outer most annular
sealing well, an intermediate annular well for the sample and the liberating agent and a center
well for the reagent which is used to trap the diffusing gas or vapour. The sealing agent (usually
2 ml.) is introduced into the outermost sealing well. An approximate amount (1 ml.) of liberating
agent (usually same as sealing agent viz. 10% H2SO4 as sealing and liberating agent for carbon
monoxide and saturated sodium carbonate solution for methanol and ethanol etc. is placed as a
pool in one half of the intermediate well i.e. in the sample well.
Trapping agent (say PdCl2 in care of carbon monoxide turning to black, acidified
potassium dichromate turning to green in case of methanol or ethanol etc.). Sample is introduced
(1 ml.) into the other half of sample well taking care that it does not mix with the liberating
agent. The cover is placed and cell is rotated to effect airtight seal. The assembly is fitted back &
forth to mix the sample and liberating agent. The cell is placed on table or water bath, if needed
for reaction. The control sample cell is also prepared using same reagent but without sample i.e.
1 ml. of water in place of sample. The color charge in central compartment is noted.
Ion Chromatography: The preferential exchange of ions on ion-exchange resins packed in the
column of ion chromatograph renders separation of anions by using mobile phases usually
buffers of diverse PH.
Extraction of Non-volatile Organic Poisons: The group includes broad spectrum of pesticides
different classes of drugs viz. acidic, basic, neutral and amphoteric, plant poisons (specially
alkaloids, glycosides etc.). Out of the above classes of poisons, pesticides account for more than
80% of fatal cases of poisoning in most of the states of India. Thus, the extraction of insecticides
requires separate discussion. At the same time the extraction of drugs of diverse classes and plant
poisons are to be elaborated for their applications
Extraction Pesticides in Matrices: The extraction of pesticides in biological materials viz.
viscera, stomach contents, gastric lavage, blood is difficult due to the interferences of fat,
degraded protein and colouring matter in the matrices. The extracts require proper clean up
except for Micellar method. The extraction in case of non-biological matrices is cumbersome and
requires either minor clean up of no clean up. The procedures may be described as follow. The
methods described hereunder are based on solvent extraction cum stripping under diverse
conditions viz. nature and condition of matrices, use of organic solvent etc.
Method – I : Biological materials (viscera, stomach content or gastric lavage etc.) are macerated
into a fine slurry by mixing with equal amount of anhydrous sodium sulphate and transferred
into a conical flask with an air condenser. 50 ml of n-hexane are added to the flask and heated on
a hot water bath for one hour. The contents are cooled and filtered. The residual slurry is
extracted twice with 25 ml portion of n-hexane. The filtered n-hexane fractioned are combined
and taken into a separating funnel. This hexane layer is vigorously shaken with 15 ml, 10 ml and
10 ml portion of acetonitrile which are previously saturated with n-hexane. The acetonitrile
layers are mixed and taken into another clean separating funnel and diluted 10 times with
distilled water. 25 ml of saturated sodium sulphate solution are added to it and extracted thrice
with 25 ml portion of n- hexane. The n-hexane layers are combined, concentrated to 5 ml by
evaporating on water bath and 5 gms of anhydrous sodium sulphate added. The extract is
evaporated as and when required for analysis.

Method – II : 50 gms of macerated tissues or biological materials are mixed with equal amount
of anhydrous sodium sulphate and 100 ml of acetone in a conical flask and then refluxed on hot
water bath for one hour. After cooling the acetone extract is filtered. The residue is extracted
twice with further 50 ml portion of acetone. The acetone fractions are combined and
concentrated by evaporation up to 50 ml for further processing (clean up). The above acetone
extract (50 ml) is taken into a separating funnel and diluted with 150 ml of water. To it 20 ml of
saturated solution of sodium sulphate is added. The contents are extracted thrice with 25 ml
portions of chloroform with gentle shaking. The chloroform extracts are combined, washed with
water – acetone mixture (1 : 1) and finally with 50 ml of water. The washed chloroform layer is
passed through anhydrous sodium sulphate and then evaporated to dryness by passing air. The
residue is ready for analysis.
Method – III : Extraction of Pesticides in stomach wash, urine and vomit:
The sample (20 ml of stomach wash or urine or 20 gms of vomit) is taken in a conical
flask. To it 50 ml of n-hexane is added. It is refluxed on a water bath for half an hour. After
cooling the liquid is filtered, mixed with 20 ml of n-hexane and taken in a separating
funnel. The n-hexane layer is separated; passed through anhydrous sodium sulphate and
evaporated to dryness by passing a current of dry air through it. The residue is ready for
analysis.
Method – IV
Extraction of Pesticides in Blood : Sample of Blood (20 ml) is mixed with 10 ml of 10%
sodium tungstate solution and 15 ml of 1(N) sulphuric acid, shaken for two minutes and then
filtered. The filtrate is kept reserved. The residue is washed with two 15 ml portions of 0.1(N)
sulphuric acid. The washing are collected, mixed with filtrate (kept reserved), transferred into a
separating funnel and extracted thrice with 20 ml portion of n-hexane. The hexane layers are
combined, passed through anhydrous sodium sulphate and the solvent is removed by passing a
stream of air as stated in the previous methods. The residue is kept reserved for analysis.
Method – V : Direct Solvent Extraction of Pesticides followed by Clean Up for Biological
Materials :
The biological materials (50 gms of viscera) are biological materials are mixed with 5 gms
of ammonium sulphate and homogenized. After addition of 100 ml of diethyl ether, the
mixture is shaken at intervals and kept overnight. It is filtered and concentrated as before.
The concentrated extract is cleaned up by passing through a chromatographic column
(diameter, 1”) containing three successive layers of different lengths viz. 2” layer of
alumina (top layer), 1” of activated charcoal (middle) and 1” layer of anhydrous sodium
sulphate (bottom) previously washed with ether. The eluate is evaporated to dryness as
before the residue is kept reserved for analysis.
Method – VI : Extraction of Pesticides by Steam Distillation cum Solvent Extraction
followed by Clean Up for Biological Materials : When the biological materials are clean and
purified i.e. less degraded and contains very little fat and colouring matter, the following method
may be carried out.
Procedure : 50 gms of biological materials are treated with a few drops of phosphoric acid
and steam distillation for 15 minutes. The distillate (100 ml) is collected and subjected to
solvent extraction with 100 ml of diethyl ether in 20 ml portion. The ethereal layers are
collected during extractions, combined and subjected to clean up by passing through
Chromatographic column as before.(method V).
Method – VII :
Isolation of Pesticides in Non Biological Materials : Matrices : 20 – 25 gms of rice or more,
if available, 100 ml of drinking water or tea or coffee or milk, wearing apparel (20 – 25
round pieces cut out from fabric, each of 1” diameter), 20 – 25 gms of soil, sand, grains or
cereals.
Procedure : For the above materials direct solvent extraction is carried out with 50 – 100
ml of diethyl ether without adding ammonium sulphate. The ethereal extract is
concentrated to 20 ml and cleaned up by column chromatography as stated above. The
ethereal extract is collected, evaporated to dryness by passing stream of air. The residue is
kept reserved for analysis.
Micellar Extraction of Pesticides in Biological Matrices:
50 gms of biological materials (viscera) are mixed with 5 gms of ammonium sulphate and
homogenized. After addition of 100 ml of diethyl ether, the mixture is shaken at intervals
and kept overnight. It is filtered. The ethereal extract is taken into a separating funnel. 10
mg of sodium lauryl sulphate (an anionic surfactant found to be most suitable out of
different classes of surfactant cationic, anionic and neutral) is added to it and stirred gently.
On setting, fat in liquid and semi-solid form is separated and taken off from the system.
The addition of surfactant is continued till all the fatty materials and proteins are separated
and settled at the bottom. The end is indicated by a charge of dark colour of ethereal layer
to colourless. The ethereal extract is shaken with 25 ml portion of water twice. The ethereal
layer is collected. In case of emulsion formation, ethereal layer is collected by breaking the
emulsion with excess ether and gentle stirring. The collected ethereal layer in dried over
anhydrous sodium sulphate to remove traces of water. The ethereal layer is decanted and
evaporated to dryness as before. The residue is kept reserved for analysis.

Extraction of Pesticides in Fruits, Vegetables, Butter fat by Universal Trace

Residue Extractor:
Procedure : The sample is extracted by direct solvent extraction with dichloromethane.
The extract is dried with granular anhydrous sodium sulphate in a column. The dried
extract thus obtained is concentrated by a stream of nitrogen. The concentrated extract is
then subjected to sweep co-distillation in the Universal Trace Residue Extractor at 230oC
for 30 minutes by passing nitrogen (230 ml / min) and using sodium sulphate 10% florisil
(1 : 1). Elution is made by 5 ml of 10% acetone in hexane or 5 ml of dichloromethane. The
extract is collected for analysis. The applications of this method to different pesticides for
their analysis in biological matrices require further standardization to set up optimum
analytical conditions to cover different classes of pesticides.

Extraction of Pesticides in Sediment, Soil, Drywates and Tissue by Accelerated

Solvent
Extraction (ASE):

Procedure : The sample is mixed thoroughly or passed through a 1 mm sieve.

Sufficient sample is introduced into the grinding apparatus to yield at least 10 –20 g after
grinding. The sample is air dried at room temperature for 48 hours in a glass tray or on
hexane cleaned aluminium foil. The drying may also be made by mixing with anhydrous
sodium sulphate until a free flowing powder is obtained ( Air drying is not recommended
for volatile pesticides Gummy, fibrous or oily materials not amenable to grinding should be
cut, shredded or otherwise separated to allow mixing. These may be grinded after mixing
with anhydrous sodium sulphate 1 : 1 proportion ). A cellulose disk is placed at the outlet
and end of the extraction cell. Approximately 10 g of each sample or 20 g of each sample
in 11 ml or 22 ml extraction cell. (Surrogate spikes and matrix spikes may be added to the
appropriate sample cell). The extraction cells were placed into the auto-sample tray and the
collection trays are loaded in appropriate number (up to 24) kef 40 ml pre-cleaned,
chapped vials with septa. The conditions for extraction in ASE are set for extraction of
pesticides by using acetone : hexane (1 : 1, v/v) as the solvent. The operating conditions
include oven temp. of 100oC, pressure at 1500 psi, oven heat time and static time each of 5
minutes and flush volume in the proportion of 60% of extraction cell volume. The extracts
are collected for analysis. The method has been validated for analysis of pesticides in soil,
sediment, dry wastes and fish tissues. However, further standardization is required for
application of ASE to biological matrices in forensic toxicological work.

EXTRACTION OF DRUGS, GLYCOSIDES AND PLANT POISONS:

This group comprises alkaloids, glucosides, barbiturates, phenothiazines, salicylates,

sulphonal groups, sulphonamides drugs of different classes (drugs of abuse), plant poisons
and certain animal poisons etc. The extraction of these poisons depends on their solubility
at different pH i.e. at low or high acidic or alkaline condition and also differential solubility
in organic solvents. The methods include Stas-Otto or Dragendorff or their different
modification, ammonium- sulphate method and modern method especially solid phase
extraction of above poisons in biological materials.

The main difficulty in all the methods for extraction of poisons in biological
materials is to get rid of fats, degraded protein and pigments which interfere with their
isolation pure form and subsequent identification and quantitation. The problem is that
quantities of these poison present kin biological matrices are small and multiple steps in the
extraction and purification may incur loss of poison giving a minus error. If not sufficiently
purified, a positive error arises. This is the reason why the result, of quantitative
determination especially the alkaloids are not dependable and the negative findings in
many cases do not represent the actual state of affairs. The quantitative result actually
represents the recovery not the exact quantity present in the tissues. However, the
percentage of recovery is improving excellently by using modifications of the existing
methods and also modern methods. The methods are described hereunder with special
reference to biological materials.
Stas-Otto Process: After extraction of insecticides from biological materials (viscera, blood,
stomach wash or vomit etc.) the homogenate is made acidic with acetic acid or tartaric acid
(pH=2). To this mixture 2-3 times of absolute alcohol are added. The contents are stirred at
frequent intervals and kept overnight. This is refluxed thereafter for 30 minutes on hot water bath
and filtered through the filter paper pulp to remove precipitated proteins. The filtrate is
evaporated on a hot water bath. To this residue is mixed thorough with 100 ml of cold distilled
water. The aqueous solution is then processed according to Stas-Otto method in the following
manner.
A. The aqueous solution is made acidic with acetic or tartaric acid if not acidic (check
up with pH paper). This is then extracted several times with diethyl ether or chloroform.
The solvent will dissolve free acid and acidic compounds such as picric acid, salicylic
acid, veronal, neutral compounds such as acetanilide and phenacetin and very weak bases
such as antipyrene, caffeine, colchicines and narcotine (but in traces).

B. The acid-aqueous part remaining after extraction with ether or chloroform is made
strongly alkaline with NaOH solution and again extracted with diethyl ether or chloroform.
Free basic substances i.e. most alkaloids except phenolic bases such as morphine and
apomorphine are isolated by NaOH and remain in aqueous solution as sodium compounds.
 C. The aqueous portion is made neutral by adding ammonium chloride or neutralizing
with dilute acids. This is made again alkaline with ammonia solution. The alkaline solution
now obtained is extracted as follows:

1. Firstly, the extraction is made with ether for apomorphine and traces of morphine.
2. This is then extracted with hot chloroform or pure amyl alcohol for morphine and narceine.

Modified Stas-Otto Method:

Procedure : 50 gms of biological materials are minced preferably in a mincing machine, mixed
with plenty of rectified spirit (about 2-3 times the weight of material) in a flask and acidified
with tartaric acid. The mixture is heated on the steam bath for 1-2 hours with thoroughly shaking
at frequent intervals. The extraction is then allowed to proceed for about 24 hours with the steam
off. It is then filtered through a flutted filter. The filtrate is evaporated and the residue is again
extracted with acidulated alcohol in the same way, filtered and washed several times with hot
rectified spirit. The combined filtrates are evaporated in a porcelain basin on the steam bath to a
syrupy consistency.
A. To the syrupy residue about 100 ml of rectified spirit is now added very slowly with
constant stirring so that insoluble matter may be granular and not gummy. It the alcohol
is added rapidly or all at a time, the insoluble matter will be gummy causing much loss
of alkaloids by enclosing them in the sticking mass. It is warmed with occasioned stirring
for about half an hour and filtered. This process is repeated once more and the combined
alcoholic extracts are evaporated almost to dryness.
B. The residue is now dissolve in about 50 ml of water acidulated with dilute sulphuric
acid and filtered after about an hour. The poisons are thus dissolved out by the aqueous
solution which is transferred to a separating funnel and extracted with a suitable solvent
such as ether, chloroform etc. in portions of about 25 ml. The solvent would take up the
following from the acid solution, colouring matters, toxic oils and resins, salicylic acid
and its derivatives (aspirin, salol etc.), barbiturates, sulphonal, acetanilide, narcotine and
alkaloids of ergot, certain glucosides such as thevetin and which has escaped initial
treatments for purification.
C. The acid aqueous solution is then rendered alkaline with a solution of sodium
carbonate or ammonia which would liberate the free base from its salt. The alkaline
solution is now extracted with chloroform in the same way as in the previous stage. It
will take up all the alkaloids except morphine (only a trace being extracted) and those
feebly basic substances which are partially extracted from the acid solution. The
extraction is separated 2 or 3 times more.
 D. If morphine is suspected, it may be extracted at this stage by amyl alcohol or
chloroform – ether (3 : 1) mixture or chloroform – alcohol (9 : 1). Of these, amyl alcohol
is the best but as it is prone to form annoying emulsions the chloroform – ether mixture
is used by many. If morphine is likely to be the only poison, the chloroform extraction
(stage D) described above may be omitted altogether. The combined chloroform or amyl
alcohol extracts are evaporated to dryness and the residue is now ready for further
purification and analysis.
E. The evaporated chloroform extract is purified by dissolving it in about 20 ml of
water acidulated with sulphuric acid and filtering through a small filter. The filtrate is
extracted with chloroform, first in acid and then in alkaline medium as in the initial
stages of extraction. These extracts are evaporated to dryness for analysis.
Further Modification of Stas – Otto Method: As there are too many steps in the extraction
of non-volatile organic poison by Stas-Otto method and also chances of loss of poison in each
of the steps, there may be even a complete failure to detect it in case of considerable loss of
poisons. The objectives of modifications are to minimize the steps as far as practicable under
special circumstances. The following modifications of the technique are sometimes necessary.

i) The alcoholic extraction of biological materials is to be carried out at room

temperature (not exceeding 40oC) i.e. without using steam bath and preferably with
absolute alcohol (to prevent hydrolysis) in place of rectified spirit for suspected
poisoning by aconite, belladonna, datura or cocaine. The evaporation of alcoholic
extract should be done under reduced pressure.
ii) The extraction with rectified spirit is to be done for 48 hours if biological materials
are preserved in saturated.
iii) For stomach contents containing too much fluid, the extraction should be done
with absolute alcohol 3 or 4 times.
iv) For stomach wash as the sample, extraction with double the quantity of absolute
alcohol acidulated by tartaric acid should be done and then allowed to evaporate on a
steam bath.
v) For filtration, Buchner funnel is preferred.
vi) To prevent loss due to emulsion formation, agitation with organic solvents, (ether or
chloroform or amyl alcohol) should be done gently in the beginning and steadily in an
violent manner thereafter (2 – 3 time in the beginning and knot exceeding 12 times at the
end). If emulsion persists, it is to be evaporated on a steam bath and the residue
taken up in fresh solvent.
Ammonium Sulphate Method:
This method is most useful for preliminary analysis (screening) of barbiturates, alkaloids
and tranquilizing drugs etc. and also identification and semi-quantitation.
Procedure : The organic materials (about 100 gms.) are cut into small pieces, macerated, mixed
with100 ml of 5 percent acetic acid and taken into a 600 ml beaker. Solid ammonium sulphate is
then added to it by frequent shaking to make a saturated solution. The about 20 gms. of solid
ammonium chloride are added in excess. The mixture is then heated in a boiling water bath for
three hours (for suspected poisoning by aconite the temperature should not exceed 600C).
The mixture is cooled slightly and filtered through the filter paper pulp. The residue
on the funnel is again extracted with two portions of 100 ml of 5 percent acetic acid and
filtered as before. The filtrates are combined and taken into a 500 ml separating funnel.
The residue slurry on filter paper is leached with 100 ml of diethyl ether and the same is
received in a cold container. The ether fraction is added to the aqueous acidic extract in the
separating funnel and shaken for 5 minutes and separated. 100 ml of ether is again added to
the acidic layer, shaken for 5 minutes and separated. The ether layers are combined. The
acidic ether extract is tested for salicylic acid, aspirin, barbiturates, meprobamates,
lysergides, benzo- diazepines etc.
The aqueous solution remaining in the separating funnel after separation of acidic
drugs is made alkaline by addition of ammonium hydroxide and extracted three time with
100 ml portions of chloroform ether mixture (1 : 3). The aqueous layer is retained for
extraction of opium alkaloids. The organic layers after separation are combined and
washed with 50 ml of water. It is extracted three times with 25 ml portions of 10%
sulphuric acid. The sulphuric acid fractions are combined and taken into another separating
funnel. (Organic layer is discarded) 50 ml mixture of chloroform – ether (1 : 3) are added
to it. Ammonium hydroxide solution (dil.) is added to make the solution alkaline shaken for
5 minutes. The organic layer is separated. The extraction is repeated thrice. The organic
layer after separation are combined, washed with l50 ml of water and then dried by passing
through anhydrous sodium subphate and evaporated to dryness. The extracted is tested for
opium, dhatura and aconite alkaloids amphetamines, meprobamate, methaqualone etc.

The acid – ether extract may be further be separated into three fractions.
i) The acid – ether fraction is shaken with 25 ml of 5% sodium bicarbonate solution. The
aqueous layer is removed and taken into another separating funnel. It is acidified with
dilute sulphuric acid and re-extracted with 25 ml of ether. This ether fraction is passed
through on hydrous sodium sulphate and then dried just to dryness. The residue (R1)
 contains salicylates.
ii) The ether layer (of acid ether fraction) after washing with sodium bicarbonate is
extracted twice with 25 ml portions of N.sodium hydroxide solution. The aqueous layer
are separated from ether layer, combined and taken into another separating funnel. It is
made acidic with dilute sulphuric acid and extracted twice 25 ml portions of ether. This
ether fraction is washed with 25 ml of water and then dried by passing through
anhydrous sodium sulphate and then evaporated to dryness. The residue (R2) contains
barbiturates in relatively purified form.
iii) The ether layer after extracting with NaOH is washed with water and then
evaporated to dryness. The residue (R3) contains meprobamate, other carbamates and
other neutral drugs.
Extraction of Drugs in Urine: Sufficient phosphoric acid or tartaric acid is added to 10 ml of
urine to adjust the PH to 3. It is then extracted with two 30 ml portions of ether. The extracts are
combined and washed with 5 ml of water. The washings are added to the sample. The aqueous
solution is retained for possible presence salicylates (Fraction A).
The etheral solution is extracted with 5 ml of 0.5 M sodium hydroxide and the extract is
retained for examination of barbiturates and weakly acid substances (Weak Acid Fraction
B – Barbiturates, Glutethimide, Paracetamol, Phenytoin, Phenylbutazone etc.).

The ethereal solution is washed with water. The washing is discarded. The ethereal
solution is then dried with anhydrous sodium sulphate and evaporated to dryness. The
residue may contain neutral drugs (Neutral Fraction C – Caffeine, Carbromal,
Chlordiazepoxide, Flurazopam, Lorazepam, Meprobamate, Methaqualone, Methyprylone,
Nitrazepam, Paracetamol).
To the aqueous solution retained after the first extraction sufficient dilute ammonia
solution is added to adjust the PH to 8 . It is extracted with two 10 ml portions of
chloroform. The combined ether extracts are washed with water, filtered. A little tartaric
acid is added to prevent the loss of volatile bases. It is evaporated to dryness. The residue
may contain basic drugs (Basic Fraction D – Amphetamine, Amitriptylene, Caffeine,
phenothiazines, Ergot Alkaloids, Morphine, Methaqualone, Flurazepam, Lorazepam etc.).
The pH of the aqueous solution obtained after extraction of Fraction D is adjusted
to PH 3 by the addition of hydrochloric acid. It is heated at 100C for 30 minutes, cooled
and extracted with two 10 ml portions of ether. The aqueous solution is kept reserved. The
combined ether extracts are washed with 5 ml of M. sodium hydroxide and evaporated to
dryness. The residue may contain benzodiazepines as benzophenones (Fraction E).
 The pH kef reserved aqueous solution is adjusted to PH 9 cooled. It is extracted
with a mixture of ethyl acetate and isopropyl alcohol (9 : 1).The solvent layer is separated
and evaporated to dryness.The residue may contain opiates (Fraction F).
Extraction of Drugs in Blood: As sample-volume in case of blood or serum or plasma is small
and only a limited number of drugs may easily be detected and identified in them, the extraction
procedure is slightly different from that for urine and stomach contents. Different fractions of
extraction viz. A, B, C, D bear the same meaning as stated in 2.9.6.3. i.e. extraction of drugs
urine. The initial extraction is carried out at PH 7.4 as many basic drugs are recovered by
chloroform extraction at this PH. As a result, the substance looked for is most likely to be found
in either fraction B or C and preparation of fraction D is only necessary either to ensure that
nothing has been missed or where no drug has been found in fractions B and C.
Procedure : 2 ml of phosphate buffer (pH = 7.4) and 40 ml of chloroform are added to 4
ml of the sample and shaken vigorously. 2 gms of anhydrous sodium sulphate are added
and again shaken again to produce a solid cake. The decanted chloroform is passed through
a filter and the cake is extracted with a further 20 ml of chloroform. The chloroform
extracts are combined.
The chloroform layer is extracted with sodium carbonate to remove salicylate
(Strong Acid Fraction A), if detected in the preliminary tests. To the chloroform layer 8 ml
of 0.5 N sodium hydroxide solution is added. The mixture is shaken and centrifuged. The
sodium hydroxide may contain barbiturates and other weakly acid substances. (Weak Acid
Fraction B).
The chloroform layer is washed with a little water. The washing is discarded. The
chloroform layer is dried with anhydrous sodium sulphate, filtered and evaporated to
dryness. The residue may contain (caffeine, carbromal, benzodiazepines, meprobamate,
phenazone etc.) neutral drugs together with a number of bases (Neutral and Basic Fraction
C). If sufficient of original sample is available, a further portion of it is made alkaline with
dilute ammonia solution and extracted with two 10 ml portions of chloroform. The
chloroform extract is dried with anhydrous sodium sulphate and evaporated to dryness. The
residue may contain basic drugs (Basic Fraction D - as stated in the previous section).
If there is not sufficient of the original sample for the extraction of basic fraction F,
the following procedure may be carried out. After fraction C has been chemically examined
by UV or Chromatographic methods, the remaining residue, if any is dissolved in
chloroform and extracted with 0.5 M sulphuric acid. This extracted portion is added to the
sodium sulphate cake retained after the first extraction (for Fraction A). It is made alkaline
with dilute ammonia solution and extracted with two 10 ml portions of chloroform. The
chloroform layers are collected, dried over anhydrous sodium sulphate and evaporated to
dryness. The residue may contain basic drugs (Basic Fraction D - as stated in the previous
section).
Solid Phase Extraction of Drugs from Urine: 5.ml of urine are added to 2 ml of phosphate
buffer (0.1 mol./L, PH 6.0) in a glass test tube and the PH is adjusted to PH 5.5 – 6.5 using 0.1
mol./L aqueous sodium hydroxide or 1 mol./L aqueous acetic acid (depending on the nature of
the drug). SPE column is inserted into vacuum manifold and washed with 1 ml of methanol and
1 ml of phosphate buffer (0.1 mol./L, PH 6.0). An 8 ml fritted reservoid is attached to the top of
the extraction column and urine is added to it. The column is dried under vacuum and washed
with 1 ml of phosphate buffer (0.1 mol./L, PH 6.0) followed by 0.5 ml of aqueous acetic acid (1
mol./L, ). The column is dried under vacuum and washed with 1 ml of hexane. The elution for
acidic and neutral drugs is made with 4 x 1 ml portions of dichloromethane. The elute is
evaporated to dryness under a stream of nitrogen at 30o – 40 oC. The residue is kept reserved for
analysis of acidic and neutral drugs. The column is then washed with methanol (1 ml) and
eluation for basic drugs is made with 2 ml of methanolic ammonium hydroxide (.2%, V/V). 3 ml
of de-ionised water is added and eluation is made with
0.2 ml of chloroform. The chloroform layer is collected and evaporated to dryness under a
stream of nitrogen as above. The residue is kept reserved for analysis of basic drugs.

Tissue Digestion using Proteolytic Enzyme: The digestion with proteolytic enzymes after give
much improved recovery and has the advantage that once the digest has been prepared,
analogous methodology to those used with plasma can be employed. It is obviously important to
ensure that use of the enzyme preparation does not introduce interferences. A further potential
problems is that conjugates and other metabolites may not survive. The procedure has been
described below.
A solution (2 gms/L) of lyophilized subtilisin in sodium dihydrogen orthophosphate /
disodium hydrogen orthophosphate buffer (7 mol./L, PH 7.4) 100 mg portions of tissue are
dissected and the excess of fluid removed by filter paper. The tissue is added to 10 ml of
tapered glass tubes and exact weights are recorded. 1 ml of subtilisin solution is added.
The tubes are sealed with ground glass stroppers and incubated fin a water bath at 50C
for 16 hours. The tubes are cooled. The contents are mixed on a vortex – mixer. Thereafter
extraction is done with 0.2 ml portions as for plasma or serum.
Micellar Extraction of Drugs in Biological Materials: 100 gms of biological materials are
finely minced in a mincing machine. An excess of rectified spirit (about 200 ml) is added to it in
a flak, mixed thoroughly and made acidic with glacial acetic acid by drop wise addition and
stirring. The mixture is then heated on a steam bath for 1 hour with through shaking at intervals
and the flask containing the mixture is kept for 24 hours at room temperature. It is then filtered
through a Buchner funnel having a bed of sand (0.3 cm) on the filter paper. The filtrate is
collected in a separating funnel. After adding of 50 ml of solvent ether, cloudiness is observed.
Fine particles of fat separate on standing which is drained off. The contents of separating funnel
is shaken with 1 mg portions of sodium lauryl sulphate when semi-solid fat separates. The
separation of fat is accelerated by addition of a few drops of water. Reddish brown oily mass of
fat settles at the bottom of the separating funnel. This is drained off. The process is separated till
the settling of fat ceased (indicated by a clear transparent extract in the separating funnel). The
contents of the separating funnel is evaporated to dryness. The residue is extracted with 20 ml
portions of water five times. The aqueous extract is made distinctly acidic by drop wise addition
of acetic acid and extracted with 80 ml of diethyl ether in 20 ml portions. The aqueous part is
kept separately. The combined ether extract as above (Acid-ether part for acidic and neutral
drugs) is washed with water till free from acid.
The washings are mixed up with the aqueous part. The combined ethereal extract is
dried over anhydrous sodium sulphate. The dried ether extract is decanted off. It is
evaporated to dryness. The residue is kept reads for analysis. The combined aqueous part is
made distinctly basic with drop wise addition of ammonia solution. It is extracted with 80
ml of chloroform in 20 ml portion. The combined chloroform extracted is washed with
water till free from alkali and dried over anhydrous sodium sulphate. It is decanted and
evaporated to dryness. The residue is kept ready for analysis (basic drugs).
Headspace procedure for Isolation of Volatiles in Biological Materials: The principle
underlying headspace analysis is that in a sealed vial at constant temperature equilibrium is
established between volatile components of a liquid sample in the vial and the gas phase above it
(the headspace). After allowing due time for equilibrium (normally 15 minutes or so) a portion of
the headspace may be withdrawn using a gas-tight syringe and injected into the GC column.
Procedure: The internal standard solution (25 mg / l ethyl benzene and10 mg / l l, 1, 2
trichloroethane) is added to a 200 μl of a mixture of expired blood and deionised water (1 :
24,v/v ) in a 7 ml glass septum vial using a semi-automatic pipette. The vial is sealed using
a crimped on PTFE- lined silicone disc. The vial is incubated at 65 oC in a heating block
and a portion (100-300μl) of headspace is withdrawn using a warmed gas-tight glass
syringe for onward analysis in gas chromatograph.
Clean-Up Procedures: In the extraction techniques described it is likely that the extract will
contain interfering substances, which will create problems in the analysis in various ways. The
occurrence is particularly so in the extracts from samples of effluent, soil, sediment and tissues
or organic matrices which contain fats, oils and other naturally occurring substances. The long
established procedure for the removal of these substances has been to pass the extract through an
alumina column and then to separate the target compounds into different batches by passing the
clean up extract through a silica column. The supply of materials by preparative thin layer
chromatography is also followed if the active components are identified by preliminary
screening. The purification in the same way may also be achieved by gas chromatography or
HPLC.
For volatile toxicants, stripping may also be done by GC-Head Space method
(Purge or Trap technique). There are different analytical conditions for the Purpose which
have been specified in literatures.
In different hyphenated techniques viz. GC-MS, LC-MS, HPLC-IR, the first technique is
applied for separation of components in the pure state for their on-line identification and
analysis by the other technique.
Recently, the clean up has been streamlined by the use of commercially available solid-
phase extraction cartridges or disc.
Different methods are described below in short.
Clean up using Alumina and Silica Column: This technique removes interfering compounds
by passing the extract through basic and acidic column and then separating active constituents
specially pesticides on a silica column.
Preparation of Solid Adsorbents:
The preparation of solid adsorbents – basic alumina, acidic alumina and silica gel is as follows.
i) Basic Alumina:
About 100 gms of alumina is placed in a silica dish, heated in muffle furnace for 4 hours
at 800oC, cooled to about 200oC and then to room temperature in a desiccator. Water
(4%, w/w) is added to the weighed portion in a stoppered flask. It is shaken well, sealed
and stored
ii) Acidic Alumina :
A porition of the alumina is washed with IM HCI by making a slurry in beaker. It is
filtered through a sinter funnel and dried in a silica dish at 150oC for 4 hours and cooled
in a desiccator. Water (4% w/w) is added to a weighed portion in a stoppered flask. It is
mixed thoroughly and

stored. These alumina preparations will slowly deactivate on exposure to air and
should be discarded after 2 weeks.
iii) Silica Gel :
 About 100 gms of silica gel is heated in a silica dish in a muffle furnace for 2 hours at
500C, cooled and then placed in a desiccator. A portion is weighed into a stoppered
glass container and water equivalent to 3% of the silica is added. The silica gel
deactivates more rapidly than the alumina and should preferably be prepared daily.
Procedure: The column is pre-washed with acetone followed by hexane and allowed to dry. The
acid / base alumina column is prepared by first adding 2 gms. of acidic alumina then 1 gm of
basic alumina to the column. The column is tapped to settle.
The silica column is prepared by adding 2.5 gms of deactivated silica gel to the
column and tapped to settle. 10 ml of hexane is passed through the column (to wet the
column) and run off in excess. Untill the hexane meniscus is level with the column
material. The organic layer (obtained after extraction of matrices for pesticides or drugs) is
then transferred on to thcolumn. The active constituents (pesticide or drugs) are then eluted
from the column by hexane or diethyl ether or suitable organic solvent. The eluate is
collected. It is then passed through the silica column and elution is made. The eluate is
collected and dried over anhydrous sodium sulphate. It is evaporated to dryness. The
residue is kept for analysis.
Modified method for Clean-Up and separation using Alumina / Silica Nitrate and
Sliica Gel:
Preparation of Solid Adsorbent:
(i) Alumina – About 100 gms of alumina is heated in a silica dish at 500oC for 4
hours and then cooled. To a weighed portion in a stoppered glass container, deionized
water equivalent to 7% (w/w) of the alumina weight. is added and the sample is agitated
to mix thoroughly. The alumina is kept in a sealed container. This adsorbent is stable for
only about a week once re- exposed to atmosphere.
(ii) Alumina / Silver Nitrate - A batch of material for adding to the column is
prepared by dissolving 0.75 gm of silver nitrate in 0.75 ml of water 4 ml of acetone is
added to it. To this solution in an unstoppered conical flask 10 gms of dried alumina is
added and shaken thoroughly. The acetone is allowed to evaporate and the preparation is
stored in the dark until ready for use. The adsorbent should be prepared freshly.
Procedure: The chromatographic column is plugged with hexane washed glass wool or
cotton wool and 15 ml of hexane is added. 1 gm of alumina / silver nitrate is poured and
allowed to settle. Then 2 gms of alumina is added and again allowed to settle. This is then
charged with a little anhydrous sodium sulphate. Hexane is run off in excess until the liquid
level is at the top of the column. The concentrated organic layer (containing pesticides or
drugs as the active constituents) is added with rinsing to the top of the column. 30 ml of
hexane or diethyl ether or any suitable organic solvent (as the case may be is passed
through the column and the eluate is collected. The eluate concentrated down to 10 ml. A
silica column is then prepared by adding 2 gms of silica to a plugged chromatographic
column with a layer of anhydrous sodium sulphate at the top. The concentrated eluate from
the alumina / silver nitrate column is added with rinsing to the silica gel column and
allowed to be adsorbed. 10 ml of hexane or diethyl ether or any suitable organic solvent or
a mixture of solvent is added to the top of the column and the eluate is collected. This is
dried over anhydrous sodium sulphate and evaporated to dryness for further analysis.
Clean – Up by a simple column Chromatographic method:
The method can be profitably employed to purify the matter after extraction in biological
materials for pesticides or drugs.
The concentrated extract (organic layer) is cleaned up by passing through a
chromatographic column (dia. 1”) containing from top alumina layer of 2”, activated
charcoal layer of 1” (middle layer) and a anhydrous sodium sulphate layer of 1” (bottom
layer) previously arranged after plugging the chromatographic column and washing bag
hexane or diethyl ether or any suitable solvent or mixture of solvents (as the case may be).
The elution is made with appropriate organic solvent. The eluate is dried over anhydrous
sodium sulphate and evaporated to dryness. The residue is kept reserved for analysis.
Clean-Up using Solid Phase Extraction (SPE) Catridges: There are a variety of catridges
available viz. Bond Elut from Analytichem International. The catridge is filled with a chemically
modified silica adsorbent and the appropriate one selected according to the nature of the material
(toxicant) viz. aminopropyl for organo-chloro compounds.
Procedure : A selective catridge is taken with a black adapter fitted on top.A 10 ml glass
syringe is fitted onto the adaptor. 5 ml kef methanol is put into the syringe. It is passed
through the tube, (tube should be wet always). The tube is then washed with 5 ml of
hexane. The syringe is detached. The concentrated organic layer containing active
constituent is added to the top of the tube and allowed to pass through it. The clean extract
is collected. The tube with the syringe is washed with the same solvent. This is added to the
clean extract as above. The combined layer is dried over anhydrous sodium sulphate. It is
then evaporated to dryness and the residue is kept reserved for analysis.
Clean-Up by Preparative TLC method: This is applicable if the identity of toxicant is
established. Thereafter the chromatogram is developed by applying the same chromatographic
condition that was used in the identification i.e. same developing solvent system, adsorbent,
temperature etc. The adjoining circular areas around the components separated at different Rf
values are scrapped off the scrapped materials at different zones are eluted with selective organic
solvent. This is collected, dried over anhydrous sodium sulphate and evaporated to dryness for
analysis.
Clean-Up by HPLC method: This is also applicable as in the case stated above i.e. the identity
of toxicant by HPLC method is to be established by noting retention time and other
chromatographic conditions viz. solvent system, flow rate, column etc. Again the same HPLC
method is employed and the separated components at various time (as per retention time
established earlier) are collected for further analysis.
Different methods of extraction / isolation of toxicants in diverse matrices and their
stripping methods have been described in the previous paragraphs. The methods are to be
employed depending on the nature of toxicant, matrices and availability of infrastructure
facilities. In the forthcoming chapter the analysis of different classes of toxicants will be
described.

GASEOUS AND VOLATILE POISONS

Analysis of gaseous and volatile poisons.

METHODOLOGY: The distillation methods for isolation of volatile poison have already been
described (section-3). The distillates in two fractions, acid steam distillate and alkaline steam
distillate are kept to undertake systematic screening and other detailed analysis as mentioned
hereunder.

Identification tests for volatiles: In biological materials (viscera), stomach contents, urine,
isolation by acid-distillation as described earlier. The distillate is subjected to the following tests:
Test for methanol:
Isolation: By acid-distillation. The distillate is subjected to the following tests.
Chromotropic Acid Test:
To 0.5 ml. of the distillate in a test tube, 0.2 ml. of 5% potassium permanganate
solution is added. After 5 minutes, saturated solution of sodium bisulphite is added drop by
drop until the permanganate colour is discharged. If a brown colour still persists, a drop of
phosphoric acid is again added to it a drop of sodium bisulphite solution. To this colourless
solution, 0.5 ml. of freshly prepared chromotropic acid solution (prepared by dissolving 5
mg. of sodium salt of chromotropic acid in 10 ml. of concentrated sulphuric acid and heating
on a water bath at 60oC for 30 minutes and cooling thereafter. A violet colour is observed.
Test for ethyl alcohol:
Isolation: Ethyl alcohol is isolated from biological materials by acid distillation. The
distillate is subjected to the following tests.
Dichromate test:
To one ml of the distillate is added 0.2 ml of 2% Potassium dichromate solution followed by
one ml of conc sulphuric acid. The yellow colour of the dichromate changes to green or
blue.
METHODS OF QUANTITATION OF ETHANOL IN BIOLOGICAL MATERIALS:
Gas Chromatographic determination of Ethanol in Blood/Urine
Isolation of Ethanol in blood : 1 ml. of blood is diluted with 4 ml. of water. It is acidified
with a few drops of 5% tartaric acid solution and then distilled. 5 ml of distillate is
collected in ice cold condition and an aliquot (10 μl) of it is injected into the gas
chromatograph as per conditions stated below.
Isolation of Ethanol in Urine: 1 ml. of urine is taken into a micro centrifuge and
centrifuged for 15 minutes. 5 μl of supernatant liquid is injected into the gas chromatograph
as per conditions stated below.
Column – Porapak θ – Polymer bead, 80 – 100 mesh, 5’ X 4 mm. (id.) glass column.
Column Temperature – 1600C Carrier Gas- Nitrogen Gas Flow - Nitrogen - 50 ml/min.
Hydrogen - 50 ml./ min. Air - 300 ml/min
Detector - F.I.D.
Quantitation :
The quantitative estimation of volatile substance is made by determining the area of the
corresponding peak and calculating the quantity of the sample from the following relation
ship.Where, Ca Cb
=
Aa Ab

Ca = Concentration of volatile substance in exhibit.

Aa = Area of peak of volatile substance present in the exhibit. Cb =
Concentration of the standard.
Ab = Peak area of the standard.
Cb is once determined by injecting 10 μl of supernatant prepared by standard blood
sample having 150 mg
Ab of volatile substance per 100 ml. of blood (the ratio remains constant)
GASEOUS POISONS: Various toxic gases are known in forensic toxicology. Although the list
includes various types of toxic gases of diverse origin or sources, cases involving a few toxic
gases are encountered in day-to-day work.
However, the table – 3.4 indicates their characteristics, origin, properties etc.
TABLE – 3.2: CHARACTERISTIC OF GASEOUS POISONS
 NAME SOURCE PROPERTIES OTHER
CHARACTERISTICS
Phosphine Phosphides used as Colourless, flammable Highly toxic, affects CNS
rodenticides, phosphorous - with odour of decaying andreacts with haemoglobin.
based industries. fish.

Identification of some Gaseous Poisons:

Method – (A): Phosphine in Biological materials: 25 gms. of biological material is taken in a

conical flask filled with a guard tube containing lead acetate soaked cotton. A few ml. of
cadmium sulphate solution is added. It is acidified with dilute sulphuric acid. The mixture is
heated gently on water bath at 40-60 °C. The gas evolved is allowed to come in contact with
AgNO3 paper. It turns grey or yellowish brown or black. (Due to reaction of phosphine with
silver nitrate solution). The presence of phosphide is indicated. The paper is dried and cut into
pieces and dissolved in dil nitric acid. The extract is evaporated to dryness for 2-3 times. The
residue is taken in a few drops of concentrated nitric acid. 1 ml. of ammonium molybdate
solution is added and warmed. The formation of canary yellow precipitate confirms the presence
of phosphide.It may also be estimated by trapping the gas in bromine water or sodium
hydrochlorite solution, and after elimination of the bromine, determination is done for the
presence of phosphate. It may be determined in gases by use of the reaction PH3 + 3 HgCl2
P(HgCl)3 + 3 HCl, the quantity of acid being proportional to the volume of phosphine present.

Method – B: By HS-GC-FPD method :

Sample preparation: Transfer 1-5 mL of sample material into 20 mL of head-space vial.

Add 10 mL of 0.1 M H2SO4 and immediately seal the vial for chromatographic analysis.
Use aqueous solution of Zn3P2 as standard and treat in the same way as described.

HS-GC-FPD analysis:

Capillary Column: HP PLOT Q (30 m × 0.32 mm × 20 µm) (Bonded polystyrene-

divinylbenzene based column) Carrier gas : Helium
Injection : Split 1:30 at room temperature
@150 °C Injection volume : 0.1 mL
Detector : Flame Photometric Detector (P-Mode) @ 310 °C
Oven Temperature : 70 °C for 0.5 min followed by 15 °C/min up to 100 °C
with an 8 min hold;
Retention time : Peak of Phosphine at 1.05 min.
Identification of carbon monoxide in blood by micro-diffusion method
  Add 2 mL of PdCl2 solution in the central well of Conway micro-diffusion cell*.
 Add 2 mL of the blood in one side of outer ring
 Add 1 mL of 10% H2SO4 in another side of outer ring
 Quickly cover the micro-diffusion cell and gently rock/rotate to mix blood
with sulfuric acid.
 Allow to diffuse for approximately 1 hour in a oven at ~50°C.
 A silver colored mirror will form in the center well of the dist in positive samples.

*[0.005 N Palladium Chloride Reagent: Weigh 0.22 g palladium chloride, transfer into a
250 mL volumetric flask and qs to volume with 0.1 N HCl and let stand overnight.
Transfer to a 500 mL volumetric flask and qs to volume with 0.1 N HCl. Store at room
temperature for up to two years. ]

ANALYSIS OF INORGANIC POISONS (CATIONS AND ANIONS)

Analysis of inorganic poisons (cations and anions)

DESCRIPTION OF METALLIC AND NON METALLIC POISONS

The important poisons in the group of metals include arsenic, antimony, mercury,
lead, thallium, zinc, manganese, barium and aluminium. The salts of these metals are toxic.
Non-metals or specifically toxic anions include borate, bromide, chlorate, cyanide, fluoride,
hypochlorite, iodide, nitrate, nitrite, oxalate, bromate, iodate, sulphide, thiocyanate etc.
Generally, the anions of the salts are responsible for the toxic action. Thus, their detection
gives a clear idea regarding the source of poison. Sometimes, toxic anions are directly used
as active species viz. cyanide, oxalate, borate etc. These cations and anions are to be
isolated especially in case of biological materials for onward detection and estimation.

There are some classical methods viz. Reinsch test, Gutzeit test, Marsh test that are
still followed for the detection of cations. The present day analysis includes micro methods
and instrumental techniques. Before elaboration, the description of cations and anions are
given below.

Detection and determination of toxic cations:

The toxic cations are to be detected in biological materials in case of fatal poisoning. This
is done by digestion (dry or wet). The extract obtained after digestion is used for chemical
test and quantitation.

ARSENIC:
Chemical Test for Arsenic:
Reinsch’s Test:
About 20 ml. of Conc. HCl (pure for toxicological work) and 100 ml. of water are taken in a
porcelain basin in which a bright copper foil (about 3” by ¼”) is placed with one of its ends
being fixed on the edge kef the basin in the form a loop. It is boiled for about half an hour to
see if the copper, basin and the acid are free from the metal to be tested (here it is arsenic).
If a stain on copper foil appears, the blank experiment is to be carried out again with fresh
materials. If the blank is negative, the suspected material (biological or non-biological) is
added and boiled for about an hour or more with occasional addition of water and acid to
make up for the loss due to evaporation. A shining steel grain stain appears in a few
minutes. Which becomes thick gradually. The stained copper strip obtained by Reinsch test
is washed cautiously with water followed by alcohol and finally with ether to remove the
adhering fat, if the matrices are biological materials. the strip is dried by keeping it between
filter paper sheets, cut in small pieces of 0.2 mm x 0.2 mm size and taken into Reinsch tube.
The tube is heated slowly on the flame of spirit lamp. The blank deposit on the copper strip
volatilization and gets deposited on the cooler part of the tube. The tube is cooled and
viewed under microscope.
Characteristic octahedral crystals of arsenious oxide are seen (sensitivity 10 g).
There are certain limitations of the test viz. negative result may be obtained if oxidizing
agent is present on As is in +5 state (it is to be reduced to +3 state) by treatment with
sodium sulphite or potassium iodide or stannous chloride or ferrous sulphate as reducing
agent. Organic arsenicals do not respond if organic matter is not destroyed. Some organic
sulphur compounds produces black stains of copper sulphide, which may be removed by
oxidation. The concentration of HCl should not be too low or too high. This test is generally
used for rapid screening of As, Sb, Hg.

Mercury

Metallic mercury, also known as quicksilver, is a liquid metal having a bright silvery
luster. It exist in nature as the metal itself and as the sulphide (cinnabar or ras sindoor).
Metallic mercury is not poisonous if taken by mouth because it is not absorbed but causes
poisoning if finely divided and inhaled, swallowed or rubbed into the skin. It vaporizes
even at room temperature to an extent sufficient to permit the inhalation of toxic amounts.
The vapour may be source of danger in industry and even in the research laboratory.
Mercury and its salts are used very extensively in the arts, commerce, dentistry and
medicine. It is used in the metallic form as inorganic mercurous (monovalent) and mercuric
(divalent) salts, and in combination with organic molecules. Mercuric compounds being
soluble are intensely poisonous.
Mercuric chloride obtained in the form of white crystalline powder or tablets are
used as a germicide, coloured by cosin, methylene blue or other dye. It has an archid
metallic taste, no smell and by far the most common cause of acute poisoning. The other
poisonous mercurical salts are mercuric oxide, mercuric ammonium chloride, mercurous
chloride or calomel, mercuric potassium iodide, mercuric nitrate and mercuric cyanide.
Fatal dose & fatal period: The fatal dose of corrosive sublimate is about 200 to 300 mg.
Death may occur within a few hours but is usually delayed for 3 to 5 days.
Chemical Tests for Mercury:
Reinsch’s Test: A silvery shining depository on the copper strip indicates the presence of
mercury. After necessary cleaning the dried shining copper strip pieces are heated slowly in a
Reinsch tube and the deposit on the cooler side is viewed under a microscope. Shining round
globules of metallic mercury are observed.

Micro Test: A portion of the stained copper strip from Reinsch test is taken into a spotted
tile. Few drops of con. Nitric acid at added to dissolve the stain on the copper strip. After
evaporation, the residue is taken in dilute hydrochloric acid and spotted on a chromatogram.
The presence of mercury is established by spraying the chromatogram with dithizone.

ANALYSIS OF NEUTRAL POISONS (ORGANIC NON-VOLATILE)

Title: Analysis of Neutral Poisons (organic non-volatile) in biological and non- biological.
ORGANOPHOSPHOROUS INSECTICIDES: These are considered as derivatives of the
corresponding acids or hydrogen phosphide (phosphine).

Classification: These compounds are the derivatives of oxy acids of phosphorous or

thiophosphoric acids. The different acids from which organo-phosphates have been derived are
as follows.

1. Phosphoric Acid.
2. Thiophosphoric Acid.
3. Dithiophosphoric Acid.
4. Miscellaneous organo phosphorous compounds

ANALYSIS OF ORGANO PHOSPHOROUS PESTICIDES:

Compounds of Forensic Interest: Dichlorovos, Phosphamidon, Mevinphos, Methyl Parathion,
Fenitrothion, Fenthion, Chlorpyriphos, Quinalphos, Diazinon, Metasystox, Dimethoate,
Malathion, Ethion, Thimet, Edifenphos etc.
The analytical steps include extraction of pesticide residue in biological and non- biological
materials, their stripping and analysis by different methods.
Isolation and stripping of Organo-phosphorous insecticides in biological and Non-
Biological Matrices:
Procedure:-
1. Biological materials (having high lipid content) are homogenized and
extracted with diethyl ether, hexane, ethyl acetate, petroleum ether or
acetone-hexane mixture (diethyl ether is mostly used) as described in
Section 2.
2. Samples with low water content or dried samples are homogenized and
extracted with binary solvent mixtures viz. hexane-acetone (1 : 1), hexane-
isopropanol (1 : 1), hexane-isopropanol (3 : 1) or methanol or acetone or
acetonitrile. Presoaking of sample with distilled water for 5 minutes may
improve the extraction efficiency.
3. Moist samples such as vegetables, fruits etc. are usually homogenized and
extracted with a binary solvent mixture such as hexane-acetone (1 : 1) or
(4 : 1), hexane-isopropanol (3 : 1) in the presence of anhydrous sodium
sulphate.

4. Soil sample is extracted with acetone-hexane, methanol-acetone or

acetonitrile by shaking.

DETECTION AND DETERMINATION OF ORGANO-PHOSPHOROUS

INSECTICIDES: The analytical methods for detection and determination of residues of
organo- phosphorous insecticides are based on chromatographic methods viz. TLC ( or
HPTLC), GLC or HPLC under diverse analytical conditions. The purified extract of
samples (biological and non-biological matrices) are subjected to analysis by any of the
above methods.

Thin layer Chromatography

TLC conditions and Spray reagents for identification of Organo -phosphorous

pesticides:

Plate : Silica gel G (thickness 0.2 mm).

Solvent System : Hexane : Acetone : : 4 : 1.
Method: Ascending
Identification :Comparing Rf values of unknown and control samples after location of
spots by any of the Spray reagents.
Chromogenic (Spray)Reagents for TLC Identification of Organo-phosphorous
insecticides:

Sraying reagent: Palladium chloride solution.

Responding Compounds : Phosphamidon, Nuvan, Fenthion (Dalf), Dasnit,Carbofuran, Zineb,
Dipteren and other phenolic compounds Propoxur (Baygon), carbaryl (Sevin) .

Detection of Dichlorovos (DDVP):

Preparation of Reagent:

1. 1% Phenylhydrazine hydrochloride Solution (w/v): 1 gm. of phenyl

hydrazine hydrochloride is dissolved in 100 ml. of distilled water and
filter.
2. 10% Hydrochloric Acid Solution: 10 ml. of 32% hydrochloric acid is diluted
to 100 ml.
Method of Spraying and Colour of Spot: The developed plate is sprayed with 1% phenyl
hydrazine
hydrochloride solution. Yellowish-red spots appear after 5 minutes. The spots turn red on
spraying
with 10% hydrochloric acid solution.
ORGANO CHLORO PESTICIDES:

Organo-chloro insecticides are being extensively used in agriculture and also

familiar in domestic applications. New varieties of these insecticides are emerging every
year. Owing to easy availability, these insecticides are frequently misused in homicidal and
poisoning cases. Accidental poisoning cases are also known. The identification and
sometimes quantitation is required which is done by TLC or HPTLC, GLC, HPLC and UV
spectroscopy after extraction of pesticide residue in biological and non-biological, matrices.
The commonly encountered Organo chloro insecticides are given below.
List of organo- chloro Insecticides
Name of Insecticide Chemical Name
1.DDT 1, 1, 1, Trichloro –2, 2 bis (p-chloro phenyl) ethane.

2. BHC Gamma isomer of 1, 2, 3, 4, 5, 6 hexachloro cyclohexane (Benzene

hexachloride).
3.Endrin. 1, 2, 3, 4, 10, 10-Hexachloro 6, 7, epoxy 1, 4, 4a, 5, 6, 7, 8,
8a octahydro 1,4,5,8 endo-exo dimethano naphthale
4. Dieldrin 1, 2, 3, 4, 10, 10-Hexachloro 6, 7, epoxy 1, 4,
4a, 5, 6, 7, 8, 8a octahydro 1,4,5,8 endo-exo dimethano napht
5. Endosulfan 6, 7, 8, 9, 10, 10-Hexachloro 1, 5, 5a, 6, 9, 9a-hexahydro 6, 9
Methano 2, 4, 3 – benzo oxathiepin – 3- oxide.

ANALYSIS OF ORGANO CHLORO PESTICIDES:

DDT, Gammaxene, Aldrin, Endrin, Endosulfan are widely used.

Isolation and Stripping of Organo chloro insecticide in Biological and Non-
Biological
Matrices: The extraction, stripping methods and selection of solvent for extraction have
already been covered in sections 2 and also under organo phosphorous compounds. The
analytical methods include TLC or HPTLC, GLC, HPLC and UV spectroscopy. A few
analytical methods or conditions of chromatography will be covered hereunder.

Detection and Identification of Organo Chloro insecticides:

TLC conditions and spray reagents for identification of organo-chloro insecticides:
Plate:Silica gel G (thickness 0.2 mm.).
Solvent system : Hexane : Acetone : : 4 : 1. Method: Ascending.
Identification:Comparing Rf values of unknown and control samples after location of spots by
any of the following spray reagents.

Chromogenic (Spray) Reagents for TLC Identification of Organo-chloro Insecticides:

Tollen’s Reagent: 10% Ammonical silver nitrate solution.

Preparation of Reagent :10 gms. of silver nitrate is dissolved in 100 ml. of water followed
by addition of a few drops of nitric acid. After some time ammonia solution is added drop wise
when a precipitate appears which dissolves on adding excess of ammonia.

Method of Spraying and the Colour of Spot : The plate is sprayed directly with the reagent
when black spots are observed.
Responding Compounds: BHC, Endosulfan, DDT, Edrin, Aldrin, Dieldrin, Toxaphen and
Heptachlor. A few of organophospho-rous compounds phorate, Sumithion, DDVP,
Phosphamidon, Phosalore also respond.

O-Toludine Reagent:
Preparation of Reagent: 0.5 gm. of O-toludine is dissolved in 100 ml. of acetone.
Mode of Spraying: The developed plate is sprayed with O-toludine reagent and then exposed to
UV irradiation for 10 minutes. Bluish green spots are obtained.
Responding Compounds: Endrin, Endosulfan, DDT, BHC, Aldrin, Dieldrin, Heptachlor,
Toxaphen.
Nickel-Amine Reagent for Specific Detection of Endosulfan.

Preparation of Reagent:
Nickel Amine ReagentEqual volumes of 5% w/v aqueous nickel chloride solution and 30%
ammonic (Sp.gr.-0.88) are mixed.

20% Sodium Hydroxide Solution: 20 gms. of sodium hydroxide is dissolved in 100 ml. of
distilled water.

Mode of Spraying: The developed plate is sprayed with 20% solution of sodium hydroxide
solution followed by nickel amine reagent. Greyish-black spots are obtained.
ORGANO CARBAMATE INSECTICIDES: The widely used carbamates include Propoxur,
Carbaryl, Carbafuran and Zineb. The mechanism of toxic manifestation in mammalian system is
similar to organo- phosphorous insecticides, but the toxicity of the former is comparatively
lesser.

Analysis of Carbamates: The analysis of carbamates is undertaken in the same way as it is done
in case of organo chloro and organo-phosphorous insecticides. The steps include extraction of
carbamates in traces in biological and non-biological matrices and their stripping for onward
analysis by chromatographic and HPLC methods.

Extraction of Carbamates in Biological and Non Biological Matrices: This is done as

described in the case of organo-phosphorous and organo-chloro pesticides. The extract is
subjected to analysis.
Detection of Carbamates :TLC conditions and Spray reagents for Identification of Carbamate
insecticides:
Plate: Silica gel G (thickness 0.2 mm.). Solvent Systems : 1.
Hexane : Acetone : : 9 : 1.
2. Hexane : Acetone : : 4: 1.
Method : Ascending.
Identification: By comparing Rf values of unknown and control samples after location of
spots by any of the spray reagents.

Chromogenic (Spray) Reagents for TLC Identification of Carbamate Insecticides.

Tollen’s Reagent:

Preparation of Reagent : 10% ammonical silver nitrate solution.

Mode of Spraying: Direct spraying with the reagent. Colour of Spot: Black.

Responding Compounds: Propoxum (Baygon), Carbaryl (Serin),Carbofuran Zinel, Dipterex and

other Phenolic compounds, Phosphamidon Demecron), Nuvan, Fenthion (Dalf, Bay tex),
Metasystox.
Alkaline Fast Blue B salt :
Preparation of Reagent : 0.1 gm. of fast blue B salt is dissolved in 100 ml. of 10% aqueous
sodium hydroxide solution (freshly prepared).
Mode of Spraying : Direct Spraying.
Colour of the Spot : Red / Violet.
Responding compounds : Propoxur, Carbaryl, Carbafuran, other Phenolic compounds.

Analysis of methyl parathion in blood and tissue by SPME-GC-NPD method*

Sample preparation: Homogenize 1 g of tissue in 2 mL of water. Transfer 300 µL of

homogenate / blood into SPME vial. Adjust the total volume up to 3 mL using distilled
water.

SPME parameters: Fiber: Polyacrylate 85 µm Extraction mode: Headspace Incubation time:15

min

Extraction temp: 70 °C
Extraction time: 20 min
Salt addition: 0.8 g (NaCl) Desorption
temp: 30 °C Desorption time: 4 min
GC-NPD analysis:

Capillary Column:EC-5 (30 m × 0.32 mm × 0.25 µm)

Carrier gas: Helium
Injection: Split less @ 230°C
Detector: Nitrogen Phosphorous Detector @ 300 °C
Oven Temperature: 120 °C for 3 min followed by 10 °C/min up to 230 °C with an 4 min
hold;
Retention time: Peak of methyl parathion at 10.8 min.
ENVIRONMENTAL CONDITIONS:
Isolation and purification of poisons may be carried out at room temperature.
METHOD OF QUALITY CHECKS USED:
Quality checks have been undertaken by one of the following methods.

1. Repeat analysis
2. Comparison with control
3. Use of two different solvent systems in TLC.
ANALYSIS OF BASIC DRUGS / POISONS ( ORGANIC NON - VOLATILES)
Title: Analysis of basic drugs / poisons (Organic-non-volatiles).

Scope: Analysis of basic drugs / poisons (Organic non-volatiles) in crime exhibits

especially biological samples (viscera, blood, urine etc.) after their extraction.

Purpose: To identify basic drugs / poisons (organic non-volatile in crime exhibit).

CLASSIFICATION: Drugs which are alkaline in nature are called basic drugs. They contain
different moieties having ring structure viz. pyridine, pyrrole, isoquinoline, quinoline i.e.
containing hetero atom N, S, O etc. in the ring. These compounds readily react with acids
forming salts. Their classification is based on their physiological action they produce as stated
hereunder.
Narcotics: Drugs which interact with those receptors in the brain which are responsible for the
transmission and response to pain are known as narcotic analgesics or simply narcotics viz.
opium alkaloid, both natural and synthetic viz. Morphine, Codeine, mono-acetyl and diacetyl
morphine. On the contrary peripheral analgesics (for example aspirin) have no abuse potential.
Now a days various designer synthetic analgesics viz. pethidine (meperidine, methadone,
fentanyl) have come up.
Deliriant: These drugs affect the vital part of brain viz. atropine, hyoscyamine, cocaine,
muscarine (fungi mushrooms).
Spinal poisons/drugs: These affect spinal tract viz. strychnine, brucine (Source Strychnos Nux
Vomica).

Cardiac poisons/drugs: They affect functioning of heart viz. Quinine, aconitine, nicotine,
digitalis glycosides.Drugs of abuse are common with the above drugs. Their identification is of
prime importance in clinical toxicology. When these drugs are consumed by the user in more
therapeutic dose, the outbreak of sign and symptoms occur and the drugs are likely to be
distributed in blood and urine. Thus, clinical toxicological cases blood and urine the main crime
exhibit. In acute cases resulting death, stomach contents and viscera are to be taken into
consideration for extraction of drugs along with blood or urine.

EXTRACTION OF DRUGS: The procedures for extraction have already been covered in
Section 2.

DETECTION AND IDENTIFICATION OF BASIC DRUGS: The identification methods

include screening tests, chemical tests, TLC, GLC, HPLC and also UV Spectrophotometry.
However, the above cited methods may be employed profitably for characterization of class and
the specific member of the class, after a particular or important group has been elaborated to
meet the requirement and these methods are applied to sample after their extraction.

Colour Tests performed with extracts for Basic drugs:

Colour tests for basic drugs performed with extract.

ALKALOIDS COLOUR DEVELOPED WITH REAGENT

MARQUIS CONC.NITRIC ACID MANDELIN

Morphine Violet Bright orange yellow Dark reddish brown

Heroin Reddish Pale yellow Reddish brown

purple

Codeine Dark violet Greenish yellow Olive green

Colour Tests with Different Reagents: Various basic drugs respond to different reagents to
produce a variety of colors, which sometimes can be useful for a screening test. The following
table can be useful to a large extent to identify the group of drugs.
TABLE: 6.1: Colour Tests with Different Reagents:

Reagent Preparation of Reagent Colour Observed Class of

Compound
Present
Formaldehyde 4 parts of sulphuric acid +6 parts of Red/Pink/Blue/ Benzodiazepines
– formalin violet/Red-
/Phenothiazine
violet /Blue- violet
Sulphuric Acid
FPN Reagent 5 ml. of ferric chloride sol- ution + Orange red / Violet red Phenothiazines
45 ml. of 20% w/v solution of / Brown red / Orange /
Perchloric acid + 50 ml. of 50% v/v Red orange / Pink
solu- tion of nitric acid. orange /Blue/ Violet /
Red Brown.
Iodoplatinate 2 ml. of 5% solution of platinic Violet / Blue-violet / Alkaloids.
Solution. chloride in 2N Hcl + 5 gms. of Brown violet / Grey
potassium iodide to 98 ml. water with violet.
stirring.
Marquis 1 volume of formalin + 9 vols of Yellow / Orange Benzodiazepines
Reagent concentrated sulphuric acid.
-do- -do- Violet ,Reddish Morphine
purple ,,Dark violet Heroin
Codeine
Dragendorff’s 1 gm of bismuth subnitrate is Orange / Red-orange / Alkaloid /
Reagent dissolved in 3 ml. of 10M Brown orange primary or
hydrochloric acid , if necessary by Precipitate. Secondary or
gentle heating. It is diluted to 20 ml. It tertiary amine.
is dissolved in 1 gm of potassium
iodide. If black precipitate of bismuth
 tri-iodide separates, it is dissolved in
2M hydrochloric acid.
Thin Layer Chromatography

TLC conditions and data for screening of some common Basic drugs:
Solvent System: Methanol : Ammonia (100 : 1.5v/v) Plate: Silica gel
G(0.2 mm thickness) Development : Ascending technique.

Spray reagent: Iodoplatinate Solution – Violet / Blue violet / Brown violet /

(for alkaloids) Grey violet spots.
Dragendorff’s Reagent – Orange Spots. (alkaloids & benzodiazepines).
FPN Reagent - Orange red / Violet red / Brown red / (Phenothiazines)Orange /
Red orange / Pink orange / Blue / Violet / Red Brown spot.
Sample: Extract of samples along with control drugs and alkaloids.
TLC conditions and data for screening of some Benzodiazepines: (8,9,10,11)
Plate: Silica gel G (0.2 mm. thickness)
Development: By ascending technique.
Solvent systems: No. 1 : Chloroform : Acetone : : 4 : 1 No. 2
: Ethyl acetate : Methanol : Strong Ammonia Solution : : : 85 : 10 : 5
No. 3 : Ethyl acetate.
Spray Reagent: Dragendorff’s Reagent – Orange, Red – orange / Brown orange spot Other
reagents viz.
Marquis Reagent or Iodo platinate & Reagent may be used.
Egs. Lorazepam, Medazepam, Nitrazepam, Nordazepam, Oxazepam, Prazepam, Temazepam,
Triazolam.

TLC conditions and data for screening of some common drugs of abuse:

Plate: Silica gel G (0.2 mm. thickness)

Development: By ascending technique.
Solvent systems: No. 1 : Cyclohexane : Tolune : Diethylamine (75 :15 : 10)
No. 2 : Methanol : Conc. Ammonia (100:1.5)
No. 3 : Chloroform : Methanol : : 90 : 10.
Spray Reagent: 1. Acidified Potassium Iodoplatinate : Violet / Blue violet / Brown violet / Grey
violet Spot.
2. Dragendorff’s Reagent – Orange / Brown Orange, Reddish Orange spot.
Egs. Methaqualone, Mecloqualone, Cocaine, Caffeine, Heroin, Diphenhydramine, Diazepam.

ANALYSIS OF ACIDIC (ORGANIC NON-VOLATILE) POISONS/ DRUG

Analysis of Acidic (organic non-volatile) poisons/ drugs in crime exhibits by different methods.

ACIDIC POISONS/DRUGS:
Acidic Drugs:Drugs which are acidic in nature are called acidic drugs. These drugs readily react
with bases forming salts. The main acidic drugs are barbiturates (substituted malonyl urea) and
salicylates etc. and a few compounds other than barbiturate viz. Glutithimide, Meprobamate etc.

Acidic Poisons: Phenolic Compounds viz. phenol, cresols, -naphthol etc.

Barbiturates:Barbiturates (salts of barbituric acid) are the drugs that are associated with criminal
poisoning cases (homicidal and suicidal) in the Indian perspective due to their easy availability.
Thus, the search for barbiturate in biological materials is of importance in case of suspected
poisoning by drugs.
Analysis of Barbiturates: As stated earlier for other toxicants under different classes ithe
preceding sections, the active constituent i.e. barbiturate is to be extracted in biological
and non-biological matrices and stripped thereafter prior to analysis for their
identification by diverse methods viz. colour test, screening by UV Spectroscopy, TLC or
HPTLC, GLC and HPLC etc.

Extraction of Barbiturates in Biological and non biological matrices: This is achieved

according to methods described in Section-2. Normally, the acid-ether part is kept reserved for
onward analysis. The extract is stripped, dried over anhydrous sodium sulphate and evaporated
to dryness as and when required for analysis.
Colour tests for the presence of Barbiturates in extracted materials: Colour Test:
The colour tests are the screening tests in the general method of analysis of toxicants. The
positive finding is a presumptive indication for any of the barbiturates necessitating
confirmed identification. However, these tests are very important.
Dille – Koppayani Test: Preparation of Reagent:
A. Cobalt Acetate Solution: 1 gm. of cobalt acetate (tetrahydrate) is
dissolved in followed by addition of 0.2 ml. of acetic acid.
B. Isopropylamine Solution: 5 ml. of isopropylamine is mixed with
100 ml. of methanol.
Test Procedure No. 1: A small amount of extracted material is placed on a spot plate. 3-4
drops of cobalt acetate solution and 3-4 drops of isopropylamine solution are added. The
appearance of a purple or blue violet colour indicates the presence of barbiturate.

Test Procedure No. 2: The residue of extract of sample is taken in 1 ml. of chloroform. To a
portion of chloroform extract of the sample, 2 drops of freshly prepared 1% cobalt
acetate in methanol is added followed by 1% lithium hydroxide in methanol drop by
drop. A blue ring at the junction indicates the presence of barbiturates.
Identification of Barbiturates by TLC: (1)
Plate : Silica gel G (0.2 mm thickness).
Development : By ascending technique.
Solvent System : System 1: Ethyl Acetate : Methanol : 5%
Ammonia (25 : 50 : 25)
System 2 : Chloroform : Acetone : : 80 : 20.
System 3: Isopropyl Alcohol : Chloroform : ammonia (45 : 45 : 10)

Spray Reagents and Colour of Spots:

Mercuric Chloride – Diphenylcarbazone Reagent: Diphenyl carbazone Solution: 0.1 gm. of
diphenyl carbazone is dissolved in 50 ml of ethanol Mercuric Chloride Solution: 0.1 gm. of
mercuric chloride is dissolved in 50 ml. of ethanol.
The mixing of A and B is in equal volume is done before spraying (Solution A is
to be replaced by a fresh bath after a short interval to get better spots.

The spraying reagent contains toxic mercuric salt and is to be done in fume chamber to avoid
hazards.

Colour of Spots: Blue violet spots on a pink background are observed for barbiturates.
Compound (Barbiturate) hRf in Solvent Systems
No. 1 No. 2 No. 3
Allobarbital. 31 50 53
Amobarbital. 40 52 74
Barbital. 33 41 51
Cyclobarbital. 35 50 59
Phenobarbital. 44 55 76
Secobarbital. 29 47 38
42 55 -

ENVIRONMENTAL CONDITIONS:

Isolation and purification of poisons may be carried out at room temperature.

LIST OF EQUIPMENTS REQUIRED FOR TOXICOLOGICAL ANALYSIS OF

DRUGS AND POISONS
Major Equipments
a) Gas Chromatography – Mass Spectrometry (GC-MS)
b) Head Space Gas Chromatography-Flame Ionization Detection (HS-GC-FID)
 c) High Performance Liquid Chromatography (HPLC)
d) Atomic Absorption Spectrophotometer
e) UV-Visible Spectrophotometer
f) FTIR Spectrophotometer
Minor Equipments
a) Thin Layer Chromatography setup
b) Hot air oven
c) Tissue homogenizer
d) Centrifuge
e) Sonicator
f) Vortex shaker
g) UV-Cabinet (254 nm, 265nm and visible lights)
h) Hot plate cum magnetic stirrer
i) Fuming hood
j) Refrigerator
k) Water bath
l) Analytical balance

TOXICOLOGICAL ANALYSIS OF DECOMPOSED MATERIAL AND BODY

REMAINING:

When extended decomposition of the remains has occurred, alternative matrices are
required. Skeletal tissue may provide an appropriate sample of choice since it is very resistant to
putrefaction.
Disintegrated viscera lead to several difficulties of toxicological examination and
complications in the elucidation of results. In decomposed bodies, the tissues are softened and
liquefied by autolysis and bacterial actions release carbohydrates, fats and proteins which are
extracted with the extracted materials in plentiful amounts and confuse the process of
examination leading to serious difficulties in the interpretation of results. Therefore, appropriate
clean-up procedures should always be applied to eliminate these materials from the extracts.
Some of the substances go through chemical changes along with the decomposition of tissues.
Hence, such substances are not detectable by chemical tests e.g. Chloral Hydrate, Sodium Nitrite,
Cocaine, Aconite, Atropine etc. Some substances are formed in the tissues by decomposition
which gives analogous chemical reaction to those obtained from drugs or chemicals such as
neurin, muscarine and mydalein.
Volatile substances are lost as a result of decomposition and Cyanide, Ethyl Alcohol,
Ketones and Sulphides may be formed from ordinary tissue components. All the same cyanide so
formed is always in trace amount, but the alcohol produced by the advanced decomposition may
be from 50-100 mg per cent. Regardless of the decomposition, many toxic substances are still
discernible, e.g. Carbon Monoxide, Cyanide, Fluoride, Barbiturates, Phosphates, Insecticides/
Pesticides, Hyocine, Strychnine, Thevetin, Cerberine, Nicotine, many other drugs and metallic
poisons.
CHALLENGES IN FORENSIC TOXICOLOGICAL EXAMINATION:

As the field of forensic toxicology expands and improves, forensic toxicologists continue
to face multiple challenges, such as the emergence of new drugs, different standards among
laboratories, and discrepancies in the interpretation of toxicological findings.