ANALYTICAL

TOXICOLOGY
Set up and Practice
 Scope of analytical toxicology
• Provide support for diagnosis, prognosis and
management of poisoning cases.
• To confirm death due to poisoning.
• Water analysis.
• Toxicological services are not available on
regular basis and where they are available, the
physician is dependent on national or regional
health laboratory established for other purposes
and operating only part of the time.
• There are however simple analytical techniques
that do not need sophisticated equipment or
expensive reagents or even a continuous supply
of electricity.
• These tests could be performed in basic
laboratories of health facilities.
 Requirements
• Trained personnel
• Basic laboratory equipments and consumables
• Inter-departmental co-ordination
• Proper sample receiving and report dispatch
methods
• Safety measures and
• Lab waste management
 Basic equipments required for
toxicological analysis
• Calibrated laboratory balances
• Bench top centrifuge
• Vortex mixer/ magnetic stirrer/ rotary mixer
• Water bath and heating block
• Butane gas burner
• Fume cupboard
• Refrigerator/ freezer
• PH meter
• Automatic/ semi automatic pipettes
• Lab glassware including volumetric appartus
and adequate cleaning facilities
• Supply of pure water
• Supply of compressed air or nitrogen
• Thin layer chromatography plates
• Facilities for developing and visualising
chromatograms
• UV spectrophotometer
• Porcelain spotting tile
 Specialized equipments
• Desirable to have
 HPLC
 GC
 Mass Spectrometry
 Radio Immuno Assay
 Enzyme Mediated Immuno Assay Technique
• There should be an access to
 Atomic Absorption Spectrophotometry
 Neutron Activation Analysis
vortex Magnetic stirrer

Rotary shaker
Elec. Weigh machine
MICROPIPPETES
centrifuge pH meter
 Water bath

Sand warmer
Hot air oven and incubator
Fuel chamber
 Practical aspects
• Health and safety in the laboratory
- Handling, storing and working with chemicals
- Awareness of Local policies and regulations.
- Written health and safety policy.

• Reagents and drug standards

-Purity(lab grade, analytical grade, general grade)
-storage methods
-statements on the reagent label (impurities)
-testing the quality
• Balances and pipettes
• Chemically pure water
• Quality assurance
-Known positive and negative specimens
should be analysed at the same time as the test
sample.
- cleaning glassware with special lab
detergent, purified water and air drying

• Recording and reporting results

 Samples
• Urine
• Stomach contents
• Scene residue
• Blood or serum
 Extraction of drug or poison
• From blood
• From urine
• From gastric lavage
 Analytical methods
• Qualitative methods
bedside colorimetric tests
card tests
• Semi quantitative methods
Thin layer chromatography (TLC)
• Quantitative methods
Ultraviolet Spectrophotometry
Gas chromatography
 COLOR TESTS
• Principle
Many drugs and poisons when present in sufficient concentration
give characteristic colors with appropriate reagents.
• merits
-relatively simple, inexpensive and quick method for qualitative
method.
-sensitive method.
• Demerits
-problem of interference
- observer variance
- color unstable and vary with concentration
TEST DONE FOR REAGENTS REQUIRED COLOR
(Sensitivity)

TRINDER’S TEST SALICYLATES 10mg/l MERCURIC CHLORIDE STRONG VIOLET

(aspirin) AQUEOUS HCL COLOR
HYDRATED FERRIC
NITRATE

O-CRESOL TEST PARACETAMOL CONC. HCL STRONG ROYAL BLUE

P-aminophenol 1mg/l 0-CRESOL SOLN. COLOR
AMMONIUM
HYDROXIDE

NEUTRAL FERRIC SALICYLATES & FERRIC CHLORIDE PERSISITENT PURPLE

CHLORIDE TEST PHENOL SOD. HYDROXIDE COLOR

FPN TEST PHENOTHIAZINES FERRIC CHLORIDE PINK, ORANGE, RED

cpz 25mg/l & SOLN OR VIOLET OR BLUE
TRICYCLICS PERCHLORIC ACID COLOR
NITRIC ACID
TEST DONE FOR REAGENTS REQUIRED COLOR
(Sensitivity)

FOLIN-CIOCALTEAU PHENOL FC REAGENT BLUE COLOR

REAGENT TEST 10mg/l SOD.HYDROXIDE

FORREST TEST TRICYCLICS(IMIPRAMI POT. DICHROMATE YELLOW –GREEN

NE AND RELATED DIL. SULFURIC ACID COLOR DEEPENING TO
COMP) PERCHLORIC ACID DARK GREEN-BLUE
25mg/l DIL. NITIC ACID
MARQUIS TEST OPIUM AND CONC. SULFURIC ACID PURPLE COLOR
DERIVATIVES FORMALIN GRADUALLY TURNS
BLUE

LEE-JONES TEST CYANIDE SOD. HYDROXIDE BLUE COLOR

10mg/l FRESH FERROUS-
SULFATE
DIL. HCL
FILTER PAPER TEST PHOSPHIDES & SILVER NITRATE SOLN. BLACKENING OF FILTER
PHOSPHORUS & LEAD ACETATE SOLN. PAPER
SULFIDES
TEST DONE FOR REAGENTS REQUIRED COLOR
(Sensitivity)

REINSCH TEST HEAVY METALS CONC. HCL PURPLE BLACK-

(ANTIMONY, DIL. HCL ANTIMONY
ARSENIC, COPPER FOIL/ MESH DULL BLACK- ARSENIC
BISMUTH, DIL. NITRIC ACID SHINY BLACK- BISMUTH
MERCURY) SILVER- MERCURY

DITHIZONE TEST LEAD CONC. HCL CHERRY RED COLOR OF

CITRATE CYANIDE SOLN. DITHIZONE LAYER
DITHIZONE IN
CHLOROFORM
QDCT IRON DIL.HCL DEEP BLUE PRECIPITATES
10mg/ml POT. FERRICYANIDE
POT. FERROCYANIDE

DICHROMATE TEST ETHANOL 2% POT. DICHROMATE GREEN OR BLUE COLOR

CONC. SULFURIC ACID (PISTA GREEN COLOR)

SCHIFF’S REAGENT METHANOL ETHYL ALCOHOL PURPLE COLOR

TEST KMnO4 3%
ORTHOPHOS. ACID 5%
OXALIC ACID 10%
SCHIFF’S REAGENT
TEST DONE FOR REAGENTS REQUIRED COLOR
(Sensitivity)

COLOR TEST FOR PARAQUAT & SODIUM DITHIONATE BLUE/BLACK- PARAQUAT

PESTICIDE DIQUAT AMMONIUM HYDROXIDE YELLOW/GREEN- DIQUAT

TEST FOR HYPOCHLORITE DIPHENYL AMINE 1% IMMEDIATE BLUE COLOR

HYPOCHLORITE SOLN. IN SULFURIC ACID

TEST FOR THIOCYANATE FERRIC CHLORIDE SOLN. BLOOD RED COLOR

THIOCYANATES

FUJIWARA TEST CHLORAL SOD.HYDROXIDE INTENSE RED/PURPLE

HYDRATE & TRICHLORO ACETIC ACID COLOR
CHLOROFORM PYRIDINE
(TRI CHLORO
COMPOUNDS)
ISONITRILE TEST CHLORINATED 20% SOD. HYDROXIDE SHUNK ODOUR IS
HYDROCARBONS PURIFIED ANILINE PERCIEVED
(CCL4,
CHLOROFORM,
 Color Tests
• Most of the reagents required for the tests can
be prepared in the lab.
• Always conduct color test with control and
blank.
 Card tests
• Drug testing facilities are now available in kit
form.
• Example:
A kit to test presence of benzodiazepine and
barbiturate.
Instant view- a multi test drugs of abuse
panel, which tests presence of 9 drugs.
THIN LAYER CHROMATOGRAPHY

• Qualitative method
• HP-TLC is a Semi quantitative method
• Based on movement by capillary action of a
liquid phase( organic solvent) through a thin,
uniform layer of stationary phase (silica gel)
held on a rigid support
(glass/aluminium/plastic)
• The compounds are separated by partition
between the mobile and stationary phases.
 Steps in TLC
• Pretreatment of the sample
-usually solvent extraction
• Preparation of TLC plate
- (silica gel 1: 2 water)or ready made plates
available
• Sample application
• Development of chromatogram
-Glass TLC development tanks
- EMA mobile phase
- uniformly saturated tank
• Visualization of chromatogram
- using iodine chamber
• Evaluation of chromatogram
-calculating Retention factor
distance travelled from the origin by the analyte
Rf=
------------------------------------------------------------
----------
distance travelled from the origin by the solvent front
 Qualitative Estimation Of Malathion From
Gastric Aspirate By TLC
• Solvent extraction of malathion from gastric aspirate (Ph 3)
using diethyl ether by liquid-liquid partition chromatography.
• Ether extract thus obtained is air dried.
• Reconstitute extract in methanol and spot on a column
marked on the plate.
• Spot standard mixture on a second column( also control on
third column).
• Develop chromatogram(10 cm run) using cyclohexane:
acetone: chloroform(saturated tank) and allow to dry.
• Visualize the chromatogram using iodine chamber.
• Evaluate the chromatogram.
 TLC
• merits
- simple, inexpensive and widely used technique
- quick method
- versatile
- almost all compounds can be evaluated.
• Demerits
-not good for volatile compounds.
-interpretation may prove difficult.
 UV/Visible Spectrophotometry
• Quantitative method
• Based on the principle that many drugs when
in solution will absorb UV radiation.
• In the spectrometer, the relationship between
the intensity of light entering and leaving a
photocell is governed by Beer-Lambert law.
• The Beer-Lambert law states that for a solution with an
absorbing solute in a transparent solvent, the fraction of
the incident light absorbed is proportional to the
number of solvent molecules in the light path.

• Evaluation by comparison with standard.

Absorption of the sample concentration of sample
--------------------------------- =
----------------------------------
Absorption of standard concentration of standard
UV Spectrophotometer
 Spectrophotometric Determination Of
Paracetamol In Plasma Or Serum
• Prerequisites: keep ready all required solutions and also prepare
standard(paracetamol) of required strength
• Add 2 ml of TCA to 1 ml of test sample and centrifuge for 5 min (step 1)
• In a separate tube add 1 ml of HCL, 2 ml sodium nitrite and
mix( nitrogen dioxide evolves out)( step 2)
• Take 2 ml supernatant from step 1 to the mixture obtained in step 2,
mix and allow to stand for 2-3 min at room temp
• Add 2 ml ammonium sulfamate soln drop by drop to remove excess
nitric acid.
• Add 2 ml sod. hydroxide and vortex mix to remove any gas bubbles
• Then, measure the absorbance against plasma blank
• Calculate plasma concentration by comparison with the results
obtained from standard soln.
Gas Chromatometer
 Poison Information Center
• ? Poison information software
• ? Toll free number
• ? personnel
 Water analysis
• Bacteriological examination
• Virological examination
• Test characteristics for drinking water
PARAMETER UNIT REQIREMENT PERMISSIBLE LIMIT
(DESIRABLE LIMIT) In the absence of
alternate source
Color - 5 25
Taste - agreeable -

Odour - unobjectionable -

Turbidity NTU 5 10

pH - 6.5-7.5 -

Total Hardness ppm 300 600

Alkalinity ppm 200 600

Chloride ppm 250 1000

Calcium ppm 75 200

Mg ppm 30 No upper limit

Total dissolved solutes ppm 500 2000

PARAMETER UNIT REQIREMENT PERMISSIBLE LIMIT
(DESIRABLE LIMIT) In the absence of
alternate source
Iron ppm 0.3 1.0

Sulfate ppm 200 400

Fluoride ppm 1 1.5

Lead ppm 0.05 No relaxation

Nitrate ppm 45 No relaxation

Mercury ppm 0.001 No relaxation

Arsenic ppm 0.05 No relaxation

Chromium ppm 0.05 No relaxation

Aluminium ppm 0.03 0.2

Residual free chlorine ppm 0.2 -