TOXICOLOGICAL ANALYSIS

specimen

DIAGNOSIS OF POISONING, is based on: 1. HISTORY & CIRCUMSTANTIAL EVIDENCES 2. CLINICAL PICTURE 3. TOXICOLOGICAL ANALYSIS

- to identify the type of poison and its amount Living Dead - vomitus (or after gastric lavage by tap water) - stomach & its content - blood ( blood alcohol, blood lead) - small & large intestine & its contents - urine (drug dependence) - part of liver with small quantity of blood - part of kidney, bladder & urine

Investigation in a case of poisoning (living)

TOXICOLOGICAL ANALYSIS Screening 1. Color tests. 2. Spectophotometry. 3. Immunologic methods: - (RIA) Radio-Immuno Assays - (EMIT) Enzyme Multiplied Immuno assay Technique VIVA 4. Chromatography: - (TLC) Thin layer chromate Confirmatory 1. GC: volatiles, gases & pesticides. 2. HPLC: Analgesics, drug of abuse and psychotropic drugs. 3. GC/MS : ** most sensitive and reliable method 4. Atomic absorption spectrophotometry used for heavy metals NON-TOXICOLOGICAL ANALYSIS 1. Kidney function tests. ( oxalaic acid ) 2. Liver function tests. ( paracetamol) 3. Blood gases, Acid-base. ( salisalyate) 4. ECG. (digitalis) 5. Radiological: X-ray chest pulmonary edema Chlorine gas Organophosphorus Carbamate insecticides Kerosene X-ray Radio opaque shadow CHIPES stomach - Chloral hydrate - Heavy metals - Iodine - Phenothiazines - Enteric Coated tab (aspirin) - Solvent (chloroform, carbon tetrachloride) Fluid level Kerosene Lead lines metaphysic (growing child) Lead poisoning CT brain scan cerebral hge CNS stimulant brain edema CO poisoning focal cerebral ischaemia CO, cyanide, methanol cerebral & cerebellar chronic ethanol abuse atrophy

Biological specimens : Urine drugs of abuse ( opiates , barbiturates ) Adv : 1. Urine sampling is non-invasive. 2. No sample preparation and easy analysis. 3. Concentration of the drugs and their metabolites are relatively high compared with blood. 4. may be detected for longer periods after intake. Disadv : substitution / adulteration/ dilution Blood Blood alcohol level + Co Hb % + met Hb% Serum acetaminophen, barbiturates, ChEII, BDZ, salicylate Hair, salive, sweat, meconium

COLOR TESTS
Principle of color test Many drugs and poisons give characteristic colors with appropriate reagents if present in sufficient concentration. The color produced usually varies in intensity proportional to the concentration. Samples used in color tests:Urine Advantages - Simple - Easy to perform (bed-side tests) - Carried directly on the available sample. Disadvantages - Screening test only - Rapid color changes - Low sensitivity - Many drugs with the same reagent give same color (non specific)

PHENOTHIAZINE SALICYLATE (ANTIPSYCHOTIC)

Sample Reagent ferric chloride FPN reagent - Ferric chloride - Perchloric acid - Nitric acid

IMIPRAMINE
(TCA)

BARBITURATE
(HYPNOTICS)

ETHYL ALCOHOL (ETHANOL)

(Potassium Permanganate Method)

METHYL ALCOHOL (METHANOL)

Chromotropic acid test (Ring test)

IRON
gastric content containing iron dilute HCL + potassium ferricyanide

Urine Forrest (imipramine) PSPN reagent - Potassium dichromate - Sulphuric acid - Perchloric acid - Nitric acid Greenish yellow Cobalt acetate in methanol, BOIL Powdered borax potassium permanganate + acid heat Potassium Permanganate + oxalic acid + chromotropic acid + sulphuric acid

Color

Violet color

Pink color

Bluish color with precipitations.

Water (colorless) Ethyl alcoholAcetaldehydeAcetic acidWater + Co2

Violet ring in the interface MethylalcoholFormaldehydeFormic acid + Chromotropic acidcompound

Blue color

BLOOD & SEMEN IDENTIFICATION

Identification of blood
1. Murder cases 2. Sexual assaults 3. Mass disaster 4. Blood transfusion or organ transplantation 5. Paternity problems 1. Screening (Preliminary/presumptive test) Phenolphtalein test Benzedine test - Oxidation reaction - Peroxide as oxidizing agent - Heme as catalyst KOH Benzedine in glacial acetic acid Blue

Identification of semen
Medicolegal importance 1. Criminal : rape, sodomy, bestiality 2. Civil : - illegal pregnancy - impotence of husband Is the stain blood/semen? 1. Physical Examination on garments - stiff starchy touch - creamy-white or yellowish white in colour Guaicum test - under UV lamp = faint bluish fluorescence *not specific - garments usually very dirty

Principle

Procedure Reagent

Result

Pink

Freshly prepared Guaiacum resin in ethanol Bluish green

2. Confirmatory 2. Microscopial Examination 1.Microscopical exam +ve = one unbroken sperm is found! Mammalian(human) Non-nucleated Rounded Biconcave Non-mammalian Nucleated Oval Biconvex Camel RBC Non-nucleated Oval Biconvex

2. Micro-chemical test Condition: 1. Stain : dried 2. Crushed very fine powder 3. Reagent : free of water (prevent dissolve of crystal)

3. Micro-chemical & Enzymatic test Microchemical Florences test Barberios test interaction between reagent + semen to Enzymatic Acid phosphatase quantitative &

Principle

4. Flame : weak (prevent clumping of crystal) & blue (prevent soot formation)

Reagent

Teichmann test (Acetic acid test) Anhydrous glacial acetic acid

Takayama test (hemochromogen crystal test) Takayama reagent - 10% NaOH - Pyridine - Saturated glucose solution - 7 volumes of distilled water Salmon pink crystals of heamochromogen Adv

Crystal

Small dark brown rhombic crystals (haematin hydrochloride crystal)

3. Spectro-scopical exam Function : - Can detect the poison - Good positive test - Very sensitive test - Very easy and give result immediately

Disadv

4. Chromatographic test Paper or thin layer chromatography

form evident coloured crystals, seen under low power microscope screening detect confirmatory choline detect spermine Fluorence reagent Saturated picric - K Iodide acid solution - iodine - water Dark brown rhombic Yellowish green crystals (larger than needle shaped haemin crystals) crystals - rapid preliminary - good positive test for seminal stain - spermine is - determination of present in both time of coitus normal & ** +ve within 12 aspermic person hours only! - determine time of coitus ( 1347hrs) - non-specific - rapid disappearance of crystal - blood/other albuminous substances may interfere false +ve

qualitative human level = 5404K units/ml animal = 27-104 units/ml vege = 48 units/ml

+ve = not necessarily indicate presence of seminal fluid high value = human origin of value in case of - aspermia - vasectomy - bestiality determine recent intercourse (1824hrs in vagina)

Precipitin test - white ring precipitate at the junction between 2 liquids

1. Blood grouping

Is it human blood/semen or not? 1. Spermato-precipitin test 2. Immuno-electrophoresis 3. Immunodiffusion 4. Antihuman globulin fixation 5. Latex agglutination test Does it belong to a certain person? 1. Grouping of semen 2. Enzyme studies 3. DNA typing

REINSCH TEST (DETECTION OF METALLIC POISON)

Advantages 1- Screening and qualitative test. 2- Easy, rapid, and cheap. 3- Highly sensitive (detect even traces). 4- Can be done directly on organic matter.
Tests are of two types: SCREENING CONFIRMATORY Good negative. Good positive. Qualitative only. Qualitative and Easy, cheap and rapid. quantitative. Ex: Reinsch test Ex: HPLC

Disadvantages 1- It is a qualitative test only (Not quantitative) 2- Can't differentiate between acute & chronic poisoning

Principle: A test for heavy metals in biological samples, using copper foil in heated hydrochloric acid. If heavy metals are present, they form visible deposits on the copper.

Confirmatory tests for detection of heavy metals = on-line analysis!! 1. Atomic absorption spectrophotometry (AAS) 2. Gas chromatography (GC) 3. Liquid chromatography 4. High performance liquid chromatogrphy .(HPLC)

Reagent = pure diluted HCl Pure : because reinsch test is a highly sensitive test, so , any impurities will give misleading results Diluted : because concentrated acid produces highly irritant fumes HCl : because it can act directly on the organic matter Steps : Dilute HCL ( 1 HCl : 2 water) Cu foil strips (Clean and bright) Boil for 5 min No impurities (i.e. reagent, beaker & cu foil are free from impurities.) Add the sample, continue boiling deposit is formed on Cu foil 5. Decantation= process of separation - done by pouring off the solution resulting from successive of mixtures. washing of the excess HCL by pouring the water on the sides of the beaker and not directly on the copper foil. 6. Descent= Removal of cu foil from the - should be by filter paper and not by your hands. beaker. 7. Dryness Should be done by gentle pressure (Not friction). to avoid removal of deposit 8. Examine COLOR of the deposit - Grey to black : Arsenic & Bismuth - Mirror-like : Mercury & Silver 9. SUBLIMATION= Transfer of a to differentiate between the 2 poisons substance from a solid state to a gas - Arsenic (As) or Bismuth (Bs) state without passing into a liquid state. - Mercury (Hg) or Silver (Ag) *done in a tube called sublimation tube
Precautions of sublimation: - sublimation tube dry and clean inclined 45 - flame weak and blue - heat the base of tube only - Opening of tube not facing anyone...!

1. 2. 3. 4. 5.

6.

Signs of complete Sublimation - Copper foils regain its original color. - Formation of sublimation line Sublimation line formed by: condensation of sublimate (vapor resulting from sublimation) in the form of crystals (its shape is specific and diagnostic for each heavy metal). - Tetrahedral & Octahedral crystals of variable sizes = arsenic - Black spherical globules of variable sizes = mercury eg : - Reinsch test. - Sublimation tube. - Crystals: Tetrahedral &octahedral crystals of variable sizes. - Poison is arsenic.

10. Examine under MICROSCOPE 11. Comment (under microscope) Reinsch test Sublimation tube Crystals Poison