الرحيم الرحمن الله بسم

العظيم الله صدق

االسراء ) (85سورة

Role of laboratory in Toxicological

Studies

Prepared by Reda M. Elsayed

29/4/2015

SAMPLING

Types of Biological samples:1- BODY FLUIDS:•Blood (plasma, serum and whole blood)•Urine, semen, saliva, gastric contents..etc.2- Tissue samples:liver, kidney, muscles….etc and

Hair.

BLOOD SAMPLES

Epindorf tubes

micropipettes

TISSUE SAMPLES

a) Biochemical and toxicological study

Homogenized by Tissue Homogenizer/ Grinder

TISSUE HOMOGENIZER

SAMPLE PRESERVATION :

b) Histopathology exam.:Organs Preserved as it is in different preservative according to type of tissue then processed by the specialist.

METHODS FOR ANALYSIS:

a- Biochemical Tests.b- Toxicological Tests.c- Histopathological Exam.

1- BIOCHEMICAL TESTS:

1. Spectrophotometery:

Used for liver and kidney functions,

glucose, pseudocholine

estrase...etc

2. Immunoassay ( ELISA and RIA):

Hormones and some enzymes….etc.

1. Spectrophotometery2. Immunoassay ( ELISA

and RIA):3. Chromatography: TLC HPLC, LC- MS GC, GC-MS

2- TOXICOLOGICAL TESTS:

SPECTROPHOTOMETERY

Bechman chemistry apparatus.

:IMMUNOASSAY TESTS

•“Immuno” refers to an immune response

that causes the body to generate

antibodies.

•“Assay” refers to a test.

Immunoassays are chemical tests used to

QUALITATIVE or SEMIQUANTITATIVE to

determine a specific substance, the

analyte, in a blood or body fluid sample,

using an immunological reaction.

RIAIn which, there is a competition between two antigens:

the labeled antigen reagent (Ag*) and

the unlabeled specimen (Ag) (or test sample analyte)

Both compete for a limited amount of antibody.

Tramadol PROCEDURE

The assay is based on the competition of

Tramadol labeled enzyme (G6PDH) and the free

drug in the urine sample for the fixed amount of

antibody binding sites.

In presence of the drug, The enzyme (G6PDH) is

free so activity is determined by the conversion

of NAD to NADH…. Colour change …positive test.

In the absence of the free drug in the sample, the

antibody binds the drug enzyme conjugate and

enzyme activity is inhibited.

Drug analyzerImmunoass

ay

ELISAAn enzyme-linked immunosorbent assay (ELISA) used to detect the presence and/or amount of a target protein of interest within an experimental sample.Detection of the target protein is made by antibodies, which make the ELISA an immunoassay. Through a series of incubation and washing steps.

IMMUNOASSAY:

Architect plus I 1000

Immuno- analyser

DETECTION AND QUANTIFICATION BY CHROMATOGRAPHY

SAMPLE PREPARATION (EXTRACTION)

Liquid-liquid extraction is a useful method to separate components (compounds) of a mixture

For example:Suppose that you have a mixture of sugar in vegetable oil (it tastes sweet!) and you want to separate the sugar from the oil. You observe that the sugar particles are too tiny to filter and you suspect that the sugar is partially dissolved in the vegetable oil.

How about add water to the mixture

Will it separate the sugar from the oil? Sugar is much more soluble in water than in vegetable oil, and, as you know, water is immiscible (=not soluble) with oil. الزيت يختلط هلبالماءThe water phase is the bottom layer and the oil phase is the top layer, because water is denser than oil.

By shaking the layers (phases) well, you increase the contact area between the two phases. The sugar will move to the phase in which it is most soluble (the water layer)

NOW THE WATER PHASE TASTES SWEET,BECAUSE THE SUGAR IS MOVED TO THE WATER PHASE UPON SHAKING.YOU EXTRACTED SUGAR FROM THE OIL WITH WATER.

In this example: water was the extraction solvent.

the original oil-sugar mixture was the solution to be extracted.

sugar was the compound (analyte) extracted from one phase to another.

the concept of liquid-liquid extraction:

Liquid-liquid extraction is based on the transfer of a solute substance from one liquid phase into another liquid phase according to the solubility.

You can use extraction to separate a substance selectively from a mixture, or to remove unwanted impurities from a solution.

In the practical use, usually one phaseis a water or water-based (aqueous)solution and the other an (organic solvent) which is immiscible with water.

The success of this method depends upon the difference in solubility of a compound in various solvents. And the choose of a suitable extraction solvent.

CHROMATOGRAPHY

1. Thin layer chromatography2. Liquid Chromatography3. Gas Chromatography (GC)

CHROMATOGRAPHIC THEORY

Chromatographic separation process

is based on the difference in the

surface interaction of the analyte

(sample) and eluent molecules

(mobile phase)

Analyte molecules while moving

through the porous packing materials

tend to interact with the surface

adsorption.

A) TLC

B) HPLC

C)GC-MS:

THEORY OF HPLC HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution.

Compounds are separated by injecting a sample mixture onto the column. The different components in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase.

COMPOSITION OF A LIQUID CHROMATOGRAPHY SYSTEM

Solvent (mobile phase)

Solvent Delivery System (Pump)

Sample Injector Column Detectors (Diode

Array) Data Collection

HPLC COLUMNS The column is one of the most

important components of the HPLC chromatograph.

It is made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30cm.

Normally, columns are filled with silica gel.

Silica is inert to most compounds and has high surface activity which can be modified easily with water and other agents. Silica can be used to separate a wide variety of chemical compounds.

TYPES OF DETECTORS Photo-Diode

Array

UV detector (200-400nm )

Fluorescence

Electrochemical

Mass-Spectrometric

IR Absorbance

Retention time (Rt):It is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the

detector) under set conditions.

CALCULATION OF RESULTS BY CALIBRATION CURVE