This presentation explains the forensic analysis of some of the toxic substances like - acids, alkalis, metals, non-metals from the biological matrices along with their identification using preliminary tests and quantification and trouble shooting in toxicological analysis.

1. INSTITUTE OF FORENSIC SCIENCE, MUMBAI
2022 - 2023
Name: Aher Sanchita Santosh
Course: M.Sc. Forensic Science
Semester: III
Roll no.: 02
Paper code: PSFSC 304
Subject: Forensic toxicology - I
Topic: Extraction of Acid & Alkalis, Metallic & Non-metallic Poisons
from Biological matrices and their Identification & Quantification,
Trouble shooting in toxicological analysis.

2. INTRODUCTION:
What does toxicological analysis mean?- “Examination of different matrices using analytical techniques for the
presence of compounds like illicit drugs, alcohol, and other toxic elements which one can’t easily find its presence in the
same.”
Basic Steps in Analysis: Extraction of active compound  Purification  Identification  Quantitation 
Interpretation / Conclusion.
Objectives of forensic toxicology:
 To establish the presence of toxicant (should know the source, entry of poison, which part is exposed, etc.).
 To utilise the different examination techniques (preliminary, chemical & instrumental) for identification of
toxicants.
 To determine what adverse effects these substances have (effects, signs, symptoms – Toxicokinetic).
 To encompass the measurement of alcohol, drugs, and other toxic substances in different matrices along with its
interpretation.

3. EXTRACTION METHODS:
What does extraction means? - “Extraction is nothing but a process where we perform an experiment and try to
completely take out the compound of our interest from the provided matrices.”
Can be mechanical or chemical extraction
What is matrix? - “Matrix is any material substance wherein the active compound of our interest may be dispersed,
accumulated, left, absorbed or chemically bound.”
Now matrices can be: Biological matrix or non-biological matrix

4. HOW TO SELECT THE EXTRACTION METHOD:
Controlling factors:
• Nature of poison, matrix or matrices
• Quantity of samples, analytical requirements, etc.
Precautions while extraction:
1. To avoid loss of active compound while extracting from the sample, it should be processed in
minimum steps.
2. To avoid interference of matrix with the active compound, proper purification of the same is required.
3. To be efficient enough while extraction and purification because it precisely and accurately determines
lower limit of detection.
4. Every biological matrices either be viscera (properly minced), stomach contents, vomit, urine, etc. are
to be examined separately.

5. EXTRACTION OF ACIDS & ALKALIS:
• By Distillation Process: (steam distillation)
o 50-100 g of sample  made consistent with 3-5 times of water and acidifying with dilute
H2SO4.
o Compounds with different boiling points can be separated using this process.
o Place the porcelain pieces in the flask to avoid bumping.
o The distillate getting collected will consists of acidic toxicants like alcohols, acetone, phenol
and derivatives, oxalic acid, etc.
o After, completion of acid distillation, flask with remaining contents is cooled and rendered
alkaline by adding NaOH solution.
o Again it is distilled in same way as stated above.
o Now the distillate consists of basic toxicants like ammonia, aniline, nicotine, etc.

6. Image Ref. https://www.google.com/url?sa=i&url=https%3A%2F%2Fmakezine.com%2Farticle%2Fhome%2Ffood-
beverage%2Fsecret-santa-john-edgar-park-all-glass-steam-still%

7. EXTRACTION OF ACIDS & ALKALIS:
• By Dialysis:
o Approx. 10 g of sample in bag shaped cellophane membrane.
o Place it in beaker of 100 ml of distilled water and slowly rotate with electrical motor or mechanical
device.
o Let it rotate for an hour and replace the water in the beaker with the fresh one and repeat the procedure
until equilibrium is attained.
o Mix these two fractions of water, evaporate to a small concentration on water bath, filter if
necessary. Filtrate is ready for further identification tests.
o Effective for extraction of toxic anions like sulphates, chlorides, fluorides, and toxic alkalis
like hydroxides of sodium and potassium. Also useful for Non-metallic poisons like Cl, I, Br,
P, etc.
Image ref. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.youtube.com

8. EXTRACTION OF ACIDS & ALKALIS:
• By Solvent Extraction (liquid-liquid extraction):
Image ref. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.chromtech.com%2Fliquid-
liquid-extraction-and-supported-liquid-extraction

9. IDENTIFICATION USING COLOUR TESTS:
• For acids:
1. Sulphuric Acid –
reaction with Barium nitrate –
produces a white ppt of barium sulphate, insoluble in water.
reaction with copper filings, mercury or chips of wood –
sulphurous acid gas is evolved.
2. Oxalic Acid –
reaction with Barium nitrate –
gives white ppt of barium oxalate, soluble in strong acids without
any effervescence and dilute acetic acid.
reaction with lead acetate –
gives white ppt of lead oxalate, soluble in nitric acid.

10. IDENTIFICATION USING COLOUR TESTS:
• For alkalis:
1. Ammonia–
Nessler’s reagent K2HgI4 having excess potash gives brown
precipitate, if ammonia is present.
2. Potassium Hydroxide –
reaction with tartaric acid – gives white crystalline precipitate
of potassium tartrate.
Quantification:
By Volumetric analysis using a solution of known concentration,
To estimate the unknown concentration of solution in presence of
Indicator.

11. EXTRACTION OF METALLIC POISONS:
• By Dry Ashing method:
Image ref. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.sciencedirect.com

12. EXTRACTION OF METALLIC POISONS:
• By Wet Digestion:
Image ref. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.youtube.com

13. EXTRACTION OF METALLIC POISONS:
• By Microwave Digestion:
Image ref. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.cannabissciencetech.com%2Fview%2Fselecting-
microwave-digestion-technology-measuring-heavy-metals-cannabis-products

14. IDENTIFICATION OF METALLIC POISONS:
• Reinsch’s Test:

15. Image ref. https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.flickriver.com

16. Crystals of Arsenic under microscope
Crystals of Mercury under microscope

17. IDENTIFICATION OF METALLIC POISONS:
• Lead -

18. IDENTIFICATION OF METALLIC POISONS:
• Gutzeit test-
Image ref.
https://www.google.com/url?sa=i&url=https
%3A%2F%2Fwww.youtube.com

19. IDENTIFICATION OF NON-METALLIC POISONS:
• Chlorine -
Image ref. https://www.google.com/url?sa=i&url=https%3A%2F%2Fin.pinterest.com

20. IDENTIFICATION OF NON-METALLIC POISONS:
• Iodine -
Image ref. https://www.google.com/url?sa=i&url=https%3A%2F%2Fbeautyofscience.pivotshare.com

21. Limitations in toxicological analysis:
 Key points –
 Not handling sample properly.
 Contamination of samples.
 On-time delivery of samples not taking place.
 Lack of knowledge of investigator.
 Manipulation due to high profile cases.
 Lack of analytical tools and techniques.
 Factors affecting stability of poisons in matrix : light, temperature, oxidation-
reduction, pH, enzymes.

22. Troubleshooting in toxicological analysis:

23. Disposal of unused samples toxicological analysis:
Biological waste- Certain biological waste can be disposed by
decontamination by autoclave, chemical disinfection or other appropriate
decontamination methods and then can go as regular trash. If not treated with
autoclave, the waste is collected in orange/red autoclave bags and closed with tape
and placed in the box-bag unit as untreated biological waste. The environmental
health and safety will pick up all box-bag units on, at least a weekly basis.
 If the solutions whether acidic or basic are to be discarded, not to spilt them
directly in the basins but they are to be firstly by neutralising it and then
discarding it. also disinfectant might be used if necessary.

24. Fatal poisoning by ingestion of a self prepared oleander
leaf infusion :
 An unusual case of poisoning by the ingestion of oleander leaves is reported. A 71 year old male laboratory
technician committed suicide at home in this unusual manner. At the death scene a steel pan and other
paraphernalia, used for the extraction of oleandrin and other cardiac glycosides from the leaves of the Nerium
oleander plant were found.

26. References:
 Extraction of poison from biological matrices
https://epgp.inflibnet.ac.in/Home/ViewSubject?catid=eCJfy23Kjy3c0vICLa6VYg==
 Tests for detection of poisons
https://epgp.inflibnet.ac.in/Home/ViewSubject?catid=eCJfy23Kjy3c0vICLa6VYg==
 A Textbook of Medical Jurisprudence and Toxicology by Rai Bahadur Jaising P. Modi, I..R.C.P. & S. (EDIN),
I.R.F.P.S (GLASGOW) – sixth editions
 The importance of microwave digestion for trace metal analysis | lab manager
https://www.labmanager.com/product-focus/the-importance-of-microwave-digestion-for-trace-metal-analysis-
27845
 Biological Waste Guide | Environmental Health and Safety https://ehs.uconn.edu/regulated-waste-
management/biological-waste-guide/#
 Problems with analysis of biological matrices https://www.slideshare.net/SanathoibaSingha/problems-with-
analysis-of-biological-matrices#
 Fatal poisoning by ingestion of Self-prepared oleander leaf infusion
https://link.springer.com/article/10.1007/s12024-020-00338-w

27. Beyond the limit, everything is toxic!
Thank you!