Extraction, Isolation and Clean-up of Poisons from Viscera
Viscera workup. Stas-Otto, ammonium sulphate, steam distillation, Conway microdiffusion, modern SPE, SPME and QuEChERS, with the Indian SOP.
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Extracting poisons from viscera requires separating target analytes from a complex biological matrix before any instrumental or chemical confirmation can proceed. The classical Stas-Otto procedure achieves this through pH-controlled liquid-liquid partitioning into four fractions: acidic and neutral organics into ether, bases into chloroform, volatiles by distillation, and metals from the digested residue. Modern Indian state forensic science laboratories have largely replaced liquid-liquid extraction with solid-phase extraction (SPE), though steam distillation and Conway microdiffusion remain the methods of choice for volatile poisons. Each extraction route is matched to the physicochemical class of the suspected poison and the matrix being analysed.
Viscera workup is the step that underpins every other analytical decision in forensic toxicology. Whatever the suspected poison, the analyst must first get it out of liver, kidney, stomach and blood before any colour test, GC-MS run or AAS quantification can proceed. Viscera extraction covers two broad generations of method: the classical procedures (Stas-Otto, ammonium sulphate, steam distillation, Conway microdiffusion) and the modern sample-preparation family (SPE, SPME, ASE, QuEChERS, SLE) that has displaced most of them in routine casework.
This topic covers the standard Indian viscera collection kit, the four Stas-Otto fractions, the volatile analytes recovered by steam distillation, the Conway microdiffusion cell, and the modern SPE and QuEChERS workflow.
By the end of this topic you will be able to:
- Describe the four Stas-Otto fractions, the reagents used at each pH step, and the classes of poison recovered in each fraction.
- Explain when steam distillation and Conway microdiffusion are preferred over solvent extraction, and name the volatile analytes each technique recovers.
- Compare SPE, SPME, ASE, QuEChERS and SLE on the basis of sorbent or solvent mechanism, sample volume requirement, and target analyte class.
- State the standard Indian viscera collection kit, the preservative used in each container, and the two exceptions that apply to suspected cyanide and ethanol cases.
- Outline the five-stage SFSL bench workflow from visual examination through confirmatory instrumentation, and identify where quality-control materials are introduced.
- Viscera
- The internal organs collected at autopsy for chemical analysis in suspected poisoning. The Indian standard set is stomach with contents, small intestine, liver and one kidney, packed in two glass jars.
- Stas-Otto procedure
- Classical 1851/1856 acid-base liquid-liquid fractionation. Acidify with tartaric acid and extract acidic and neutral organics into ether, then alkalise and extract bases into chloroform. Yields four fractions used to separate barbiturates, phenols, alkaloids and volatiles.
- Conway microdiffusion cell
- Two-well glass dish. Sample plus releasing reagent in the outer well, trapping reagent in the inner well, sealed lid. Volatile poison (HCN, CO, alcohols) diffuses across the air gap and is captured for colorimetry.
- Steam distillation
- Co-distillation of volatile poisons with water vapour at temperatures below their pure boiling points. Used for ethanol, methanol, phenol, chloroform, nitrobenzene, formaldehyde and (with H2SO4) cyanide.
- SPE
- Solid-phase extraction. Sample is loaded onto a packed sorbent cartridge (C18, mixed-mode MCX/MAX, polymeric Oasis HLB), washed and selectively eluted. The dominant modern clean-up step in Indian SFSL toxicology.
- SPME
- Solid-phase microextraction. A polymer-coated fibre is exposed to headspace or sample, analytes adsorb, the fibre is then thermally desorbed in a GC injector. Ideal for volatiles, no solvent needed.
- QuEChERS
- Quick, Easy, Cheap, Effective, Rugged, Safe. Acetonitrile partitioning with MgSO4 and NaCl, followed by dispersive SPE clean-up using PSA and C18. Originally a pesticide-residue method, now used broadly in forensic toxicology.
- ASE
- Accelerated solvent extraction (Dionex ASE). Pressurised solvent at elevated temperature pushes the analyte off the tissue matrix in minutes, replacing overnight Soxhlet.
- SLE
- Supported liquid extraction. Diatomaceous earth columns absorb the aqueous sample as a thin film and a single pass of organic solvent elutes the analyte. Single-pass replacement for shake-flask liquid-liquid.
Why viscera matter and how they reach the lab
In a suspected poisoning death, the autopsy surgeon collects viscera for chemical examination. Liver concentrates lipophilic drugs and metals, kidney holds water-soluble metabolites awaiting excretion, stomach and intestine retain unabsorbed parent compound, and blood gives the circulating concentration at the moment of death.
Section 194 of the Bharatiya Nagarik Suraksha Sanhita 2023 (BNSS) mandates an inquest in unnatural deaths and requires viscera preservation in suspected poisoning. The collection is logged on Form 21, sealed with the police seal in cloth and wax, and dispatched under the mortuary register and the chain of custodyentry.
The standard kit:Jar 1 holds about 200 g of stomach with its contents, the upper small intestine and about 30 mL of gastric fluid.Jar 2 holds about half of the liver and one whole kidney. Preservative is saturated sodium chloride in both jars.Blood(about 10 mL) goes into a separate tube of sodium fluoride and potassium oxalate; NaF blocks bacterial breakdown of ethanol, critical for the analysis of ethyl alcohol in beverages, blood and breath.
Two exceptions apply.For suspected cyanide NaCl interferes with the colour tests, so viscera go in preservative-free and refrigerated.For suspected ethanol NaF alone is the preservative, since alcohol partitions out of saturated NaCl. The book chapter on post-mortem and viscera collection in poisoningcovers the full Form 21 workflow.
Classical extraction: the Stas-Otto and ammonium sulphate methods
The single most tested classical procedure is the Stas-Otto fractionation published by Jean Servais Stas in 1851 (for the Bocarmé nicotine case) and refined by Friedrich Otto in 1856. The principle is acid-base liquid-liquid partition: weak acids and bases swap between the aqueous and an organic phase depending on pH, so adjusting pH pulls different classes into different fractions.
The viscera are minced, mixed with rectified spirit and a little tartaric acid, warmed to denature proteins, filtered, and the filtrate is evaporated to a syrup. The syrup is taken up in water, acidified with tartaric acid and shaken with diethyl ether. The ether layer carries acidic and neutral organics (Fraction A: barbiturates, salicylates, phenols). The aqueous layer is then alkalised with sodium bicarbonate or sodium carbonate and shaken with chloroform (often chloroform plus isopropanol), which carries the bases (Fraction B: strychnine, morphine, atropine, nicotine, cocaine, quinine). Volatile poisons are recovered separately by distillation (Fraction C); the protein residue is digested for metals (Fraction D).
The ammonium sulphate precipitation method is the protein-stripping cousin. Saturated ammonium sulphate is added to the homogenate to salt out proteins; the supernatant is filtered, and the small-molecule poisons are extracted with an organic solvent at the appropriate pH. Less laborious than full Stas-Otto, useful when viscera are heavily decomposed.
Both methods are slow and solvent-heavy, which is why Indian SFSLs have moved most casework to SPE. The classical procedures still get tested because every Indian textbook (Modi, Parikh, Reddy, Nandy) leans on them, and the chemistry teaches the underlying acid-base reasoning. The book chapter on classical extraction: Stas-Otto and ammonium sulphatewalks through the bench recipes.
Distillation, microdiffusion and dialysis
When the suspected poison is volatile, Stas-Otto is the wrong tool. Three older techniques hold their place.
Steam distillation drives steam through the macerated viscera in an acidified flask; the volatile analyte co-distils with water below its pure boiling point. The distillate is condensed and analysed by colour test, GC-FID or GC-MS. It is the standard recovery method for ethanol, methanol, phenol, chloroform, nitrobenzene and formaldehyde and (with concentrated H2SO4 added to liberate HCN from cyanide salts) for cyanide.
The Conway microdiffusion cell is a small two-well glass dish with a lid. Sample plus releasing reagent in the outer well, trapping reagent in the inner well, lid sealed, cell left at room temperature. The volatile diffuses across the air gap and is captured for colour or titrimetric measurement. Used routinely for carbon monoxide(released by K3Fe(CN)6, trapped in PdCl2),hydrogen cyanide(released by H2SO4, trapped in NaOH, developed by König or picrate), and alcohols(released by warming, trapped in K2Cr2O7). Big advantage over distillation: 0.5 to 1 mL of sample is enough.
Dialysis moves small-molecule poisons across a semipermeable membrane while keeping proteins behind. Slow but gives a clean aqueous extract; used today mainly when SPE cartridges clog on greasy tissue.
For metals the residue from any of these workups goes through dry ashing or wet digestionbefore AAS or ICP-MS quantification. The book chapter on distillation, microdiffusion and dialysis techniquescovers the full chemistry.
Modern clean-up: SPE, SPME, ASE, QuEChERS and SLE
The modern Indian SFSL toxicology bench runs on cartridges and dispersive sorbents, not separating funnels. Five techniques carry almost all current casework.
Solid-phase extraction (SPE)is the workhorse. The aqueous extract is loaded onto a packed sorbent cartridge, the matrix is washed off, and the analyte is eluted with a small volume of selective solvent. Dominant sorbents are C18(reversed-phase, general drugs),mixed-mode MCX(basic drugs: amphetamines, opiates, benzodiazepines),mixed-mode MAX(acidic drugs: barbiturates) and polymeric Oasis HLB(broad-spectrum default for unknown screens).
Solid-phase microextraction (SPME)exposes a polymer-coated fused-silica fibre to the sample or its headspace; analytes adsorb, the fibre is then thermally desorbed in a GC injector. Solvent-free, ideal for volatiles (alcohols, accelerants) off blood or tissue homogenates.
Accelerated solvent extraction (ASE / Dionex)pressurises a solvent above its atmospheric boiling point at 80 to 200 degrees Celsius, pushing the analyte off the matrix in minutes instead of overnight Soxhlet. Used for pesticide residues in fatty tissue.
QuEChERS("quick, easy, cheap, effective, rugged, safe") is the pesticide-residue method that has spread into wider toxicology. Shake tissue with acetonitrile salt out with anhydrous MgSO4 and NaCl to drive analytes into the organic layer, then clean with dispersive SPE using PSA(removes acidic interferences and fatty acids) and C18(removes lipids). Single-tube, minimal-solvent.
Supported liquid extraction (SLE)uses diatomaceous earth columns that hold the aqueous sample as a thin film while organic solvent flows through. Single-pass replacement for shake-flask liquid-liquid, no emulsions. The book chapter on modern extraction: SPE, SPME and accelerated solventcompares them in full.
Bench workflow and quality control at the Indian SFSL
A realistic SFSL viscera workup runs five steps in order.Visual and olfactory exam first: kerosene smell, garlic smell of phosphide, bitter-almond smell of cyanide, and the colour of stomach contents (yellow for picric acid, blue-green for copper sulphate, red for nitric acid) are recorded before any reagent touches the sample.
Second, colour and spot screens narrow the field (Reinsch for heavy metals, Marquis and Mecke for alkaloids, Trinder for salicylates), and an immunoassay panel(ELISA, lateral-flow) gives a rapid yes/no across opiates, amphetamines, benzodiazepines, cannabinoids and cocaine. Third, if any screen fires, the sample is extracted using the appropriate method Stas-Otto for an unknown alkaloid, steam distillation for a suspected volatile, SPE for a known drug class, QuEChERS for pesticides, dry ashing for metals.
Fourth, the extract goes on a confirmatory instrument GC-MS for general drugs and pesticides, LC-MS-MS for thermally labile and polar drugs, AAS or ICP-MS for metals. Fifth, the analyst signs and seals the report.
Quality control is what wins the case in court. Every run carries a blank viscera control(extracted from known clean tissue) to flag contamination, spiked recovery controls to prove extraction efficiency, and deuterated or 13C internal standards added at the extraction step (not at injection) to correct for losses. The Indian institutional anchors examiners like to test are the DFSS Forensic Toxicology SOP (2020 / 2022)CFSL Hyderabad and CFSL ChandigarhAIIMS Forensic Medicine and Toxicology and the state SFSL toxicology divisions. The book chapter on biological, non-biological and viscera matricescatalogues which matrix gives which answer.
What is the standard Indian viscera kit for a suspected poisoning autopsy?
Why is the Stas-Otto procedure still taught when most Indian SFSLs run SPE now?
What does QuEChERS stand for and what is its workflow?
What is a Conway microdiffusion cell used for in forensic toxicology?
Which poisons are recovered by steam distillation from viscera?
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