Forensic Decomposition Stages

Properties of Poisons

History of Arsenic Poisoning

• Many ways arsenic disrupts biochemical functions include oxidative stress, mimicking phosphorus, and enzyme inhibition.
• The Mary Blandy case was the first to utilize chemical testing where arsenic was used to poison her father, who disapproved of her marriage.
• Mathieu Orfila, known as the "Father of Toxicology," helped develop tests for detecting blood in a forensic context and was among the first to use a microscope to assess blood and semen stains.
• He was also famous for his use and understanding of the Marsh test.

Marsh Test

• A test for arsenic poisoning where a suspected sample of arsenic is mixed with H₂SO₄ and Zn, which produces a bubbling gas.
• It is allowed to build up and then released through ignition by a flame proximal to a porcelain dish.
• Arsnic is present if metal deposits form on the dish.
• Zinc reduces As³⁺ to As³⁻
• Arsenic protonated in an acid forms arsine gas, which thermally decomposes to produce As(g).

Toxicity

• Paracelsus stated that "all things are poison, and nothing is without poison; only the dose permits something not to be poisonous."
• Concerns focus on relatively benign substances of pollutants in food, water, and air rather than lethal quantities.
• Contemporary toxicology focuses on the global spread of contaminants and chemicals, increasing chemical contaminant diversity, and complications presented by subtle yet enduring toxic responses.

Chemical Exposure

• When a chemical (toxic or benign) contacts a biological organism, the contact is defined as an exposure.
• A dose is when a chemical is administered medically.

Toxicokinetics

• The movement of an exogenous chemical from absorption through distribution to its final disposition via metabolism and excretion.
• It relies on quantifying the time course of the toxicant in the body.
• It reflects how the body handles toxicants as indicated by the plasma concentration at various time points.
• The end result is a biologically effective dose of the toxicant, which is the amount of absorbed compound reaching targets or sites of action to cause a biologic effect.
• Effects are a result of the interaction of the biologically effective dose of the ultimate active form of the toxicant with a molecular target in the body.

Steps of Toxicokinetics

• Absorption: Entry of toxicants through external membrane barriers into circulation.
• Distribution: Movement of toxicants through circulatory fluids to specific organs and tissues within the body. Some prodrugs can be bioactivated here.
• Metabolism: Biochemical processes that convert the original (parent) toxicant to various metabolites.
• Elimination: Removal of the toxicant or metabolites from the body.

Toxicodynamics

• Adverse effects produced by the xenobiotic and the mechanism by which they are produced.
• Glycoalkaloids and organophosphates (etc.) inhibit acetylcholinesterase, resulting in the accumulation of acetylcholine.
• It causes constructive propagation of nerve signals.
• AcH does not broken down and it disrupts call membranes by binding to cholesterols

Toxicological Process

• Toxicokinetics -> metabolism -> initiation -> toxicodynamics

Toxicity Determinants

• Largely dictated by a substance's physiochemical properties, including lipophilicity, acid-base properties, and size, which determine if a molecule can diffuse across all membranes and reach its target.
• A route of exposure can also have a profound effect on toxicity.

Lipophilicity

• Partition coefficient (Kp, Kow): relative lipophilicity vs. hydrophilicity.
• Kow = [organic phase] / [aqueous phase]. Measured via molar concentration in a 2-phase solvent mixture at equilibrium.
• Log Kow is proportional to lipophilicity; a larger Log Kow indicates a more lipophilic substance.
• Weak organic acids are mostly neutral at pH < pKa. Weak organic bases are mostly neutral at pH > pKa.

Hydrophilic Toxins

• Lipophilic substances can permeate cell membranes easily; some hydrophilic substances cross membranes through different mechanisms.
• Some metal ions pass through protein channels intended for other metals--molecular mimicry.
• Lead enters through calcium channels, and mercury binds to sulfur on cysteine to mimic methionine.
• Hydrophilic substances absorb poorly dermally and generally need to be ingested or inhaled.

Dose-Response Relationship

• Tests the capacity of a chemical to produce a discrete endpoint in a population.
• Focuses on the rate in a population rather than individual responses.
• Produces a sigmoidal curve.

Threshold Concentration

• Can be determined by a dose-response relationship.
• It is the concentration at which the probability of an adverse impact is low enough to be deemed acceptable.
• A threshold dose is a mathematical construct derived from NOAEL values determined from laboratory experiments on animals.

Exposure Limits

• Steps for setting exposure limits:
  • Identify critical effects (NOAEL).
  • Calculate the threshold for critical effects.
  • Apply uncertainty factors to account for inter-species and inter-individual differences.
  • Exposure limit equates to NOAEL divided by uncertainty factors UF₁ x UF₂.

Fat Soluble Compounds

• Fat-soluble compounds, like vitamin A compounds, are hard to excrete.
  • Deficiency leads to impaired vision.
  • High levels can lead to death and organ development issues.

Risk

• Risk depends on chemical concentration, route of exposure, and inherent toxicity.
• LD₅₀: A dose that causes death in 50% of the population. It cannot alone predict what is deadliest, as the probability of exposure also matters.

TCDD Poisoning

• Polyhalogenated organic compounds are significant environmental pollutants.
• Viktor Yushchenko was suspected of acute TCDD poisoning, which caused chloracne and scarring on his face; his levels were 50,000-fold higher than the general population.

Molecules of Murder

• When tied to a crime, one must use analytical techniques for detection and quantification.
• They must be strategically chosen and answer legal relevance questions in a timely manner.

Individualization

• Paul Kirk defined "criminalistics" as the science of individualization.
• Chemical identification serves to establish the structure of an unknown compound. Forensics seeks to link chemical traces to a given person, forming a chemical fingerprint.
• Mixture composition and impurity profile can help forensic chemists individualize a sample.
• Isotope ratio MS can be employed if batch samples are very pure; it looks at characteristic variation on a molecular level.
• Individualization fallacy is when a match doesn't mean the suspected object/person is the donor. There is also a random match probability.

Detection and Quantitation and Chemistry

• Identification, separation, and quantification of chemical evidence can help answer questions of interest to a legal system.
• Assistance in investigation and prosecution of criminals or absolve an innocent person from suspicion while being paramount in the resolution of complex criminal cases.

Qualitative Chemical Analysis

• Provides confirmation on the presence of the chemical in a sample, such as colorimetric tests like sodium rhodizonate (Pb, Ba).
• It generally does not provide useful information on the quantity of the chemical.

Quantitative Chemical Analysis

• Provides information on the amount of analyte in the sample.
• It can be performed using a wide variety of methods and instrumentation.

Chromatography

• Chromatography was founded by Michael Tswett in 1906. Forensic samples are predominantly complex mixtures that require separation.
• It enables the separation, identification, and purification of the mixture's components for qualitative and quantitative analysis.

Gas Chromatography

• A chromatographic technique where volatile compounds are separated; samples are vaporized without thermal decomposition.
• Retention is where smaller, more volatile compounds elute first.
• Higher BP = Stronger IMFs result in a longer tr.
• Lower BP = Weaker IMFs result in a shorter tr.
• Peaks in a chromatogram must be resolved sufficiently for identification and quantitation.
• Factors affecting resolution: column parameters, selectivity (SP), and flow rate.
• Advantages include high resolution, low LODs, speed, accuracy, and reproducibility.
• Disadvantages include that it is limited to volatile compounds, often has decomposition, is destructive, incompatible with aqueous samples, and requires extensive prep.

High-Performance Liquid Chromatography

• A technique where solubilized compounds are separated and analyzed.
• Isocratic elution involves a constant solvent composition.
• Gradient elution involves solvent composition changes, which can increase resolution and run efficiency.
• Normal phase: non-polar MP, polar SP
• Reverse phase: polar MP, non-polar SP, which is the method of choice for pharmaceuticals.
• Under the same conditions, an analyte has a characteristic tr to aid that can aid in identification.
• Factors affecting HPLC resolution: to, column parameters, particle size, SP, MP, and flow rate.
• Advantages include retention time performance, ability to use non-volatile cmpds, flexible mobile phase, and less sample prep - but they disadvantages include that EXPENSIVE, slower, incompatible with solvent analytes, and is laborious
• Mass Spectrometry is used to identify and quantify a wide spectrum of analytes.

Mass Spectrometry (MS)

• Measures mass-to-charge ratio (m/z).
• Gold Standard = ChromatographyMS. Offers enhanced selectivity as it conveys information about the molecular weight (MW) of the analytes.
• While two analytes may share retention time, it is unlikely for two analytes to share both tr and MW.
• Ionization techniques are soft or hard:
  • Soft ionization preserves the parent peak (no fragmentation).
  • Hard ionization fragments, which is important for analyzing analytes with the same MW (ions accelerated to have same Ex).
• This allows the analyzer to start separating based on m/z.
• Deflection: A magnetic field will deflect ions based on their mass and charge.
• Detection is where a detector quantifies ions and produces a signal.

Peak Types in MS

• Base peak: most abundant ion to which the spec. is normalized.
• Molecular ion peaks corresponds to MW.
• Daughter ions: From fragmentation.
• Tandem MS involves multiple MS connecting to one another or a single MS through multiple analyzers in series.
• Initial steps involve isolating the parent ion which subjected to fragmentation and producing product ions.
• This greatly enhances selectivity and facilitates structural elucidation while mass spectra are compared to those found it spectral libraries by which compounds are identified.

Derivatization

• Many compounds contain polar groups, limiting compatibility with GC/MS.
• Derivatization converts a compound to a product to confer desirable volatilizy characteristics.
• Examples include silylation, acylation, and alkylation which is useful when a parent compound shows poor diagnostic ions for MS.
• Mass spectra with ions of higher m/z ratios and abundance can be obtained, thus increasing specificity.

Internal Standards

• Structurally similar but not identical to the analyte of interest.
• Quantitative MS relies on IS; the identification of the analyte includes peak shape, injection variance, normalization of recovery differences, and normalization of ionization effects.

Spectroscopy

• Relies on an analyte's interaction with light.
• Non-destructive. Specs split energy reflected by the analyte into different λ to produce a spectrum.
• Measures the amount of UV-Vis light absorbed. Absorption maximum (λmax) can characterize a compound and can be compared to reference spectra qualitatively.

IR Spec (Infrared Spectroscopy)

• Irradiates sample to stimulate vibrations, useful for structural info.
• Only vibrations resulting in dipole moment changes produce bands.
  • Symmetrical - no bands.
  • Asymmetrical - w bands, very low resolution.
• Groups absorb at different characteristics wavenumbers
• Instrumentation is inexpensive and portable
• Analysis often performed on the scene
• Can be used quantitatively.

Raman Spectroscopy

• Illuminating w/ laser and measuring the photons that undergo inelastic (Raman) scattering.
• Measures changes in polarizability.
• Allows visualization of homonuclear bonds
• Provides high structural resolution while using quantitative measurements for real- time results.
• More expensive, but can be used on-site and requires little or no prep.

Decomposition

• A complex chemical process that begins immediately after death. Soft tissues decompose into simple organic matter over time.
• Proteins, lipids, sugars, nucleic acids, and bone decompose into a variety of products.
• Decomp. Chemistry can be used in tandem with forensic disciplines to extract information needed to establish a post-moterm interval(PMI).
• Synergistic relationship between entomology and decomp chemistry.
• Chemical studies that observe the fate of remains in humans help locate clandestine graves and estimate post-burial intervals.
• Dr. Vass demonstrated in the 2000s chloroform is formed by post mortem decomp.
• Stages of decomposition:
  1. Fresh: Onset of autolysis and mortis, minimal odor.
  2. Bloat: Onset of putrefaction and gas accumulation.
  3. Active decay: Deflation of body, high rate of dissolution, and a complex odor.
  4. Advanced decay: Reduction in moisture but remains there, tissue and odors are reduced
  5. Dry remains: Hair, cartilage, and bone remain from soil and have a reduced odor.

Decomposition Breakdown

• Breakdown in a controlled situation occurs, with degradation occuring in the body of the subject.
• Generally done with O species, enzymes, micro organisms or bacteria
• A study of samples of fluids or volatiles are collected above human matrix

Types of Decomposition

• Decamp will begin very quickly after death where tissue will undergo stages of decay; preskeletonization is when tissue is present and postskeletonization is after tissue has decayed leaving skeleton.
• In preskeletonized tissues, the first identifiable process of decamp is autolysis.
• High concentrations of ATP is found in high concentrations and no to low 0₂
• Intracellular pH decreases where lysosomes become pyruvate.
• Volatile fatty acids are produce from the breakdown of muscles and fatty lipids.
• Lots of putrescine and cadaverine exists
• Non- chromatographic studies include ninhydrin and ninhydrin testing. Release of nitrogen in a body body to locate graves Ninhydrin mechanism is the nucleophilic addition of mino acid to the keytone, Dimerisation with creates colour

Adipocere

• Adipocere occurs when neutral fats are metabolized and occur in decomposed remains and surround soil
• DRIFT(IR) is specific to identify carbynyl bands with increasing free fatty acid

Static Headspace

• A headspace above samples is removed after the head or evidence is removed by syringe and analysed.
• Analysis is nondestructive, and the sample can be measured using little material and be injected into GC samples

Dynamic Headspace

• Gas is collect and collected using analysis using purges flow
• Purages flow must trap a sorbent, passive concentration will happen if
• Sorbent are more laborers and quantitative during analysis

Virtreous Humor

• Virtious fluid is often use during electrolyte analysis to increase death levels with liner relationships. -Flame photo metry follows this method and UV analysis for simple effective measure

Ion Chromatography

• Similar in function to LCMS, where the SP charge allows to analysis ions
• Analytes are eluted during the runs of samples, and concentrations are analysed and displayed across column in data

Hypoanthine

• Purine based derivates are formed during degridation.
• Compound are known to have liner relation with in levels.

Fatty Acids in relation to decomp

• Short chain FFAs is associated to decomp and in microbial soil samples that are analyzed from derivation and matrix issue

VOC in Decomp

VOC has profiles for gases that emulated during after death due to different decomposition stage. Common VOC during decomp incluce DMDS, Toluene, hexane, 1,2 ,2.4 trimethylbenzene, 2 pronepne, 3 petanone and xylence. Benzene derivatives have most studies, and combined with death to analyse and controls HS are analysed for tube, and lungs analysed and analysed with GC, GCMS

Analysis Using Analytical and sampling methods

• Small scale collection is achieved and can be used for high throughout throughput
• Voc is sample collection can be use 2DGC by colleting sampling and sorbent is added
• SPME

Analysis of collection

• Soil, water, and air are all important analysis samples. Matrix effects and partitioning occur and are analysed PMI process of analysis and consideration
• PMI is the study or decomposition of subject in which animal and human tissues need to be analyzed
• sampling can depend on location of sites, geography and environmental facturs

Animal vs Plant studies

• Plant and animal relationship are key and used during the study of decay and effects through weight ,hair ,muscles
• Plants is often used for smaller sample sizes

Canines as sniffer Technology

1. Sniffer Dogs are used for technology by seeking clandestine bodies for remains depending and analysis using enviro effects. Scientific animal are analysed.

2. The technology can be used to analyze what the level for analysis

Evednce

• Evedice in study of plant matter for for analysis and to reduce to efeects.
• There can be feeding analysis for homgenours and distributed matrix.
• Test are often not palartabe test.

Toxins

• Compounds that toxic, naturally are synthetic dangerous.
• Occuring during reaction of the effects that attack
• Cytotoxin reaction is a cause of failure.
• The cause failure and reaction will give movement of signals
• Serine proyeeses mimics lead to unsatblie effects for plasma.
• Metalloproteinases lead to bleeking. .

Block Mamba

Neuro toxins, cardiootins, and toxic effects are analyzed.

Explore the characteristics, processes, and analytical techniques used in forensic decomposition studies. Understand postmortem changes, microbial activity, and chemical analysis methods. Includes the role of base peaks and analytical techniques.