Unit 6
Sampling in Analytical Toxicology
Introduction
An analytical sample is a representative part of a material or
substance that is extracted for testing or calibration.
The process of collecting and testing samples is called analytical
sampling, and it is a critical step in analytical chemistry.
Postmortem is an examination of a dead body to determine the cause
of death.
Introduction…
Sampling is a critical step in analytical toxicology, and the
accuracy of the results depends on the quality of the samples
provided.
It involves obtaining a specimen from a body, environment, or
substance to detect, identify, and quantify toxins or poisons.
Sample is a small part or quantity intended to show what the
whole is like.
Laboratory sample is a sample that is collected for analysis in a
laboratory.
Laboratory samples can come from a variety of sources, including:
• Human body: Blood, urine, saliva, sputum, feces, semen, and
other bodily fluids and tissues
• Oil-bearing rocks: Samples from exploratory drilling projects
• Viral swabs: Samples from infectious viruses
Types of Samples
Samples in toxicological studies can be taken from a variety of
sources, depending on the context of the investigation:
a) Biological Samples:
Blood: Commonly used to detect drugs, alcohol, and poisons.
• It can provide information about recent exposure.
Urine: Frequently used for detecting metabolites of drugs and
toxins, particularly useful for chronic or recent exposures.
Hair: Long-term exposure can be assessed through hair analysis,
especially for substances like drugs, heavy metals, or environmental
toxins.
Saliva and Sweat: Used in cases where non-invasive sampling is
desired, such as drug testing.
Tissues and Organs: Post-mortem toxicology involves sampling
tissues (e.g., liver, kidney) to assess the cause of death or poisoning.
b) Environmental Samples:
Air: Sampling for volatile compounds or airborne
pollutants (e.g., pesticides, solvents).
Water: Common for environmental toxicology, such
as detecting chemical contaminants or toxins in
drinking or recreational water.
Soil and Sediments: Used for determining
environmental contamination, including metals,
pesticides, or organic compounds.
c) Food and Consumer Products: Sampling food,
beverages, cosmetics, or pharmaceuticals for contaminants
or adulterants.
Sampling Techniques
The specific technique used will depend on the sample type and what
is being tested.
Common sampling methods include:
a) Forensic and Clinical Sampling:
Venipuncture: For blood collection, it’s important to use proper
technique to avoid hemolysis and contamination.
Urine Collection: Involves techniques to prevent tampering (e.g.,
supervised collection).
Hair Analysis: Requires cutting hair close to the scalp for accurate
representation of long-term exposure.
Post-mortem Sampling: Collecting samples from the deceased
can involve blood, vitreous humor, and tissue specimens to assess
cause of death or poisoning.
b) Environmental Sampling:
Grab Sampling: Single-time point sampling, often used for
measuring volatile compounds or air pollutants.
Composite Sampling: Involves pooling multiple samples from a
specific area over a period to assess average contamination levels.
Passive Sampling: Uses a device to capture pollutants over a
period, which is ideal for assessing low-level contaminants.
Handling and Preservation: are essential to maintain the integrity of
a sample, prevent degradation, and avoid contamination.
Factors to consider include:
a) Temperature Control: Some toxins or drugs degrade rapidly, so
samples must be refrigerated or frozen immediately after collection.
Blood: Should be collected in anticoagulant tubes and processed
quickly.
Urine and Hair: Often stored at low temperatures to prevent
bacterial growth or degradation of compounds.
b) Storage Containers: Use containers that are chemically inert to
avoid interaction with the sample.
Glass vs Plastic: Glass containers are preferable for some
substances, as plastic can absorb or adsorb certain analytes.
c) Time Sensitivity: Toxicological analysis is often time-sensitive.
Chain of Custody: Particularly in forensic toxicology, it is critical
to establish a documented chain of custody to maintain the integrity
of the sample and prevent contamination or tampering.
Sample Preparation in Toxicological Analysis
Sample preparation is a crucial step in toxicological analysis as it
directly influences the accuracy, sensitivity, and reliability of
analytical results.
The process involves various steps to extract, purify, and
concentrate the substances of interest from complex biological or
environmental samples.
Toxicological samples often contain a wide range of interfering
substances (e.g., proteins, lipids, salts), so preparation techniques
are employed to isolate the target analytes and minimize
contamination or interference.
Homogenization
Why it's needed: Biological samples like blood, tissues, or
organs can have varying levels of toxins throughout, and solid
tissues (e.g., liver, kidney) often need to be homogenized to
ensure even distribution of analytes before analysis.
How it works: Samples are mechanically broken down using a
homogenizer or a blender to create a uniform solution, making it
easier to extract the analytes.
Extraction
Extraction involves separating the toxic substance or its
metabolites from the biological matrix.
This is one of the most important steps because it allows for the
isolation of analytes from complex matrices (e.g., blood, urine,
tissue, environmental samples) that may contain interfering
compounds.
Common extraction techniques include:
a) Liquid-Liquid Extraction (LLE):
How it works: The sample is mixed with two immiscible solvents
(e.g., water and an organic solvent like chloroform or ethyl
acetate).
The target analytes preferentially partition into the organic layer,
which is then separated and evaporated to concentrate the
analytes.
Advantages: Simple and effective for a wide range of compounds.
Disadvantages: Can be labor-intensive, uses large amounts of
solvents, and may not be suitable for very small concentrations.
Liquid-Liquid Extraction
b) Solid-Phase Extraction (SPE):
How it works: The sample is passed through a column packed with
a solid-phase material (e.g., silica, polymer beads).
The analytes bind to the stationary phase, and then they are eluted
using a solvent that selectively releases the target substances.
Advantages: More efficient and selective than LLE, uses smaller
amounts of solvents, and is more automated.
Disadvantages: Can be expensive and requires specialized
equipment.
c) Solid-Phase Microextraction (SPME):
How it works: A fiber coated with an adsorbent material is exposed
to the sample (usually a liquid or vapor phase).
The target analytes adsorb onto the fiber, which is then analyzed
using techniques like Gas Chromatography-Mass Spectrometry
(GC-MS).
Advantages: Minimal sample handling, no solvents, and can be
used for volatile and semi-volatile compounds.
Disadvantages: Limited to certain types of analytes and requires
highly sensitive instruments.
d) Protein Precipitation:
How it works: In biological samples (e.g., blood, urine), proteins
are a major matrix component.
To separate drugs or toxins from proteins, a precipitating agent
(e.g., methanol, acetonitrile) is added, which causes the proteins to
clump and fall out of solution.
The supernatant containing the analytes is then collected.
Advantages: Simple and quick.
Disadvantages: May not be effective for all types of analytes, and
the precipitation process may not fully remove all proteins,
potentially causing matrix effects.
4. Derivatization
Why it's needed: Some compounds in biological samples may
not be readily detectable or sufficiently stable for analysis by
certain instruments.
Derivatization is a chemical process that modifies analytes to
make them more amenable to detection.
How it works: A chemical reagent is used to react with the target
analyte, forming a derivative that has enhanced properties for
analysis, such as increased volatility, better stability, or higher
sensitivity in detection.
Example: Acylation or alkylation is often used to modify polar
drugs or metabolites, making them more suitable for Gas
Chromatography (GC) analysis.
Advantages: Improves detectability, sensitivity, and separation.
Disadvantages: Adds an additional step in the process, may
introduce errors if not done carefully.
5. Filtration and Centrifugation
Why it's needed: Biological samples often contain particulate
matter, such as cells, fat, or other debris, that can interfere with
analysis.
How it works: Filtration or centrifugation is used to remove solid
components from the sample.
Centrifugation involves spinning the sample at high speeds to
separate the components based on their density, with the heavier
particles (e.g., blood cells or tissue debris) settling at the bottom.
Filtration: The sample is passed through a fine filter to remove
particulate matter.
Centrifugation: Separates heavier particles based on their size
and density, leaving a clear supernatant containing the analytes.
Advantages: Quick and effective.
Disadvantages: Some analytes may be lost during filtration or
centrifugation if they are bound to particulate matter.
6. Concentration and Evaporation
After extraction, the sample is often too diluted for analysis,
especially when the analytes are present in trace amounts.
Evaporation or concentration steps are used to reduce the sample
volume, increasing the concentration of analytes to detectable
levels.
How it works: The sample is evaporated under reduced pressure
(using a rotary evaporator) or by gentle heating, leaving behind a
concentrated residue of the target analytes.
Advantages: Effective for low-concentration samples.
Disadvantages: Loss of volatile compounds can occur if the
process is not carefully controlled.
7. Final Filtration or Clean-up
After the extraction and concentration steps, the sample may still
contain unwanted substances (e.g., lipids, salts, or other matrix
components) that can interfere with analysis. Final clean-up steps
help remove these contaminants.
How it works: The sample may be passed through an additional
solid-phase clean-up step or liquid-liquid extraction to further
purify the analytes.
Advantages: Improves the purity of the sample and reduces matrix
interference.
Disadvantages: Adds additional time and complexity to the
preparation process.
Column chromatography sample clean up
8. Calibration and Internal Standards
Before analysis, the preparation of calibration standards and the
use of internal standards is essential to ensure accurate
quantification of analytes:
Calibration Standards: Known concentrations of the target
analytes are prepared to create a calibration curve that is used to
quantify the analytes in the unknown samples.
Internal Standards: These are compounds similar to the target
analytes but are not present in the sample.
They are added to the sample to compensate for losses during
extraction and analysis.
Quality Assurance in Sampling
Sampling procedures must be rigorously controlled to ensure the
reliability and reproducibility of the results.
This involves:
• Standard Operating Procedures (SOPs): Ensuring consistent
methods of sample collection and handling.
• Blanks and Controls: Including negative and positive controls or
blank samples to detect contamination or interference in the
analysis.
• Calibration of Instruments: Ensuring that any equipment used to
collect or analyze samples is calibrated and functioning properly.
Challenges in Toxicological Sampling
Sampling in toxicology presents several challenges, including:
Matrix Interference: Complex samples (e.g., blood or urine) may
contain interfering substances that make it difficult to detect the toxin
of interest.
Volatile Compounds: For substances that are volatile (e.g., alcohol,
solvents), ensuring proper containment during sample collection is
crucial.
Detection Limits: Some toxins may be present at very low levels,
requiring sensitive equipment and proper sample concentration
techniques.
Legal and Ethical Considerations: Especially in forensic
toxicology, proper legal protocols must be followed to ensure that
sampling is done in compliance with regulations.
Techniques for Analysis Post-Sampling
Once the sample has been collected and preserved, various analytical
techniques can be used to detect and quantify toxic substances,
including:
Chromatography (e.g., Gas Chromatography (GC), Liquid
Chromatography (HPLC)): Used to separate components in the
sample for further analysis.
Mass Spectrometry (MS): Often coupled with chromatography to
identify the molecular structure of the analytes.
Immunoassays: These may be used for detecting specific toxins
or drugs (e.g., ELISA for drug screening).
Spectrophotometry: Used for detecting certain chemicals,
especially metals or ions.
Example of a Sampling Process in Analytical Toxicology
Scenario:
Investigating a Possible Drug Overdose
• A forensic toxicologist is tasked with investigating a suspected
drug overdose in an individual found unconscious in a public
place.
• The toxicologist needs to collect and analyze biological samples
to confirm the cause of intoxication and identify any substances
that may have contributed to the overdose.
1. Initial Assessment and Sampling Plan
The forensic toxicologist reviews the case details and prepares for
sampling.
They decide to collect the following samples:
Blood: To assess the presence of drugs, alcohol, or poisons and
determine the concentration of those substances.
Urine: To identify drugs or metabolites that might be excreted in
the urine.
Vitreous Humor (Post-mortem): If the individual is deceased,
vitreous humor is often analyzed as it contains high concentrations
of substances at the time of death.
2. Sample Collection
Blood Collection:
The toxicologist chooses a clean venipuncture site on the patient (e.g.,
the antecubital fossa) and uses sterile techniques to withdraw blood
into appropriate tubes (e.g., EDTA or heparin tubes for whole blood).
The blood is then labeled with the date and time of collection, and
it is transported immediately to the lab under refrigerated
conditions to prevent degradation of volatile substances.
Urine Collection:
A urine sample is collected in a sterile container, following the
appropriate protocol to ensure the sample is not tampered with.
The urine is also labeled and stored in a cool, dark place to
minimize breakdown of any analytes (e.g., drug metabolites).
Vitreous Humor (Post-mortem):
If the individual has passed away, a post-mortem sample is obtained,
ideally by a medical examiner or forensic pathologist.
Vitreous humor is collected from the eye and placed into a sterile
container for analysis.
This sample is also labeled and stored appropriately to ensure it
can be tested without degradation.
3. Sample Handling and Preservation
The blood and urine samples are immediately placed on ice or
refrigerated to preserve the integrity of the substances in the
samples.
Volatile drugs, such as alcohol, are particularly sensitive to
temperature and will degrade quickly if not handled properly.
The vitreous humor is stored in sealed containers to prevent
contamination, and it is refrigerated if needed.
4. Analysis in the Lab
Once the samples are received at the laboratory, they undergo a series
of analytical tests to identify and quantify any toxic substances:
Blood Analysis:
The toxicologist begins by screening the blood sample using Gas
Chromatography-Mass Spectrometry (GC-MS) to identify and
quantify any drugs, alcohol, or poisons present.
Common substances tested for include opioids (e.g., heroin,
fentanyl), benzodiazepines, alcohol, or stimulants (e.g., cocaine,
methamphetamine).
Urine Analysis:
Urine is tested using Liquid Chromatography-Mass Spectrometry
(LC-MS) to identify drug metabolites.
The toxicologist may look for traces of illicit drugs, prescription
medications, or common over-the-counter substances.
Post-mortem Analysis:
The vitreous humor sample is analyzed similarly, often focusing on
substances that are retained longer in vitreous humor post-mortem
(e.g., alcohol or certain drugs) that can help determine the cause of
death.
5. Reporting Results
After completing the analysis, the toxicologist interprets the
results, taking into account the concentrations of identified
substances in the blood, urine, and vitreous humor.
If fentanyl was detected at a high concentration in the blood, it
could explain the overdose and help establish the cause of death
in a forensic case.
The urine analysis might show metabolites of heroin, which could
further support the overdose scenario.
The vitreous humor could confirm the concentration of alcohol in
the deceased's system, helping to corroborate or challenge the
initial forensic findings.
The toxicologist will prepare a report that outlines the substances
detected, their concentrations, and how they may relate to the cause
of death or the specific nature of the overdose.
