Sample collection, storage and preparation Flashcards
what are 8 sample collection considerations?
ease of collection
matrix interferences
parent drug and/or metabolites
detection/analysis time
stability of the drugs in the sample
putrefaction
potential for automation analysis
reference data
post mortem forensic toxicology
Establish the cause and mode of intoxication/death through the analysis of various fluids and tissues during autopsy
sample collection post mortem (9)
blood urine vitreous humour liver gastric contents bile hair lung fluid kidney
ante-mortem forensic toxicology - human performance
Evaluating the role of a compound in the modification of human behaviour, usually applied to traffic safety and the respective operation of a motor vehicle, as well as doping in sport.
ante-mortem forensic toxicology - xenobiotics testing
Establish prior use or abuse of selected compounds through the analysis of body fluids usually urine. Results from these tests are usually applied to the workplace setting.
sample collection ante-mortem
blood exhaled air urine hair saliva
blood sample collection time?
28 hours ish
saliva sample collection time?
a few days
urine sample collection time?
from a couple hours to days
weeks for marijuana
hair sample collection time
from a couple weeks to months/years
sample collection urine
non invasive
collected in post mortem investigations since some toxins show in higher levels in urine
Sample is checked for adulteration by checking the pH, creatine, specific gravity and for any unusual colour or smell
Poor correlation between drug concentrations in urine and drug effects
sample collection saliva
Non invasive
Easy to collect
Simple matrix – little interference
Indicative of recent drug use
Many different drugs can be determined
sample collection blood
The most satisfactory method for obtaining samples is from venous puncture of the femoral vein
Invasive
Post-mortem blood specimen are taken from two sites
- Heart
- Peripheral (femoral vein)
High correlation between blood drug concentration and the effects of the drug
Whole blood, plasma and/or serum
Dried blood-spots (DBS)
sample collection hair
Preferably collected from the back of the skull where the average hair growth is fairly constant
In cases with a suspicion of a recent poisoning, analyses of plucked hair may be better
Interval for most drugs during which blood, urine and cut hair may all be negative
Drugs only present in extremely low concentrations
Controversy between active and passive drug use
Good timeline for drug usage
what are the 4 routes of entry for drugs in hair
During formation of shaft (anagen phase)
Diffusion from blood stream
Diffusion from secretions
External examination
sample collection bile
Can be useful where morphine, benzodiazepines and chlorpromazine are suspected toxins
These toxins are concentrated by the liver and excreted into the gall bladder
Direct collection of bile into a bottle is advised because bile is too viscous to be drawn by a needle
sample collection gastric content
Typically done in a sudden death in which the deceased has large quantities of a lethal agent in the stomach
Contents should be empties into a wide mouth jar
In the case of suicide, large amounts of pills can be found in the gastric tract
sample collection brain
Useful to assess the impact on the overall body burden
Can establish dose of cocaine in body at time of death
Complex matrix that requires extensive sample clean-up and preparation
2 advantages of blood
detect parent compound
correlation between amount of drug and blood concentration
4 disadvantages of blood
limited volume
low concentrations of basic drugs and some other poisons
complex matriculates - interferences
invasive method
4 advantages of urine
often large volume
high concs of many poisons
simpler matrix than blood
non-invasive method
2 disadvantages of urine
parent drug might be present in low concentration
no or little correlation between amount
advantage of gastric contents
may contain large amounts of poison (if ingested)
2 disadvantages in gastric components
variable sample
no use if inhaled or injected
advantage of hair
usually available even if decomposition advanced
2 disadvantages of hair
high sensitivity needed
only gives exposure data for the weeks/months before death
4 advantages of saliva
often large volumes
high concentrations of many poisons
simpler matrix than blood
non-invasive
2 disadvantages of saliva
parent drug might be present in low concentration
no or little correlation between amount
2 advantages of liver
parent drug and metabolites can be found
high concentrations in comparison to other tissues
disadvantage of liver
extensive sample prep required
advantage of bile
useful for morphine, benzodiazepines and chlorpromazine
disadvantage of bile
difficult to sample
3 advantages of brain
useful for assessing the overall body burden
unaffected by trauma to abdomen and chest
establish cocaine dose
2 disadvantages of brain
extensive sample prep required
little inartistic significance
sample storage considerations
Stability in simple matrix
Preservations in test tube to prevent putrefaction of blood
Tissue stored same as blood but no preservatives in container
Volatiles need to be stored properly (e.g. sealed, cooled container)
what should samples not be stored in
plastic
what are the two reasons why samples need to be stored properly before analysis
to prevent analyte degradation
to prevent analyte formation
sample storage HCN
formed when certain fuels burn
asphyxiant gas
- prevents chemical respiration
- suffocates even though one can breath
highly unstable in blood
- degrades rapidly
- whole blood needs to be frozen
why do we undertake sample preparation
detection techniques are often not responsive to the analyte in the form its present in the sample
results may also be distorted by interfering compounds
what may sample prep involve
dissolution extraction reacting with another chemical species filtering dilution
matrix effects
matrix are the components of a sample other than the target analyses
matrix can severely affect the quality of the results
dilute and shoot
sample prep
no sample prep - just dilution
used for simple sample matrixs
fast and simple
too crude
headspace GC
sample prep
no sample prerp- just heating
used for liquid or solid matrixs
- analyte needs to be more volatile than matrix
not suitable for thermally instable compounds
too crude
precipitation
Used for blood sampled and other protein-rich samples
Simple method for removing proteins
- Better than heating or cooling sample due to higher efficiency
Something is added to the sample to make the proteins precipitate
- Salts – sulphates or ammonium
- Organic solvent
Precipitated proteins are centrifuged
- Supernatant then collected
Liquid-liquid extraction
One of the most widely employed and useful technique
Used for simple matrices such as urine or serum and plasma
Simple and straightforward technique, also reasonably effective
At least 2 phases of liquids
- Selective partitioning of analytes versus contaminants between the 2 phases
Immiscible solvents are mixed – one containing the analyte
The 2 phases are agitated, by vortexing or shaking, to bring about substantial physical mixing
After agitation, the phases are allowed to separate
The phase containing the analytes is removed either by careful pipetting or by “freeze-pour”
- Placing the samples in a freezer to freeze the aqueous layer, after which the organic layer may be poured off
Example: hexane and acetonitrile for barbiturates in serum
- Polar drugs end up in the acetonitrile
- Lipids from serum will partition in the hexane
Could add buffers or pH modifiers (acids, bases)
Low extraction efficiency
solid phase extraction
Perhaps the most powerful sample prep technique
- High selectivity
- Flexibility
- High automation potential
Chromatographic sorbent in a column format
Available in variety of format to accommodate different sample sizes and applications
SPE column
the extraction device used to execute the SPE protocol. Also called cartridge or extraction plate
sorbent
packing used to implement the SPE procedure. also called stationary phase
matrix
Liquid present in the SPE sorbent bed at any time. May be either a protocol solvent or the sample itself
retention
Occurs when the analytes are attracted to and held by the active chemistry within the sorbent bed
elution
Disruption of the attractive interaction between the analytes and the sorbent bed, resulting in the analytes being released from the sorbent and out of the column
breakthrough
Analyte passing through the SPE column unretained during sample application, especially when the desired result is retention
capacity
mass of retained compounds that may be held by given mass of sorbent
SPE
a sample is passed through the column bed and analytes are retained
SPE cartridge is washed to remove interferences, and the purified analytes subsequently eluted from the column
- Sample matrix liquid passes through
May also be used to retain interferences, allowing analytes to pass unretained through the sorbent bed
SPE 4 steps
conditioning
sample addition
washing
elution
SPE advantages
high output
only small amount of sample needed
several different sorbents avaliable - high specificity
commercial kits available
can be automated
solid phase micro-extraction
Fibre coated in adsorbent
- Polymer (liquid)
- Sorbent (solid)
Can be used on liquid or gas phase sample
Fibre is then inserted into the injection port of the GC
- Heating causes analytes to desorb and enter GC column
Fast and simple
In most cases, no solvent needed
