Sample collection, storage and preparation

Question 1

what could samples be in analytical toxicology

Answer 1

body fluids
drugs
sediment and water samples for ecotoxicology

Question 2

body fluids should be treated as what

Answer 2

highly contagious

Question 3

timing of sample and selection should be what

Answer 3

timing of sample should be critical due to degradation and selection of appropriate specimens is very important

Question 4

considerations in sample collection

Answer 4

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

Question 5

3 distinct areas of forensic toxicology

Answer 5

post mortem

ante mortem- human performance

ante mortem xenobiotics testing

Question 6

post mortem

Answer 6

Establish the cause and mode of intoxication/death through the analysis of various fluids and tissues during autopsy

Question 7

sample collection of post mortem

Answer 7

suicides, motor vehicle crashes, industrial accidents- blood, urine, vitreous humour, liver

homicides and or suspicious- blood urine vitreous humour, gastric contents, bile liver, hair

drug related- blood, liver, urine, vitreous humour, gastric contents, bile, liver, hair

volatile substance abuse- blood, urine, vitreous humour, lung fluid, or tied off lung, liver

heavy metal poisoning and exposure to other poisons- blood urine vitreous humour, liver, hair, kidney

Question 8

ante mortem- human performance

Answer 8

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

Question 9

ante mortem xenobiotics testing

Answer 9

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.

Question 10

ante mortem sample collection

Answer 10

motor vehicle offences, doping, workplace testing- blood, exhaled air, urine

heavy metal poisoning and exposure to other poisons- blood urine and hair

drug abuse- blood urine saliva hair

volatile substance abuse- blood urine exhaled air

Question 11

sample collection timeline

Answer 11

blood- within 24hrs
salival- hrs to days
urine- a few hrs to a few days (weeks for marijuana)
hair- weeks to months

Question 12

sample collection urine

Answer 12

• Mostly used for drug screening
• Also collected in post-mortem investigation since some toxins show in higher levels in urine
• Sample is checked for adulteration by checking pH, creatine, specific gravity and for any unusual colour or smell
• Poor correlation between drug concentration in urine and drug effects

Question 13

sample collection saliva

Answer 13

• Often used in drug screening
• Easy to collect
• Simple matrix
• Indicative of recent drug use
• Many different drugs can be determined

Question 14

sample collection in blood

Answer 14

• The most satisfactory method for obtaining samples is from venous puncture of the femoral vein
• Post mortem blood specimen are taken from two sites: heart and peripheral (femoral vein) should collected at every autopsy
• High correlation between blood drug concentration and the effects of the drug
• Whole blood, plasma and or serum
• Dried blood spots (DBS)

Question 15

sample collection hair

Answer 15

• Preferably collected from the back of the skull where the average hair growth is fairly constant
• In cases with 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

Question 16

four routes of entry for drugs

Answer 16

• During formation of shaft (anagen phase)
• Diffusion from blood stream
• Diffusion from secretions
• External contamination

Question 17

sample collections bile

Answer 17

• 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

Question 18

sample collection gastric content

Answer 18

• Typically done in a sudden death in which the deceased has large quantities of a lethal agent in the stomach
  • Contents should be emptied into a wide mouth jar
  • In the case of suicide, large amounts of pills can be found in the gastric tract

Question 19

sample collection brain

Answer 19

• 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

Question 20

blood vs urine

Answer 20

ADVANTAGES
Blood- detect parent compound - correlation between amount of drug and blood conc.

urine- often large volume - high conc of many poisons - simpler matrix than blood - non-invasive method

DISADVANTAGES
blood- limited volume - low conc of basic drugs and some other poisons - complex matrix interferences - invasive method

urine- parent drug might be present in low conc - no or little correlation between amount

Question 21

Gastric vs Hair vs Saliva

Answer 21

ADVANTAGES
Gastric- may contain large amounts of poison

Hair- usually available even if decomposition advanced

Saliva - often large volume - high conc. of many poisons - simpler matrix than blood - non-invasive method

DISADVANTAGES
Gastric- if available, variable sample - no use if inhaled or injected

Hair- high sensitivity needed - only gives exposure data for the weeks/months before death

saliva- parent drug might be present in low conc - no or little correlation between amount

Question 22

liver vs bile vs brain

Answer 22

ADVANTAGES
liver- parent drug and metabolites can be found - high conc. in comparison to other tissues

bile- useful for morphine, benzodiazepines and chlorpromazine

brain- useful for assessing the overall body burden - unaffected by trauma to abdomen and chest - establish cocaine dose

DISADVANTAGES
liver- extensive sample preparation required

bile- difficult to sample

brain- extensive sample preparation required - little intrinsic significance

Question 23

sample storage considerations

Answer 23

• Stability in sample matrix
• Preservatives in test tube to prevent putrefaction of blood
• Tissue stored same as blood but no preservatives in container
• Volatiles need to be stored properly

Question 24

sample storage HCN

Answer 24

• HCN was the major killer during a fire in 1998
• HCN is formed when certain fuels burn
• HCN is an asphyxiant gas (suffocates as it prevents the cellular chemical respiration)
• Highly unstable in blood, degrades rapidly
  (Whole blood needs to be frozen, fridge is not cool enough)

Question 25

sample preparation

Answer 25

- Detection techniques are often not responsive to the analyte in the form its present in the sample - The results may also be distorted by interfering compounds – matrix effects Sample preparation may involve - Dissolution - Extraction - Reacting with another chemical species - Filtering - Dilution

Question 26

dilute and shoot

Answer 26

``` • No sample preparation – just dilution – Reduce matrix effects - Used for simple sample matrices – E.g. urine - Fast and simple • Sometimes too crude ```

Question 27

Head space and GC

Answer 27

• No Sample preparation – just heating – Reduce matrix effects - Used for liquid or solid matrices – Analyte needs to be more volatile than matrix - Not suitable for thermally instable compounds • Sometimes too crude

Question 28

precipitation

Answer 28

- Used for blood samples 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 (anion) or ammonium (cation) – Organic solvent – e.g. acidic methanol - Precipitated proteins are centrifuged - Supernatant then collected

Question 29

liquid- liquid extraction (LLE)

Answer 29

- 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” (freezes aqueous layer and organic layer can be poured off)\ - Example: hexane and acetonitrile for barbiturates in serum - Could add buffers or pH modifiers (acids, bases) - Low extraction efficiency

Question 30

solid phase extraction (SPE)

Answer 30

• preparation technique commonly used today – High selectivity – Flexibility – High automation potential - Chromatographic sorbent in a column format • Available in a variety of format to accommodate different sample sizes and applications

Question 31

SPE terminology SPE column

Answer 31

– The extraction device used to execute the SPE protocol. Also called cartridge or extraction plate

Question 32

SPE terminology sorbent

Answer 32

– Packing used to implement the SPE procedure. Also called stationary phase.

Question 33

SPE terminology matrix

Answer 33

liquid present in the SPE sorbent bed at any time. May be either a protocol solvent or the sample itself

Question 34

SPE terminology retention

Answer 34

– Occurs when the analytes are attracted to and held by the active chemistry within the sorbent bed

Question 35

SPE terminology elution

Answer 35

– 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

Question 36

SPE terminology breakthrough

Answer 36

– Analyte passing through the SPE column unretained during sample application, especially when the desired result is retention

Question 37

SPE terminology capacity

Answer 37

– Mass of retained compounds that may be held by a given mass of sorbent

Question 38

SPE

Answer 38

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 May also be used to retain interferences, allowing analytes to pass unretained through the sorbent bed

Question 39

SPE steps

Answer 39

conditioning sample addition washing elution

Question 40

SPE advantages

Answer 40

- High output - Only small amount of sample needed - Several different sorbents available- high specificity - Commercial kits available - Can be automated

Question 41

solid phase micro extraction (SPME)

Answer 41

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