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Flashcards in Sample collection, storage and preparation Deck (41):
1

what could samples be in analytical toxicology

body fluids
drugs
sediment and water samples for ecotoxicology

2

body fluids should be treated as what

highly contagious

3

timing of sample and selection should be what

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

4

considerations in 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

5

3 distinct areas of forensic toxicology

post mortem

ante mortem- human performance

ante mortem xenobiotics testing

6

post mortem

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

7

sample collection of post mortem

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

8

ante mortem- 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

9

ante mortem 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.

10

ante mortem sample collection

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

11

sample collection timeline

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

12

sample collection urine

- 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

13

sample collection saliva

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

14

sample collection in blood

- 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)

15

sample collection hair

- 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

16

four routes of entry for drugs

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

17

sample collections 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

18

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 emptied into a wide mouth jar
• In the case of suicide, large amounts of pills can be found in the gastric tract

19

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

20

blood vs urine

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

21

Gastric vs Hair vs Saliva

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

22

liver vs bile vs brain

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

23

sample storage considerations

- 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

24

sample storage HCN

- 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)

25

sample preparation

- 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

26

dilute and shoot

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

27

Head space and GC

• 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

28

precipitation

- 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

29

liquid- liquid extraction (LLE)

- 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

30

solid phase extraction (SPE)

• 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

31

SPE terminology SPE column

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

32

SPE terminology sorbent

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

33

SPE terminology matrix

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

34

SPE terminology retention

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

35

SPE terminology 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

36

SPE terminology breakthrough

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

37

SPE terminology capacity

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

38

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

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

39

SPE steps

conditioning

sample addition

washing

elution

40

SPE advantages

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

41

solid phase micro extraction (SPME)

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