Analytical Toxicology

Question 1

What are the steps in undertaking an analytical toxicology investigation?

Answer 1

Pre analytical
Analytical
Post analytical

Question 2

What happens during the pre analytical step?

Answer 2

Obtain details of suspected poisoning episode
Obtain the patients medical and occupational history
Decide the priorities for an analysis

Question 3

What happens during the analytical step?

Answer 3

Perform the agreed analysis

Question 4

What happens during the post analytical step?

Answer 4

Interpret the results

Question 5

Questions asked when conducting analytical toxicology?

Answer 5

Which sample should be collected/is relevant?
How should it be interpreted?
Which analytical technique should be chosen?
Qualitative and quantitative analysis - screening tests or target analysis?
Disposition of xenobiotics in the body - look for parent compound or metabolite?

Question 6

What is phase 1 metabolism?

Answer 6

Functionalisation - chemical modification of the original xenobiotic molecule by oxidation, reduction or hydrolysis (may activate the compound)

Question 7

What is phase 2 metabolism?

Answer 7

Conjugation - conjugation reactions, in which a second, hydrophilic molecule such as glucuronic acid is added to the molecule (toxic effects will be lower change structure of molecule)

Question 8

What is phase 3 metabolism?

Answer 8

The products of phase 2 biotransformations may be father metabolised in what is sometimes termed phase 3 reactions

Question 9

What is the point of metabolism?

Answer 9

Makes the molecule more polar so easier to be excreted by urine, half life is shorter

Question 10

What is metabolism catalysed by?

Answer 10

Enzymes

Question 11

Examples of phase 1 metabolism?

Answer 11

Oxidation, reduction, hydrolysis, hydration, dehalogenation

Question 12

Examples of phase 2 metabolism?

Answer 12

Sulphation, glucuronidation, glutathione conjugation, acetylation, amino acid conjugation, methylation

Question 13

What is the organic solvent water partition coefficient P?

Answer 13

P - ratio of the concentration of unionised compound X in organic solvent ( eg octane, chloroform) to the concentration of compound X in water

Question 14

What is P a measure of?

Answer 14

The hydrophobicity and hydrophilicity of chemicals - higher partition to organic solvent higher value of P and higher hydrophobicity for unionised compounds only, most drugs and toxins will be charged at particular conditions

Question 15

What is the organic solvent water partition coefficient D?

Answer 15

D - ratio of the sum of the concentrations of all forms (ionised and unionised) of the compound X in organic solvent (eg octanol, chloroform) in each of the two phases - higher partition organic solvent higher D value higher hydrophobicity

Question 16

What is logD dependent on?

Answer 16

pH dependent, hence one must specific the pH at which the logD was measured
log P = log D at any pH for unionisable compounds

Question 17

What is logKoW?

Answer 17

Octanol water partition coefficient - value changes on pH for charged molecules focus on particular pH, if in non charged state, probability of partitioning into organic state higher

Question 18

Absorption values for high P values?

Answer 18

High absorption happens for higher P values - more hydrophobic compounds easily transported and distribution round the body

Question 19

Characteristics of lipophilic xenobiotic substance?

Answer 19

Rapidly absorbed
Rapidly and widely distributed in body tissues
Extensively metabolised before it is excreted

Question 20

What is the acid dissociation constant Ka?

Answer 20

Equilibrium constant for the following reaction of a weak acid HA, the smaller the value of pKa the stronger the acid

Question 21

What is pKa?

Answer 21

pKa is defined as the pH where chemical exists as 50% ionised and 50% unionised

Question 22

When are acids most highly ionised?

Answer 22

Acids are most highly ionised at high pH (pH > pKa)

Question 23

When are bases most highly ionised?

Answer 23

Bases are most highly ionised at low pH (pH < pKa)

Question 24

What information do dissociation constants give on the fate of chemicals?

Answer 24

Living organisms (sorption, distribution, metabolism and excretion) 
Environment (sorption, distribution, bioaccumulation)

Question 25

What do rapid screening techniques provide?

Answer 25

Rapid analysis, no sample preparation required, low sensitivity and selectivity, usually allow for detection only of any drugs/poisons in the samples

Question 26

What do advanced analytical techniques provide?

Answer 26

Takes time, require sample preparation, very sensitive and selective, allow for identification and quantification of drugs/poisons

Question 27

What are examples of rapid screening techniques?

Answer 27

Colour tests, immunoassay techniques

Question 28

What are examples of advanced analytical techniques?

Answer 28

HPLC, GC, MS, NMR

Question 29

Steps in analytical procedure?

Answer 29

Sample collection, sample preservation, sample preparation, sample analysis

Question 30

What happens during sample collection?

Answer 30

Need to collect a representative sample, when sample is collected need to decided how much is needed, more than one sample needed, mix together to make composites, needs to be as limited as possible any intervention could lead to errors in analysis

Question 31

What happens during sample preservation?

Answer 31

To ensure that sample retains its physical and chemical characteristics, acidify or freeze sample so microbes won't be active, often compounds we wan aren't stable they are subject to hydrolysis and photochemical reactions or metabolise to something else, so we need to make sure time between collections and preparation is as short as possible, preserve to make sure sample is stable

Question 32

What happens during sample preparation?

Answer 32

To separate analytes from the matrix to improve the analysis and detectability (eg homogenisation, extraction, concentration, clean ups hydrolysis of conjugates, derivatisation), often samples aren't in the right form for analysis analytes need to be in the correct matrix, structure of molecules and matrix is changed to make it amenable for detections helps with cleaning up matrix, biggest issue is too many interferences affecting sample - so remove interferences to get nice analysis

Question 33

What happens during sample analysis?

Answer 33

Qualitative (colour tests, immunoassays) and quantitative (big machines such as NMR etc) analysis

Question 34

What is important about the steps in analytical procedure?

Answer 34

Not all steps many not be required for all tests, depends on the instruments used, sample collected, analytes wanted, and so these all require different steps

Question 35

Steps for extraction?

Answer 35

Extraction, concentration and clean up

Question 36

What happens during extraction?

Answer 36

To present samples directly to the chromatographic system

Question 37

What happens during concentration?

Answer 37

To increase the concentration of analytes in the extract - (machines cannot detect concentration of collected sample so often have to get analytes out of matrix into another solution and concentrate it, increase concentration of analyte increase choice of success of analytic process

Question 38

What happens during clean up?

Answer 38

To remove potentially interfering matrix compounds (particularly important for GC LC/MS) solvent and analytes give best results but often not the case since interferences often sneak through, clean up will try to reduce this to give nice measurement

Question 39

Extraction procedures for non/semi volatile compounds from liquids?

Answer 39

Liquid liquid extraction Solid phase extraction Solid phase microextraction

Question 40

What happens in liquid liquid extraction?

Answer 40

Two immiscible liquids put two together shake, non polar parts partition into organic phase, simple but limited since polar compounds unlikely to partition to organic phase

Question 41

Extraction procedures for non/semi volatile compounds from solids?

Answer 41

``` Soxhlet extraction Ultrasonic extraction Supercritical fluid extraction Accelerated solvent extraction Microwave assisted extraction ```

Question 42

Whats different about accelerated solvent extraction?

Answer 42

It is accelerated by high pressure and temperature

Question 43

Extraction procedures for volatile compounds from solids and liquids?

Answer 43

Static headspace extraction Dynamic headspace extraction - purge and trap Solid phase micro extraction

Question 44

How are oxazepam and temazepam metabolised and excreted?

Answer 44

Oxazepam and temazepam are conjugated (sulphate and glucuronide conjugates) and excreted in urine at almost the same rate as they are generated

Question 45

How does pH control metabolism?

Answer 45

pH controls how, why and where different molecules are distributed so acidic molecules will be absorbed in basic conditions, basic molecules will be absorbed in acidic conditions

Question 46

How to prove administration of cocaine?

Answer 46

To prove administration of cocaine look for benzoylecogonine (an inactive compound), logKow goes down, OCH3 goes to OH makes it more amenable for excretion with urine

Question 47

Why won't heroin be found in urine?

Answer 47

Most hydrophobic molecule so very quickly metabolised so won't find this in urine

Question 48

Importance of 6-acetylmorphine in metabolism of heroin?

Answer 48

Not all metabolites are useful, sometimes have to use minor metabolites such as 6-acetylmorphine, only 1% of heroine gets transferred into this - really powerful tools needed to find this

Question 49

Why is morphine not the compound of interest to prove administration of heroin?

Answer 49

Cannot be compound of interest since could be present due to morphine or codeine consumption, can't prove heroine abuse