Drug Bioanalysis 2 Flashcards
What are tracer techniques?
Analytical techniques used to detect and measure tracers (tracer substances) in a variety of investigative biological studies
> commonly applied in biostudies of drugs and drug metabolites
- Involve application of a tracer
- Tracer: PURPOSELY LABELLED ‘REPLICA’ OF A PARTICULAR CHEMICAL SUBSTANCE
- Tracers are only distinguished from their ‘parent’ compound by the presence of a specifically substituted marker atom(s)
For Tracers;
A) What do they function as
B) what are some of their properties
C) What is some of the common isotopic labels used in the majority of tracers
D) How do atom labels hamper/negate detection of tracers
E) How should labels be incorporated
F) Where are the sites that requier caution to isotopic labelling
G) What the types of isotopic labels used in tracers?
A)
- TRACER FUNCTIONS AS A ‘REPLICA/SUBSTITUTE’ FORM OF A PARTICULAR ‘PARENT’ COMPOUND
B)
- TRACERS RETAIN THE SAME CHEMICAL, PHYSICAL, BIOCHEMICAL AND BIOLOGICAL PROPERTIES AS THEIR ‘PARENT’ COMPOUNDS
- Virtually identical to their parent compound –> except they have been specifically labelled with a substitute marker atom
- Marker atom: consist of various isotopes for element found in the parent compounds
- Marker atom serves as a label and means for easy detection and measurement of the tracer within biological systems/media
C)
- Isotopic labels: C, H, N and O ISOTOPES
- Can use >1 isotopic label to give a ‘multi-labelled’ tracer
- presence of an isotopic label should NOT alter the properties of the ‘parent’ substance
- tracers should be easily synthesized
D)
- When atom labels are placed in ionisable, chemically and metabolically labile sites
E)
- LABELS MUST BE INSERTED INTO NON-IONISABLE, CHEMICALLY AND METABOLICALLY STABLE SITES WITHIN THE ‘PARENT’ MOLECULE
F)
See attached image
G)
Radioisotopes and Stable Isotopes
For radioisotope labels;
A) what are they used for
B) Why must caution be used
C) what properties make for a easy detection radiolabel?
D) should they emit radiation?
E) Give a few example
A)
- Used to produce radiolabelled tracers (radiotracers)
B)
- they undergo nuclear decay to give stable nuclei and emit nulcear radiation (α, β + γ )
C)
- Display a relatively high specific activity i.e. a small amount of tracer should emit a reafily detectable amount of radiation
D)
- yes, but must be non-damaging or non-lethal to biological systems
E)
See attached image
How can radiotracers (radioisotopes) be analysed?
- Liquid scintillation counting (LSC)
- Autioradiography
- Positron emission tomagraphy (PET)
For Liquid scintillation counting (LSC);
A) What it used for
B) What does it detect
C) What type of data does it provide?
D) Disadvantages?
E) Technique? What is used?
A)
- used for detection and measuring radiolabelled compounds in biological media and fluids
- Also used for in vitro and simple in vivo tracer analysis e.g. in bacterial cell cultures
B)
- Used mostly to detect ‘low energy’ β-emitters e.g. Tritium (3H), Carbon-14 containing radiotracers
C)
- Provides qualitative and quantitative data
D)
- general requirement for lengthy sample preparation prior to analysis
E)
- Technique involves the application of a fluor (phosphor) agent which emits light energy (photons) when energetically stimulated by nuclear radiation
- Commonly used fluor is 2,5-Diphenyloxazole (PPO)
What are the three steps for LSC?
- Liquid scintillation cocktail
> Radioactive sample is added to scintillation cocktail
- Transfer of β-Radiation
> Beta particles are emitted which cause solvent molecules to become excited
> The energy of the solvent molecules is transferred to the fluor molecules, which in turn emit light
- Detection of the tracer
> Jar is placed inside a scintillation counter, which captures and digitizes the light photons
> Amount of radioactivity present is measured i.e. amout of radiolabelled tracer in sample
For autoradiography;
A) Describe what it is
B) What substances it used for
C) What is typically used for
D) Good qualitative results?
E) Give an example
A)
- Analytical technique involving exposure of radiolabelled materials to X-ray photographic media (film or emulsion)
- Employed mostly for the bioanalysis of tissue/organ sections and various cell cultures
B)
- Used primarily for β and γ emitting substances
C)
- Typically used for the visualisation of a radiolabelled tracer’s in vivo location
D)
- yes, but poor quantitative results
E)
See attached image
For Positron Emission Tomagrophy (PET);
A) What is it
B) Which radiotracers is it used for the exclusive study of
C) Which isotopes are generally the most short lived and expensive radioisotopes?
D) What useful information does it provide
E) Disadvantages
F) Give an example
A)
- Technique for bioanalysis of positron (anti-electron) emitting radiolabelled substances using an X-ray scanner/imager
- Similar to Computerised Axial Tomagraphy (CAT)
B)
- 11C, 13N, 15O and 18F positron emitting isotopic labels
C)
- positron emitting isotopic labels
D)
- provide useful information on ‘living systems’
- obtain ‘real-time’ in vivo tracer information
- used for dynamic drug and receptor ligand studies - most notably neural (brain) receptor studies
E)
- Typically expensive and limited by radioisotope issues
F)
- See attached image
For stable isotopic labels;
A) what do they do?
B) What is the difference between radioisotopes and stable isotpic lables
C) How are they different fron the parent compound
D) Give some examples
A)
- Used to produce stable (heavy) isotopically tracers
B)
- stable isotopic labels do NOT undergo any inherent degradation or change
C)
- Comprise of ‘heavier’ isotopic forms of the elements originally found in the ‘parent’ compound
D)
How to analyse stable isotopes
- Mass spectroscopy –> GC/LC-MS
- NMR spectroscopy
- Optical spectroscopy
What are some common types of pharmaceutical studies involving tracers?
- Elucidation of biochemical and/or metabolic pathways for drug substances, medicinal natural products and drug excipients
- Pharmacokinetic Studies
- Drug Receptor and Transporter studies
- Drug Metabolite Studies
- Drug Dosage Studies
For absorption studies
A) What type of drugs are involved?
B) Qualitative or quantitative information?
C) Invitro or invivo drug studies
Data obtained is correlated with that gathered from drug distributions tudies
A)
- Drug dosage studies
- Can employ either radiolabelled or stable isotope drugs
B)
- Requires reliable quantitative information e.g. using a radiolabelled drug and LSC method
C)
- Both
For Distribution studies;
A) What drugs does it use/ methods
B) Invivo or invitro
C) Qualitative or quantitative
A)
- Uses mostly radiolabelled drugs and detection method based on autoradiography or PET
B)
- Consist of in vivo drug studies predominantly
- may involve real-time drug studies e.g. in vivo monitoring of slow-release drugs
C)
- Requires reliable QUALITATIVE results
- QUANTITATIVE drug data an also be obtained by using standard calibration methods
For metabolism studies;
A) What drugs is it used for
B) In vivo or in vitro
C) What does it involve
D) how can drug metabolites be detected/identified
E) how can quantitative data be obtained (LSC and HPLC-MS methods)
A)
- heavy or radiolabelled drugs
B)
- Used for both
C)
- Involve extraction of biological samples and bioanalysis of biological fluids mostly e.g. plasma, urine, bile extract, gastric fluid
D)
- detected/identified via mass spectrometry and NMR spectroscopy
E)
- LSC and HPLC-MS methods
For excretion studies
A) What is it for
B) What drugs does it use
C) what types of bioanalysis does it involve
D) Quantitative or qualitiative
Data obtained is normally correlated with that gathered from drug absorption studies
A)
- Used to obtain information on the ultimate fate of administered drugs and drug metabolites
B)
- Typically utilise radiolabelled drugs
C)
- Mostly involve bioanalysis of urine, faeces and also occasionally expired air
D)
- Generally require QUANTITATIVE drug data e.g. utilising LSC method
