Drugs Analysis and Therapeutic Drug Monitoring Flashcards
What are the requirements for measuring drugs in biological matrices?
There must be a high sensitivity as often very low concentrations are used
Must have good selectivity so it can be specific for the drug and not its metabolites and similar looking compounds etc
What are the two main analytical techniques for drug analysis?
Immunoassays such as radioimmunassay, enzyme immunassay, Enzyme linked immunosorbent assay
Chromatographic techniques such as Gas chromatography, High pressure liquid chromatography and various detectors
What are the requirements of a radio immunassay?
There must be a specific antibody which binds to the drug with high affinity (this usually is requires an adjuvant to be bound to the drug)
A radioactively labelled drug
A method for separation of AB-bound and free drug in solution
Radioactivity counting equipment
What are common methods of separating Ab-bound drug from free drug for a radioimmunassay?
Activated charcoal, polyethylene glycol, second Ab method
How is a radioimmunassay performed?
A solution is made up containing a known amount of high affinity antibodies, a known amount of radioactively labelled drug and the sample containing unlabeled drug
The antibody will then bind both the unlabeled and labeled drug with both drug-forms competing for its binding sites
The bound and unbound drug are then separated and the radioactivity counted
The radioactivity level can then be compared to a calibration curve to determine drug concentration
What are the advantages of a radioimmunassay?
Great sensitivity and large sample capacity
What are the disadvantages of a radioimmunassay?
It can only work for a single drug
There may be cross-reactivity with metabolites
The counting equipment is expensive
The radioactivity means there is potential health risks
Due to the last separation phase complete automation may be difficult
What are the differences between a radio-immunassay and an enzyme immunassay?
An enzyme immunassay uses a drug conjugated to an enzyme instead of a radioactively labeled drug, this enzyme will be in activated if the antibody binds to that drug allowing enzymatic activity to be used as a measure drug concentration
There is no need for a separation phase
What advantages does an enzyme-immunassay have over a radio-immunassay?
Avoids radioactivity reducing health risks
Does not require a separation phase allowing for more automation
Retains the specificity and selectivity of a radioimmunassay
How can chromatography be used for drug analysis?
It separates compounds based on physicochemical properties such as ionic charge, molecular weight, lipid solubility and pKA
How does chromatography work?
There is a stationary phase and mobile phase and separation occurs by compounds either binding to or exclusion from the stationary phase
What is the major drawback of traditional chromatography?
It is very slow
What are the drawbacks of gas chromatography?
The drugs must be volatile and stable at high temperatures of 300-400 degrees
This gives a very limited range of drugs for which this technique can be used
How does high pressure liquid chromatography work?
The stationary phase has smaller particles that are more tightly packed, this results in high pressures being able to drive the compound through column faster
What is the purpose of an internal standard?
It is a compound with a similar structure to the drug of interest
It can be added at a known concentration to a biological matrix
this allows for correction for loss of drug in the clean up
What are the advantages of high-pressure liquid chromatography?
It has a wide applicability and versatility
Can quantitate several drugs/metabolites in a several sample
Has linear calibration curves
High specificity with good accuracy and precision
What are the disadvantages of high pressure liquid chromatography?
There is an extraction and clean-up procedure required
It cannot be fully automated
It is time consuming taking 5-10 minutes a sample
The equipment and columns are expensive
What is the gold standard for drug analysis?
LCMS/GCMS
What are the advantages of LCMS/GCMS?
The use of a mass spectrometer measures mass to harge ratio of individual molecules that have been converted into ions
It is the most selective and sensitive drug analysis available
There is structure identification and confirmation
Can be used to confirm positive tests
What is precision?
The closeness of replicate determination with the acceptable limit of variability being 10% intra-assay and 15% inter assay
What is accuracy?
The closeness of the determined value to the true value with an acceptable deviation of 15% from the true value
What is sensitivity with regards to a bioassay?
The minimum quantifiable concentration
Or the lowest concentration that can be measured with acceptable accuracy (20% precision and accuracy)
What is specificity with regards to a bioassay?
The ability of the assay to measure only what it is intended to measure
What is stability with regards to a bio assay?
The stability of the drug in a matrix and the effects of time, temperature and storage conditions as samples are typically stored and if this is for a long period of time the drug may break down causing inaccurate results
What is therapeutic drug monitoring?
The use of blood/plasma drug concentrations as a tool for individualization of drug therapy
This is necessary to ensure optimal drug response
What is the target concentration intervention strategy?
Pick a target concentration (usually mid-point therapeutic range)
Determine the patients clearance and volume of distribution
Calculate the loading dose and dose regimen
Evaluate drug effects at steady state
If there are problems:
Revise clearance and recalculate dose
Go to step 4
What are the typical properties of drugs which require therapeutic drug monitoring?
A narrow therapeutic index
Large variability in their pharmacokinetics between patients
A high risk of causing sever toxicity
What patient factors would result in therapeutic drug monitoring being required?
Therapeutic failure when the patient is definitely taking the drug
Suspected non-compliance
Suspected drug-drug interactions in multiple drug therapy
Major organ failure
