Drugs Analysis and Therapeutic Drug Monitoring

Question 1

What are the requirements for measuring drugs in biological matrices?

Answer 1

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

Question 2

What are the two main analytical techniques for drug analysis?

Answer 2

Immunoassays such as radioimmunassay, enzyme immunassay, Enzyme linked immunosorbent assay
Chromatographic techniques such as Gas chromatography, High pressure liquid chromatography and various detectors

Question 3

What are the requirements of a radio immunassay?

Answer 3

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

Question 4

What are common methods of separating Ab-bound drug from free drug for a radioimmunassay?

Answer 4

Activated charcoal, polyethylene glycol, second Ab method

Question 5

How is a radioimmunassay performed?

Answer 5

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

Question 6

What are the advantages of a radioimmunassay?

Answer 6

Great sensitivity and large sample capacity

Question 7

What are the disadvantages of a radioimmunassay?

Answer 7

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

Question 8

What are the differences between a radio-immunassay and an enzyme immunassay?

Answer 8

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

Question 9

What advantages does an enzyme-immunassay have over a radio-immunassay?

Answer 9

Avoids radioactivity reducing health risks
Does not require a separation phase allowing for more automation
Retains the specificity and selectivity of a radioimmunassay

Question 10

How can chromatography be used for drug analysis?

Answer 10

It separates compounds based on physicochemical properties such as ionic charge, molecular weight, lipid solubility and pKA

Question 11

How does chromatography work?

Answer 11

There is a stationary phase and mobile phase and separation occurs by compounds either binding to or exclusion from the stationary phase

Question 12

What is the major drawback of traditional chromatography?

Answer 12

It is very slow

Question 13

What are the drawbacks of gas chromatography?

Answer 13

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

Question 14

How does high pressure liquid chromatography work?

Answer 14

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

Question 15

What is the purpose of an internal standard?

Answer 15

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

Question 16

What are the advantages of high-pressure liquid chromatography?

Answer 16

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

Question 17

What are the disadvantages of high pressure liquid chromatography?

Answer 17

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

Question 18

What is the gold standard for drug analysis?

Answer 18

LCMS/GCMS

Question 19

What are the advantages of LCMS/GCMS?

Answer 19

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

Question 20

What is precision?

Answer 20

The closeness of replicate determination with the acceptable limit of variability being 10% intra-assay and 15% inter assay

Question 21

What is accuracy?

Answer 21

The closeness of the determined value to the true value with an acceptable deviation of 15% from the true value

Question 22

What is sensitivity with regards to a bioassay?

Answer 22

The minimum quantifiable concentration

Or the lowest concentration that can be measured with acceptable accuracy (20% precision and accuracy)

Question 23

What is specificity with regards to a bioassay?

Answer 23

The ability of the assay to measure only what it is intended to measure

Question 24

What is stability with regards to a bio assay?

Answer 24

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

Question 25

What is therapeutic drug monitoring?

Answer 25

The use of blood/plasma drug concentrations as a tool for individualization of drug therapy
This is necessary to ensure optimal drug response

Question 26

What is the target concentration intervention strategy?

Answer 26

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

Question 27

What are the typical properties of drugs which require therapeutic drug monitoring?

Answer 27

A narrow therapeutic index
Large variability in their pharmacokinetics between patients
A high risk of causing sever toxicity

Question 28

What patient factors would result in therapeutic drug monitoring being required?

Answer 28

Therapeutic failure when the patient is definitely taking the drug
Suspected non-compliance
Suspected drug-drug interactions in multiple drug therapy
Major organ failure