Drug Analysis and Toxicology Method Validation

Absorption

The process of taking in or incorporating one substance into another

Biological absorption

Substances are taken in by and moved around an organism by various processes eg carrier system, passive diffusion.

Systemic Absorption

Substances are distributed around the body once they enter the blood stream - into tissues and organs that could be far away from the point of entry.

Absorbed dose

Amount taken up by organism into organs or tissues of interest. The full amount administered may not be absorbed.

Active metabolite

Metabolite with pharmacological or toxic activity.

Metabolism

Sum total of all physical and chemical processes that take place within an organism to break down and build up substances.

Adulterations

The act of making something poorer in quality by adding another substance.

Drug Testing

The analysis of biological samples to detect the presence of drugs.

Tissue Selection

Selection of tissue depends on the type of poison and subject circumstances (living or dead).

Blood

Used to detect, quantify and interpret drug and toxicant concentrations. Can establish recent drug ingestion - as it is still in circulation. Help to determine what effect this concentration of this drug may have had. Concentrations change depending on time after ingestion and death - movement.

Urine

Used for workplace drug testing. Sample that can be collected easily but mainly contains inactive drug metabolites. Metabolites specific to diferent drugs - Indicates that has been in the blood previously, has been metabolised and is now being excreted.

Hair

Non-invasive sample that provides a long time window for drug history. Affected by genetics, hair colour, hair damage. Very low concentrations of the dug.

Nails can also be used for even longer time window - but less understood how they process toxins

Liver

Primary solid tissue choice for post mortem toxicology.

Drugs are mainly metabolised here, they are in high concentrations

Reflects what is circulating in the body and can comment on the likelihood of producing an effect.

Vitreous humour

Sample from deceased only; interpretation limited but protected from microorganisms, damage and decomposition. The concentration of drugs here tend to mirror blood concentrations.

Breast milk

Determines neonate exposure but is invasive to collect.

Gastric content

Useful for indicating recent ingestion or delayed gastric emptying.

Can reveal original form (pills) and amount ingested before metabolism takes place.

Method validation

Proving a specific method is reliable, accurate and suitable for its purpose

Method validation process

• Define intended use: Sample type, analyte, purpose

• Experimentation and data analysis: Conduct studies and measure validation parameters

• Record and report all data, prove fitness for purpose

Method validation parameters

Accuracy

Precision

Linearity

Interference/specificity

LOD/LOQ

Verification

Evaluation experiments conducted to determine whether certain specifications are met.

Demonstration

Evaluation studies conducted by a laboratory to show that it can obtain expected performance or results previously documented.

Evaluation

A generic name for any study that measures the performance capabilities of an assay.

Validation

Initial evaluation experiments conducted to determine whether a new test system meets laboratory requirements.

Allowable Error (EA)

The amount of error that can be tolerated without invalidating the usefulness of the result.

Decision Limit (Xc)

Any concentration of analyte that is critical for medical interpretation. Concentration benchmarks such as detection limits, dose selection, exposure limits

Performance Standards

Criteria for acceptable results in testing.

Interference

Non-target analytes affecting detection or quantitation.

Precision

Reproducibility of results expressed as SD or CV%.

Linearity

Proportionality between test results and analyte concentration.

Accuracy

Closeness of a measured value to the true value.

LOQ

Lowest detectable analyte amount meeting accuracy requirements.

Ion Suppression

Reduced ionisation due to co-eluting compounds. reduces analyte signals

Ion Enhancement

Increased ionisation caused by co-eluting compounds. increasing analyte signals.

Recovery

the proportion of the known amount of a substance that is successfully extracted from a sample matrix and measured by the analytical process. optimum recovery is 100%

Recovery Calculation

Set 1: Drug X in mobile phase (no matrix, no extraction)

Ratio: A3/A4

Set 2: Known amount of Drug X spiked into blank matrix, then extracted. Internal standard added after extraction

A1/A2

Recovery %: (A1/A2)/(A3/A4) X 100

A1: peak area of drug x in extracted matrix (set 2)

A2: peak area of internal standard in set 2

A3: peak area of drug x in set 1

A4: peak area of internal standard in set 1

Calibration Model

A method to relate the instrument response (e.g., peak area) to known concentrations of an analyte. Used to determine unknown sample concentrations.

Calibration curve and equation of a line.

Carryover

Residual analyte from a previous sample that contaminates the next sample, causing inaccurate measurements. Common causes include incomplete system flushing, sticky or high-concentration analytes, and adsorption to tubing or vials. Prevent by washing the injection path, using blanks between samples, and optimising sample prep. Can lead to false positives or overestimated concentrations.

R2 Criteria

Correlation coefficient should exceed 0.975.