How Drug Tests Work: Immunoassays and GC-MS

Millions of drug tests are carried out every year: for job applicants, athletes, drivers, hospital patients and people on probation. Most follow the same two-stage pattern. A cheap, fast screening test sorts samples into “negative” and “possibly positive”, and every possible positive is sent for a slower, highly specific confirmatory test. Understanding why the system works this way needs a little biochemistry, a little analytical chemistry and some statistics.

What is actually being measured?

When a drug enters the body, the liver starts converting it into other compounds called metabolites, which are easier to excrete. Tests often look for these metabolites rather than the original drug, because:

• metabolites may stay in the body for longer, widening the detection window
• a metabolite proves the drug was actually taken into the body (not just present as contamination on the sample)

Some examples:

Drug	Main target in urine
Cannabis (THC)	THC-COOH, a carboxylic acid metabolite
Cocaine	benzoylecgonine
Heroin	6-monoacetylmorphine (specific to heroin) and morphine
Amphetamines	the parent drug itself

Which sample?

Sample	Typical detection window	Notes
Urine	days (longer for heavy cannabis use)	most common; easy to collect; relatively high concentrations
Blood	hours to a day or two	best reflects current effect, e.g. for driving
Oral fluid (saliva)	hours to a day or two	used in roadside tests; hard to cheat
Hair	months	drug trapped in the growing hair shaft; about 1 cm per month
Sweat patch	days to weeks	worn continuously

Stage 1: Immunoassay screening

Screening tests use antibodies, proteins made by the immune system that bind very tightly to one particular molecular shape. Laboratories produce antibodies that recognise a drug or its metabolite.

Most drug screens are competitive immunoassays. The principle:

1. The test contains a limited number of antibodies, plus a labelled version of the drug (tagged with an enzyme, a dye or a fluorescent group).
2. The drug from the sample and the labelled drug compete for the antibody binding sites.
3. The more drug there is in the sample, the less labelled drug can bind.
4. The amount of bound (or unbound) label is measured, and this indicates how much drug was present.

How a dipstick test shows a result

In the familiar lateral flow cup or strip, the urine soaks along a membrane carrying coloured particles attached to antibodies.

• No drug in the sample: the antibody particles are free to bind to drug molecules fixed in a line on the strip, so a coloured test line appears.
• Drug present above the cut-off: the sample drug has already occupied the antibodies, so they can’t bind at the test line, and no line appears.

This surprises many people: on most drug test strips, a line means negative. A separate control line must always appear, to show the test has worked.

Cut-off concentrations

A screen doesn’t say “yes” to any trace of drug. It’s calibrated to a cut-off concentration, often expressed in ng/mL (nanograms per millilitre). Below the cut-off, the result is reported negative. Cut-offs are set high enough to avoid positives from tiny, innocent exposures (such as breathing in second-hand smoke) while still detecting real use.

Why screening tests produce false positives

Antibodies recognise shape, and other molecules with similar shapes can sometimes bind too. This is called cross-reactivity. Examples that have been reported for some immunoassays include:

• some cold and decongestant medicines cross-reacting with amphetamine tests
• certain antibiotics and other prescription drugs affecting opiate screens
• poppy seeds genuinely containing small amounts of morphine and codeine, which can be enough to exceed some opiate cut-offs

Because of this, a positive screen is only ever a presumptive result. It is never reported as a final positive without confirmation. The same principle runs through forensic chemistry generally.

Stage 2: Confirmation by GC-MS or LC-MS/MS

Confirmation uses chromatography coupled to mass spectrometry, which separates and identifies molecules by their properties rather than by shape recognition.

GC-MS

1. The sample is prepared: the drug is extracted from the urine using a solvent or a solid-phase extraction cartridge, removing salts, urea and proteins. See solubility and polarity.
2. Many drugs are chemically derivatised (converted to a more volatile form) so they can travel through a gas chromatograph.
3. The gas chromatograph separates the compounds. Each has a characteristic retention time.
4. The mass spectrometer fragments each compound, producing a pattern of ions that acts like a fingerprint.

A result is confirmed only if the retention time and the ratios of several characteristic ions match a reference standard analysed in the same batch.

LC-MS/MS

Increasingly, labs use liquid chromatography with tandem mass spectrometry. It handles drugs that are too polar or heat-sensitive for GC, often without derivatisation. The first mass analyser selects the drug’s molecular ion; this is broken into fragments; the second analyser measures specific fragments. Monitoring these transitions is extremely selective and sensitive. See HPLC for the chromatography part.

Quantitation with internal standards

Confirmatory methods also measure how much is present. Labs add a known amount of an isotope-labelled internal standard, a version of the target molecule where some hydrogen atoms are replaced by deuterium (²H). It behaves almost identically in extraction and chromatography but has a higher mass, so the mass spectrometer can tell them apart. Comparing the two signals corrects for losses during sample preparation. This builds on calibration curves and on isotopes.

Stopping cheating: specimen validity

Some people try to dilute or adulterate samples. Labs check:

• temperature of a freshly collected urine sample (it should be close to body temperature)
• creatinine concentration and specific gravity, which are abnormally low in heavily diluted urine
• pH, since adding acids or bases pushes it outside the normal range. See the pH scale.
• oxidising adulterants such as nitrites or bleach

Sports drug testing

Anti-doping labs work at the extreme end of analytical chemistry. They look for hundreds of substances, including hormones that the body also makes naturally. For testosterone, for example, labs measure the ratio of testosterone to a related natural compound; an abnormal ratio triggers isotope ratio mass spectrometry, which can tell synthetic testosterone (made from plant sources, with a slightly different carbon-13 to carbon-12 ratio) from the body’s own.

Breath alcohol: a different approach

Roadside alcohol testing doesn’t use antibodies. Handheld devices typically use a fuel cell, in which ethanol from the breath is oxidised at a platinum electrode, producing a current proportional to the alcohol concentration. Evidential machines at police stations often use infrared absorption by the C–H bonds of ethanol. See infrared spectroscopy.

Key takeaways

• Drug tests usually target metabolites, which last longer and prove the drug was processed by the body.
• Screening immunoassays use antibodies and competition with a labelled drug; on most strips, a test line means negative.
• Cross-reactivity means screens can give false positives, so they’re only presumptive.
• GC-MS and LC-MS/MS confirm identity and measure concentration, using isotope-labelled internal standards.
• Specimen validity tests, cut-offs and chain of custody make the whole system reliable.