Methods of Drug testing

There is a large degree of variation (in terms of expense, accuracy, timelines, sensitivity and selectivity) when it comes to different methods of drug testing. Drug testing laboratories will generally use a cheap method of screening for initial tests, and then re-test positive samples using a more accurate and expensive method. The screening methods that are used most often involve immunochemical testing. These are known as thin-layer chromatography (TLC) and immunoassays.


Immunoassays are a type of drug screening that returns either a yes or no outcome. The results are highly sensitive and specific. There are three types of immunoassays that are in common use. These are: 1) enzyme immunoassay (EIA), 2) fluorescence polarization immunoassay (FPIA) and 3) radioimmunoassay (RIA). These three tests are all based on the same general principle. Binding antibodies are used to detect specific drugs or groups of drugs. If a urine or hair sample contains a drug and it is combined with a solution including the drug's antibody, then it will bind.

The principle being used here is similar to the "rabbit test" used for detecting pregnancy. The drugs that are sought are linked to a large molecule and then injected into a sheep or rabbit. The immune system of the animal then develops antibodies to fight the drug. These antibodies are collected, purified and then used in immunoassay testing. A competition for the antibody binding agent takes place between the sample drug and the tagged drug that's being tested.

Limitations of Immunoassay Testing

Because immunoassay tests only produce a yes or no outcome, they can only provide an estimate as to the quantity of the drug in the individual's system. Another significant limitation of this method is cross-reactivity, which occurs when other substances and legal drugs bind with the testing antibodies. This can produce a false positive result which shows the presence of a metabolite that is not actually in the sample.

Specificity and Cross Reactivity

When it comes to immunoassay, the term "specificity" refers to the way in which the antibody recognizes the drug or group of drugs that is being tested. The term "cross-reactivity" refers to the way that the antibody recognizes other substances that aren't the specific drug that is being tested. This means that an antibody with a high degree of specificity will have low cross-reactivity. Cross-reactivity levels vary significantly between different immunoassays. Each of the three commonly used methods mentioned above are considered to have high degrees of specificity when compared to other screening tests, but nevertheless they are not perfect. When an immunoassay is being used to test for amphetamines for instance, it may find drugs that are structurally similar to amphetamines, including a variety of over-the-counter medications like ephedrine, phenylpropanolamine and sympathomimetic amines. This means that other tests, such as a GC/MS chemical testing, need to be carried out in order to confirm a positive result. The unfortunate reality however, is that these second tests don't always occur. Sometimes the sample is simply retested using the same method as the original test, or it fails to be retested at all.

Enzyme Immunoassay (EIA)

With EIA testing, any drugs that are found in the sample are labeled using an enzyme. The enzyme attaches itself to the antibody and in this state it is inactive. If there are drugs in the sample, then the enzyme becomes displaced from the antibody and is activated. The extent of this reaction will depend on the quantity of the drug present in the sample.

There is another test commonly known as EMIT (Enzyme Multiplied Immunoassay Test) that was developed in Palo Alto by the Syva Corporation. This test involves a modification of the way that an enzyme acts in relation to its substrate, lysozyme. The drug (or a metabolite) is injected into an animal, typically with several other chemicals. The animal's immune system is then prompted into producing certain chemicals, known as antigens, that become bound to the drug. The antigens are extracted and purified from the blood of the animal. The enzymes (or lysozymes) are attached to the drug or metabolite that was initially injected, which may be methadone, morphine or an amphetamine. When the antibody to the drug is put into the same solution as the drug-enzyme, the enzyme's functionality is inactivated. However, if the drug being tested is contained in a particular urine sample, the antibody's bind to the enzyme will reduce because it will also want to bind with the drug itself. When a drug enzyme is unbound it becomes active and the solution is cleared as the bacterial suspension is lost. The clearing of the solution refers to the change in the way it absorbs light. This is measured using an instrument known as a spectrophotometer.

The Radioimmunoassay (RIA)

One example of a popular radioimmunoassay is "Abuscreen", developed by Hoffman-LaRoche. This RIA is comparable to the EMIT in that the technology is similar and it also relies upon antibodies that have been specially produced for the purpose of the test. The main difference between these tests is that the RIA uses radioactive isotopes (known as "tracers") as a label and measurement for the results. The quantity of the drug contained in a sample is measured by how much tracer is needed to displace the drugs that have bound themselves to the antibody. The level of radiation that is emitted by the tracer and antibody is measured with a gamma counter.

The advantage of RIA is that it produces more accurate results that EMIT, however it is also more expensive and the radioactivity means that specialized equipment and laboratories are required. The fact that EMIT and RIA methods of testing are both versions of immunoassay means that testers should not use one method to confirm the results of the other. A better approach would be using a nonimmunological method (like gas chromatography for example) as a second confirmatory test.

The biggest disadvantage of using immunoassays is typically the fact that they have low levels of specificity. This is because there are not very many antisera that can be specifically applied to a single compound. Fluorescent polarization immunoassay (or FPIA) is a method that has much higher levels of specificity and sensitivity. FPIA was developed for drug testing purposes in 1986 by Abbott Labs.

Confirmatory Tests

One a positive test result is returned using a screening method, it should not be assumed to be 100% accurate. A second and more accurate testing method using different chemical principles has to be applied in order to confirm the results.

Gas Chromatography

There are two additional testing methods which use a tube made of either glass or metal that has been filled with a substance which has a certain polarity. These methods are called either Gas Chromatography (GC) or Gas Liquid Chromatography (GLC).

These tests involve vaporizing the sample and then injecting it into the column as a steady gas flow. Identical compounds will interact with the packing inside the column in the same way meaning that the progress through the tube at an identical rate. At the end of the column there is a detector which records and quantifies the data. The period between the injection and the recording of a response by the detector is known as the "retention time".

When substances in the same column have identical retention times, it is regarded as strong proof of identical substances. The GC and GLC tests rely upon standard references in order to calibrate the results.

Gas Chromatography / Mass Spectrometry (GC/MS)

GC/MS tests use advanced technology that is superior in analyzing toxicology results. For this reason GC/MS tests should always be used to confirm positive test results. As the name suggests, GC/MS involves two different techniques, first gas chromatography and then mass spectrometry. The main reason behind conducting a confirmation test for a positive result is to eliminate the possibility of human error. Using the GC/MS testing method has the highest levels of sensitivity and specificity. It is an expensive testing method that requires certain skills in order to perform the analysis. This means that it is generally only used for confirmatory tests or tests that are likely to be used in court and therefore must satisfy forensic requirements.

The GC/MS method is applicable for gathering both quantitative and qualitative results. The sensitivity levels are generally measured in nanograms or pictograms, depending on the particular drug that is being tested. GC/MS testing has a higher degree of specificity than TLC methods, and has between 100 and 1,000 times more sensitivity. It is easily able to identify the majority of organic molecules, including many popular recreational drugs like marijuana, heroin and cocaine.

Drug testers can apply the GC/MS method (as well as GLC and certain RIA methods) to all bodily fluids, including blood and serum. The level of a drug in the blood is a very important measurement as this is the only level that represents the person's level of intoxication and this therefore has the greatest legal significance.

The GC/MS method involves combining a hair specimen or a urine sample with a specific organic solvent. The drug is then concentrated by evaporating the organic solvent before the GC/MS process begins. This process involves two distinct steps. First, a gas chromatograph is used to separate the drugs, and then a mass spectrometry is conducted on the separated drugs.

In the gas chromatography phase, a gas (generally helium) is forced through a silica column that has a polymer layer made of cross linked silicone. The vaporized drugs interact with the polymer causing them to separate. The time it takes each one to arrive at the column's end is referred to as the "retention time" (RT).

After the sample has been separated into its various compounds using gas chromatography, the substances are then identified as the leave the gas chromatograph using mass spectrometry. This process involves firing an electron beam at each of the components. This breaks the components into different fragments and forces them to move through a magnetic field. The molecule that makes up a drug will always split into identical fragments. This is referred to as the drug's mass spectrum. Each drug has its own unique mass spectrum, similar to a person's fingerprint. A computer program is then used to compare and analyze the mass spectrum. This program can identify the parent compound, as well as the probable fragments. When the program returns a match of 98% it is considered to confirm the existence of the particular compound.

GC/MS is a very specific drug detection method, however these tests are very expensive in terms of both the equipment required and the expert analysis that is needed in order to understand the results. GC/MS involves using state-of-the-art technology which is very accurate. This level of accuracy means that they are frequently used in forensic, clinical, pharmaceutical and industrial laboratories.

The second part of the process involves inserting the silica column into the mass spectrometer. The mass spectrometer is made up of a vacuum chamber and quadropoles which surround the column of the gas chromatograph. As the drugs leave the column they are ionized by electrons and then forced to enter the quadropoles. The fragments are separated into different quadropoles according to their molecular weight and charge. The fragments are then converted into electrical pulses using an ion detector and the relevant information is feed into a special computer system. The mass spectra results are then produced to represent the original molecule.

The high costs and required expertise has mean that GC/MS systems have not been available for many laboratories. However, improvements in the technology have made a lot of the process more automated which has brought down the costs and made GC/MS much more affordable in recent years. A lot of labs have purchased the Hewlett-Packard gas chromatograph which can be connected to the Hewlett-Packard Mass Selective Detector. This is a very accurate and reliable GC/MS system, as long as the tester has sufficient training and is using the equipment properly.

Thin-Layer Chromatography (TLC)

Thin-Layer Chromatography is being used less often since the 1990's, but you may still find references to TLC when reading about drug tested. TLC is a type of chromatography which involves extracting urine with a reagent and then subjecting the urine extraction to a procedure that is designed to separate it into its distinctive components. In TLC, the drug test results are found by analyzing the drug's migration pattern on a thin, absorbent glass plate that is typically coated with silica. A solution is sprayed onto the glass plate and this solution has different reactions when it comes in contact with various drugs. The reaction creates colored spots in patterns that signify particular drugs.

A drop of the extracted urine is put onto the TLC plate in a process known as "spotting". The plate is then emerged in a solvent and the solvent climbs the plate through the absorbent capillaries. The rising solvent brings with it any drugs that are contained in the urine. Specific drugs always create the same migration patterns. Once the plate dries it is analyzed and the presence of specific drugs can be confirmed. For example, if there is cocaine in the urine sample, then spraying the visualization solution onto the plate will reveal a particular spot that shows the movement of the cocaine. The location of the spot is given an "Rf" number. This refers to the ratio of the distance that the drug traveled to the distance that the solvent travelled. The measurements for each of these distances begin at the point of origin which is the point at which the urine extract sample was spotted. To make the analysis easier, ultraviolet lights are often used to illuminate the plate. It is generally expected that identical molecules will have the same Rf migration patterns and produce identical colors. However, TLC testing frequently results in false positive due to the fact that some prescription and legal over-the-counter drugs have identical migration patterns as illegal drugs. It is important that TLC results are analyzed by an experienced expert and positive results are confirmed using a more accurate method.

TLC is frequently used by drug treatment programs, drug detox clinics, methadone programs, industrial screening and drug testing prison inmates or parolees.

Problems with TLC Drug testing

The results that can be ascertained from TLC drug testing are qualitative, which means that they return either a yes or no answer in relation to drug positivity. When a positive result is returned it is not able to be quantified. This situation is the same for immunoassay tests, however using tests with varying cut-offs can produce a quantitative result. The sensitivity of TLC testing is much lower than other types of tests. When the substance abuse levels are low, TLC is less accurate in detecting results. This makes the results of TLC screenings confusing. Whether or not a particular sample is classified as positive will depend on the level of concentration of the particular drug in the sample. This is also known as the sensitivity cut-off value. Most TLC tests will have a sensitivity cut-off of between 1,000 and 2,000 nanograms per milliliter. However, there are some drugs that will only be detectable with a concentration level over 2,000 ng/ml and so these drugs can be difficult to find using a TLC test. Immunoassay tests, on the other hand, frequently have cut-offs of 100 ng/ml or lower. When a TLC test produces a negative result it may actually mean that the TLC testing technique lacks the sensitivity to detect the concentration of the drug. Like other immunoassay tests, TLC also has a low level of specificity. It is commonly used for broad drug screening because it's quick, cheap and there is no need for technical equipment.

Medical situations often rely upon TLC to show cases of high-level drug use and instances where the body contains toxic drug dosages. TLC is perfectly suited for the emergency room when the drugs consumed are often unknown and the medical staff need a fast measurement of the toxicity levels.

Do It Yourself Tests

1987 was the first year that do it yourself drug tests became commercially available. The American Drug Screens of Dallas developed one of the first drug testing kits which was known as "AWARE". These do it yourself drug tests kits came with mailing tubes and specimen containers. It was designed to assist parents who wanted to know whether or not their children were using drugs. The parents collected samples which were then sent away to the processing laboratory. The test results were mailed to the parent within two weeks.

There are now many companies that offer testing through the Internet. If you are concerned about the results of a drug test then you may wish to use an independent testing service or buy a kit that you can use at home. People who are planning on passing drug test by using switched or altered samples may want to test their plans on an independent testing service before sitting the real test. A selection of products are available for sale from

Specificity and Sensitivity

The specificity of a drug test refers to how accurately it can distinguish between different drugs. The sensitivity, on the other hand, refers to the smallest quantity of a drug that a test is able to find in a urine sample. The sensitivity level of a test should be appropriately related to the purpose of the test.

Cut-Off Level

The drug test's sensitivity is directly related to its cut-off level and the detection limit. The cut-off level refers to the value that the specimen must record in order to be considered positive. Anything below the cut-off level will be considered a negative result. Screening and confirmatory tests will have different sensitivity and specificity levels which in turn means that their cut-off concentration levels will also be different.

Generally speaking, the initial screening test will be broad and cheap. These types of tests include RIA, TLC and EMIT. They are quite sensitive and they are able to detect most types of drugs or drug metabolites. These tests only return a positive or negative answer. A positive result should always be confirmed using a different type of test. The best test to use for confirmation purposes is the GC/MS method, but this is a lot more costly. The specificity level of the confirmatory test should be higher than the initial test. When one account orders a large amount of a similar test they will generally receive a discount proportional to the volume. Alternatively, when a test has been specifically designed to meet strict standards, they will obviously be more expensive.

Confirming a Positive Result

When a particular urine sample returns a positive result, it should always be re-analyzed using a different test. The sample should return a positive result to both tests before a positive result is definitively recorded. This is known as the "confirmation" of a test result.