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Urine Drug Testing Methods


Drug test performed by immunoassay are designed to classify substances as either "present" or "absent" according to predetermined cutoff thresholds. Definitive identification of a specific drug and/or its metabolite(s) requires more sophisticated tests, such as Gas Chromatography Mass Spectrometry (GC/MS), or in our Partner Laboratory case, the labs are outfitted with the “Gold Standard” of analytical chemistry technology including the latest high resolution Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) equipment offering unmatched accuracy in urine drug testing.  The urine drug screening method chosen depends upon the question that needs to be answered.


The immunoassay drug tests, which are designed to classify substances as either "present" or "absent" according to a predetermined cutoff threshold, are the most commonly used methods. Immunoassays are based on the principle of competitive binding, using antibodies to detect the presence of a particular drug or metabolite in a urine sample. A known amount of an antibody and the drug or metabolite that has been labeled with an enzyme are added to the urine sample. The drug or metabolite in the sample competes with the labeled drug or metabolite for binding sites on the antibody to form antigen-antibody complexes. The amount of enzyme-labeled antigen that binds with the antibody is inversely proportional to the amount of the drug and/or its metabolite(s) in the sample.

The biggest advantages of immunoassays are their ability to quickly and simultaneously detect drugs in urine as well as their minimal cost. The biggest disadvantage of immunoassays is that they vary in the type of compounds that can be detected. Some immunoassays detect specific drugs, while others identify only classes of drugs. An immunoassay's ability to detect drugs will vary according to the drug's concentration in the urine and the assay's cutoff concentration. Any response above the cutoff level is reported as positive, and any response below the cutoff is reported as negative. (So for example, if the cutoff is set at 50 ng/mL, a result of 49 ng/mL will be reported as negative.) Immunoassays are also subject to cross-reactivity. In other words, substances with similar, and sometimes dissimilar, chemical compositions might cause a test to appear positive for the target drug. Because of the potential for cross-reactivity, samples that test positive for classes of drugs by immunoassay might need to be retested in the laboratory using an alternative method if specific identification of the drug is required.


A number of single-use immunoassay devices are commercially available for point of care (POC) testing for classes of commonly misused drugs. POC devices typically use immunochromatographic methods that produce visually read results. Used alone, POC testing by immunoassay is often inadequate in patient-centered urine drug testing, since most physicians want to identify the presence of a specific drug or metabolite, and not merely the drug class.


Most POC tests are based on competitive binding to antibodies by drug(s) present in the urine and a drug conjugate that is bound to a porous membrane. In the absence of the drug in the sample, a limited number of dye-conjugated antibodies bind the immobilized drug conjugate, forming a colored line (negative result) in the test window. When the amount of drug in a urine sample is equal to or greater than the cutoff concentration of a particular device, the drug saturates the antibody and prevents the antibody from binding the immobilized drug conjugate, so no line forms in the window (positive result). But this is a counterintuitive response. So some POC devices now operate more logically and produce a color for a positive result. POC devices have several advantages. They have a rapid turnaround time, are portable, are less expensive, and are seemingly easy to use. One particularly useful role for POC testing is to provide on-the-spot identification of illicit drug use.


Potential disadvantages of POC devices include the subjective nature of the qualitative assays, lack of adequate quality assurance and quality control (for example, the integrity of the test reagents following transportation and storage), data management issues, a limited menu of detectable drugs offered, and lack of evidence that using POC devices improves patient outcomes when compared with laboratory testing. Training of users should include quality issues and recognition of any device limitations. In contrast to testing laboratories, POC devices purchased from a manufacturer might not include independent scientific support, although most manufacturers offer toll-free hotlines for consultation. For these reasons, physicians should evaluate POC devices carefully before using them routinely. Physicians should also use such devices with caution to prevent misinterpretation of the results because most tests do not provide numbers on sensitivity or specificity.


Generally, more definitive laboratory-based procedures–such as GC/MS, LC/MS, and LC-MS/MS are necessary to identify specific drugs in the following three circumstances.

  • To specifically confirm the presence of a given drug, for example, that morphine is the opiate causing a positive immunoassay response

  • To identify drugs not included in an immunoassay test

  • When results are contested comparing qualitative and quantitative screening



The Need for UDT

How UDT Screen Works

The Science of Urine

UDT Methods

Process of UDT

Commonly Abused Drugs

UDT Law in Florida

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