The Initial Test
Every urine specimen must be analyzed using an initial test (i.e., an immunoassay test) that has been approved for commercial distribution as an in vitro diagnostic test by the Food and Drug Administration (FDA). A number of different immunoassay techniques are available to screen for the five drug classes [e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), kinetic interaction of microparticles in a solution (KIMS), and fluorescence polarization immunoassay (FPIA)].
The initial test is used to eliminate "negative" urine specimens from further consideration and to identify the presumptively positive specimens that require confirmation or further testing. A negative specimen is any specimen that contains no drug or whose apparent concentration of analyte is less than the cutoff concentration for that drug or drug class.
Some laboratories may conduct a second initial test prior to gas chromatography/mass spectrometry (GC/MS) confirmation in an effort to enhance the specificity of the assay. If the laboratory uses a second initial test to further identify a specimen as positive prior to GC/MS confirmation, the second initial test is subject to the same criteria and the Mandatory Guidelines requirements as the first initial test.
Immunoassays use antibodies to detect the presence of a drug or metabolite in urine. Antibodies are proteins that chemically bind with specific substances called antigens (i.e., a drug or drug metabolite). In immunoassay tests, a known amount of an antibody is added to the urine specimen. In addition, a known amount of labeled drug or drug metabolite (antigen) is added to the specimen. Any drug or drug metabolite present in the specimen will compete with the labeled drug or metabolite to bind with the antibodies forming antigen-antibody complexes. The amount of labeled antigen that is able to bind with an antibody is a function of the amount of drug or drug metabolite in the urine. Chemical spectrophotometric endpoints of these reactions are used to semi-quantitatively identify drugs and/or metabolites in each urine specimen.
The Validity Test
Specimen validity testing refers to testing conducted by a laboratory to identify any attempt to tamper with a specimen. This includes testing to identify adulteration (e.g., putting a substance into a specimen that is designed to mask or destroy the drug or drug metabolite that the specimen may contain or to adversely affect the assay reagent) or substitution (e.g., diluting a urine specimen with a liquid to effectively decrease the concentration of a drug below the cutoff concentration, or replacing a valid urine specimen with a Drug-Free specimen).
An extensive literature review was conducted to assist HHS and DOT in determining the normal ranges for the routine clinical measurements that could be conducted on urine specimens. Standards were developed as to what were forensically sound criteria for classifying urine specimens as substituted and adulterated.
The Confirmatory Test
All specimens identified as positive on the initial test must be confirmed positive using gas chromatography/mass spectrometry (GC/MS) before a positive result can be reported.
GC/MS is a combination of two different analytical techniques. Gas chromatography physically separates the various substances that have been extracted from a specimen (such as urine). Mass spectrometry is the technique used to provide a positive identification of substances that were separated by the gas chromatograph. In general, GC/MS analysis involves using a solid phase or solvent-solvent extraction procedure to extract a drug from most other components of a urine specimen, and after the extraction procedure the extract is injected into the GC/MS to perform the final separation, identification, and quantification of the specific drug or drug metabolite present in the urine specimen.
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