Cotinine Test Specifications

For Determination of Smoking Status Only.

The COT One Step Cotinine Test Device (Urine) is a lateral flow chromatographic immunoassay for the detection of Cotinine in human urine at a cut-off concentration of 200 ng/mL. This test will detect other related compounds, please refer to the Analytical Specificity table in this package insert.

This assay provides only a preliminary analytical test result. A more specific alternate chemical method must be used in order to obtain a confirmed analytical result. Gas chromatography and mass spectrometry (GC/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

Cotinine is the first-stage metabolite of nicotine, a toxic alkaloid that produces stimulation of the autonomic ganglia and central nervous system when in humans. Nicotine is a drug to which virtually every member of a tobacco-smoking society is exposed whether through direct contact or second-hand inhalation.  In addition to tobacco, nicotine is also commercially available as the active ingredient in smoking replacement therapies such as nicotine gum, transdermal patches and nasal sprays. In a 24-hour urine, approximately 5% of a nicotine dose is excreted as unchanged drug with 10% as cotinine and 35% as hydroxycotinine; the concentrations of other metabolites are believed to account for less than 5%.¹ While cotinine is thought to be an inactive metabolite, it’s elimination profile is more stable than that of nicotine which is largely urine pH dependent. As a result, cotinine is considered a good biological marker for determining nicotine use.  The plasma half-life of nicotine is approximately 60 minutes following inhalation or parenteral administration.² Nicotine and cotinine are rapidly eliminated by the kidney; the window of detection for cotinine in urine at a cutoff level of 200 ng/mL is expected to be up to 2-3 days after nicotine use. The COT One Step Cotinine Test Device (Urine) is a rapid urine screening test that can be performed without the use of an instrument. The test utilizes a monoclonal antibody to selectively detect elevated levels of Cotinine in urine. The COT One Step Cotinine Test Device (Urine) yields a positive result when the Cotinine in urine exceeds 200 ng/mL.

The COT One Step Cotinine Test Device (Urine) is an immunoassay based on the principle of competitive binding. Drugs which may be present in the urine specimen compete against the drug conjugate for binding sites on the antibody.  During testing, a urine specimen migrates upward by capillary action. Cotinine, if present in the urine specimen below 200 ng/mL, will not saturate the binding sites of antibody coated particles in the test device. The antibody coated particles will then be captured by immobilized Cotinine conjugate and a visible colored line will show up in the test line region. The colored line will not form in the test line region if the

Cotinine level exceeds 200 ng/mL because it will saturate all the binding sites of anti-Cotinine antibodies. A drug-positive urine specimen will not generate a colored line in the test line region because of drug competition, while a drug-negative urine specimen or a specimen containing a drug concentration less than the cut-off will generate a line in the test line region. To serve as a procedural control, a colored line will always appear at the control line region indicating that that proper volume of specimen has been added and membrane wicking has occurred.

The test device contains mouse monoclonal anti-Cotinine antibody-coupled particles and Cotinine-protein conjugate. A goat antibody is employed in the control line system.

• Do not use after the expiration date.

• The test device should remain in the sealed pouch until use.

• All specimens should be considered potentially hazardous and handled in the same manner as an infectious agent.

• The used test device should be discarded according to local regulations.

Store as packaged in the sealed pouch either at room temperature or refrigerated (2-30°C). The test device is stable through the expiration date printed on the sealed pouch. The test device must remain in the sealed pouch until use. DO NOT FREEZE.  Do not use beyond the expiration date.  The urine specimen must be collected in a clean and dry container. Urine collected at any time of the day may be used. Urine specimens exhibiting visible precipitates should be centrifuged, filtered, or allowed settle to obtain a clear supernatant for testing.

Urine specimens may be stored at 2-8°C for up to 48 hours prior to assay. For prolonged storage, specimens may be frozen and stored below -20°C. Frozen specimens should be thawed and mixed before testing.  

                                                      ATERIALS Materials Provided

• Test devices • Droppers • Package insert

• Specimen collection container  • Timer

Allow test device, urine specimen to reach room temperature (15-30°C) prior to testing.

1.Bring the pouch to room temperature before opening it. Remove the test device from the sealed pouch and use it as soon as possible.

2.Place the test device on a clean and level surface. Hold the dropper vertically and transfer 3 full drops of urine (approx. 100 µL) to the specimen well (S) of the test device, and then start the timer. Avoid trapping air bubbles in the specimen well (S). See the illustration below.

3.Wait for the colored line(s) to appear. The result should be read at 5 minutes. It is important that the background is clear before the result is read. Do not interpret the result after 10 minutes.

(Please refer to the illustration above)

NEGATIVE:* Two lines appear. One colored line should be in the control line region (C), and another apparent colored line should be in the test line region (T). This negative result indicates that the Cotinine concentration is below the detectable level (200 ng/mL).
*NOTE: The shade of color in the test line region (T) may vary, but it should be considered negative whenever there is even a faint line.
POSITIVE: One colored line appears in the control line region (C). No line appears in the test line region (T). This positive result indicates that the Cotinine concentration exceeds the detectable level (200 ng/mL).
INVALID: Control line fails to appear. Insufficient specimen volume or incorrect procedural techniques are the most likely reasons for control line failure. Review the procedure and repeat the test using a new test device. If the problem persists, discontinue using the lot immediately and contact your local distributor.

A procedural control is included in the test. A colored line appearing in the control line region (C) is considered an internal procedural control. It confirms sufficient specimen volume, adequate membrane wicking and correct procedural technique. 

1.The COT One Step Cotinine Test Device (Urine) provides only a qualitative, preliminary analytical result. A secondary analytical method must be used to obtain a confirmed result. Gas chromatography/mass spectrometry (GC/MS) is the preferred confirmatory method.^1,2

2.It is possible that technical or procedural            errors, as well as other interfering substances in the urine specimen may cause erroneous results.

3.Adulterants, such as bleach and/or alum, in urine specimens may produce erroneous results regardless of the analytical method used. If adulteration is suspected, the test should be repeated with another urine specimen.

4.A positive result indicates only that the presence of Cotinine is above the cut-off concentration.  It does not indicate or measure level of consumption.