Chromogenic substrate to detect urine adulteration

Details Chromogenic substrate to detect urine adulteration Chromogenic substrate to detect urine adulteration

2001-02-27

2003-01-07

Leary, Louise N. (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving oxidoreductase

C435S004000, C435S283100, C435S970000, C436S111000

Reexamination Certificate

active

06503726

ABSTRACT:

FIELD OF THE INVENTION
The invention is directed generally to a composition and method that detects whether a urine sample submitted for drug testing contains an oxidant chemical adulterant.
BACKGROUND
Individuals may be requested or required to provide a urine sample that will be tested for the presence of drugs of abuse or metabolites of drugs of abuse. An initial or screening test is frequently performed first. A positive result is usually confirmed by a method different from that used for initial testing and usually having greater sensitivity and specificity. An initial negative test, however, is usually not confirmed. Thus, an individual who is fearful of a positive result in an initial screening test may alter his or her urine sample to prevent detection of the drug or drug metabolite.
One method of altering a urine sample is by diluting the sample so that the drug or drug metabolite concentration is below the detection threshold in a screening test. For example, water and/or saline may be added to the sample to dilute the drug or its metabolite to a concentration that is less readily detected by the screening test. To detect this type of alteration, the urine sample is frequently assayed to determine if physiological parameters such as creatinine concentration, pH, and specific gravity are within normal ranges, or if these parameters are abnormal due to the presence of diluent.
Chemical adulterants may be added to the sample to chemically convert a drug metabolite to a less detectable or non detectable product. Such chemicals include nitrite and chromate. The presence of chemical adulterants is more difficult to assess, since tests for the specific chemicals must be performed. For example, a group of adulterants has recently been developed to chemically modify 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (&Dgr;
9
THC), a metabolite of marijuana. These adulterants prevent recognition of &Dgr;
9
THC by drug screening and/or confirmatory assays, but do not otherwise affect the assays. These adulterants do not alter physical and/or chemical properties of the sample, such as pH and specific gravity, which are commonly monitored to detect urine adulteration.
As each new chemical adulterant is recognized and identified, tests are developed for identification of the specific adulterant. However, with the development of multiple adulterants, each of which is chemically distinct and each of which is capable of destroying or masking THC metabolites, the process of identifying adulterated urine samples becomes increasingly difficult. Multiple tests must be performed on each sample to assure detection of all chemically adulterated samples. Furthermore, there is a period of time for each adulterant during which samples containing that adulterant are not detected because the test-specific adulterant has not yet been identified and/or confirmed.
Thus, there is a need for a composition and method that detect a group of adulterants that are added to urine to prevent detection of marijuana use.
SUMMARY OF THE INVENTION
The invention is directed to compounds and methods that detect a group of adulterants in urine which are designed to oxidize, and thus prevent detection of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (&Dgr;
9
THC).
The invention is also directed to a device to detect adulteration of a biological sample with a chemical oxidant that prevents detection of marijuana use. The sample is contacted with a pad containing a substrate for an enzyme that forms a colored product in the presence of the oxidant. The pad may be affixed to a backing to form a test strip, and may have a sample application site. The chromogenic substrate on the pad may be 3,3′,5,5′-tetramethylbenzidine, diaminobenzidine, 3-amino-9-ethylcarbazone, 4-chloro-1-napthol, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, and ortho-phenylenediamine.
The invention is also directed to a method to detect an oxidant adulterant added by an individual to a urine sample to prevent detection of marijuana use by the individual. In the method, a sample is reacted with a chromogenic substrate for a peroxidase, then is monitored for production of a chromogen, which indicates that an oxidant adulterant was present in the sample. The oxidant chemical may be nitrite, such as potassium nitrite or sodium nitrite, or chromium VI such as pyridinium chlorochromate, or peroxidase and hydrogen peroxide (H
2
O
2
). The sample may be monitored on a test strip containing the chromogenic substrate, or by a clinical analyzer set at a wavelength to monitor production of the chromogen.
The invention will be further appreciated with reference to the following detailed description and examples.
DETAILED DESCRIPTION
The inventive compositions and methods detect members of the group of adulterants that chemically modify 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (&Dgr;
9
THC). Individuals adulterating urine samples with these chemicals likely appreciate that oxidized THC metabolites are not detected by routine screening tests such as immunoassays, and/or confirmatory tests such as analysis by gas chromatography/mass spectrometry (GC/MS). However, such oxidizing agents can be easily detected using standard reagents that react in colorimetric assays to detect peroxidase enzyme activity. These reagents, which are dyes, can either be used in solution or applied to a solid surface to detect the presence of oxidizing adulterants in human urine, such as adulterants added to evade detection of marijuana use.
Generally, a variety of urine adulterants are sold by entities which seek to aid drug abusers in evading detection by urine drug tests. Such adulterants are sold under trade names such as “Whizzies”®, “Klear”® and “Urine Luck”®, but the nature of the adulterant is not generally disclosed. A number of new adulterants have also been introduced via the Internet; the advent of each new adulterant provides a window of time during which users of the adulterant are not detected due to the lack of test methods. Moreover, the increasing number of adulterants has made detection of adulterated samples difficult and costly because of the need for a number of specific tests required to detect adulteration.
The most recent adulterants introduced have been those which prevent detection of the drug of abuse or its metabolite by chemical modification of the drug. These adulterants include sodium or potassium nitrite (Klear®) and pyridinium chlorochromate (Urine Luck®, whose active ingredient is hexavalent chromium (CrVI)). In each case, the new adulterant was designed to evade existing adulteration test methods, the nature of the adulterant was not divulged by the vendor, and identification of the adulterant required significant effort on the part of drug testing laboratories and/or manufacturers of tests to detect drugs of abuse.
Tests have been developed to detect nitrite as an adulterant. These tests are based on the well-known Griess reaction with sulfanilamide and napthylethylenediamine. Tests have also been developed to detect chromium (VI) as an adulterant, based on the reaction of chromium (VI) with 1,5-diphenylcarbazide. These tests have been developed commercially and are sold as Nitrite-Detects and Chromate-Detects assays, respectively (Microgenics Corp., Fremont, Calif.). Each of these tests is specific for the target analyte; for example, the Griess reaction does not detect chromium (VI), whereas 1,5-diphenylcarbazide does not react with nitrite. Samples which test positive by these screening methods are then confirmed for the presence of the drug and/or its metabolite by more specific assays such as ion chromatography, atomic absorption spectroscopy (AAS) or high performance liquid chromatography (HPLC).
Most recently, a new urine adulterant has become available which also acts to chemically modify &Dgr;
9
THC such that it is not detected by immunoassays and/or other confirmatory methods. This adulterant, termed “Stealth®,” is available via the Internet but its chemical identity is currently not widely known.

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