I've been compiling information on chemical adulterants that can be added to urine to interfere with immunoassay based drug screens. A really good review article on this subject was published in late 2007. The article examines a number of commonly used commercial and OTC adulterants, their mechanism of action, specific drug metabolites that they interfere with, and the ease at which the testing lab can detect the presence of the adulterant.
The purpose of this post is two-fold; I want to share the information provided by this article, and I also want to ask the ADD community to help brainstorm other chemicals that would also be effective adulterants, AND that would not alter urine in a way that would make it obvious to the testing lab.
Article Ref: Dasgupta, A. 2007. The effects of adulterants and selected ingested compounds on drugs-of-abuse testing in urine. Am. J. Clin. Pathol. 128:491-503.
Background.
Modern urine drug testing guidelines require the use of a three step approach before a test result is considered positive. The initial step comprises a routine urinalysis that measures a number of physical properties of the urine sample to confirm the integrity of the sample, and to exclude certain disease processes that could alter test results. Once urine integrity has been confirmed, the second step is to conduct a screening test that checks for the presence of multiple drugs of abuse simultaneously, and typically yields a dichotomous result (e.g. positive or negative). Finally, a confirmation test is carried out to confirm if a positive is true or false, and/or to quantify the level of the drug present in the urine specimen.
The ideal place to interfere with the testing process lies in the second (screening) step. Interfering with the urine integrity is easily detectable (see below). The third (confirmatory) step uses GC/MS, and can detect parts per billion quantities of drug/drug metabolite. By maintaining sample integrity (step one) but interfering with sample screening (step two), there is no reason for the testing lab to continue on to GC/MS (very costly in comparison to the other steps).
Urinalysis - Sample integrity.
Routine urinalysis is carried out on all urine samples before proceeding to the drug screening test. A number of urine parameters are measured, but I'll simply provide details about the tests relevant to the drug screening process:
a) Specific Gravity - a measurement of the total number of dissolved solutes within a urine sample. Probably the most important part of routine urinalysis in drug testing -- it provides information about how concentrated/dilute urine is, and is the simplest way to tell if the urine sample has been diluted with an external source of water. Significant oral water consumption, while effective in diluting urine and decreasing drug concentration to below detectable thresholds, usually invalidates the sample due to the drop in specific gravity. Many labs will consider diluted urine to be a screening positive, and will lower the threshold of their detection cutoff limit to compensate for the degree of dilution. Normal urine specific gravity is between 1.010 and 1.030.
b) pH. Normal physiological urine pH is between ~4.5 and ~7.5. Addition of strong acids or strong bases to a urine sample is an excellent way to destroy drug metabolites -- but do so only at pH's outside the physiological range. While it is possible to say basify to pH 14, then back-titrate to physiological pH, I can't think of a way of doing this without drastically increasing the specific gravity (acid + base -> aqueous salt -> increased SG)
c) Creatinine and Urea – Main nitrogenous waste products of metabolism. Used to quantify kidney function, but also help to confirm that the urine sample is in fact urine.
It is important that any chemical adulterant that is used maintain the normal physiological properties of the urine. To recap: 1) Not increase or decrease the specific gravity, 2) maintain pH within physiological ranges, 3) not alter the concentration of urea or creatinine within the sample. If any of these parameters are interfered with, the sample can be altered
Immunoassay - Sample screening.
Immunoassays such as ELISA, EMIT, etc are the most commonly employed method of drug screening. Immunoassays use antibody molecules (IgG; Immunoglobulin G) that specifically bind the drug molecule in the sample. The general workup of these tests are as follows:
The urine is applied to wells that have been cut into a polyethylene plastic 'plate'. Each well contains a primary binding antibody that is specific for each drug being tested. If the drug is present in the urine, the molecule binds to the antibody in the well. The plate is washed with buffered saline, and any bound drug molecules remain in the wells, and everything else is washed away. A second antibody specific for the drug is then applied to the well. If the drug molecule is present in the well, this antibody binds as well, making a 'sandwich'. The secondary antibody is conjugated to an enzyme that can catalyze a reaction that produces a colour change when a specific chemical substrate is added. The substrate is added; if the drug molecule was added, a colour change appears (as the secondary antibody is present, as is the enzyme bound to it that catalyzes the reaction). If no drug molecule is present, there is no colour change.
There are four places in this assay that I've identified as having potential for chemical interference. Chemicals could be spiked into the urine sample that would do the following:
Mechanism #1) Destroy the drug/metabolite before handing off the urine sample. No drug/metabolite, no chance of positive.
Mechanism #2) Destroy the primary (binding) antibody in the first step of the immunoassay when the urine is added to the polyethylene wells. No binding antibody, no way for the drug/metabolite to bind.
Mechanism #3) Detach the binding antibody from the polyethylene well when the urine is added. Afiak, the binding antibody sticks to the polyethylene through electrostatic interactions, and not by some sort of chemical/covalent linkage.
Mechanism #4) Interfere chemically with the antibody-drug molecule binding so that unbound drug washes away unbound before the secondary antibody is added.
Chemical additives described that fit one of the above criteria:
a) Papain - A protease in papaya fruit that breaks down peptide bonds, including those in IgG, and can also degrade esters. In theory this should fit criteria #2, but the enzyme is too bulky to get into the right conformation to cleave the antibody bound to the polyethylene. However, papain rapidly cleaves
the ester bond in 11-nor-carboxyTHC and nordiazepam, and will cause a negative for THC/diazepam in most tests when added to the urine sample. PROs: Can't be detected by most labs. CONs: doesn't work for all tests, only works for a limited number of drugs.
b) Pyridinium chlorochromate (PCC). Common organic reagent for oxidation of hydroxy groups, and found in some commercial urine adulterants ("Urine Luck"). Mechanism #1; destroys the drug/metabolite. Works with low concentrations (2 mmol/L final conc. in urine). Provides false positives for THC and opiates (except between pH 5 and 7) but not amphetamines or cocaine. PROS: Works really, really well. CONS: Victim of its own success. The presence of PCC in urine is now screening for in many labs.
c) Potassium Nitrite. Common food additive. Chemical oxidizer. Decomposes THC metabolites providing false positives (Mech #1) . PROS: Can be found commercially ("KLEAR"). Urine can be tested for nitrite contamination, but many drugs and diseases (eg urinary tract infection) cause a nitrite presence in the urine normally. CONS: Takes 4-24 hours for complete degradation of THC. Only works for THC.
D) Hydrogen peroxide + Peroxidase. Oxidizer + enzyme that speeds up the oxidation process. Available in commercial products ("Stealth"). Degrades LSD and opiates (Mech. #1) . PROS: Works rapidly. Total destruction of drugs/metabolites (samples turn up GC/MS negative). Techniques to detect it are under development, but not widely used. CONS: Only works for low or moderate concentration of drugs in urine.
E) Glutaraldehyde. Disgustingly smelly toxic stuff used in medicine as a tissue fixative and disinfectant. Available OTC in some wart treatments. Denatures proteins, rendering antibodies unable to bind the drugs (Mech. PROS: Interferes with antibodies so basically gives false negatives for any drug being tested. CONS: Commonly tested for. Also, requires 1-2% concentration to work effectively, and it can be smelled at this conc.
F) Tetrahydrozoline. Alpha agonist found in most OTC eye drops. Destroys THC metabolites. PROS: No good technique for detecting its presence in urine. CONS: Only works for THC.
So now is the part where I ask you all to brainstorm
The majority of these adulterants work through mechanism #1 above, i.e. destroying the drug being tested. Imho the optimal adulterants are chemicals that interfere with the antibody rather than individual compounds, as they render the entire screening test useless. Remember the rules; it can't be a strong acid or base, react with creatinine or urea, or alter the specific gravity significantly.
Thanks guys!
The purpose of this post is two-fold; I want to share the information provided by this article, and I also want to ask the ADD community to help brainstorm other chemicals that would also be effective adulterants, AND that would not alter urine in a way that would make it obvious to the testing lab.
Article Ref: Dasgupta, A. 2007. The effects of adulterants and selected ingested compounds on drugs-of-abuse testing in urine. Am. J. Clin. Pathol. 128:491-503.
Background.
Modern urine drug testing guidelines require the use of a three step approach before a test result is considered positive. The initial step comprises a routine urinalysis that measures a number of physical properties of the urine sample to confirm the integrity of the sample, and to exclude certain disease processes that could alter test results. Once urine integrity has been confirmed, the second step is to conduct a screening test that checks for the presence of multiple drugs of abuse simultaneously, and typically yields a dichotomous result (e.g. positive or negative). Finally, a confirmation test is carried out to confirm if a positive is true or false, and/or to quantify the level of the drug present in the urine specimen.
The ideal place to interfere with the testing process lies in the second (screening) step. Interfering with the urine integrity is easily detectable (see below). The third (confirmatory) step uses GC/MS, and can detect parts per billion quantities of drug/drug metabolite. By maintaining sample integrity (step one) but interfering with sample screening (step two), there is no reason for the testing lab to continue on to GC/MS (very costly in comparison to the other steps).
Urinalysis - Sample integrity.
Routine urinalysis is carried out on all urine samples before proceeding to the drug screening test. A number of urine parameters are measured, but I'll simply provide details about the tests relevant to the drug screening process:
a) Specific Gravity - a measurement of the total number of dissolved solutes within a urine sample. Probably the most important part of routine urinalysis in drug testing -- it provides information about how concentrated/dilute urine is, and is the simplest way to tell if the urine sample has been diluted with an external source of water. Significant oral water consumption, while effective in diluting urine and decreasing drug concentration to below detectable thresholds, usually invalidates the sample due to the drop in specific gravity. Many labs will consider diluted urine to be a screening positive, and will lower the threshold of their detection cutoff limit to compensate for the degree of dilution. Normal urine specific gravity is between 1.010 and 1.030.
b) pH. Normal physiological urine pH is between ~4.5 and ~7.5. Addition of strong acids or strong bases to a urine sample is an excellent way to destroy drug metabolites -- but do so only at pH's outside the physiological range. While it is possible to say basify to pH 14, then back-titrate to physiological pH, I can't think of a way of doing this without drastically increasing the specific gravity (acid + base -> aqueous salt -> increased SG)
c) Creatinine and Urea – Main nitrogenous waste products of metabolism. Used to quantify kidney function, but also help to confirm that the urine sample is in fact urine.
It is important that any chemical adulterant that is used maintain the normal physiological properties of the urine. To recap: 1) Not increase or decrease the specific gravity, 2) maintain pH within physiological ranges, 3) not alter the concentration of urea or creatinine within the sample. If any of these parameters are interfered with, the sample can be altered
Immunoassay - Sample screening.
Immunoassays such as ELISA, EMIT, etc are the most commonly employed method of drug screening. Immunoassays use antibody molecules (IgG; Immunoglobulin G) that specifically bind the drug molecule in the sample. The general workup of these tests are as follows:
The urine is applied to wells that have been cut into a polyethylene plastic 'plate'. Each well contains a primary binding antibody that is specific for each drug being tested. If the drug is present in the urine, the molecule binds to the antibody in the well. The plate is washed with buffered saline, and any bound drug molecules remain in the wells, and everything else is washed away. A second antibody specific for the drug is then applied to the well. If the drug molecule is present in the well, this antibody binds as well, making a 'sandwich'. The secondary antibody is conjugated to an enzyme that can catalyze a reaction that produces a colour change when a specific chemical substrate is added. The substrate is added; if the drug molecule was added, a colour change appears (as the secondary antibody is present, as is the enzyme bound to it that catalyzes the reaction). If no drug molecule is present, there is no colour change.
There are four places in this assay that I've identified as having potential for chemical interference. Chemicals could be spiked into the urine sample that would do the following:
Mechanism #1) Destroy the drug/metabolite before handing off the urine sample. No drug/metabolite, no chance of positive.
Mechanism #2) Destroy the primary (binding) antibody in the first step of the immunoassay when the urine is added to the polyethylene wells. No binding antibody, no way for the drug/metabolite to bind.
Mechanism #3) Detach the binding antibody from the polyethylene well when the urine is added. Afiak, the binding antibody sticks to the polyethylene through electrostatic interactions, and not by some sort of chemical/covalent linkage.
Mechanism #4) Interfere chemically with the antibody-drug molecule binding so that unbound drug washes away unbound before the secondary antibody is added.
Chemical additives described that fit one of the above criteria:
a) Papain - A protease in papaya fruit that breaks down peptide bonds, including those in IgG, and can also degrade esters. In theory this should fit criteria #2, but the enzyme is too bulky to get into the right conformation to cleave the antibody bound to the polyethylene. However, papain rapidly cleaves
the ester bond in 11-nor-carboxyTHC and nordiazepam, and will cause a negative for THC/diazepam in most tests when added to the urine sample. PROs: Can't be detected by most labs. CONs: doesn't work for all tests, only works for a limited number of drugs.
b) Pyridinium chlorochromate (PCC). Common organic reagent for oxidation of hydroxy groups, and found in some commercial urine adulterants ("Urine Luck"). Mechanism #1; destroys the drug/metabolite. Works with low concentrations (2 mmol/L final conc. in urine). Provides false positives for THC and opiates (except between pH 5 and 7) but not amphetamines or cocaine. PROS: Works really, really well. CONS: Victim of its own success. The presence of PCC in urine is now screening for in many labs.
c) Potassium Nitrite. Common food additive. Chemical oxidizer. Decomposes THC metabolites providing false positives (Mech #1) . PROS: Can be found commercially ("KLEAR"). Urine can be tested for nitrite contamination, but many drugs and diseases (eg urinary tract infection) cause a nitrite presence in the urine normally. CONS: Takes 4-24 hours for complete degradation of THC. Only works for THC.
D) Hydrogen peroxide + Peroxidase. Oxidizer + enzyme that speeds up the oxidation process. Available in commercial products ("Stealth"). Degrades LSD and opiates (Mech. #1) . PROS: Works rapidly. Total destruction of drugs/metabolites (samples turn up GC/MS negative). Techniques to detect it are under development, but not widely used. CONS: Only works for low or moderate concentration of drugs in urine.
E) Glutaraldehyde. Disgustingly smelly toxic stuff used in medicine as a tissue fixative and disinfectant. Available OTC in some wart treatments. Denatures proteins, rendering antibodies unable to bind the drugs (Mech. PROS: Interferes with antibodies so basically gives false negatives for any drug being tested. CONS: Commonly tested for. Also, requires 1-2% concentration to work effectively, and it can be smelled at this conc.
F) Tetrahydrozoline. Alpha agonist found in most OTC eye drops. Destroys THC metabolites. PROS: No good technique for detecting its presence in urine. CONS: Only works for THC.
So now is the part where I ask you all to brainstorm
The majority of these adulterants work through mechanism #1 above, i.e. destroying the drug being tested. Imho the optimal adulterants are chemicals that interfere with the antibody rather than individual compounds, as they render the entire screening test useless. Remember the rules; it can't be a strong acid or base, react with creatinine or urea, or alter the specific gravity significantly.Thanks guys!
