Urinary adulterants and drugs-of-abuse testing

In the U.S., amphetamines, cocaine, cannabinoids, opiates, barbiturates, benzodiazepines, methadone, methoqualone and phencyclidine (PCP) are the most widely abused drugs. The windows of detection of these drugs/metabolites, as well as the sensitivities of their screening immunoassays, are given in

A negative urine drug test does not mean that the person who donated the urine has never abused any drug. If the drug exposure occurred before the drugs window of detection, the test may be negative, or the concentration of drug may be so low that it is below the tests sensitivity and thus cannot be positively identified. Designer drugs like 3,4-methylenedioxyamphetamine (MDA) and 3.4-methylenedioxymethamphetamine (MDMA, Ecstasy) are also abused. 

Screening and confirmation of abused drugs

Immunoassays are available for quick screening of abused drugs in urine. The most commonly screened drugs are amphetamines, opiates, benzodiazepines, cocaine, methadone, barbiturates, methaqualone, PCP, tetrahydrocannabinol (THC) and propoxyphene. Usually, positive drug screen results are confirmed by GC/MS. 

Federal guidelines for cutoff limits and other issues

On Sept. 15, 1986, President Reagan issued Executive Order No. 12564 which directed federal agencies to achieve a drug-free work environment. Each agency was directed to develop criteria for drugs-of-abuse testing. The Department of Health and Human Services (DHHS, formerly NIDA), assumed the responsibility for developing guidelines for drugs-of-abuse testing. The federally mandated testing process includes the proper collection of the urine specimen, the chain of custody procedure and specimen analysis. Urine sample testing, performed by a Substance Abuse and Mental Health Services Administration (SAMHSA) certified laboratory, begins with screening for illicit drugs by an FDA-approved immunoassay. Confirmation, if needed, is done by GC/MS. The SAMHSA positive cutoff values for urine drugs-of-abuse testing are also given in Table 1.¹ 

Drug testing programs in the U.S are either federally mandated or nonmandated. In the first group (e.g., Department of Transportation), the employer is required by federal regulation to drug test its employees. In the second category, employers choose to test for reasons other than the federal requirements. Some private employers that are not mandated to test under federal authority have instituted employee drug testing programs to create drug-free workplaces. Both programs also formalized the role of the medical review officer (MRO), a specialist who is an integral part of a drug testing program, determines the cause of positive drug test results (interference, other prescription drugs, etc.) and counsels the employee.

Common adulterants used to mask drug testing

Accurate screening immunoassay results are very important in drugs-of-abuse testing because, if the immunoassay screen is negative, the GC/MS confirmation step is not performed. People try to undermine their drug tests by adding adulterants to their urine specimens. Several adulterants can cause false negative results in drug tests by interfering with the screening immunoassays. Common adulterants use to mask drug testing are as follows:^2,3

1. Table salt (sodium chloride).

2. Household vinegar (acetic acid).

3. Liquid laundry bleach (sodium
hypochlorite).

4. Concentrated lemon juice.

5. Goldenseal tea (produces dark urine).

6. Diluted urine (creatinine below 15
mg/mL).

7. Visine eye drops (interfere mostly with EMIT assay).

Household vinegar and concentrated lemon juice, which make the urine acidic, can be easily detected by checking pH of the specimen. Table salt increases specific gravity of urine. The presence of Visine eye drops in urine cannot be detected by specimen integrity testing. 

• Amphetamines: Sodium chloride at concentration of 75 g/L caused a false negative EMIT result in urine containing 1,420 ng/mL of amphetamine. Drano, at a concentration of 18 mL/L masked amphetamines present at a concentration of 1,800 ng/mL in a urine specimen analyzed by the EMIT assay. 
• Barbiturates: Sodium chloride, liquid hand soap and Drano masked barbiturates concentrations up to 1,450
  ng/mL. 
• Benzodiazepines: Visine, hand soap and Drano caused false negative tests with benzodiazepines at concentrations less than 6,500
  ng/mL. 
• Cocaine: Drano and sodium chloride mask cocaine screens at benzoylecgonine concentrations up to 1,180 ng/mL. 
• Opiates: Drano and sodium chloride interfered with opiate immunoassays. Urine samples with opiate concentrations up to 2,700 ng/mL tested negative in the presence of 125 mL/L of Drano. Sodium chloride interfered when opiate concentrations were below 780 ng/mL. 
• Marijuana: Sodium chloride, Drano, soap, Goldenseal tea and vinegar all interfered with this immunoassay (negative interference). The presence of this drug can be masked by these adulterants.²

Although the fluorescence polarization immunoassay (FPIA) is less subject to interference from adulterants when compared to the EMIT assay, some interferences have been reported. Though sodium chloride caused negative interferences with all drugs tested by EMIT, it caused a slight decrease in measured concentrations of benzodiazepines by FPIA. Sodium bicarbonate caused false positive with the EMIT opiate assay and PCP assay by FPIA. Hydrogen peroxide caused false positive benzodiazepine result by FPIA.³

Both the collection site and laboratory have a number of mechanisms to detect potentially invalid urine specimens. The temperature of the urine specimen immediately following collection should be 90.5-98.9F. The urine specific gravity should be between 1.005-1.030 and pH should be between 4.0-10.0. The creatinine concentration should be 20-400 mg/dL. However, some drug testing laboratories consider a creatinine concentration of 15 mg/dL as the lower end of cutoff concentration. Adulteration with sodium chloride at a concentration necessary to produce false negative immunoassay results always produces a specific gravity over 1.035.

Adulterants like Urine Luck, UrinAid, Klear and Whizzies can also mask the presence of illicit drugs in urines. The presence of these compounds in urine cannot be detected by the routine specimen integrity tests.⁴ (See Table 2.) 

Table 1.

Adulteration of urine by Urine Luck

Wu, et al. reported that the active ingredient of Urine Luck is 200 mmol/L of pyridinium chlorochromate (PCC), which causes a decrease in response rate for all EMIT II drug screens and for the Abuscreen morphine and THC assays. In contrast, the Abuscreen amphetamine assay produced a higher response rate in the presence of PCC, and PCC had no effect on the Abuscreen results of benzoylecgonine and PCP. PCC adulteration of urine did not alter the GC/MS confirmation of methamphetamine, benzoylecgonine and PCP. However, apparent concentrations of opiates and THC were reduced.⁵

Spot tests for detecting PCC in urine

Spot test 1

Wu, et al. also described the spot test protocol for detection of PCC in urine. The indicator solution contains 10 gm/L of 1, 5-diphenylcarbazide in methanol. The indicator, which detects the presence of chromium ion, is colorless when prepared. Two drops of indicator solution, when added to 1.0 mL of urine, produces a reddish-purple color in the presence of PCC and thus indicates a positive test.⁵ 

Spot test 2

• Stock solution: 1 percent potassium iodide in distilled water.
• Procedure:

1. In a test tube add approximately 200 mL (approximately six to seven drops from a transfer pipette) of stock potassium iodide solution. 
2. Add about 100 mL (approximately three to four drops) of urine specimen suspected of PCC adulteration.
3. Add two drops of 2N hydrochloric acid. There is an immediate release of iodine from the colorless potassium iodide solution if PCC is present in the urine. Shaking of this solution with n-butanol results in the transfer of iodine in the organic phase. If no nitrite is present, the potassium iodide solution remains colorless. No interference is seen in the presence of high glucose or ketone bodies.⁴

Spot test 3

Addition of four to five drops of 3 percent hydrogen peroxide to approximately 200 mL (approximately six to seven drops from a transfer pipette) of urine adulterated with PCC caused rapid formation of a dark brown color due to reduction of heptavalent chromium by hydrogen peroxide, which became a dark brown precipitate upon standing. Unadulterated urine turned colorless after addition of hydrogen peroxide.⁴

Nitrite adulteration of urine

Klear is a widely available commercial adulterant that is marketed for clearing all positive drug tests results from urine. This product, packaged in two tubes containing 500 mg white crystalline material, readily dissolves in urine with no change in the color or temperature of the urine. This product may cause false negative results in the GC/MS confirmation for marijuana. ElSohly first reported this product as potassium nitrite and provided evidence that nitrite leads to the decomposition of 9-THC ions and its internal standard. The authors further reported that using a bisulfite sample pretreatment step⁶ could eliminate this interference. 

ElSohlys research group further investigated the effect of nitrite on immunoassay screening of other drugs, including cocaine metabolites, morphine, THC metabolites, amphetamine and phencyclidine. Nitrite at a concentration of 1.0 M had no effect on the Abuscreen assay. At a higher nitrite concentration, the amphetamine assay becomes more sensitive and THC assay becomes less sensitive. The GC/MS analysis of benzoylecgonine, morphine, amphetamine and phencyclidine were not affected by the addition of Klear, while recovery of THC metabolite was significantly reduced. This interference can be eliminated by bisulfite treatment.⁷

Whizzies, which also contains potassium nitrite, is available on the Internet. Nitrite in urine may arise in vivo from bacterial infections and is also found in urine in low concentrations. In urine specimens cultured positive for microorganisms, the concentration of nitrite was below 36 mg/mL. Patients receiving medications, such as nitroglycerine, isosorbide dinitrate, nitroprusside and ranitidine, may have increased nitrite levels in their blood. Nitrite concentrations were below 36 mg/mL in specimens cultured positive for microorganisms, and nitrite concentrations were below 6 mg/mL in patients receiving medications that are metabolized to nitrite. In urine specimens adulterated with nitrite, nitrite concentrations were 1,910-12,200 mg/ml.⁸ The authors analyzed nitrite concentrations in urine utilizing a Lachat QuickChem AT automated continuous flow analyzer (Lachat Instruments, Milwaukee, WI) using a protocol approved by the U.S Environmental Protection Agency.

Spot tests for nitrite 

Spot test 1 

• Stock solution: 2 percent potassium permanganate in distilled water, 2 N hydrochloric acid.
• Procedure:

1. In a test tube add about 200 mL (approximately six to seven drops from a transfer pipette) of stock potassium permanganate solution.
2. Add about 100 mL (approximately three to four drops) of urine specimen suspected of nitrite adulteration.
3. Add two drops of 2N hydrochloric acid.

If nitrite is present, the pink permanganate solution turns colorless with effervescence immediately after addition of the hydrochloric acid. This is due to reduction of the heptavalent manganese ion of potassium permanganate by nitrite. The presence of very high glucose in urine (glucose > 1,000 mg/dL) and ketone bodies may cause false positive results, with the solution turning colorless after nitrite is present, the solution turns colorless immediately.⁴

Spot test 2 

• Stock solution: 1 percent potassium iodide in distilled water.
• Procedure:

1. In a test tube add approximately 200 mL (approximately six to seven drops from a transfer pipette) of stock potassium iodide solution. 
2. Add about 100 mL (approximately three to four drops) of urine specimen suspected of nitrite adulteration.
3. Add two drops of 2N hydrochloric acid. If nitrite is present in the urine, there is an immediate release of iodine from the colorless potassium iodide solution. Shaking of this solution with n-butanol resulted in the transfer of iodine in the organic phase. If no nitrite is present, the potassium iodide solution remains colorless. No interference is seen from the presence of high glucose or ketone bodies.⁴

Glutaraldehyde as an adulterant to urine

Glutaraldehyde can mask the presence of illicit drugs in urine.⁹ This product, available under the trade name of UrinAid, is manufactured by Byrd Laboratories (Topanga, CA) and sold for $20 to $30 per kit. Each kit contains 5 mL solution of glutaraldehyde. Additionally, a 10 percent solution of glutaraldehyde is available from pharmacies as over-the-counter medication for the treatment of warts. Glutaraldehyde at a concentration of 0.75 percent to 2 percent by volume can lead to false negative screening results on the EMIT II drugs-of-abuse screening assay. The assay for cocaine (as benzoylecgonine) was mostly affected.

AdultaCheck 4 Test strips for detection of urine adulteration

AdultaCheck 4 test strips and AdultaCheck level 1 and 2 controls (Chimera Research and Chemical Inc., Tampa, FL) can be used to detect common adulterants in urine. The strips consist of four individual tests for creatinine, nitrite, glutaraldehyde and pH. Creatinine testing is used to determine if a specimen is diluted. A nitrite pad checks for the presence of commercial adulterants containing nitrite (Klear and Whizzies). The nitrite levels detected by this method are well above the nitrite levels found in urinary tract infections and from medication use. 

The glutaraldehyde pad detects glutaraldehyde (Urine Luck). A pH over 10 is indicative of household bleach or Drano. King reported that AdultaCheck 4 is an excellent way to detect contamination in the urine specimen.¹⁰ Recently, Peace and Tarnai evaluated three on-site adulterant detection devices for specimen integrity checks of urine specimens submitted for drugs-of-abuse testing. Intect7 simultaneously tests creatinine, nitrite, glutaraldehyde, pH, specific gravity, pyridinium chlorochromate (PCC) and bleach. Mask Ultra Screen checks for creatinine, nitrite, glutaraldehyde and pH. AdultaCheck 4 tests for the presence of creatinine, nitrite, glutaraldehyde and pH. The authors concluded that Intect7 is most sensitive in detecting adulterants like Stealth, Urine Luck (PCC), Clean ADD-IT-ive (contains glutaraldehyde) and Klear (contains nitrite).¹¹

Conclusions

Adulterants impose a new challenge in the testing for abused drugs. Routine specimen integrity testing involving pH, creatinine, specific gravity and temperature is not adequate to detect the presence of recently introduced adulterants like Urine Luck, Klear and Stealth. These agents can cause false negatives in the immunoassay screening step. Because GC/MS confirmation is only done in immunoassay positive specimens, true presence of a drug can be missed if these newly introduced agents are used for adulteration. Fortunately, spot tests have been introduced, and several strip tests are available for further validation of specimen integrity. A detail specimen integrity check can be done using these spot tests or new test strips in specimens submitted for pre-employment screen or other sensitive cases where the person has a strong motivation to beat the drug test.

Dr. Amitava Dasgupta is a member of the Department of Pathology and Laboratory Medicine at the University of Texas-Houston Medical School in Houston.

References

1. Dasgupta A. Drugs of abuse analysis. In: Meyers RA ed. Encyclopedia of analytical chemistry Vol 2. Baffins Lane, Chichester: John Wiley & Sons Ltd. 2000:1238-1257.

2. Mikkelsen SL, Ash O. Adulterants causing false negative in illicit drug test. Clin Chem. 1988: 34; 2333-2336.

3. Warner A. Interference of household chemicals in immunoassay methods for drugs of abuse. Clin Chem. 1989: 35; 648-651.

4. Dasgupta A, Wahed A, Wells A. Rapid spot test for detecting the presence of adulterants in urine specimens submitted for drug testing. Am J Clin Pathol. 2002; 117: 325-329.

5. Wu A, Bristol B, Sexton K, Cassella-McLane G, Holtman V, Hill DW. Adulteration of urine by Urine Luck. Clin Chem. 1999: 45; 1051-1057.

6. ElSohly MA, Feng S, Kopycki WJ, Murphy TP, Jones AB, Davis A, Carr D. A procedure to overcome interferences caused by adulterant Klear in the GC-MS analysis of 11-nor-D9-THC-9-COOH. J Anal Toxicol. 1997: 20; 240-242. 

7. Tsai SC, ElSohly MA, Dubrovsky T, Twarowska B, Towt J, Salamone SJ. Determination of five abused drugs in nitrite-adulterated urine by immunoassay and gas chromatography-mass spectrometry. J Anal Toxicol. 1998: 22: 474-480.

8. Urry F, Komaromy-Hiller G, Staley B, Crockett D, Kushnir M, Nelson G, Struempler R. Nitrite adulteration of workplace drug testing specimens: sources and associated concentrations of nitrite and distinction between natural sources and adulteration. J Anal Toxicol. 1998: 22; 89-95.

9. George S, Braithwaite RA. The effect of glutaraldehyde adulteration of urine specimens on Syva EMIT II drugs of abuse assay. J Anal Toxicol. 1996: 20; 195-196.

10. King EJ. Performance of AdultaCheck 4 test stripes for the detection of adulteration at the point of collection of urine specimens used for drugs of abuse testing. J Anal Toxicol. 1999: 23; 72.

11. Peace MR, Tarnai LD. Performance evaluation of three on-site adulterant detection devices for urine specimens. J Anal Toxicol. 2002; 26: 464-470.
February 2003: Vol. 35, No. 2

© 2003 Nelson Publishing, Inc. All rights reserved.