marijuana

Illicit drug abuse remains a serious public health issue. According to the 2013 National Survey on Drug Abuse and Health, an estimated 24.6 million Americans age 12 years and older were current illicit drug users—9.4% of the U.S. population. Marijuana was the most commonly abused illicit drug, followed by cocaine, heroin, and hallucinogens.

Federal guidelines define an adulterated specimen as a urine specimen containing either a substance that is not a normal constituent or an endogenous substance at a concentration that is not a normal physiological concentration. Pre-employment screening programs typically do not involve direct supervision of specimen collection, so employment candidates may attempt to cheat drug testing by adulterating specimens. This makes it essential for laboratories to identify pre-analytically any such adulterated specimens.

Ways of Cheating a Drug Test

Usually people try to cheat drug testing by three different ways: substituting their urine with synthetic urine or drug-free urine purchased from a clandestine source; drinking a commercially available product to flush out drugs; or adding an adulterant in vitro to the urine specimen after collection.

Synthetic urine is difficult to detect because it has similar pH, creatinine, and specific gravity to normal urine. Specific tests are needed to identify compounds that are normal constituents of human urine but not found in synthetic urine, such as cortisol. Commercially available products that adulterate urine or flush out drugs can be classified under two broad categories. The first includes fluids or tablets that, along with drinking large amounts of water, dilute urine. Common products are Absolute Detox XXL drink, Absolute Carbo Drinks, Ready Clean Drug Detox Drink, Fast Flush Capsules, and Ready Clean Gel Capsules.

The second category of products is in vitro urinary adulterants that are added to urine after collection. Examples include Stealth (peroxidase and peroxide), Klear (nitrite), Clean ADD-IT-ive (glutaraldehyde) and Urine Luck (pyridinium chlorochromate [PCC]). In addition, iodine is a strong oxidizing agent and may potentially destroy abused drugs, especially marijuana metabolites (2). Research also indicates that papain with intrinsic ester hydrolysis ability could significantly reduce the concentration of 11-nor-9-carboxy-9-tetrahydrocannabinol (THC-COOH), a metabolite of marijuana, if added to the urine specimen in vitro (3).

Household Chemicals as Urinary Adulterants

Would-be drug test cheaters might try adulterating their specimens with household chemicals, but most can be detected by specimen integrity testing. Both collection sites and laboratories have at their disposal a number of mechanisms to detect potentially invalid specimens. The temperature, for instance, should be within 90.5–98.9°F. The 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 cutoff. One common adulterant, sodium chloride, always produces a specific gravity greater than 1.035 if added at a concentration necessary to produce a false-negative result.

Unfortunately, specimen integrity testing doesn’t detect all adulterants. For example, it won’t pick up adulteration of urine with Visine eye drops, isopropanol, or other urinary adulterants. However, effective spot tests and special urine dipsticks are available (See Table 1).

Flushing, Detoxification Agents, and Diuretics

Flushing and detoxification agents are frequently advertised as effective means of passing drug tests. Many of these products contain caffeine or other diuretics to increase the output of urine, as well as sugar and natural or artificial flavoring agents. The objective is to produce diluted urine so that concentrations of abused drugs and or metabolites fall below the recommended cutoff concentrations.

Cone et al. evaluated the effect of excess fluid ingestion on false-negative marijuana and cocaine urine test results by studying the ability of Naturally Clean Herbal Tea, goldenseal root, and hydrochlorothiazide to cause false negative results. Volunteers drank one gallon of water, herbal tea, or took hydrochlorothiazide 22 hours after smoking marijuana cigarettes or intranasal administration of cocaine. Their creatinine levels dropped below the cutoff 2 hours after intake of excessive fluid. Marijuana and cocaine metabolite levels (as measured by both enzyme multiplied immunoassay technique [EMIT] and fluorescence polarization immunoassay [FPIA]) decreased significantly and frequently switched from positive to negative in subjects after consuming 2 quarts of fluid. Even excess water was effective in diluting a urine specimen to cause false negative results, although herbal tea diluted urine faster compared to water alone (5).

Using Spot Tests

When specimen integrity testing cannot detect an adulterated specimen, laboratories can employ a variety of effective spot tests.

• Urine Luck Wu et al. reported that the active ingredient of “Urine Luck” was PCC, a strong oxidizing agent, which at a concentration of 100 gm/L, caused significantly decreased response rate for all EMIT II drug screens, indicating the possibility of false-negative results. In contrast, for the Abbott Abuscreen test, only morphine and marijuana assays were affected, but a false-positive result was observed with the amphetamine assays. This adulteration of urine did not alter GC/MS confirmation of methamphetamine, benzoylecgonine, and phencyclidine, but apparent concentrations of opiates and THC-COOH were significantly reduced. Wu et al. also described a simple spot test using 1,5-diphenylcarbazide in methanol (10 gm/L) to detect the presence of PCC in urine, in which a reddish purple color developed in the presence of PCC (6). Moreover, adding a few drops of 3% household hydrogen peroxide solution to approximately 0.5 mL of urine specimen caused immediate development of a dark brown color and dark brown precipitate if PCC was present in the urine. As a strong oxidizing agent, PCC could also liberate iodine from potassium iodide solution in acidic medium (7). Notably, several other adulterants available online contain PCC. 

• Nitrite Containing Agents Products such as “Klear,” which contains potassium nitrite, can cause interference in GC/MS confirmation of THC-COOH. However, a bisulfite step at the beginning of sample preparation can eliminate this problem (8). Nitrite in urine may arise in vivo in patients receiving medications such as nitroglycerin, isosorbide dinitrate, and nitroprusside, or due to urinary tract infection. However, concentrations of nitrite usually are below 36 µg/mL in such specimens, while nitrite concentrations are 1,910–12,200 µg/mL in urine specimens adulterated with nitrite (9). Nitrite can be easily detected by simple spot tests. Addition of a few drops of a nitrite-adulterated urine specimen to 0.5 mL of 1% potassium permanganate solution, followed by addition of a few drops of 2N hydrochloric acid, turned the pink permanganate solution colorless with effervescence. Another spot test to detect nitrite used 1% potassium iodide solution. Adding a few drops of nitrite adulterated urine to 0.5 mL of potassium iodide solution, followed by addition of a few drops of 2N hydrochloric acid, resulted in immediate release of iodine from the colorless potassium iodide solution. If any organic solvent, such as hexane, was added the iodine was readily transferred in the organic layer giving the layer a distinct color of iodine (7). Nitrite could also be detected by diazotizing sulfanilamide and coupling the product with N-(1-napthyl) ethylenediamine. 

 • Stealth Stealth is an adulterant which consists of two vials, one containing a powder (peroxidase) and another vial containing a liquid (hydrogen peroxide), both added to the urine. Stealth is capable of invalidating immunoassay screening of THC-COOH, LSD, and opiates using both Roche ONLINE assays and Microgenics CEDIA assays if these drug or metabolites are present in modest concentrations (125–150% of cutoff values). In addition, GC/MS confirmation could be affected (10). Valtier and Cody described a rapid spot test to detect the presence of Stealth in urine. Addition of 10 µL of urine to 50 µL of tetramethylbenzidine working solution, followed by addition of 500 µL of 0.1 M phosphate buffer solution caused the specimen to turn dark brown. Peroxidase activity could also be monitored by using a spectrophotometer (11). Our investigation showed that if a few drops of a urine specimen adulterated with Stealth were added to potassium dichromate followed by a few drops of 2N hydrochloric acid, a deep blue color developed immediately, which usually faded with time. 

• Glutaraldehyde Glutaraldehyde containing products were one of the first that appeared in the market to invalidate drugs of abuse testing. Glutaraldehyde solutions are also available in hospitals and clinics as a cleaning agent. Glutaraldehyde at a concentration of 0.75% volume could lead to false-negative screening results for a cannabinoid test using the EMIT II drugs of abuse screen. Amphetamine, methadone, benzodiazepine, opiate, and cocaine metabolite tests are also affected at glutaraldehyde concentration between 1 and 2% using EMIT II immunoassays. Wu et al. described a simple fluorometric method for the detection of glutaraldehyde in urine. When 0.5 mL of urine was heated with 1.0 mL of 7.7 mmol/L potassium dihydrogen phosphate (pH 3.0) saturated with diethyl-thiobarbituric acid for 1 hour at 96–98°C in a heating block, a yellow green fluorophore developed if glutaraldehyde was present. Shaking the specimen with n-butanol resulted in the transfer of this adduct to the organic layer which could be viewed under long wavelength UV light. Glutaraldehyde in urine can also be estimated using a fluorometer (14). 

• Zinc Sulfate: A New Urinary Adulterant Although not widely used, zinc sulfate is an effective urinary adulterant that could invalidate all drug tests using EMIT assay. Currently there is no suitable method for detecting zinc sulfate in adulterated urine. Therefore, two rapid spot tests to detect the presence of zinc sulfate in urine were developed. Addition of 3–4 drops of 1N sodium hydroxide solution to approximately 1 mL of urine containing zinc sulfate resulted in formation of a white precipitate, which was soluble in excess sodium hydroxide. In the second spot test, addition of 3–4 drops of 1% sodium chromate solution to 1 mL of urine containing zinc sulfate followed by addition of 4–5 drops of 1N sodium hydroxide led to formation of a yellow precipitate (zinc chromate) (15).

Testing Urine Specimens for Adulterants

Specially designed urine dipsticks such as AdultaCheck 4, AdultaCheck 6, or Intect 7 can be used to detect many adulterants in urine. AdultaCheck 6 detects creatinine, oxidants, nitrite, glutaraldehyde, pH, and chromate. The Intect 7 test strip for checking adulteration in urine is composed of seven different pads to test for creatinine, nitrite, glutaraldehyde, pH, specific gravity, bleach, and PCC.

Guidelines from the Substance Abuse and Mental Health Services Administration require additional tests for urine specimens with abnormal physical characteristics or ones that show characteristics of an adulterated specimen during initial screening or confirmatory tests. A pH less than 3 or more than 11, and nitrite concentrations greater than 500 mg/mL indicate the presence of adulterants. A nitrite colorimetric test or a general oxidant colorimetric test should be performed to identify nitrite.

The presence of chromium (VI) in a urine specimen also is indicative of adulteration at a cutoff concentration of 50 mg/mL. The presence of chromium in a urine specimen could be confirmed by a chromium colorimetric test or a general test for the presence of oxidant. A confirmatory test should be performed using multi-wavelength spectrophotometry, ion chromatography, atomic absorption spectrophotometry, capillary electrophoresis, or inductively coupled plasma mass spectrometry.

Elemental halogens, such as pure bromine or iodine, can also be used as adulterants. The presence of these halogens should be confirmed by a halogen colorimetric test or a general test for the presence of oxidants. Confirmatory tests may employ multi-wavelength spectrophotometry, ion chromatography, atomic absorption spectrophotometer, capillary electrophoresis, or inductively coupled plasma mass spectrometry.

To detect glutaraldehyde, laboratories should use a general aldehyde test or the characteristic immunoassay response in one or more drug immunoassay tests for initial screening. Similarly, the presence of PCC should be confirmed using a general test for the presence of oxidant and a GC/MS confirmatory test. Finally, surfactant should be verified by using a surfactant colorimetric test with a greater than or equal to 100 mg/mL dodecyl benzene sulfonate equivalent cutoff.

Conclusion

It is essential for laboratories to detect adulterated urine in the pre-analytical step, as many adulterants invalidate immunoassay screening tests. Although routine specimen integrity tests can detect most of the household adulterants except Visine eye drops and alcohol/isopropanol, adulterants containing strong oxidizing agents such as potassium nitrite, pyridinium chlorochromate, or Stealth require a different approach. Spot tests, specially designed urine dipsticks, as well as more analytically sophisticated methods such as chromatographic methods, are available in the toxicology laboratory to identify these adulterants. If a urine specimen is adulterated it must be documented and reported, but no further testing is necessary.

References

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Amitava Dasgupta, PhD, DABCC, FACB, is a professor of pathology and laboratory medicine at the University of Texas-Houston Medical School. He is the medical director of laboratory services of TIRR-Memorial Hospital as well as director of clinical chemistry and point-of-care testing at Memorial-Hermann Hospital in Houston. He has published 209 peer-reviewed papers in various journals and is on the editorial board of five journals.
+EMail: Amitava.Dasgupta@uth.tmc.edu