Clinical Chemistry / SPOT TESTS TO DETECT ADULTERANTS IN URINE SPECIMENS

Rapid Spot Tests for Detecting the Presence of Adulterants in Urine Specimens Submitted for Drug Testing
Amitava Dasgupta, PhD, Amer Wahed, MD, and Alice Wells, MT(ASCP)
Key Words: Urine adulteration; Spot tests; Nitrite; Pyridinium chlorochromate

Abstract
Several adulterants are used to mask tests for abused drugs in urine. Adulterants such as Klear and Whizzies contain potassium nitrite, and Urine Luck contains pyridinium chlorochromate (PCC). The presence of these adulterants cannot be detected by routine specimen integrity checks (pH, specific gravity, and temperature). We developed rapid spot tests for detecting these adulterants in urine. Addition of 3% hydrogen peroxide in urine adulterated with PCC caused rapid formation of a dark brown color. In contrast, unadulterated urine turned colorless when hydrogen peroxide was added. When urine contaminated with nitrite and 2 to 3 drops of 2N hydrochloric acid were added to 2% aqueous potassium permanganate solution, the dark pink permanganate solution turned colorless immediately with effervescence. Urine contaminated with nitrite liberated iodine from potassium iodide solution in the presence of 2N hydrochloric acid. Urine adulterated with PCC also liberated iodine from potassium iodide in acid medium but did not turn potassium permanganate solution colorless. Urine specimens from volunteers and random urine samples that tested negative for drugs did not cause false-positive results. These rapid spot tests are useful for detecting adulterated urine to avoid falsenegative drug tests.

Drugs of abuse continue to be a serious problem in the United States. Workplace urine drug testing is an important step by many employers in the public and private sectors to ensure a drug-free work environment. Urine is the preferred specimen for the testing for abused drugs because the collection process is noninvasive. Moreover, drug metabolites can be detected for a longer time in urine than in serum. Immunoassay results are very important in the testing for abused drugs because if the immunoassay screen is negative, the gas chromatographymass spectrometry (GC/MS) confirmation step is not performed. Employees who test positive in workplace drug testing may experience untoward consequences, including dismissal from the job. As a result, some people attempt to escape detection of abused drugs by adding adulterants to urine.1 Some in vitro adulterants can invalidate drug testing by interfering with the immunoassay detection system, or they may chemically modify the drug metabolite to a compound not recognized by the antibody of the immunoassay.1 Several adulterants, including table salt, vinegar, liquid laundry bleach, concentrated lemon juice, Golden Seal tea, and Visine eyedrops are used to invalidate a drug test.2,3 Fortunately, all these compounds except Visine eyedrops can be detected during urine specimen integrity tests (creatinine, pH, temperature, and specific gravity). Commercial products are available to help individuals pass a drug test. Products like Klear and Whizzies contain potassium nitrite, and the presence of such adulterants in urine cannot be detected by routine specimen integrity tests. Klear comes in 2 microtubes containing 500 mg of white crystalline material, which readily dissolves in urine with no change in color or temperature of the urine. This adulterant can successfully mask the presence of tetrahydrocannabinol (THC) in
Am J Clin Pathol 2002;117:325-329
325

American Society for Clinical Pathology

Dasgupta et al / SPOT TESTS TO DETECT ADULTERANTS IN URINE SPECIMENS

urine in both immunoassay screening and GC/MS confirmation, causing a false-negative result and permitting the person to pass a drug test despite smoking marijuana.4,5 However, GC/MS confirmation of benzoylecgonine, morphine, amphetamine, and phencyclidine (PCP) was not affected by nitrite adulteration. Another product in aiding a person to beat a drug test is Urine Luck. Wu et al6 identified the active ingredients of Urine Luck as pyridinium chlorochromate (PCC). This adulterant can cause a decrease in response rate for all abused drugs in the EMIT II immunoassays (Dade-Behring, Deerfield, IL). Moreover, the recovery of opiates and THC also was reduced substantially in the GC/MS confirmation.6 Addition of Urine Luck to urine caused darkening of the yellow color, but not sufficiently to cause suspicion of the presence of adulterants. Moreover, presence of the active component of Urine Luck cannot be detected by routine specimen integrity checks. We developed rapid spot tests to detect the presence of these adulterants in urine and report our findings.

pipette) caused rapid formation of a dark brown color, and a dark brown precipitate appeared after the specimen was allowed to stand for 30 to 60 seconds. Spot Test 2 The stock solution was 1% potassium iodide in distilled water, and the procedure was as follows: 1. Place approximately 200 L of stock potassium iodide solution (approximately 6-7 drops from a transfer pipette) in a test tube. 2. Add 100 L of a urine specimen (approximately 3-4 drops) suspected of PCC adulteration to the same test tube. 3. Add 2 drops of 2N hydrochloric acid to the same test tube. Immediate release of iodine from the colorless potassium iodide solution was observed owing to the presence of PCC in the urine. Shaking of this solution with n-butanol resulted in the transfer of iodine in the organic phase. If no PCC was present in the urine, the potassium iodide solution remained colorless. Spot Tests for Nitrite Spot Test 1 The stock solution was 2% potassium permanganate in distilled water and 2N hydrochloric acid. The procedure was as follows: 1. Place 200 L of stock potassium permanganate solution (approximately 6-7 drops from a transfer pipette) in a test tube. 2. Add 100 L of a urine specimen (approximately 3-4 drops) suspected of nitrite adulteration to the same test tube. 3. Add 2 drops of 2N hydrochloric acid. The pink permanganate solution turns colorless with effervescence immediately after addition of hydrochloric acid if nitrite is present. Spot Test 2 The stock solution was 1% potassium iodide in distilled water. This spot test is the same as spot test 2 for detecting the presence of PCC in urine. To study the interference of high glucose and ketone bodies with these spot tests, we used several urine specimens known to contain high amounts of ketone bodies and glucose as screened in routine urinalysis by dipsticks.

Materials and Methods

Potassium nitrite and PCC were purchased from Sigma Chemical (St Louis, MO). Potassium permanganate, potassium iodide, 3% hydrogen peroxide and n-butanol were obtained from Aldrich Chemical (Milwaukee, WI). Drugsof-abuse urine controls (SENTRY) were obtained from J and S Medical Associates (Framingham, MA). Klear and Urine Luck were bought from Internet sites. Some of the urine specimens used for this study were submitted routinely to our laboratory for drugs-of-abuse analysis. We used leftover specimens after performing and reporting tests to the ordering physicians. These specimens otherwise would have been discarded. We also used urine specimens from volunteers who have no history of abusing drugs. For preparing adulterated urine, we added PCC or potassium nitrite in proportion to the urine volume as reported in the literature.4,6 We also used Klear and Urine Luck for adulteration of urine specimens. We added PCC or potassium nitrite to urine control samples for drug-of-abuse testing and compared screening results with unadulterated controls using an immunoassay. We used Abuscreen (Abbott) and an AxSYM analyzer (Abbott Laboratories, Abbott Park, IL) for the screening for abused drugs in urine. Spot Tests for Detecting PCC in Urine Spot Test 1 Addition of 4 to 5 drops (approximately 150 L) of 3% hydrogen peroxide to approximately 200 L of urine adulterated with PCC (approximately 6-7 drops from a transfer
326

Results
The addition of 4 to 5 drops 3% hydrogen peroxide solution in an aliquot of approximately 200 L of urine adulterated with PCC turned the urine dark green and then dark
American Society for Clinical Pathology

Am J Clin Pathol 2002;117:325-329

Clinical Chemistry / ORIGINAL ARTICLE

Table 1 Effect of the Urine Adulterant Pyridinium Chlorochromate (PCC, Urine Luck) on the Fluorescence Polarization Immunoassay Screen for Drugs of Abuse*
Drug Concentration, ng/mL (Semiquantitative) Specimen Control 1 Amphetamine 1,249 Positive Cocaine 338 Positive 378 Positive 336 Positive 401 Positive 353 Positive 860 Positive 908 Positive 911 Positive 829 Positive 933 Positive Opiate 402 Positive 102 Negative 39 Negative 39 Negative 37 Negative 990 Positive 847 Positive 718 Positive 54 Negative 39 Negative PCP 39 Positive 42 Positive 36 Positive 39 Positive 42 Positive 101 Positive 102 Positive 103 Positive 98 Positive 102 Positive Cannabis 64 Positive 21 Negative 12 Negative 10 Negative 8 Negative >135 Positive 53 Positive 43 Negative 22 Negative 12 Negative Barbiturates Benzoylecgonine Methadone 303 Positive 241 Positive 226 Positive 259 Positive 233 Positive 1,086 Positive 1,016 Positive 983 Positive 839 Positive 930 Positive 235 Positive 213 Positive 217 Positive 219 Positive 206 Positive 930 Positive 932 Positive 956 Positive 923 Positive 866 Positive 513 Positive 502 Positive 516 Positive 503 Positive 518 Positive 991 Positive 1,020 Positive 1,100 Positive 1,084 Positive 1,034 Positive

Plus 50 mg/mL PCC 2h 1,236 Positive 6h 1,378 Positive Plus 100 mg/mL PCC 2h 1,228 Positive 6h 1,229 Positive Control 2 1,864 Positive Plus 50 mg/mL PCC 2h 1,783 Positive 6h 1,956 Positive Plus 100 mg/mL PCC 2h 1,946 Positive 6h 2,069 Positive

PCP, phencyclidine. * 2 h and 6 h are the times after the addition of PCC. Boldface indicates drug testing became negative in the presence of adulterant; the initial test was positive.

brown. A brown precipitate appeared after approximately 30 to 60 seconds after the addition of hydrogen peroxide. In contrast, in unadulterated urine, the addition of 4 to 5 drops of 3% hydrogen peroxide resulted in fading of the yellow color, and some urine specimens turned colorless. Although 3% hydrogen peroxide solution is effective for detecting the presence of PCC in urine, it is not effective for detecting the presence of nitrite in urine. However, the second spot test we developed using potassium iodide is effective for detecting the presence of both PCC and potassium nitrite in urine. Both potassium nitrite and PCC-contaminated urine are capable of oxidizing iodide ion to iodine. The characteristic color of free iodine is readily detectable, and iodine liberated from potassium iodide is readily soluble in an organic solvent such as n-butanol. It also is possible to distinguish between PCC and potassium nitrite as urine adulterants by using the third spot test we developed using potassium permanganate solution. Potassium nitrite can readily turn the deep purple of potassium permanganate solution colorless in acid medium owing to the conversion of a heptavalent manganese ion to a bivalent manganese ion. However, PCC did not change the deep purple potassium permanganate to colorless. If PCC was present, a dark brown color developed on addition of potassium permanganate and
American Society for Clinical Pathology

acid. Such color formation also was observed in some unadulterated urine specimens. We tested 100 control urine specimens (negative for abused drugs and had no adulterants) to ensure the reliability of the 3 spot tests we developed. We observed no false-positive reactions, indicating that if PCC or nitrite were not present in urine, the spot test results would be negative. The presence of a high amount of glucose and ketone bodies (4+ on dipstick) did not interfere with the spot tests using hydrogen peroxide or potassium iodide. However, high amounts of glucose and ketone bodies in urine turned the dark purple potassium permanganate to colorless after standing for approximately 3 to 5 minutes. In contrast, if nitrite was present as an adulterant, the permanganate solution turned colorless rapidly (within 1 minute). PCC is effective for decreasing the response rate for PCP and opiate assays using the Abuscreen Table 1. Even a moderate concentration of PCC is capable of successfully masking a positive response of both opiate and PCP in control 1 after 2 hours of incubation of PCC with the control. Interestingly, the high control (control 2) was affected equally by moderate PCC concentrations, but a longer incubation time of 6 hours was needed. Addition of Urine Luck
Am J Clin Pathol 2002;117:325-329
327

Dasgupta et al / SPOT TESTS TO DETECT ADULTERANTS IN URINE SPECIMENS

Table 2 Effect of the Urine Adulterant Potassium Nitrite (Klear, Whizzies) on the Fluorescence Polarization Immunoassay Screen for Drugs of Abuse*
Drug Concentration, ng/mL (Semiquantitative) Specimen Control 1 Amphetamine 1,249 Positive Cocaine 338 Positive 370 Positive 361 Positive 356 Positive 365 Positive 860 Positive 812 Positive 877 Positive 914 Positive 835 Positive Opiate 402 Positive 396 Positive 406 Positive 402 Positive 395 Positive 990 Positive 1,000 Positive 881 Positive 910 Positive 936 Positive PCP 39 Positive 39 Positive 38 Positive 27 Positive 23 Negative 101 Positive 93 Positive 103 Positive 98 Positive 93 Positive Cannabis 64 Positive 57 Positive 43 Negative 49 Negative 40 Negative >135 Positive >135 Positive 112 Positive 122 Positive 85 Positive Barbiturates 303 Positive 240 Positive 262 Positive 269 Positive 289 Positive 1,086 Positive 941 Positive 943 Positive 1,038 Positive 964 Positive Benzoylecgonine Methadone 235 Positive 242 Positive 235 Positive 243 Positive 229 Positive 930 Positive 967 Positive 907 Positive 1,004 Positive 1,019 Positive 513 Positive 511 Positive 499 Positive 499 Positive 504 Positive 991 Positive 1,042 Positive 992 Positive 1,019 Positive 1,017 Positive

Plus 10 mg/mL nitrite 2h 1,234 Positive 6h 1,317 Positive Plus 25 mg/mL nitrite 2h 1,168 Positive 6h 1,180 Positive Control 2 1,864 Positive Plus 10 mg/mL nitrite 2h 1,858 Positive 6h 1,850 Positive Plus 25 mg/mL nitrite 2h 1,795 Positive 6h 1,753 Positive

PCP, phencyclidine. * 2 h and 6 h are the times after the addition of PCC. Boldface indicates drug testing became negative in the presence of adulterant; the initial test was positive.

also produced a lower response of opiate and PCP in screening assays, as expected. Whizzies is another urine adulterant available from the Internet. This adulterant also contains potassium nitrite. Our results indicate that nitrite is effective for masking THC in immunoassay screens using Abuscreen, even at low nitrite concentrations, although 6 hours was needed for this process. In contrast, if a higher amount of nitrite was present, only 2 hours were needed for a false-negative THC value Table 2. The concentrations of nitrite we used in this study were as described by ElSohly et al4 and Tsai et al,5 and our results are in agreement with their findings. When we added Klear to urine control samples, we observed similar results, and the result for THC became negative. Our results are in agreement with ElSohly et al,4 who first demonstrated that the addition of authentic Klear in urine samples positive for THC can result in false-negative immunoassay and GC/MS confirmation results. To ensure that the in vivo nitrite concentration did not produce a positive spot test result, we supplemented urine specimens from volunteers to achieve final nitrite concentrations up to 100 g/mL. We observed no positive response in the spot test results, indicating that in vivo nitrite production does not interfere with our spot tests.
328

Discussion
Specimen integrity checks are performed routinely at collection sites to ensure specimen integrity before submitting specimens to the laboratory for testing for drugs of abuse. The temperature, for instance, should be 32.5C37.2C. The specific gravity should be 1.005 to 1.030, and the pH should be between 4.0 and 10.0. The creatinine concentration should be 20 to 400 mg/dL (1.8-35.4 mmol/L), because if urine is diluted with tap water to beat a drug test, the creatinine concentration should fall below 20 mg/dL. Adulteration with sodium chloride at a concentration necessary to produce a false-negative result in immunoassay screening always produces a specific gravity more than 1.035. Although the presence of common adulterants, such as table salt, vinegar, liquid laundry bleach, and lemon juice, can be detected by routine specimen integrity checks, the presence of Klear, Whizzies, and Urine Luck cannot. Wu et al6 described a spot test for the detection of PCC, the active component of Urine Luck, in adulterated urine. The indicator solution contains 10 g/L of 1,5-diphenylcarbazide in methanol. The indicator detects the chromium ion and is colorless when prepared. When 2 drops of indicator solution
American Society for Clinical Pathology

Am J Clin Pathol 2002;117:325-329

Clinical Chemistry / ORIGINAL ARTICLE

were added to 1.0 mL of urine, a reddish purple color developed if PCC was present in the urine. We showed that 3% hydrogen peroxide solution is an effective way for detecting urine adulterated with PCC. Moreover, 3% hydrogen peroxide solution is cheap and readily available in drugstores. Wu et al6 described in detail the effect of PCC adulterant in urine on the EMIT II immunoassay. Response rates were decreased for all EMIT II drug assays at a PCC concentration of 100 g/L. The PCC did not affect the GC/MS recovery of methamphetamine, benzoylecgonine, and PCP or their deuterated internal standards, but the GC/MS recovery of opiates and THC was reduced significantly. Therefore PCC, the major component of Urine Luck, is effective at producing a false-negative result if present in the urine as an adulterant. We showed that PCC also is effective at decreasing the response in Abuscreen. We chose 2 time parameters (2 and 6 hours) of incubation with PCC before analysis because at least 2 hours are required for urine specimens to reach the laboratory from collection sites. We also chose 6 hours because approximately 6 hours are required from the time of collection to analysis if the specimen is from a different hospital. Longer delays are possible in urine drug screens for other organizations, especially if the organization does not have a drug analysis facility and specimens are sent to a reference laboratory. Only emergency department requests for abused drug testing are performed as stat tests in our laboratory, but it is unlikely for an overdosed patient to adulterate the urine specimen. Klear is widely available to clear all positive drug test results from urine. This product comes in 2 microtubes containing 500 mg of white crystalline material. This product readily dissolves in urine with no change in the color or temperature of the urine, and it may cause false-negative GC/MS results for confirmation of marijuana. ElSohly et al4 first reported this product as potassium nitrite and provided evidence that nitrite leads to decomposition of ions of 9-THC and its internal standard. The authors further reported that using a bisulfite step at the beginning of sample preparation could eliminate this interference.4 Tsai et al5 further studied the effect of nitrite on immunoassay screening for other drugs, including cocaine metabolites, morphine, THC metabolites, amphetamine, and PCP. Nitrite at a concentration of 1.0 mol/L had no effect on the Abuscreen assay. At a higher nitrite concentration, the amphetamine assay became more sensitive and the THC assay became less sensitive. The GC/MS analyses of benzoylecgonine, morphine, amphetamine, and PCP were not affected, while recovery of THC metabolites was reduced significantly. Again, this interference can be eliminated by bisulfite treatment.5 The spot tests we developed can identify nitrite as an adulterant, and, if necessary, the bisulfite step may be initiated to avoid a false-negative urine drug test result.
American Society for Clinical Pathology

Nitrite in urine may arise in vivo and is found in urine in low concentrations. Patients receiving medications such as nitroglycerine, isosorbide dinitrate, nitroprusside, and ranitidine may have increases nitrite levels in the blood. However, concentrations of nitrite were below 36 g/mL in specimens that cultured positive for microorganisms, and nitrite concentrations were below 6 g/mL in patients receiving medications that are metabolized to nitrite. On the other hand, nitrite concentrations were 1,910 to 12,200 g/mL in urine specimens adulterated with nitrite.7 We described spot tests to detect adulterants that otherwise cannot be detected by routine urine integrity tests. These tests may be useful to eliminate false-negative drug test results.
From the Department of Pathology and Laboratory Medicine, University of Texas-Houston Medical School. Address reprint requests to Dr Dasgupta: Dept of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, 6431 Fannin, MSB 2.292, Houston, TX 77030.

References
1. Wu A. Integrity of urine specimens submitted for toxicological analysis: adulteration, mechanism of action and laboratory detection. Forensic Sci Rev. 1998;10:47-65. 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. ElSohly MA, Feng S, Kopycki WJ, et al. A procedure to overcome interference caused by the adulterant Klear in the GC-MS analysis of 11-nor-Delta9-THC-9-COOH [letter]. J Anal Toxicol. 1997;21:240-242. 5. Tsai SC, ElSohly MA, Dubrovsky T, et al. Determination of five abused drugs in nitrite-adulterated urine by immunoassay and gas chromatographymass spectrometry. J Anal Toxicol. 1998;22:474-480. 6. Wu A, Bristol B, Sexton K, et al. Adulteration of urine by Urine Luck. Clin Chem. 1999;45:1051-1057. 7. Urry F, Komaromy-Hiller G, Staley B, et al. 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.

Am J Clin Pathol 2002;117:325-329

329