Novel Spot Tests for Detecting the Presence of Zinc Sulfate in Urine, a Newly Introduced Urinary Adulterant to Invalidate Drugs of Abuse Testing

Transcription

1 Novel Spot Tests for Detecting the Presence of Zinc Sulfate in Urine, a Newly Introduced Urinary Adulterant to Invalidate Drugs of Abuse Testing Kerry J. Welsh, MD, PhD, Jennifer E. Dierksen, MD, Jeffrey K. Actor, PhD, and Amitava Dasgupta, PhD From the Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX. CME/SAM Key Words: Spot test; Zinc sulfate; Adulterated urine; False negative DOI: /AJCP2FJ9VBPXJYTQ ABSTRACT Objectives: To find a suitable method for detecting zinc sulfate in adulterated urine. Methods: Two rapid spot tests to detect the presence of zinc sulfate in urine were developed. Results: Addition of 3 to 4 drops of 1N sodium hydroxide solution to approximately 1 ml of urine containing zinc sulfate led to the formation of a white precipitate, which was soluble in excess sodium hydroxide. In the second spot test, addition of 3 to 4 drops of 1% sodium chromate solution to 1 ml of urine containing zinc sulfate followed by the addition of 4 to 5 drops of 1N sodium hydroxide led to formation of a yellow precipitate (zinc chromate). Detection limit of these visual spot tests was 10 mg/ml of zinc sulfate in urine. Twenty drug-free urine specimens and urine containing high amounts of sugar or reducing substances were tested with no false-positive spot test results observed. However, if lead is present in high amounts in urine, it may cause false-positive spot test results. When aliquots of urine controls for drugs of abuse testing were supplemented with different amounts of zinc sulfate, false-negative drug test results were observed except for amphetamine. Zinc sulfate also falsely reduced measured urine alcohol level in urine. Conclusions: Zinc sulfate can invalidate urine drug and alcohol testing but can be detected using the novel spot tests developed. Upon completion of this activity you will be able to: list common adulterants used for beating drug tests. define role of newly introduced urinary adulterant zinc sulfate in invalidating immunoassays used for screening abused drugs in urine. defining role of zinc sulfate in causing falsely lowered urine alcohol level. apply newly developed spot tests for zinc sulfate to screen urine specimens submitted for drugs of abuse testing. identify which urine specimens (emergency room patient versus physician office) should be subjected to spot test. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 595. Exam is located at Alcohol and drug abuse is a significant public health issue in the United States and many countries worldwide. It is estimated that 18 million Americans abuse alcohol, while an estimated 22.6 million Americans (8.9% of the US population) 12 years or older are current illicit drug users. 1 Illicit drugs abused by Americans include marijuana/ hashish, cocaine (including crack), heroin, hallucinogens, inhalants, or prescription-type psychoactive drugs obtained illegally without a valid prescription. Marijuana is the most commonly used illicit drug; cocaine abuse continues to be widespread among the general population. 1 On September 15, 1986, President Reagan issued Executive Order directing all federal employees involved in law enforcement, national security, protection of life and property, public health and safety, and other functions requiring a high degree of 572 Am J Clin Pathol 2013;140: DOI: /AJCP2FJ9VBPXJYTQ Welsh_ indd 572

2 public trust to be subjected to mandatory drug testing. Following this executive order, the US Department of Health and Human Services developed guidelines, and Congress passed the Drug Free Workplace Act in Urine is the preferred specimen in preemployment drug testing, and urine specimens are screened for the presence of abused drugs using immunoassays approved by the Food and Drug Administration. No further testing is performed if a urine screen is negative. However, confirmation should be performed for immunoassay-positive specimens by a second technique, preferably by gas chromatography/mass spectrometry (GC/ MS). 2 Workplace drug testing is also common among private employers because such practice can prevent accidents. Gerber and Yacoubian 3 assessed the impact of drug testing within the construction industry and concluded that companies with drug testing programs experienced a 51% reduction in injury incident rates within 2 years of implementation. People try to beat drug tests by either ingesting a detoxifying agent or adding adulterant in vitro to a urine specimen after collection. Specimen integrity testing (ph, creatinine, specific gravity, and temperature) is useful in identifying these individuals trying to beat the drug test. A detoxifying agent results in diluted urine that can be identified by low creatinine in urine. Several household chemicals are used to invalidate drugs of abuse testing in urine. The most commonly used chemicals include table salts (sodium chloride), vinegar, Drano, Joy, hand soap, liquid laundry bleach, denture cleansing tablet, lemon juice, ascorbic acid, hydrogen peroxide, and rubbing alcohol (isopropyl alcohol), but specimen integrity testing can identify all such adulterants except Visine eye drops. 4 Various urinary adulterants available on the Internet, such as pyridinium chlorochromate, glutaraldehyde, and potassium nitrite, have been introduced, and these adulterants can successfully invalidate the immunoassay for various drugs of abuse as well as for GC/MS confirmation of the marijuana metabolite (tetrahydrocannabinol carboxylic acid). However, these adulterants can be detected by spot tests or by using specially designed urine dipsticks, such as AdultaCheck 6 (Sciteck Diagnostics, Arden, NC) and Intect 7 (Branan Medical Group, Irvine, CA) Zinc sulfate has recently been promoted as an effective urinary adulterant to invalidate drug tests by several websites that provide tips to beat drug testing. Venkatratnam and Lents 13 reported that zinc sulfate is an effective urinary adulterant that can produce false-negative results using an enzyme-linked immunosorbent assay for methamphetamine, cocaine (tested as benzoylecgonine), and marijuana. Zinc sulfate at a concentration of 10 to 15 mg/ml is sufficient to achieve false-negative results. The authors further commented that no method is available to detect the presence of zinc sulfate in adulterated urine, including the recently introduced AdultaCheck 10 dipstick for detecting urinary adulterants. 13 Therefore, novel spot tests were developed to detect the presence of zinc sulfate in urine. The data presented here also indicate that zinc sulfate may invalidate additional drugs of abuse testing and showed negative interference (falsely lower values) with urine alcohol testing. Materials and Methods Zinc sulfate, sodium hydroxide, and sodium chromate were obtained from Sigma (St Louis, MO). In addition, albumin (Sigma), creatinine (Fisher Scientific, Waltham, MA), urea (Fisher Scientific), calcium chloride (Fisher Scientific), magnesium sulfate (Fisher Scientific), cobalt chloride (Sigma), copper sulfate (Sigma), ferric chloride (Fisher Scientific), nickel sulfate (Sigma), and lead nitrate (Sigma) were also purchased to perform potential interferences of these substances with spot tests for zinc sulfate. Drugs of abuse tests were performed using reagent kits (EMIT II Plus) obtained from Siemens Diagnostics (Deer Park, IL), and tests were run using the Vista 1500 analyzer (Siemens Diagnostics) with the protocol recommended by the manufacturer. Urine alcohol level was also determined by using the Vista 1550 analyzer with an enzymatic alcohol assay kit obtained from Siemens Diagnostics. Intect 7 urine test strips were purchased from Branan Medical Group. Urine pools were prepared from specimens submitted to our hospital laboratory for drugs of abuse testing. These specimens were retained for a week after reporting results to ordering physicians before being used for this investigation; all specimens were deidentified and considered discarded specimens according to the protocol approved by the Institutional Review Board at the University of Texas Houston Health Science Center for research using leftover specimens. Stock solutions containing 1% sodium chromate and 1N sodium hydroxide were prepared in deionized water. These 2 solutions were used for the 2 spot tests developed for detecting the presence of zinc sulfate in urine. For the first spot test, addition of 3 to 4 drops (approximately 50 µl) of 1N sodium hydroxide solution to approximately 1 ml of urine containing zinc sulfate led to formation of a white precipitate that was soluble in excess sodium hydroxide (additional drops of sodium hydroxide; µl) Image 1. In the second spot test, addition of 3 to 4 drops of 1% sodium chromate solution to 1 ml of urine containing zinc sulfate followed by addition of 4 to 5 drops of 1N sodium hydroxide led to formation of a yellow precipitate (zinc chromate) (Image 1). In both spot tests, appearances of precipitates are different from urinary sediment. Aliquots of positive control urine containing various abused drugs at approximately 20% more than cutoff concentrations were supplemented with 10, 15, 30, and 50 mg/ Am J Clin Pathol 2013;140: DOI: /AJCP2FJ9VBPXJYTQ 573 Welsh_ indd 573

3 Welsh et al / Novel Spot Tests to Detect Zinc Sulfate in Urine A B C D Image 1 Spot tests to detect zinc sulfate (ZnSO4) in urine. A, Urine containing ZnSO4. B, Sodium chromate spot test. C, Sodium hydroxide spot test. D, Precipitate in the sodium hydroxide spot test becomes soluble with excess sodium hydroxide. Table 1 Cutoff Concentrations and Upper Limits of Detection of Various Immunoassays for Drugs of Abuse on the Vista 1500 Analyzer Drug Cutoff Concentration, Analytical Measurement ng/ml Range, ng/mla Amphetamine 1,000 Barbiturates 200 Benzodiazepines 200 Opiates 300 Benzoylecgonine 300 Marijuana metabolite 50 Methadone 300 Phencyclidine 25 Propoxyphene 300 a Semiquantitative concentration Am J Clin Pathol 2013;140: , , ml of zinc sulfate and reassayed for all drugs of abuse using the Vista 1500 analyzer and EMIT II Plus reagent kits. The cutoff concentration and analytical measurement range of the drugs of abuse analyzed are listed in Table 1. However, the concentrations produced for these drugs are semiquantitative. Results were thus measured before and after supplementation with zinc sulfate for comparison. In another set of experiments, a urine pool was prepared from specimens that tested negative for drugs and then supplemented with various drugs of abuse (Sigma) in concentrations approximately 20% to 30% above the cutoff concentrations; drugs of abuse testing were then conducted. Aliquots of the pool were further supplemented with 10, 15, 30, or 50 mg/ml of zinc sulfate followed by repeated drugs of abuse testing to compare with the original values. In addition, aliquots of discarded patient DOI: /AJCP2FJ9VBPXJYTQ Welsh_ indd 574

4 urine specimens that tested positive for cocaine (tested as benzoylecgonine), opiates, marijuana, or benzodiazepines were supplemented with zinc sulfate, and drug tests were repeated to determine the effect of zinc sulfate on urine specimens that originally tested positive for abused drugs. Finally, the effect of zinc sulfate on the urine alcohol test was performed using 3 specimens that tested positive for alcohol. The alcohol assay on the Vista analyzer is an enzymatic assay using alcohol dehydrogenase enzyme. The analytical measurement range of this assay is 20 to 300 mg/dl of alcohol concentration and produces a quantitative result. To investigate the sensitivity of our spot test to detect zinc sulfate, we supplemented aliquots of a negative urine pool with 0.1, 0.5, 1, 5, 10, 15, 30, and 50 mg/ml of zinc sulfate. Both spot tests were then conducted to investigate whether these spot tests could detect the presence of zinc sulfate at lower concentrations than needed to invalidate immunoassays. These specimens were also tested by Intect 7 urine test strips to evaluate whether Intect 7 could detect zinc sulfate in urine. Twenty negative urine specimens were tested with our spot tests to ensure that the tests did not produce falsepositive results. Urine specimens with a high glucose content or positive for ketone bodies were tested with the spot tests to ensure that they did not produce false-positive results in the presence of high glucose and ketone bodies. Furthermore, the possible interference of high levels of albumin, creatinine, urea, calcium chloride, magnesium sulfate, cobalt chloride, copper sulfate, ferric chloride, nickel sulfate, and lead nitrate was assessed by addition of these agents to urine at concentrations of 10 mg/ml, followed by performing the spot tests. Results Venkatratnam and Lents 13 demonstrated that zinc sulfate at a concentration of 10 to 15 mg/ml is effective in interfering with cocaine, methamphetamine, and tetrahydrocannabinol tests. Aliquots of a negative urine pool were supplemented with increasing amounts of zinc sulfate (0.1, 0.5, 1, 5, 10, 15, 30, and 50 mg/ml) to investigate if the spot tests developed could detect the presence of zinc sulfate in urine at lower concentrations than needed to invalidate drug testing. Unambiguous positive responses with both spot tests were observed when zinc sulfate was present at 10 mg/ml, indicating that these novel spot tests are effective in detecting the presence of zinc sulfate in urine at the concentration required to invalidate drug tests. Because these spot tests are visual, requiring subjective interpretation, the detection limit was established at 10 mg/ml of zinc sulfate concentration because changes are obvious at this concentration. In the presence of excess zinc sulfate (50 mg/ml), almost 1 ml of sodium hydroxide was needed for dissolving white precipitate. For the sodium chromate spot test, an excessive amount of yellow precipitate was formed if zinc sulfate was present in excessive concentration (50 mg/ml). In contrast, the presence of zinc sulfate in urine was not detected using Intect 7 test strips even with 50 mg/ml of zinc sulfate. Venkatratnam and Lents 13 studied the effect of zinc sulfate on testing for 3 drugs of abuse. However, 9 drugs (amphetamine, barbiturates, opiates, benzodiazepines, marijuana metabolite, methadone, benzoylecgonine, phencyclidine, and propoxyphene) are routinely analyzed in our institution. Therefore, the effect of zinc sulfate on testing all these drugs was investigated. Zinc sulfate at a concentration of 10 mg/ml, as reported by Venkatratnam and Lents, was used for this study, as well as increasing zinc sulfate concentrations. When aliquots of drug of abuse control urine containing drugs at approximately 20% above the cutoff concentration were supplemented with 10 mg/ml of zinc sulfate, only benzoylecgonine and phencyclidine tests became negative. However, in the presence of 15 mg/ml of zinc sulfate, opiate and marijuana metabolite tests were also negative in addition to benzoylecgonine and phencyclidine. All drugs of abuse tests were negative except for amphetamine with the highest concentration of zinc sulfate (50 mg/ml) Table 2. Similar results were observed when increasing concentrations of zinc sulfate were added to aliquots of a drug-free urine pool supplemented with abused drugs (or metabolites) followed by repeated drugs of abuse testing. Testing for marijuana metabolite showed negative results in the presence of 10 mg/ml of zinc sulfate, and all drugs tests were negative except for amphetamine with 50 mg/ml of zinc sulfate Table 3. In addition, 4 urine specimens that tested positive for cocaine (tested as benzoylecgonine), opiates, benzodiazepines, and marijuana (tested as marijuana metabolite) were selected. Two specimens were specifically selected for each drug in which the semiquantitative drug levels were 20% to 50% above the cutoff concentrations and another 2 specimens for each drug in which the drug levels were at the upper end of the analytical measurement range or above it. Aliquots of each specimen were supplemented with zinc sulfate and retested. When any drug was present 20% to 50% above the cutoff, zinc sulfate was able to produce negative results at various concentrations. However, when a drug was present at a level near the upper end of the analytical measurement range or above the range, zinc sulfate even at a 50 mg/ml concentration was not able to produce negative results, although semiquantitative drug values were significantly reduced. Therefore, zinc sulfate is an effective in vitro adulterant if a drug is present modestly over the cutoff concentration. Because urine alcohol testing is also a frequently ordered test in our laboratory, the effect of zinc sulfate on the determination of alcohol concentration using the Vista 1500 analyzer was investigated. Urine alcohol testing produces a Am J Clin Pathol 2013;140: DOI: /AJCP2FJ9VBPXJYTQ 575 Welsh_ indd 575

5 Welsh et al / Novel Spot Tests to Detect Zinc Sulfate in Urine Table 2 Effect of Adding Zinc Sulfate to Aliquots of Urine Drugs of Abuse Controls on Drugs of Abuse Testing Using the Vista 1500 Analyzer Drug Concentration, ng/ml (Semiquantitative) Specimen Amph Barb Opiate Benz THC MTD BE PCP PPX Control 1,201 (Pos) 333 (Pos) 384 (Pos) 313 (Pos) 58 (Pos) 373 (Pos) 313 (Pos) 29 (Pos) 386 (Pos) + 10 mg/ml ZnSO 4 1,167 (Pos) 313 (Pos) 323 (Pos) 259 (Pos) 55 (Pos) 314 (Pos) 267 (Neg) 22 (Neg) 346 (Pos) + 15 mg/ml ZnSO 4 1,082 (Pos) 303 (Pos) 56 (Neg) 232 (Pos) 44 (Neg) 301 (Pos) 240 (Neg) 19 (Neg) 338 (Pos) + 30 mg/ml ZnSO 4 1, (Pos) <50 (Neg) 161 (Neg) 39 (Neg) 302 (Pos) 151 (Neg) <5 (Neg) 320 (Pos) + 50 mg/ml ZnSO 4 1,013 (Pos) <20 (Neg) <50 (Neg) <30 (Neg) <15 (Neg) 295 (Neg) <35 (Neg) <5 (Neg) 110 (Neg) Amph, amphetamines; Barb, barbiturates; BE, benzoylecgonine, major metabolite of cocaine; Benz, benzodiazepines; MTD, methadone; Neg, negative test result (value below cutoff concentration of a drug); PCP, phencyclidine; Pos, positive test result; PPX, propoxyphene; THC, tetrahydrocannabinol (marijuana metabolite); ZnSO 4, zinc sulfate. Table 3 Effect of Negative Urine Supplemented With Various Drugs and Increasing Concentrations of Zinc Sulfate on Drugs of Abuse Testing Using the Vista 1500 Analyzer Drug Concentration, ng/ml (Semiquantitative) Specimen Amph Barb Opiate Benz THC BE PCP PPX Urine supplemented with drugs 1,271 (Pos) 262 (Pos) 596 (Pos) 297 (Pos) 57 (Pos) 535 (Pos) 50 (Pos) 498 (Pos) + 10 mg/ml ZnSO 4 1,138 (Pos) 238 (Pos) 323 (Pos) 252 (Pos) 25 (Neg) 414 (Pos) 38 (Pos) 448 (Pos) + 15 mg/ml ZnSO 4 1,118 (Pos) 231 (Pos) 291 (Neg) 263 (Pos) <15 (Neg) 384 (Pos) 29 (Pos) 412 (Pos) + 30 mg/ml ZnSO 4 1,032 (Pos) 67 (Neg) <50 (Neg) 32 (Neg) <15 (Neg) 252 (Pos) <5 (Neg) <5 (Neg) + 50 mg/ml ZnSO 4 1,016 (Pos) <20 (Neg) <50 (Neg) <30 (Neg) <15 (Neg) <35 (Neg) <5 (Neg) <0 (Neg) Amph, amphetamines; Barb, barbiturates; BE, benzoylecgonine, major metabolite of cocaine; Benz, benzodiazepines; Neg, negative test result (value below cutoff concentration of a drug); PCP, phencyclidine; Pos, positive test result; PPX, propoxyphene; THC, tetrahydrocannabinol (marijuana metabolite); ZnSO 4, zinc sulfate. Table 4 Effect of Adulteration of Alcohol-Positive Urine With Zinc Sulfate on Alcohol Testing Using the Vista 1500 Analyzer Specimen Alcohol Concentration, mg/dl Specimen mg/ml ZnSO mg/ml ZnSO mg/ml ZnSO mg/ml ZnSO 4 Specimen mg/ml ZnSO mg/ml ZnSO mg/ml ZnSO mg/ml ZnSO 4 38 Specimen mg/ml ZnSO mg/ml ZnSO mg/ml ZnSO mg/ml ZnSO 4 ZnSO 4, zinc sulfate. quantitative result in contrast to urine drug testing, which provides a semiquantitative result. Significant declines in urinary alcohol concentration in the presence of zinc sulfate using the Vista 1500 analyzer were observed Table 4. The specimen containing 25 mg/dl of alcohol produced negative results (<20 mg/dl, the detection limit of the assay) in the presence of 15 mg/ml of zinc sulfate. The specimen containing 76 mg/ dl of alcohol showed a negative result in the presence of 50 mg/ml of zinc sulfate. However, the specimen containing 130 mg/dl of alcohol tested positive even in the presence of 50 mg/ml of zinc sulfate, although the value was significantly reduced. To our knowledge, negative interference of zinc sulfate in urine alcohol testing has not been reported. Specimen integrity testing (ph, creatinine, temperature, and specific gravity) is useful in identifying various household chemicals that are added to urine to invalidate drugs of abuse testing. The change in specific gravity of urine upon the addition of increasing amounts of zinc sulfate was investigated. Unfortunately, specific gravity did not change significantly even after supplementing urine specimens with 50 mg/ml of zinc sulfate, the highest zinc sulfate concentration studied. For example, the specific gravity of a urine specimen was The specific gravity increased to only in the presence of 10 mg/ml of zinc sulfate, and the specific gravity was in the presence of 50 mg/ml of zinc sulfate, which was significantly below the cutoff. Therefore, specimen integrity testing cannot identify the presence of zinc sulfate in urine. In addition, we observed insignificant lowering of urine ph in the presence of zinc sulfate (even 50 mg/ml), indicating that specimen integrity testing is not useful in detecting urine specimens adulterated with zinc sulfate. Our results are consistent with observations by Venkatratnam and Lents. 13 To ensure that ketone bodies or high glucose if present in urine do not interfere with these spot tests, we evaluated urine specimens that showed high glucose content or were positive for ketone bodies using our spot tests. No positive 576 Am J Clin Pathol 2013;140: DOI: /AJCP2FJ9VBPXJYTQ Welsh_ indd 576

6 response was observed, indicating that high glucose or ketone bodies do not interfere with these spot tests. Furthermore, 20 negative urine specimens were tested with both spot tests, and no false-positive response was observed. The potential interference of albumin, creatinine, urea, calcium, magnesium, cobalt, copper, iron, nickel, and lead also was assessed Table 5. High levels (10 mg/ml) of albumin, creatinine, and urea did not interfere with either spot test. Calcium, cobalt, copper, iron, and nickel resulted in the formation of precipitates with the sodium hydroxide spot test; however, the precipitates formed were not soluble in excess sodium hydroxide. Only lead produced a false-positive spot test. We selected a 10 mg/ ml concentration of all these interfering substances because the detection limit of zinc sulfate spot tests is 10 mg/ml. However, these high concentrations for these interfering substances are unlikely in a clinical situation. Discussion After the US Congress passed the Drug Free Workplace Act in 1988 that resulted in Mandatory Guidelines for Federal Workplace Drug Testing Programs, there have been many scientific and legal challenges to the validity of urine drug testing. In response, the Substance Abuse and Mental Health Services Administration (SAMHSA) put forth many revisions, strict procedural guidelines, and specimen validity testing criteria to manage suspicious and adulterated samples during and after urine collection. 14 Because many adulterants can invalidate immunoassay screening of various drugs, it is important to identify such adulterated specimens prior to testing; if immunoassay screening is negative, GC/MS identification is not conducted. According to SAMHSA guidelines, no further testing is necessary if a specimen is adulterated. Consequently, a job can be denied to an individual submitting an adulterated specimen. Venkatratnam and Lents 13 already demonstrated that zinc sulfate can interfere with methamphetamine, cocaine, and marijuana testing. Moreover, the authors commented that no test is currently available that can detect the presence of zinc sulfate in adulterated urine. Therefore, 2 rapid spot tests were developed in this report for detecting the presence of zinc sulfate in urine. In addition, our results also indicate that zinc sulfate at 10 mg/ml can invalidate the phencyclidine test. Furthermore, opiate, marijuana metabolite, benzoylecgonine, and phencyclidine tests were negative with the addition of 15 mg/ml of zinc sulfate. At a 50 mg/ml zinc sulfate concentration, barbiturate, benzodiazepine, opiate, marijuana metabolite, methadone, benzoylecgonine, phencyclidine, and propoxyphene tests were also negative. However, in contrast to the observations of Venkatratnam and Lents, the amphetamine test was not affected in this investigation. This may be related to the use of a different immunoassay for testing amphetamine. Our study indicated that zinc sulfate is effective in producing false-negative test results with 8 common drugs of abuse tests. In addition, zinc sulfate can significantly interfere with the urine alcohol test, producing falsely lower values. However, zinc sulfate was ineffective in invalidating drug testing when drugs were present at a very high concentration in urine (near the upper end of the analytical measurement range or above). Since zinc sulfate is an effective adulterant, it is important to identify it prior to immunoassay screening. The Adulta- Check 10 urine test strip is not effective in identifying zinc sulfate. 13 The study reported here further indicates that another urine test strip, Intect 7, is also not capable of detecting the presence of zinc sulfate in urine even if present in a very high concentration. However, these novel spot tests can effectively identify the presence of zinc sulfate in urine. The first spot test is fairly specific for identifying zinc ion in urine because zinc ion has the unique feature of forming a white precipitate of zinc hydroxide in the presence of sodium hydroxide, which is soluble in excess sodium hydroxide. The second spot test consists of a yellow precipitate generated due to the formation of zinc chromate; this test is also specific for zinc because the Table 5 Visual Changes of Various Substrates (10 mg/ml) Using the Spot Tests for Zinc Sulfate Substrate Sodium Hydroxide Spot Test Sodium Chromate Spot Test Albumin No effect No effect Creatinine No effect No effect Urea No effect No effect Calcium chloride White precipitate, insoluble in excess NaOH No effect Cobalt chloride Blue precipitate, insoluble in excess NaOH Rust color precipitate Copper sulfate Blue precipitate, insoluble in excess NaOH Green precipitate Ferric chloride Red precipitate, insoluble in excess NaOH Red color Lead nitrate a White precipitate, soluble in excess NaOH Yellow precipitate Magnesium sulfate Cloudy, no precipitate No effect Nickel sulfate White precipitate, insoluble in excess NaOH No effect NaOH, sodium hydroxide. a Results similar to zinc sulfate spot test, but lead salt concentration of 10 mg/ml in urine is highly unlikely even after lead poisoning. Am J Clin Pathol 2013;140: DOI: /AJCP2FJ9VBPXJYTQ 577 Welsh_ indd 577

7 Welsh et al / Novel Spot Tests to Detect Zinc Sulfate in Urine other chromate salts that are insoluble in water have a much darker yellow color, although some chromate salts such as potassium chromate are water soluble and yellow. Only lead was found to produce a false-positive result with the spot tests we developed. Fortunately, elevated blood lead levels are rare, occurring in approximately 6.3 per 100,000 adults. 15 In addition, urine lead levels in adults with lead poisoning were reported to be significantly lower than the levels needed to cause a false-positive spot test in this investigation. 16 We did not investigate the exact mechanism by which zinc sulfate interferes with both drugs of abuse testing and alcohol testing. In EMIT II Plus assays of drugs of abuse and the enzymatic alcohol assay, the signal is measured at 340 nm, which is generated due to conversion of nicotinamide adenine dinucleotide (NAD) into NADH. Possibly zinc sulfate interacts with NADH and, through the redox reaction, converts it back to NAD, and thus absorption at 340 nm is reduced. Novel spot tests for detecting zinc sulfate in urine are not conclusive for the presence of zinc ion in the urine specimen. Further testing to confirm the presence of excess zinc in urine may be needed to meet specific medical and legal situations. The presence of a high amount of zinc in urine can be confirmed by atomic absorption spectrophotometry. More recently, inductively coupled plasma mass spectrometry (ICP-MS) has been found useful for unambiguous identification and quantification of zinc and other elements in urine. 17 Therefore, ICP-MS can also be used for further testing of urine specimens tested positive by spot tests. It was concluded that the 2 novel tests developed are effective in identifying the presence of zinc sulfate as an adulterant in urine. 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; amitava.dasgupta@uth.tmc.edu. References 1. US Department of Health and Human Services. Results From the 2010 National Survey on Drug Use and Health: National Findings. Washington, DC: US Department of Health and Human Services, Substance Abuse and Mental Health Services Administration (SAMHSA), Office of Applied Studies; Bush D. The US mandatory guidelines for federal workplace drug testing programs: current status and future considerations. Forensic Sci Int. 2008;174: Gerber JK, Yacoubian GS Jr. An assessment of drug testing within the construction industry. J Drug Educ. 2002;32: Warner A. Interference of household chemicals in immunoassay methods for drugs of abuse. Clin Chem. 1989;35: Wu A, Bristol B, Sexton K, et al. Adulteration of urine by Urine Luck. Clin Chem. 1999;45: Wu AH, Forte E, Casella G, et al. CEDIA for screening drugs of abuse in urine and the effect of adulterants. J Forensic Sci. 1995;40: Goldberger BA, Caplan YH. Effect of glutaraldehyde (UrinAid) on detection of abused drugs in urine by immunoassay [letter]. Clin Chem. 1994;40: Wu A, Schmalz J, Bennett W. Identification of Urin-Aid adulterated urine specimens by fluorometric analysis [letter]. Clin Chem. 1994;40: Peace MR, Tarnai LD. Performance evaluation of three on-site adulteration detection devices for urine specimens. J Anal Toxicol. 2002;26: King EJ. Performance of AdultaCheck 4 test strips for the detection of adulteration at the point of collection of urine specimens used for drugs of abuse testing. J Anal Toxicol. 1999;23: Dasgupta A, Wahed A, Wells A. Rapid spot tests for detecting the presence of adulterants in urine specimens submitted for drug testing. Am J Clin Pathol. 2002;117: Dasgupta A, Chughtai O, Hannah C, et al. Comparison of spot tests with AdultaCheck 6 and Intect 7 urine test strips for detecting the presence of adulterants in urine specimens. Clin Chem Acta. 2004;34: Venkatratnam A, Lents NH. Zinc reduces the detection of cocaine, methamphetamine, and THC by ELISA urine testing. J Anal Toxicol. 2011;35: Phan HM, Yoshizuka K, Murry DJ, et al. Drug testing in the workplace. Pharmacotherapy. 2012;32: Centers for Disease Control and Prevention. Adult blood lead epidemiology and surveillance United States, MMWR Morb Mortal Wkly Rep. 2009;58: Balali-Mood M, Shademanfar S, Rastegar Moghadam J, et al. Occupational lead poisoning in workers of traditional tile factories in Mashhad, northeast of Iran. Int J Occup Environ Med. 2010;1: Wang J, Harald Hansen E, Gammelgaard B. Flow injection on-line dilution for multi-element determination in human urine with detection by inductively coupled plasma mass spectrometry. Talanta. 2001;55: Am J Clin Pathol 2013;140: DOI: /AJCP2FJ9VBPXJYTQ Welsh_ indd 578