No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Abstract
Adulteration and substitution (of urine by other fluids) are practices conducted by individuals who undergo urine drug testing for the purpose of invalidating results of such tests. Drug testing laboratories that are certified by the Substance Abuse and Mental Health Services Administration will soon be required to perform tests for the presence of these adulterants and substitution. Mandatory tests include urine creatinine, specific gravity, pH, and analysis for oxidizing adulterants. This article will describe the current practices of urine adulteration and substitution, and summarize the new proposed guidelines for the laboratory tests that are used for detecting adulterants.
... Urine substitution is achieved through the replacement of a urine specimen with that of drug-free urine [27], some nonurine liquids such as water or saline [28], or commercially available synthetic urine that possesses the same essential characteristics of human urine, eg, correct pH, specific gravity, and creatinine concentration [29]. Urine substitution can be facilitated by the use of some sophisticated devices including the Butt Wedge™, the Urinator™, and the Whizzinator™, or in extreme cases, by catheterization [1,27,29]. ...
... Hypochlorite was found to directly affect immunoassay reagents, resulting in erroneous and in some cases false negative test results for FPIA assay [82], EMIT assay [75], CEDIA assay [60], and KIMS assay [83]. Hypochlorite has also been found to cause degradation of opiate analytes in urine, which were confirmed by GC-MS analysis [28,29]. ...
... Nitrite successfully masked the detection of BZE by immunoassay screening methods, but did not interfere with GC-MS confirmation as reported in a study by Tsai et al. [76]. Nitrite-containing commercial products such as Klear ® and Whizzies ® were reported as effective masking agents for the detection of opiates [28,29,39]. Potassium nitrite, under acidic environment (pH < 7), was shown to be effective in masking the detection of 6-MAM by the CEDIA immunoassay [44]. ...
Urine drug testing plays an important role in monitoring licit and illicit drug use for both medico-legal and clinical purposes. One of the major challenges of urine drug testing is adulteration, a practice involving manipulation of a urine specimen with chemical adulterants to produce a false negative test result. This problem is compounded by the number of easily obtained chemicals that can effectively adulterate a urine specimen. Common adulterants include some household chemicals such as hypochlorite bleach, laundry detergent, table salt, and toilet bowl cleaner and many commercial products such as UrinAid (glutaraldehyde), Stealth® (containing peroxidase and peroxide), Urine Luck (pyridinium chlorochromate, PCC), and Klear® (potassium nitrite) available through the Internet. These adulterants can invalidate a screening test result, a confirmatory test result, or both. To counteract urine adulteration, drug testing laboratories have developed a number of analytical methods to detect adulterants in a urine specimen. While these methods are useful in detecting urine adulteration when such activities are suspected, they do not reveal what types of drugs are being concealed. This is particularly the case when oxidizing urine adulterants are involved as these oxidants are capable of destroying drugs and their metabolites in urine, rendering the drug analytes undetectable by any testing technology. One promising approach to address this current limitation has been the use of unique oxidation products formed from reaction of drug analytes with oxidizing adulterants as markers for monitoring drug misuse and urine adulteration. This novel approach will ultimately improve the effectiveness of the current urine drug testing programs.
... Sodium hydroxide is a caustic strong base. It causes change to alkaline pH in urine samples, then it effects the drug binding and solubility and produces false negative urine results [13]. ...
... It was suggested that GC-MS confirmation test results aren't effected by non-oxidizing adulterans. Adding sodium hydrosulfide or sulfamic acid to the GC-MS method can help remove excess oxidizing additive and may prevent further oxidation of unchanged opium analytes in the sample, thus the accuracy of GC-MS results is less affected by oxidizing adulterans [13]. ...
... Sodium hydroxide is a caustic strong base. It causes change to alkaline pH in urine samples, then it effects the drug binding and solubility and produces false negative urine results [13]. ...
... It was suggested that GC-MS confirmation test results aren't effected by non-oxidizing adulterans. Adding sodium hydrosulfide or sulfamic acid to the GC-MS method can help remove excess oxidizing additive and may prevent further oxidation of unchanged opium analytes in the sample, thus the accuracy of GC-MS results is less affected by oxidizing adulterans [13]. ...
... These results were in agreement with Uebel and Wium, (2002) who reported that drano is very effective in masking cannabis detection by immunoassay techniques. Also in consistent with Bronner et al., (1990) and Wu, (2003) who stated that hand soap detergent adulteration has caused false-negative results across a variety of drug assays using the cloned enzyme donor immunoassay (CEDIA) including screens for THC, amphetamine, barbiturates, cocaine, opiates, and PCP. Effect of different adulterants on THC-COOH detection and quantification by GC-MS: ...
... Another reagent, acidified potassium dichromate, will give a deep blue color change that fades over time when exposed to urine containing stealth (Caitlin et al., 2007). (Wu, 2003) 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 a 7.7-mmol/L concentration of potassium dihydrogen phosphate (pH3.0) ...
... In the current research, dilution was the most common procedure of tampering of urine samples from both pre-employment/workplace and the suspected specimens settings. Diluting urine sample is often the easiest way to make a false negative drug test result [15] samples delivered to toxicology laboratory for drug abuse testing were diluted (creatinine <4 mmol/L) [16]. ...
... In fact, it is known that a large intake of fluids before the urine analysis reduces drugs of abuse levels, as well as urine creatinine levels, which allow possible counterfeit samples to be identified (5,14,39). In our population, the percentage of diluted samples was 3.8%, and positive/non-negative samples had lower creatinine than the overall average (97.6 vs.126.72 mg/dL; p-value=0.0002), ...
BACKGROUND:
Previous reports revealed poor performance in identifying drugs of abuse users through first-level workplace drug testing (WDT), based on urine samples. In a cross-sectional study, we evaluated: (i) the effect of creatinine normalization of drug values from diluted urine samples (creatinine levels ≤20 mg/dL) on the prevalence of drug users; (ii) the independent procedure-related predictors of positivity and dilution.
METHODS:
Workers' urine samples were collected at the workplace or at our certified laboratory between 2008 and 2012. All samples were analysed for drugs of abuse by immuno-enzymatic method in our laboratory, according to the Italian WDT law. Detectable drugs of abuse concentrations lower than the positive cutoff values were normalized based on mean levels of urinary creatinine. Detectable concentrations of drugs were confirmed by GC/MS. Multivariate logistic regression was used to detect independent procedure-related predictors of positive and diluted urine samples.
RESULTS:
Of the 3080 urine samples screened, 51 (1.7%) were found positive for some drugs of abuse (26 cannabinoids and 16 cocaine) and 116 (3.8%) were diluted. Seventeen out of 23 diluted urine samples with detectable concentrations of cannabinoids or cocaine were found positive after urine creatinine normalization and GC/MS confirmed both negative and positive results. This increased the percentage of positivity for cannabinoids and cocaine from 1.35% to 2.09% (+55%, p=0.0005), which is closer to the expected prevalence of drug users based on Italian self-reported surveys. Collection of samples in the laboratory was an independent predictor of positivity (OR=2.33, 95%CI 1.27-4.28) and diluted urine sample (OR=1.65, 95%CI 1.04-2.61).
CONCLUSIONS:
Efficacy of first-level WDT could be improved by well-controlled pre-analytical procedures and urine creatinine normalization of detected concentrations of drugs of abuse.
Aim:
Currently, procedures that identify the drugs 'destroyed' in adulterated urine specimens are very limited. This study aimed to determine the effect of pyridinium chlorochromate (PCC) on routine opiate assays and identify reaction products formed. Results/methodology: Opiate-positive urines adulterated with PCC (20 and 100 mM) were analyzed using CEDIA(®) immunoassay and GC-MS. Urine and water samples spiked with 6-monoacetylmorphine, morphine and its glucuronides (10 µg/ml) and PCC (0.02-100 mM) were monitored with LC-MS, and the products characterized.
Conclusion:
PCC significantly decreased the abundance of morphine, codeine and IS. Adulterated water and urine samples containing 6-monoacetylmorphine, morphine and morphine-3-glucuronide yielded morphinone-3-glucuronide, 7,14-dihydroxy-6-monoacetylmorphine, 7,8-diketo-6-monoacetylmorphine and 7,8-diketo-morphine (tentative assignment). Reaction pathways may be different in the two matrices.
Manipulations of urine sample by urine substitution, urine dilution, and urine adulteration with highly oxidative chemicals to escape detection in doping control analysis have been reported in the past. Adulteration of urine with oxidising chemicals such as potassium permanganate, cerium ammonium nitrate, pyridinium chlorochromate etc can lead to considerable changes in the endogenous steroidal profile parameters and thus mask the abnormality in steroidal profile following steroid abuse. In this study we have identified the formation of two stable oxidation products on reaction of potassium permanganate with testosterone, an important endogenous urinary steroid. Isolation and characterisation of these oxidation products were performed using chromatography and spectroscopy and the products were elucidated as 4α,5α-dihydroxytestosterone and 4β,5β-dihydroxytestosterone. Formation of these two molecules in human urine after adulteration with potassium permanganate has been demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis. The products 4α,5α-dihydroxytestosterone and 4β,5β-dihydroxytestosterone have not been previously reported in urine and hence has the potential to be included in routine drug testing program for monitoring possible testosterone abuse and permanganate adulteration of urine.
Drug testing programs are established to help achieve a drug-free work environment, promote fair competition in sport, facilitate harm minimization and rehabilitation programs, better manage patient care by clinicians and service law enforcement authorities. Urine remains the most popular and appropriate testing matrix for such purposes. However, urine is prone to adulteration, where chemicals, especially oxidizing chemicals, are purposely added to the collected urine specimens to produce a false-negative test result. This article will describe the effect of various popular oxidizing adulterants on urine drug test results, the countermeasures taken by laboratories in dealing with adulterated urine samples and the prospect of developing more robust and economical methods to combat urine adulteration in the future.
RATIONALEPyridinium chlorochromate (PCC) is the active ingredient of 'Urine Luck', a commercially available in vitro adulterating agent used to conceal the presence of drugs in a urine specimen. The exposure of codeine and its major glucuronide metabolite codeine-6-glucuronide (C6G) to PCC was investigated to determine whether PCC is an effective masking agent for these opiate compounds.METHODS
Following the addition of PCC to both spiked and authentic codeine and C6G-positive urine specimens, the samples were monitored using liquid chromatography/mass spectrometry (LC/MS). Stable reaction products were identified and characterized using high-resolution MS analysis and, where possible, nuclear magnetic resonance (NMR) analysis.RESULTSIt was determined that PCC effectively oxidizes codeine and C6G, thus altering the original codeine-to-C6G ratio in the urine specimen. Four reaction products were identified for codeine: codeinone, 14-hydroxycodeinone, 6-O-methylcodeine and 8-hydroxy-7,8-dihydrocodeinone. Similarly, three reaction products were identified for C6G: codeinone, codeine and a lactone of C6G (tentative assignment).CONCLUSIONS
Besides addressing the complications added to interpretation, more investigation is warranted to further determine their potential for use as markers for monitoring the presence of codeine and C6G in urine specimens adulterated with PCC. Copyright © 2014 John Wiley & Sons, Ltd.
Concerns regarding specimen integrity have long been a major issue of urine drug testing due to acts of urine adulteration. At a high concentration, in vitro urine adulteration using sodium hypochlorite (bleach) produced false-negative results for 3,4-methylenedioxymethamphetamine (MDMA) in CEDIA® immunoassay screening with strong negative readings. However, these strong negative readings may act as a warning sign for further investigation of the sample where the detection of a unique marker in the form of N-chloroMDMA will suggest urine adulteration via bleach. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified N-chloroMDMA is a major product formed between hypochlorite and MDMA in urine. N-chloroMDMA was found stable at 4 °C for at least 10 h, but decomposed over time at room temperature (20 °C) with MDMA being identified as one of its main decomposition products.
Commercially available snack bars and other foodstuffs prepared from pressed hemp seeds were ingested by volunteers. Urine specimens were collected for 24 h after ingestion of the foodstuffs containing hemp seeds and tested for marijuana using an EMIT immunoassay and gas chromatography-mass spectrometry (GC-MS). Specimens from individuals who ate one hemp seed bar demonstrated little marijuana immunoreactivity, and only one specimen screened positive at a 20-ng/mL cutoff. Specimens from individuals who ate two hemp seed bars showed increased immunoreactivity, and five specimens screened positive at a 20-ng/mL cutoff. A single specimen yielded a quantitative GC-MS value (0.6 ng/mL), but it failed to meet reporting criteria. Several specimens from individuals who ate three cookies made from hemp seed flour and butter screened positive at both 50- and 20-ng/mL cutoffs. Two specimens produced quantitative GC-MS values (0.7 and 3.1 ng/mL), but they failed to meet reporting criteria. Several specimens also tested positive with an FDA-approved on-site marijuana-screening device. Hemp seeds similar to those used in the foodstuffs did not demonstrate the presence of marijuana when tested by GC-MS. In this study, ingestion of hemp seed food products resulted in urine specimens that screened positive for marijuana. No specimens gave a GC-MS quantitative value above the limit of detection for marijuana.
The active ingredient in the commercial workplace urine drug-testing adulterant, Klear, was previously determined to be nitrite ion. Nitrite adulteration compromises the confirmation of some drugs, notably the marijuana metabolite. A previously reported bisulfite step overcomes some nitrite adulteration, but it cannot do so in every case, which leaves the laboratory to report the specimen as not suitable for testing. Unlike many other adulterants, nitrite is found in normal urine at low concentrations. In order to defend a report of nitrite adulteration, it is necessary to provide evidence that the amount of nitrite in a workplace urine specimen could not arise by normal means. The objectives of this study were to identify all sources of nitrite in urine and the range of concentrations associated with these sources and to determine if nitrite adulteration can be supported based upon a quantitative result. The scientific literature was reviewed for internal and external sources of nitrite and their concentration ranges and are reported. The following specimens were obtained and nitrite concentrations measured by a spectrophotometric method: clinical specimens nitrite positive by test strip (< 15 micrograms/mL); specimens culture positive for nitrate-reducing microorganisms (< 36 micrograms/mL); specimens from patients on medications that may metabolize to nitrite (< 6 micrograms/mL); and drug-test specimens, both negative (< 130 micrograms/mL) and others that appeared to be adulterated with nitrite (range 1910-12,200 micrograms/mL, mean 5910). The literature and the nitrite measurements of this study indicate a substantial difference between concentrations from natural sources compared with adulteration. A quantitative measurement of nitrite by a well-structured assay can provide scientifically valid and forensically defensible proof of adulteration with a nitrite-containing substance.
Nitrite ion has been identified as the active ingredient of two commercial adulterants that could cause discrepant results between the immunoassay screening and gas chromatographic-mass spectrometric (GC-MS) confirmation of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in urine. Procedures to chemically convert the nitrite ion at the beginning of sample preparation for GC-MS analysis may not overcome all nitrite adulteration cases because portions of the THCCOOH might have been lost between the time of sample collection and the time of analysis. This study was conducted to further investigate the influence of both urine sample matrix and the duration of nitrite exposure on nitrite interference of THCCOOH detection. Forty clinical "THC-positive samples" that had been screened and confirmed positive for the presence of THCCOOH were spiked with 0.15M or 0.3M of nitrite. The levels of THCCOOH at various time intervals after nitrite spiking were monitored by instrument-based cannabinoids immunoassays (Syva EMIT d.a.u. and/or Roche Abuscreen ONLINE assays) and by an onsite THC immunoassay (Roche ONTRAK TESTSTIK). Results from this report demonstrate that the two outstanding "urine specimen factors" that dictated the effectiveness of the nitrite adulteration were urinary pH and the original drug concentration before nitrite spiking. Significant decreases in the immunoassay results could be observed within 4 h of nitrite treatment in the majority of samples with acidic urinary pH values. Regardless of their original concentration of THCCOOH (GC-MS ranging from 33 to 488 ng/mL), all of the 20 samples that had acidic pH values gave negative immunoassay results 1 day after nitrite adulteration. In contrast, the immunoassay results of samples with neutral or basic pH values were less affected by nitrite exposure in the same studies. Approximately two-thirds of the samples with pH values greater than 7.0 remained immunoassay-positive 3 days after nitrite spiking. Nevertheless, some of the adulterated urine that showed no change in immunoassay results might exhibit significant decrease in GC-MS recoveries even with bisulfite treatment, collaborating with the observations that a portion of samples screened positive with THC immunoassay in the laboratory could fail to confirm with GC-MS analysis. The decrease or loss of immunoassay detectable cannabinoid cross-reactives in acidic "THC-positive samples" can be attenuated by chemically increasing the pH value of the samples to the basic pH range.
A GC/MS method for the simultaneous determination of morphine, codeine, and O-6-monoacetylmorphine as well as other semisynthetic opiates in urine is described. The method employs 2H6-acetic anhydride to acetylate free hydroxyl groups on all of the opiates. As a result, the same hydrolysis and extraction
procedure is used for all analytes. Quantitation is achieved using gas chromatography/mass spectrometry in the electron impact
mode and with selected ion monitoring of the three most characteristic ions for each analyte. Ratios of these three ions are
used for verifying identity while the molecular ions are used for quantitation. The detection limit for all analytes is less
than 10 ng/mL.
The effect of various adulterants on radioimmunoassay (RIA) tests for amphetamine, cannabinoids, cocaine (metabolite), phencyclidine (PCP), barbiturate, and morphine was evaluated. A total of 16 readily available agents, at concentrations ranging from 1 to 25%, were tested with negative and positive urine samples. The results demonstrate that both false positive and false negative RIA results can be produced by easily achievable levels of different agents in urine specimens. Of the RIA tests evaluated, the one for cannabinoids was most sensitive to the presence of adulterants, whereas the PCP assay was most resistant. Although the presence of some adulterants could be detected by measuring the pH or specific gravity of the urine, other agents, at concentrations sufficient to alter the RIA results, could not be as easily detected. Gas chromatography/mass spectrometry (GC/MS) analysis of adulterated samples with false positive RIA results showed no detectable drug, indicating that the adulterants interfered with the RIA test itself. In contrast, GC/MS analysis of samples with false negative RIA results confirmed that at least some adulterants acted by reducing the drug concentrations in the specimen.
Marijuana is confirmed and quantitated in urine as 11-nor-delta 9-THC-9-COOH by GC/MS in the selected ion monitoring mode after extraction from urine. The addition of varying amounts of household bleach causes significant decreases in the quantitated amounts of tetrahydrocannabinol (THC). Urine samples containing various known amounts of THC (25-219 ng/mL) were spiked with increasing amounts of household bleach (0-64 microL/mL of urine). The samples were extracted using a solid-phase extraction procedure, derivatized, and analyzed by GC/MS. The area counts for the deuterated internal standard versus the native drug, as well as the quantitation for the various spiked samples, were compared with those of the non-adulterated samples. The results demonstrated that there was an inverse relationship between the amount of bleach used and the amount of THC recovered. The area counts for both the deuterated and native THC decreased as the amount of bleach increased. There appeared to be a consistent decrease in the ratio of native to deuterated THC, suggesting that the bleach affected the native drug more than the deuterated compound. The same decrease in THC concentrations were noted when the samples were assayed by an RIA methodology (Roche Abuscreen) as well as by an FPIA assay (Abbott TDx).
With the increased availability of nitrite-containing commercial products that are used for the adulteration of urine samples in the workplace, it is necessary for laboratories to be able to detect and confirm the presence of nitrite in these samples. We have developed a method to confirm the presence of nitrite in urine samples. The method uses the IonPac AS 14 analytical column with the Dionex series 45001 Bio LC system equipped with an anion self-generating suppressor and conductivity detector. Using a single-point calibration, the method is linear and accurately quantitates nitrite to 12,000 microg/mL. The limit of detection is 30 microg/mL, and the day-to-day precision of the assay has a coefficient of variation (CV) of 4.3% at 1200 microg/mL and 3.8% at 2700 microg/mL of nitrite.
Normal human urine contains many anions and cations. Ionic concentrations in urine have classically been determined by spectrophotometry of color reactions, flame emission spectrophotometry, atomic absorption spectrophotometry, high performance liquid chromatography, or potentiometry with ion-specific electrodes. Capillary ion electrophoresis (CIE) is a form of capillary electrophoresis which uses the differential electrophoretic mobility of ions to perform a separation of an ionic mixture. Various salts can be added to urine specimens to abnormally elevate ionic concentrations and interfere with either immunoassay urine drug screening procedures or gas chromatographic/mass spectrometric confirmation techniques. Application of CIE for the direct detection of endogenous anions and anionic adulterants in human urine specimens was the purpose of this investigation. CIE was performed using a Waters Quanta 4000 Capillary Electrophoresis System with either direct or indirect ultraviolet absorption detection at 254 nm. CIE of 30 random normal urine specimens and 21 urine specimens suspected of adulteration was performed. Duplicate aliquots were assayed by CIE and by colorimetric technique for nitrite. Sixteen specimens had elevated concentrations of nitrite and/or nitrate. The correlation coefficient between nitrite CIE and colorimetric results was 0.9895. Three specimens had detectable concentrations of chromate and were suspected of being adulterated with "Urine Luck," an adulterant found to contain chromate. Two specimens suspected of being adulterated with bleach were found to only contain chloride, sulfate, and phosphate. CIE is applicable to forensic analysis of urine anion concentrations. CIE can easily quantitate numerous endogenous anions and offers a method to detect and/or confirm anion adulteration of urine specimens.
Stealth is an adulterant used to avoid detection of drug abuse. The product does have an effect on the ability to detect several drugs of abuse, including the opiates morphine and codeine. It has previously been shown that low concentration (2500 ng/mL morphine) samples adulterated with Stealth tested negative by both Roche OnLine and Microgenics CEDIA immunoassays, but those spiked with higher concentrations (6000 ng/mL of codeine and morphine glucuronide) were positive. Initial results showed confirmation analysis was also sometimes negatively impacted by this adulterant. Urine samples were spiked with 6000 ng/mL of codeine and/or morphine glucuronide to assess the effect of Stealth. Each individual sample was split into separate aliquots. One aliquot of each was adulterated with Stealth following package directions. The samples were then tested by immunoassay and gas chromatography-mass spectrometry (GC-MS). The control and adulterated aliquots were positive by both immunoassays. Results of GC-MS analysis of the Stealth-adulterated aliquots following standard procedures using deuterated internal standards proved unsuccessful in several cases. In 4 of 12 cases (33%), neither the drugs nor internal standards were recovered despite repeated attempts. In one other sample, recovery was dramatically reduced, making accurate quantitation impossible, whereas the unadulterated aliquots of the same samples posed no problem with recovery. Addition of sodium disulfite to the aliquots prior to extraction allowed recovery of the drugs and internal standards from all samples. Analysis of the samples showed the concentration of morphine and codeine decreased in some by as much as 17 and 30%, respectively. In other cases, there was essentially no difference in the concentration seen before and after adulteration, with or without disulfite treatment. Unless the initial concentration of opiate is near the cutoff, samples containing opiates are likely to be immunoassay positive, it is important to consider this procedure as an option for samples that screen positive but the opiates and their respective internal standards are not recovered for GC-MS analysis.