Effects of Adulterants on Analytical Tests

Effects of Selected Adulterants on Drug Tests

Numerous studies document the effects of suspected adulterators and commercial adulterant products on analytical tests for drugs of abuse. In 1976, Kim and Cerceo added sodium chloride to drug-positive urine specimens and obtained negative results using an immunoassay method for drugs of abuse (enzyme-multiplied immunoassay technique – EMIT). An l988 study by Mikkelsen and Ash evaluated the effects of a variety of chemicals purported to be used as adulterants on EMIT. The authors also monitored the pH, relative density, and appearance (i.e., color and turbidity) of adulterated specimens that gave false-negative results. Another study by Cody and Schwarzhoff evaluated a variety of potential adulterants with regard to their effect on screening tests, in this case, radioimmunoassay. Similarly, a 1989 study by Warner evaluated effects of various chemicals on immunoassay methods including Fluorescence Polarization Immunoassay FPIA, radio-immunoassay RIA, and EMIT. Schwarzhoff and Cody investigated the effects of 16 various substances used as adulterants on FPIA. Wu et al. evaluated the effects of 10 adulterant substances on another immunoassay method, cloned enzyme donor immunoassay (CEDIA). A 1994 study by Baiker et al. investigated the effects of bleach on the analysis of tetrahydrocannabinol carboxylic acid (THCA), using small amounts of bleach that were not detectable by odor or altered pH. The analytical methods evaluated in this study included FPIA, RIA, and GC-MS.

As described before, initial attempts at adulteration utilized readily available materials intended for other purposes. In many cases, these adulterants needed to do nothing more than prevent the sample from being tested because, initially, there were no consequences to adulterating a sample. In many cases, just causing the sample to physically look or smell different was sufficient to prevent its being tested. Whether or not it actually had an impact on test results was not important. Many of the products had significant negative impacts on the integrity of the testing process if the samples were, indeed, tested. Many were no surprises because of their obvious strong nature, such as Drano™ and bleach. Others were difficult to identify and actually had a negative effect on the testing process. These included Visine or small amounts of blood, small enough volume that the urine sample did not appear abnormal. In another study by Cody small amounts of simple table salt, that could be easily embedded under a fingernail and then “washed” into the urine specimen, a process that could be easily accomplished without careful observation of the individual providing the sample, were evaluated. The use of such unsophisticated adulteration attempts are not common today because the program used the information regarding these adulterants to set rules that prevented many from being used (i.e., washing hands before providing the sample measuring temperature, examination of the sample for appearance and odor).

Glutaraldehyde was first identified as the ingredient in a commercial adulterant product called UrinAid. The effects of glutaraldehyde on 4 different immunoassays were evaluated in a 1994 study by Goldberger and Caplan. The methods evaluated were EMIT, FPIA, kinetic interaction of micro particles in a solution (KIMS), and RIA They found RIA to be the least affected of the assays evaluated. They found the adulterant had varying degrees of effect depending on the assay resulting in both false-negative results for samples that should have been found to be positive and several examples of individual samples that were false-positive, particularly the PCP assay. A 1996 study by George and Braithwaite evaluated the effects of glutaraldehyde on the Syva EMIT II immunoassay. Those authors showed the effect of glutaraldehyde was concentration-dependent, ranging from virtually no effect to causing false-negatives with all drugs analyzed. Conversely, Wong, using the Monitect PC II test strip, saw no effect on the expected positive results at a concentration of 2.5% v/v), a concentration that George and Braithwaite found to affect all EMIT drug assays. The drug concentrations varied between the studies, some higher and some lower, which may have contributed to the results in addition to the basic difference in the assay principle.

Pyridinium chlorochromate was first identified as the ingredient in a commercial adulterant product called Urine Luck. Wu et al. evaluated the effects of pyridinium chlorochromate on 2 immunoassay methods (i.e., Syva EMIT II, Roche Online) and on GC-MS assays for drugs of abuse. The authors also described tests that could be used to detect the adulterant: a spot test for chromate ions and a GG-MS procedure for pyridine. The assay for pyridine had little practical application because pyridine is normally found in the body and there are no studies to demonstrate normal versus “adulterated” levels as have been developed for other adulterants. The adulterant did affect all EMIT and Roche Online assays tested giving lower readings for all but amphetamines where the results appeared to be increased. They found no impact on GC-MS analysis except for decreased recovery for THCA and opiates. A 2000 study by Paul et al. documented the effects of pyridinium chlorochromate on tests for drugs of abuse and described a method for the quantitative detection of chromium VI in urine.

Nitrite was an ingredient in several commercial adulterant products. Lewis et al. performed a study to show the reactions of nitrite and THCA using HPLC and negative electro-spray mass spectrometry. The authors described a procedure for the addition of potassium carbonate to urine specimens to prevent nitrite from affecting THCA analysis. Tsai et al. documented the effects of nitrite on immunoassays and GC-MS tests for cocaine metabolite (benzoylecgonine), morphine, marijuana metabolite (11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid, THCA), amphetamine, and phencyclidine PCP. A 2000 study by Thai et al. documented the effects of nitrite on 2 immunoassay methods (i.e., Syva EMIT and Roche Online, as well as a tetrahydrocannabinol (THC) point of collection test (i.e., Roche ONTRAK TESTSIK THC and a THCA GC-MS assay. Paul and Jacobs evaluated the effects of several oxidizing adulterants, including nitrite, on THCA analysis by GC-MS.

Peroxide and peroxidase were ingredients identified in the commercial adulterant product Stealth. In 2001, Cody and Valtier tested urine samples adulterated with Stealth using 2 immunoassay techniques: enzyme immunoassay (Online) and CEDIA. The authors also performed specimen validity testing on the samples, including pH, specific gravity, color creatinine, chloride, urea, blood, glucose, and nitrite. Analysis showed that none of these parameters exceeded established normal clinical values. Samples containing THC acid metabolite benzoylecgonine, morphine, secobarbital, PCP, amphetamine, and lysergic acid diethylamide (LSD) then tested and showed Stealth to cause samples positive for the marijuana metabolite (11-nor-∆9-tetrahydrocannibinol-carboxylic acid), LSD, and opiate (morphine) at 125 to 150% of cutoff to screen negative. Analysis of THCA, LSD, and morphine at median concentrations of these analytes at the laboratory were also analyzed and gave the same results. The authors also described a simple analytical procedure for identification of the presence of the Stealth adulterant using 10 µL of urine in a test tube containing 50 µL of TMB substrate reagent in 500 µL of 0.1 M phosphate buffer pH 7. Cody et al. also evaluated the effects of Stealth on GC-MS assays for marijuana metabolites and opiates, including a procedure to allow identification of morphine by GC-MS in the presence of this adulterant. The effect of Stealth on morphine was concentration-dependent, showing that at very high concentrations the amount of drug exceeded the oxidation capacity of the adulterant. The authors modified the extraction procedure by the addition of 2.5 mg/mL sodium disunite to the aliquots before extraction, which was able to afford essentially full recovery of the analyses including the internal standard, from the sample. The effect of Stealth using the Monitect PCll device was evident for THC cocaine, and PCP screens in the 2002 work of Wong but not opiate or amphetamine assays.

Papain, an adulterant investigated by Burrows et al., is a common ingredient in commercial meat tenderizers. This study evaluated the effects of papain on FPIA for multiple drugs, on a GC-MS assay for THCA, and on an HPLC-UV assay for nordiazeparn.

There have also been studies evaluating the effects of adulterants of on-site point-of-collection immunological tests for drugs of abuse and evaluating the effectiveness of on-site devices for determining specimen validity. At this time, these on-site tests are not allowed for federal workplace drug-testing programs, but they are used for private sector workplace programs. In 2004, HHS published proposed revisions to the Mandatory Guidelines to allow the use of point-of-collection tests for federal employee testing and requested public comments concerning the proposed revisions. The process is continuing at this time.

Effects of Commercial Adulterant Products on Drug and SVT Testing Using Control Materials

Since April 2001, the DWP has supported the investigation by the NLCP of the effects of various commercial adulterant products on initial and confirmatory drug tests used for regulated specimens. In this investigation, the adulterant product was added to a 90 mL certified negative urine sample as well as a 90-mL certified negative urine sample fortified at twice the HHS initial test cutoff for the relevant drug class, with selected drug analytes representing each of the mandated HHS drug classes. The analytes included THCA, benzoylecgonine, morphine, acetylrnorphine 6-AM, phencyclidine, and methamphetamine. A paired set of control reference urine specimens was prepared by taking 90 mL of certified negative and fortified negative urines and adding volumes of water identical to the volumes of the adulterants added to the adulterated urine specimens. The adulterated and ‘diluted” control reference urines were stored refrigerated for 24 h before being sent via FedEx to be analyzed on the third day after preparation. The samples were tested at several certified laboratories, using the same initial and confirmatory drug tests as used for regulated specimens. The immunoassay reagents, extraction procedures, and/or GC-MS methods differed among the laboratories selected to perform the analyses but represented all immunoassay techniques used as primary or secondary procedures (Dade Behring Enzyme Immunoassay, Microgenics DRI Enzyme Immunoassay, Microgenics CEDIA, Roche KIMS, and Abbott FPIA). This investigation also evaluated the effects of these various commercial adulterant products on specimen validity tests used by the testing laboratories for regulated specimens (i.e., pH, creatinine, specific gravity, and oxidant). If the product was determined to affect the initial drug test, confirmatory drug test, or validity test results creatinine, specific gravity, pH or oxidant), the components of the adulterant were identified. The results of the study through 2007 are presented in Tables 5.2, 5.3 and 5.4. The specimen results for the adulterated urines were compared to their “diluted” control reference and adulterant effects were determined by relative suppression of measured drug or change in SVT response. The degree of dung suppression was categorized as within ranges (<20%, ≥20%, ≥50%, and ≥80%), and changes in SVT response were defined as more than ±5.0 mg/dL for creatinine, more than ±0.005 for SpGr, more than ±0.5 for pH, and more than 200 mcg/mL for relative oxidant activity as compared to nitrite. These products preferentially affected analyses of opiates and cannabinoids and the CEDIA initial drug test was most sensitive to the effects of the adulterants.

Those products that had effects on drug analyses or produced non-normal SVT results were analyzed for their components. The results of the component analyses are presented in Table 5.5.

Evaluation of these data demonstrated that Adulterant products containing nitrite affected CEDIA assays, opiate and cannabinoid initial drug tests, morphine, 6-AM, and THCA confirmation assays as well as pH and oxidant SVT tests. It was also determined that adulterant products containing chromium VI affected cannabinoid initial drug tests and THCA confirmation as well as pH SVT tests. In addition, it was determined that sulfate and halogen containing adulterant products affected cannabinoid initial drug tests and THCA confirmation as well as pH and SpGr SVT tests. Adulterant products containing iodate affected CEDIA assays, opiate and cannabinoid initial drug tests, morphine, 6-AM, and THCA confirmation assays as well as creatinine and pH SVT tests. Many of the adulterant products contained acid, some relating to their anionic counter-ions and others reacted with acid to stabilize the adulterant.

Over this investigational period only one substitution product was identified as being used to subvert drug-testing results. This product was Minute-man, whose initial testing results were drug-negative, creatinine 185 mg/dL, pH 6.1, and no abnormal oxidant activity. Subsequent testing of a bottle found in a restroom trash container resulted in non-detectable amounts of potassium, phosphate, and uric acid and a chloride level 20 times the upper reference range. All of these values were inconsistent with normal human urine.

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