Adulteration Of Urine By "Urine Luck"

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Abstract

Background: In vitro adulterants are used to invalidate assays for urine drugs of abuse. The present study examined the effect of pyridinium chlorochromate (PCC) found in the product "Urine Luck".

Methods: PCC was prepared and added to positive urine controls at concentrations of 0, 10, 50, and 100 g/L. The controls were assayed for methamphetamine, benzoylecgonine (BE), codeine and morphine, tetrahydrocannabinol (THC), and phencyclidine (PCP) with the Emit II (Syva) and Abuscreen Online (Roche) immunoassays, and by gas chromatography/mass spectrometry (GC/MS). Two tests were also developed to detect PCC in urine: a spot test to detect chromate ions using 10 g/L 1,5-diphenylcarbazide as the indicator, and a GC/MS assay for pyridine. We tested 150 samples submitted for routine urinalysis, compliance, and workplace drug testing for PCC, using these assays.

Results: Response rates decreased at 100 g/L PCC for all Emit II drug assays and for the Abuscreen morphine and THC assays. In contrast, the Abuscreen amphetamine assay produced apparently higher results, and no effect was seen on the results for BE or PCP. The PCC did not affect the GC/MS recovery of methamphetamine, BE, PCP, or their deuterated internal standards, but decreased GC/MS recovery of the opiates at both intermediate (50 g/L) and high (100 g/L) PCC concentrations and apparent concentrations of THC and THC-d3 at all PCC concentrations. Two of 50 samples submitted for workplace drug testing under chain-of-custody conditions were positive for PCC, whereas none of the remaining 100 specimens submitted for routine urinalysis or compliance drug testing were positive.

Conclusions: PCC is an effective adulterant for urine drug testing of THC and opiates. Identification of PCC use can be accomplished with use of a spot test for the oxidant.

Workplace urine drug testing continues to be an important program for employers in the private and public sectors. Under the guidelines established by the Substance Abuse and Mental Health Services Administration, testing involves a two-step process of screening by immunoassay, and confirmation by gas chromatography/mass spectrometry (GC/MS)1 (1). Employees who test positive for a drug class may suffer untoward consequences, ranging from denial of employment to suspension from work and dismissal. As a result, some individuals attempt to conceal their drug abuse by drinking large volumes of liquids to dilute urine drug concentrations (in vivo adulteration) or by adding foreign substances to urine after collection to invalidate testing procedures (in vitro adulteration) (2). Some in vitro adulterants act by interfering with the immunoassay detection scheme. Others convert the target drug to compounds that do not bind to the antibodies used in the immunoassays or that produce negative results in subsequent confirmation testing (2). Some states have outlawed the use of urine drug testing adulterants. Commercial products are available to assist an individual in "passing a drug test". One of the earliest products was "UrinAid". The active ingredient was glutaraldehyde, which interfered with screening immunoassays by producing final absorbance rate readings that were lower than those of true negative urine samples (3). The presence of glutaraldehyde can be detected by direct colorimetric analysis for aldehydes. "Klear" or "Whizzies" are commercial adulterants that contain potassium nitrate and cause interferences in the GC/MS analysis of marijuana [tetrahydrocannabinol (THC)] metabolites (4). Their presence can be suspected by a low or absent recovery of added deuterated THC internal standard (IS) and confirmed by direct measurement using colorimetric procedures (5). The latest product is "Urine Luck". The active ingredient of this adulterant is 200 mmol/L pyridinium chlorochromate (PCC). The present studies were conducted to determine the conditions under which Urine Luck interferes with screening and confirmation analyses for drugs of abuse.

Materials and Methods

materials
Urine Luck was purchased through the Internet from Spectrum Laboratories. Commercial urine quality-control materials were from Bio-Rad Laboratories (Lyphochek) and Euro/DPC Ltd. (CONDOA Level II). Two different controls were used because they exhibited different pH- buffering capacities. The pH of the Bio-Rad control was adjusted to 6.0 and 8.0 before the addition of PCC. The pH of the CONDOA was adjusted to 6.0 before adulteration. All pH measurements were performed on a digital meter. For the reaction-vs-time study, the CONDOA control was used without prior pH adjustment (pH 5.9). Adulterated urine controls were stored at room temperature before analysis. The controls nominally contained 1200 μg/L methamphetamine; 360 μg/L total morphine; 360 μg/L benzoylecgonine (BE), a cocaine metabolite; 60 μg/L 9-carboxy-THC (THC-COOH); and 30 μg/L phencyclidine (PCP). Except for the opiates, for which the cutoff concentration has been raised to 2000 μg/L, these concentrations exceeded the current Substance Abuse and Mental Health Services Administration cutoff concentrations by ∼20—25%. Because the control material did not contain codeine, we supplemented a drug-free urine sample (pH 6.0) with a codeine calibrator to a final concentration of 500 μg/L and examined the effect of PCC. Immunoassays for barbiturates, benzodiazepines, methadone, propoxyphene, and other drugs of interest were not tested because these drugs are not part of the Federal workplace drug testing panel. A 200 mmol/L solution of PCC (Sigma Chemical Co.) was prepared in saline. The pH of this solution was <1.0. Control samples were supplemented with 10, 50, and 100 g/L PCC. These concentrations were selected because the instructions accompanying Urine Luck call for dilution of 90—150 mL (3—5 oz) of urine with the vial containing 7 mL of the adulterant (final urine PCC concentration, 47—78 g/L). To identify the active component of Urine Luck, 200 mmol/L potassium chlorochromate (Sigma) and pyridine (Aldrich) were also obtained and prepared separately. Pyridine (10 g/L) was added to one vial of the Bio-Rad Level II control. Chlorochromate (10 g/L) was also added to a separate vial of the Level II control.

immunoassays
Unadulterated and adulterated quality controls were tested for five drug classes with the Emit II (Syva) and Abuscreen Online (Roche Diagnostics) assays on Hitachi 717 and Cobas Integra chemistry analyzers, respectively. Both reagents were used according to the manufacturers' recommendations (no extra dilutions made). For Emit II, the absorbance rates were recorded relative to the cutoff calibrator, which was set to zero. Under these conditions, any positive absorbance rate was indicative of a positive drug result. For Abuscreen, the approximate concentrations of drugs were recorded in μg/L. To simulate routine workplace drug testing procedures, adulterated samples were stored at room temperature for a minimum of 2 days before screening or confirmation testing.

spectrometric analysis and spot test for the adulterant
Absorbance curves for blank urine and that adulterated with 50 g/L PCC were measured on a scanning ultraviolet-visible spectrophotometer (Cary 219; Varian). The wavelength was scanned from 380 to 620 nm, using a 1-cm pathlength quartz cuvette. A spot test was developed using an indicator solution of 10 g/L 1,5-diphenylcarbazide in methanol. This indicator detected the presence of chromate anions and was colorless when first prepared (6). Two drops of the indicator were added to 1.0 mL of urine. A positive result was the production of a reddish-purple color.

gc/ms
Samples were assayed quantitatively by GC/MS analysis in the selected-ion monitoring mode, using procedures in routine use at the Hartford Hospital Forensic Toxicology Laboratory. A summary of the specific conditions used is shown in Table 1⇓ . Drug assays were conducted on a Hewlett-Packard 5890A gas chromatograph 5970B mass selective detector.

A GC/MS assay was developed for the detection of pyridine. To a 5-mL urine sample, 4.0 g of potassium carbonate was added with intermittent mixing. Acetone (0.5 mL) was added and mixed for 5 min. Under these high salt conditions, acetone forms a separate (top) layer with aqueous urine. The samples were centrifuged at 1000g. A 100-μL aliquot of the acetone layer was placed into a vial for GC/MS analysis. A Finnigan Mat ITS40 GC/ion trap mass spectrometer was used for pyridine analysis. A 1-μL sample was analyzed (splitless injection) on a 0.25 mm diameter DB-5 capillary GC column (J&W Scientific). The initial column temperature was 35 °C, which was ramped to 120 °C at 15 °C/min, and then to 280 °C at 50 °C/min, and held for 1 min. The temperatures of the injector and transfer lines were 275 and 280 °C, respectively. Full-scan electron ionization spectra were collected from m/z 30 to 200 atomic mass units at a scan rate of 600 ms/scan. All processed spectra were background subtracted. A drug-free urine sample supplemented with 2.5 and 5g/L PCC was used to make the reference calibrators.

urine samples
Fifty urine samples submitted for routine workplace drugs-of-abuse analysis were assayed using the spot test. Some of these samples were suspected of having been adulterated with PCC on the basis of an unusual color or low pH. Samples positive for the spot test (putatively because of chromate ions) were assayed by GC/MS for pyridinium. As controls, 50 urine samples submitted for routine urinalysis testing at Hartford Hospital and 50 urine samples submitted for compliance toxicology testing were also assayed using the spot tests. There was no reason for donors of these samples to adulterate their urine with PCC.

nitrite and chromium analysis
Four urine samples positive for the spot test and negative for pyridinium by GC/MS were further tested for nitrites, using a qualitative urinalysis dipstick (Chemstrip; Roche Diagnostics) and quantitative nitrite assay (nT; Chimera Chemical) on an automated chemistry analyzer (Hitachi 717; Roche). Nitrite is absent in unadulterated urine. These samples were also sent to National Medical Services (Willow Grove, PA) for chromium analysis using graphite furnace atomic absorption spectroscopy.

Results

The spectrophotometric absorbance curve of a 50 g/L solution of PCC in saline is shown in Fig. 1⇓ (spectrum A). A clearly defined inflection occurs in this spectrum at 440 nm. Spectrum B in Fig. 1⇓ is the spectrum of an unadulterated urine specimen. Spectrum C in Fig. 1⇓ is the spectrum of the same specimen adulterated with 50 g/L PCC. Other than a large increase in the absorbance at 440 nm, there are no distinguishing features in the absorbance spectrum of the adulterated sample. A similar spectrum would be expected with a urine sample that contains hemoglobin because of the presence of the Soret band at 400—440 nm (7). The adulterated urine itself appears a little more orange than normal, but was within the realm of possible urine colors. Spectrum D in Fig. 1⇓ is the spectrophotometric absorbance curve for a urine sample adulterated with 50 g/L PCC and reacted with the indicator. A major absorbance peak is present at 550 nm, which does not interfere with the native absorbance of the urine or urine to which the adulterant was added (Fig. 1⇓ , spectra A and C). The indicator is colorless and contributes nothing to the visible spectrum. In the absence of the adulterant, the spectrum of a urine specimen containing the indicator is identical to that of urine without the indicator.

The effects of PCC adulteration on immunoassay screening using the Emit II and Abuscreen systems are shown in Tables 2 and 3. There was no effect in either system for the BE assay except for a minor (10%) drop in the absorbance rate and concentration at the highest adulteration concentration (100 g/L). There was no significant change in the results of the BE GC/MS analysis (Fig. 2⇓ ). Only at high PCC adulterant concentration (100 g/L) did the amphetamine assay give a response rate less than the threshold value for Emit II (Table 2⇓ ). For Abuscreen, the apparent concentration was actually higher with increasing PCC concentrations. Although we were not able to determine why this occurred, the increase had been reported previously with the in vitro adulterant nitrite (8). A reduction in the absorbance rate was observed for the Emit II PCP assay. GC/MS analysis showed <10% interference for methamphetamine and no interference for PCP (Fig. 2⇓ ). These data suggest that the low pH of the highly adulterated samples was likely the mechanism for the interference in the Emit II assay, rather than a chemical alteration to the target drug. The Emit II package insert list an operational immunoassay pH range of 4.5—8.0. Urine specimens with pH values below (or above) this range will interfere with the activity of the enzyme label, glucose-6-phosphate dehydrogenase. It is unclear why low pH did not interfere with the BE assay to the same extent. The Abuscreen apparently was less subject to interferences by extremes in pH. (No restrictions for pH were listed on the manufacturer's package insert.)

Urine Luck had a greater effect on the results of the opiate and THC immunoassays. For morphine, there was a significant drop in the morphine signal at 50 g/L PCC, whereas at 100 g/L PCC, the result was below the 300 μg/L cutoff concentration. A similar effect in the Emit absorbance rate was also observed for codeine. GC/MS analysis (Fig. 2⇑ ) confirmed these findings: morphine decreased in concentration from 391 μg/L (blank) to 11 μg/L (100 g/L PCC, with ion ratios outside acceptable limits), whereas a more modest drop was observed for codeine (589 to 368 μg/L, respectively). The recoveries of the deuterated IS for morphine and codeine were unaffected by the presence of PCC. We were not able to determine what morphine or codeine were converted to by the PCC adulterant.

For THC, the destruction of the drug begins at the lowest adulterant concentrations and was complete at 50 g/L for both the Emit II and the Abuscreen assays (Tables 2⇑ and 3⇓ ). GC/MS results also confirmed that the drug was degraded by PCC. Moreover, the deuterated IS added to the GC/MS assay was also progressively destroyed in the presence of increasing concentration of the adulterant, as shown in Table 4⇓ . Unlike the other assays, the mechanism of interference for this adulterant appeared to be a combination of a suboptimum pH and oxidation effects. The GC/MS assay for the THC-COOH metabolite was particularly vulnerable to degradation because the solid-phase extraction for THC metabolites was conducted at low pH (2.0 ± 0.5), further facilitating the oxidation of the drug and its IS.

The effect of 10 g/L PCC adulterant on the Emit II results as a function of time after adulteration are shown in Table 5⇓ . There was no additional degradation in the resulting absorbance rates beyond the first few hours.

The colorimetric spot test (screening assay) for PCC detects the presence of chromate. In unadulterated urine, the total chromium concentration (Cr+3 and Cr+6) is 1.9—38.4 nmol/L (9). Even in toxic overdoses, urine concentrations are far below the values found in PCC-adulterated samples (10 mmol/L for a 50 g/L solution). The spot test was not sensitive enough to detect chromate ions in unadulterated urine samples. Under physiologic urine pH conditions, the color development took 5—7 min for completion and remained stable for hours (data not shown). Under increasing acid conditions, the reaction was faster, although the resulting color faded after 1 h. When this spot test was used on 50 samples submitted for toxicologic analysis, a positive result was obtained for 9 urine samples (Table 6⇓ ). Sufficient amounts of urine for GC/MS analysis were available on six of these samples. Pyridine was identified in two of these six samples. Fig. 3⇓ is a representative GC/MS analysis of an adulterated urine sample submitted for toxicology testing. Under alkaline extraction conditions, pyridinium was dissociated from the metal complex to form free pyridine. The major features were the M+1 ion at m/z 80, the M ion at m/z 79, and a fragment ion at m/z 52. Ions at m/z 43 and 59 were produced from residual bleed from the acetone solvent, which could not be background corrected. The spot test was negative for all 100 samples submitted for routine urinalysis and compliance drug testing.

We determined that the active ingredient of Urine Luck was the chlorochromate anion, and not pyridine or its combination with chlorochromate. A 10 g/L solution of either PCC or potassium chlorochromate reduced the THC Emit II response for the control from −31 ΔA/min for the unadulterated urine, to −60 and −54 ΔA/min, respectively, for the chlorochromate-adulterated samples. A 10 g/L solution of pyridine alone produced no change in response relative to the unadulterated urine (−25 ΔA/min).

Discussion

Detecting the presence of adulterants is essential in reducing the rate of false-negative results for urine drugs-of-abuse testing. Nitrites can be readily detected by a urinalysis dipstick test or through direct chemical analysis. Because nitrites do not interfere with immunoassays, samples containing concentrations of THC above the threshold will be routinely assayed for the THC-COOH metabolite by GC/MS. A urine sample that exhibits little or no recovery of a deuterated IS or target drug is highly suggestive of an adulterant such as nitrite (4).

Using elemental analysis, mass spectrometry (for pyridine), ion chromatography (for chloride), and inductively coupled plasma atomic emission spectroscopy, the active ingredient in Urine Luck was determined to be PCC. Urine samples adulterated with PCC tested by GC/MS will produce results that are similar to those for nitrite, i.e., low or no recovery of the IS or target drug. This adulterant differs from nitrite, however, because at high concentrations, the chemical also produces negative immunoassay screening results. Thus, drug testing laboratories unaware of the presence of PCC will not perform GC/MS analysis, and the use of PCC may escape detection. Its presence can be suspected by the presence of an abnormally low pH or the appearance of an orange tint to the urine.

The spot test described in this report may be a simple screening test that can be used for further testing of suspected urine samples. This test is not specific for chromate ions because molybdenum, mercury, and vanadium salts can also produce a positive reaction (6). However, it is unlikely that a spot test will be positive under normal physiologic or pathologic conditions or after environmental exposure because the expected metal concentrations in these urines would be substantially lower than those in situations of in vitro adulteration (9). For laboratories performing a large volume of forensic urine drug testing, an instrument-based colorimetric assay may be more convenient, although none are commercially available at this time. The specificity of the spot test for PCC is of concern because we were able to confirm only one-third of the positively screened samples for pyridine. It may be possible that other oxidizing adulterants yet unknown to toxicology laboratories are being used that contain metal ions or substances capable of oxidizing drugs of abuse. Both PCC and potassium chlorochromate react with the nitrite urinalysis dipstick to produce a purple color that is darker than the color that occurs for nitrites; therefore, the dipstick method could be used as an alternative or in addition to the spot test.

Confirmation of the presence of pyridine may be performed by direct GC/MS analysis for pyridine. When the DB-5 column was used, pyridine eluted very early in the gas chromatogram. Use of a more polar column, such as carbowax, will likely enable solvents such as pyridine to be retained longer on the column. However, these polar columns are not routinely used for drug-of-abuse testing and, therefore, are less readily available. Although the molecular ion of pyridine is m/z 79, we also consistently observed the M+1 ion. This suggested that some chemical ionization putatively occurred, with saturation of ions within the source. Because the adulterant was not tested on the mass selective detector, it is unknown whether this phenomenon was specifically related to testing using the ion trap.

The combination of a colorimetric assay for chromate and GC/MS assay for pyridine is forensically acceptable for the detection of PCC. Each uses a different analytical technique, and each targets a different chemical aspect of the adulterant. With routine screening, manufacturers might opt to remove Urine Luck from the market, as was the case for UrinAid. Undoubtedly it will be replaced with another adulterant.

Source, Graphs and Figures: Adulteration of Urine by
 
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