Lessons learned from a case of tert-butyl glucuronide excretion in urine - "New" psychoactive alcohols knocking on the back door?

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a marker of recent ethanol consumption or ethanol exposition. tert-Butanol is primarily used as a solvent and intermediate chemical. Like tert-amyl alcohol, tert-butanol is discussed in Internet forums as ethanol replacement. We discuss false-positive immunological EtG screenings by excretion of different alcohol glucuronides (EtG homologs), mainly tert-butyl glucuronide in urine of a polytoxikomanic in-patient. METHODS: Three consecutive urine samples from an in-patient with a long history of multiple substance abuse including solvents were analyzed by DRI EtG enzyme immunoassay (ThermoFisher Scientific Microgenics) on a Beckman Coulter AU680 analyzer, an in-house LC-MS/MS for EtG, 1-propyl, 2-propyl, 1-butyl, 2-butyl, and tert-butyl glucuronide, and an in-house headspace GC-FID of free congener substances methanol, 1-propanol, 2-butanone, 2-butanol, isobutanol, 1-butanol, 3-methyl-1-butanol, 2-methyl-1-butanol, and additionally for ethanol, acetone, 2-propanol, tert-butanol and 2-methyl-2-butanol. RESULTS: EtG immunoassay yielded two positive urine samples (0.2 and 0.6mg/L or 0.1 and 0.2mg/g creatinine; cut-off 0.1mg/L) which were tested EtG negative by LC-MS/MS (cut-off 0.1mg/L) but positive for tert-butyl glucuronide (3.7 and 27.1mg/L), 2-butyl glucuronide (1.1 and 3.5mg/L), and 2-propyl glucuronide (0.1 and 0.4mg/L). Headspace GC-FID detected tert-butanol (0.97 and 4.01mg/L), methanol (0.96 and 0.62mg/L), 2-butanone (0.84 and 1.65mg/L), and 2-butanol (0.04 and 0.09mg/L), but no ethanol and no 2-methyl-2-butanol. CONCLUSION: Cross-reaction of EtG homologs, mainly tert-butyl glucuronide after suspected tert-butanol or isobutane abuse, explains the false-positive EtG immunoassay findings. Future investigations could address the usefulness of alcohol glucuronides (EtG homologs) in urine as (a) biomarkers of an exposition to alkans or their corresponding alcohol metabolites and (b) as markers for using "old"-well known alcohols like tert-butanol or tert-amyl alcohol as easy to obtain, cheap, potent and "undetectable" ethanol replacements or "New" Psychoactive Alcohols.

Excessive urinary excretion of isopropyl glucuronide after isopropanol abuse.

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a marker of alcohol consumption. We present a case of a false-positive immunological EtG screening result due to excessive isopropyl glucuronide excretion in urine of an alcohol-dependent patient with a history of industrial cleaning fluid abuse. METHODS: EtG screening was done with the Microgenics DRI EtG enzyme immunoassay on a Beckman Coulter AU680 analyzer according to the testkit instructions. Confirmatory analysis was done by LC-MS/MS for EtG, 1-propyl (syn. n-propyl), 2-propyl (syn. isopropyl), 1-butyl, 2-butyl, and tert-butyl glucuronide. Both methods were validated according to the Guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh, Germany). RESULTS: EtG screening by immunoassay was positive, approx. 860mg/L or approx. 1540mg/g creatinine (forensic cut-off 0.1mg/L, clinical cut-off 0.5mg/L). LC-MS/MS confirmatory analysis was negative for EtG (<0.05mg/L; forensic cut-off 0.1mg/L), but strongly positive for 2-propyl glucuronide (approx. 1100mg/L or 2000mg/g creatinine; cut-off 0.1mg/L). 1-propyl, 1-butyl, and tert-butyl glucuronide were negative (<0.05mg/L; cut-off 0.1mg/L), 2-butyl glucuronide was 0.1mg/L (cut-off 0.1mg/L). CONCLUSION: Consumption of household and industrial chemicals with short chain aliphatic alcohols should be considered a rare but potential source of false-positive EtG immunoassay results. Glucuronides from frequently used short chain aliphatic alcohols, like 1-propanol (syn. n-propanol) and 2-propanol (syn. isopropanol) as the most important disinfectant components, should be included into EtG confirmatory analysis. This will be helpful not only for the assessment of the source for remarkable EtG immunoassay results, it can also contribute to a more specific diagnosis in cases with suspected intoxication by consumer or industrial chemical products. Excessive urinary 2-propyl glucuronide (syn. isopropyl glucuronide) concentrations should be considered a marker of isopropanol intoxication.

Cross-reaction of propyl and butyl alcohol glucuronides with an ethyl glucuronide enzyme immunoassay.

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a marker of recent alcohol consumption. Using immunoassays for EtG screening without confirmatory analysis bears a risk of getting false-positives as shown for trichloroethyl glucuronide from chloral hydrate medication and 1-propyl glucuronide from propanol-based hand disinfection. The aim of the study was to check whether glucuronides of frequently used aliphatic short chain alcohols aside from EtG and 1-propyl glucuronide can cross-react with the DRI(®) Ethyl Glucuronide Assay. METHODS: Aliquots of EtG-free urine were individually spiked with methyl ß-D-glucuronide, 1-propyl ß-D-glucuronide, 2-propyl ß-D-glucuronide, 1-butyl ß-D-glucuronide, 2-butyl ß-D-glucuronide, and tert-butyl ß-D-glucuronide. To check the response rate of the DRI(®) Ethyl Glucuronide Assay to its target analyte, EtG was also added to a native EtG-free urine sample. The spiked alcohol glucuronide concentrations (seven levels up to 10mg/L) and the DRI(®) Ethyl Glucuronide Assay results were evaluated by Passing-Bablok regression analysis. The 95% confidence interval ranges for the slope of the regression function were considered a measure of cross-reaction of the individual alcohol glucuronides with the enzyme immunoassay. RESULTS: 2-Propyl glucuronide showed a cross-reactivity of 69-84% at the 95% probability level, methyl glucuronide, 1-propyl glucuronide, and 1- and 2-butyl glucuronide of 4-9%, and tert-butyl glucuronide almost no cross-reactivity. The response rate for EtG was 87-94% at the 95% probability level. CONCLUSION: The DRI(®) Ethyl Glucuronide Assay shows cross-reaction rates with aliphatic short chain alcohol glucuronides aside from EtG which bear a risk of getting false-positives regarding ethanol consumption. Mass spectrometric detection of EtG is mandatory for confirmation of positive immunological EtG screenings.

Inhalation but not transdermal resorption of hand sanitizer ethanol causes positive ethyl glucuronide findings in urine.

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a specific marker of recent ethanol consumption. There is an ongoing debate about whether inhalation or transdermal resorption of sanitizer ethanol is the underlying cause for positive EtG findings after hand disinfection. METHODS: Desderman(®) pure (Schülke & Mayr GmbH, Norderstedt) with 78.2g 96% (v/v) ethanol/100g and approx. 10% 2-propanol was used for multiple hand disinfection without and under an exhauster. Simulating a common working day in a clinic, 5 co-workers of our lab used the sanitizer 32 fold within 8h and 2 persons were merely exposed to the sanitizer vapor but without any dermal sanitizer contact. Any additional ethanol intake or exposition was reliably excluded. Spot urine was collected at baseline, after 1, 2, 4, 6 … 14, and finally 24h after the first sanitizer use. A validated LC-MS/MS was used for MRM and MS(3) of EtG and qualitative analyses of ethyl sulfate and 2-propyl glucuronide. RESULTS: Multiple hand disinfection caused positive EtG findings of up to 2.1mg/L or 1.7mg/g creatinine in 4 out of 5 test persons and even of 0.6mg/L or 0.8mg/g for 2 controls which were merely exposed to the sanitizer vapor but without any sanitizer contact. EtG results between the clinical (0.5mg/g) and the forensic (0.1mg/g) cut-off were obtained even 6h after the last sanitizer exposition. An exhauster prevented the sanitizer vapor inhalation and reduced the EtG excretion to mostly below the detection limit of 0.02mg/g. The maximum value was 0.09mg/g. Ethyl sulfate and 2-propyl glucuronide (2-PpG) were detectable only in the EtG positive samples. 2-PpG is a metabolite of 2-propanol, which is quite frequently used in disinfectants. Thus, the detection of this substance can be used in cases of odd EtG results as an indicator of (unintended) sanitizer exposition. CONCLUSION: Ethanol from hand sanitizers is predominantly incorporated by the respiratory tract but not via the skin. It can cause a distinct ethyl glucuronide excretion and thus analytically true-positive but forensically false-positive EtG findings in the urine of ethanol abstaining persons. Since accidental ethanol inhalation can occur quite frequently in the working place or even private household, such a situation should always be considered when EtG is used as a marker of recent ethanol consumption.

Ethyl glucuronide identified in commercial hair tonics.

BACKGROUND: Ethyl glucuronide (EtG) in hair is considered as a specific marker of ethanol consumption. Prompted by a report of positive EtG hair testings due to hair treatment with an EtG containing hair lotion, commercially available herbal hair tonics from supermarkets, drug-stores, and health food stores were analyzed for the presence of EtG and ethyl sulfate (EtS). METHODS: LC-MS/MS (QTRAP 5500 mass spectrometer) was done in multiple reaction monitoring (MRM), enhanced product ion (EPI) and MS(3) mode. The lower limit of quantitation was 0.05 mg/L for EtG and the cut-off for the detection of EtS 0.01 mg/L. RESULTS: Altogether 11 hair tonics from 8 manufacturers were tested, with 1 product in 3 different lots. EtG ranged between 0.07 and 1.06 mg/L (7 products from 4 manufacturers) and was almost identical in the 3 lots of 1 product (1.01-1.06 mg/L). EtS was found in 3 out of the 11 hair tonics. CONCLUSIONS: EtG is quite frequently present in commercially available herbal hair tonics. Using EtG in hair as a marker of alcohol (ab)use, one has to consider external sources of EtG and has to assess the use of hair care products, esp. if the patient denies any ethanol intake. Whether EtS is a more reliable alcohol (ab)use marker, as sometimes discussed, should be critically assessed against the background of its broad use in large amounts in industrial chemistry.

False-positive ethyl glucuronide immunoassay screening caused by a propyl alcohol-based hand sanitizer.

BACKGROUND: Urine ethyl glucuronide (EtG) is considered as a specific marker of recent ethanol consumption. We describe false-positive DRI(®) EIA EtG enzyme immunoassay results caused by propyl glucuronides in urine after using a propanol-based hand sanitizer. METHODS: EtG screening was done with the DRI(®) EIA EtG assay (Microgenics), using a cut-off of 0.5 mg/L as recommended by the manufacturer and of 0.1 mg/L as demanded by the German Regulations for Reissuing Drivers Licenses. Confirmatory EtG analysis was done with the ClinMass(®) EtG LC-MS/MS testkit (Recipe), extended by the mass transitions 235.1→75.1, 235.1→85.1, and 235.1→113.1 for the detection of the 1- and 2-propyl glucuronides. Self-experiments were done by staff members of our lab (n=7), using 3 mL Sterillium(®) Classic Pure (30 g/100 g 1-propanol and 45 g/100 g 2-propanol) for hand sanitation every quarter of an hour for 8 h according to DIN EN 1500:2011-05 with and without an exhauster and by passive inhalation of the sanitizer vapor. Spot urine samples were taken immediately before and up to 24 h after the first sanitizer use. RESULTS: False-positive immunoassay results of up to 4 mg/L or 2.3 mg/g creatinine were obtained after normal use of the sanitizer and also after passive inhalation of the sanitizer vapor (up to 0.89 mg/L or 0.61 mg/g). Immunoassay results were positive even after 4-fold use of the sanitizer (up to 0.14 mg/L or 0.38 mg/g) and up to 6 h after the last sanitizer contact (maximum 0.63 mg/L and 0.33 mg/g for sanitizer users and 0.25 mg/g after passive inhalation). Spiking of EtG-free urine with 1-propyl glucuronide (Athena Environmental Sciences) between 0.05 and 10 mg/L clearly demonstrated a cross reaction of the immunoassay of approx. 10% as compared to EtG. LC-MS/MS of urines with a positive immunoassay EtG result did not show EtG signals, but distinct signals of 1-propyl glucuronide (n-propyl glucuronide) and 2-propyl glucuronide (iso-propyl glucuronide). An exhauster effectively prevented the inhalation of the sanitizer vapor, the formation of propyl glucuronides and thus false-positive DRI(®) EIA EtG screening results, proving that propyl alcohols are almost exclusively taken up by respiration. CONCLUSIONS: The widespread use of propanol-containing products such as hand sanitizers may lead to sufficient uptake of propyl alcohols and excretion of significant amounts of propyl glucuronides to yield false-positive DRI(®) EIA EtG screening results. Thus, positive EtG immunoassay results have to be controlled by mass-spectrometry, in clinical cases at least if ethanol intake is denied by the patient.