Ethyl Glucuronide, Ethyl Sulfate, and Ethanol in Urine after Intensive Exposure to High Ethanol Content Mouthwash

Transcription

1 Ethyl Glucuronide, Ethyl Sulfate, and Ethanol in Urine after Intensive Exposure to High Ethanol Content Mouthwash Gary M. Reisfield 1, *, Bruce A. Goldberger 1,2, Amadeo J. Pesce 3, Bridgit O. Crews 3, George R. Wilson 4, Scott A. Teitelbaum 1, and Roger L. Bertholf 5 Departments of 1 Psychiatry and 2 Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida; 3 Millennium Research Institute, San Diego, California; and Departments of 4 Community Health and Family Medicine and 5 Pathology, University of Florida Health Science Center, Jacksonville, Florida Abstract To determine the degree of ethanol absorption and the resultant formation and urinary excretion of its conjugated metabolites following intensive use of high ethanol content mouthwash, 10 subjects gargled with Listerine antiseptic 4 times daily for 3¼ days. First morning void urine specimens were collected on each of the four study days and post-gargle specimens were collected at 2, 4, and 6 h after the final gargle of the study. Urine ethanol, ethyl glucuronide (EtG), ethyl sulfate (EtS), and creatinine were measured. Ethanol was below the positive threshold of 20 mg/dl in all of the urine specimens. EtG was undetectable in all pre-study urine specimens, but two pre-study specimens had detectable EtS (6 and 82 ng/ml; 16 and 83 µg/g creatinine). Only one specimen contained detectable EtG (173 ng/ml; 117 µg/g creatinine). EtS was detected in the urine of seven study subjects, but was not detected in the single specimen that had detectable EtG. The maximum EtS concentrations were 104 ng/ml and 112 µg/g creatinine (in different subjects). Three subjects produced a total of eight (non-baseline) urinary EtS concentrations above 50 ng/ml or 50 µg/g creatinine and three EtS concentrations exceeding 100 ng/ml or 100 µg/g creatinine. In patients being monitored for ethanol use by urinary EtG and EtS concentrations, currently accepted EtG and EtS cutoffs of 500 ng/ml are adequate to distinguish between ethanol consumption and four times daily use of high ethanol content mouthwash. Ethanol abstinence is required by substance use monitoring programs for professionals, in certain workplace and legal contexts, and other zero tolerance situations. Measurement of ethanol in breath or body fluids is an insensitive indicator of abstinence because ethanol is rapidly metabolized to acetaldehyde and is detectable in blood and breath for only a few hours after consumption. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor but longer-lasting metabolites of ethanol, and are being used as biomarkers of recent (prior two to five days) ethanol use. However, determining the threshold urinary concentrations of EtG and EtS that reliably distinguish between deliberate ethanol use and incidental exposure to ethanol-containing consumer products (e.g., hand sanitizers, mouthwashes, and food) has been problematic. There is no uniformly applied threshold; laboratories and regulatory authorities (1) have used thresholds from 100 to 1000 ng/ml (2 4). Skipper et al. (5) introduced EtG testing in the United States and have suggested that even a 1000 ng/ml threshold may be too low to rule out incidental ethanol exposures. Indeed, we recently reported a maximum urinary EtG concentration exceeding 2000 ng/ml after intensive handwashing with an ethanol-containing hand sanitizer (6). The aim of this study was to determine the highest concentrations of EtG and EtS that could be produced with frequent, but plausible, use of high ethanol content mouthwash. Introduction * Author to whom correspondence should be addressed: Gary M. Reisfield, MD, University of Florida College of Medicine, 8491 NW 39th Avenue, Gainesville, FL garyr@ufl.edu. Materials and Methods Study protocol The protocol was approved by the University of Florida Institutional Review Board (UFJ ) and informed consent was obtained from all study subjects. Subjects were recruited through personal communications with the investigators. The subjects included six females and four males. Subjects were compensated for their involvement in the study. 264 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher s permission.

2 Study participation was offered to individuals between the ages of 18 and 70, inclusive. Any history of ethanol use disorder, allergy or sensitivity to ethanol, hepatic or renal dysfunction, diabetes mellitus, and symptoms of urinary tract infection were exclusionary criteria. Subjects were required to abstain from ethanol use for five days prior to the study. Instructions included the avoidance of ethanol-containing hand sanitizers and, to as great a degree as possible, ethanol-containing foods, beverages, and cosmetics. Ten subjects were recruited, enrolled, and completed the study. On Day 1 of the study, subjects provided a first morning void urine specimen. Subjects then gargled with 20 ml of Listerine ( Original unflavored product; 26.9% ethanol) antiseptic for 30 s after breakfast, lunch, and dinner, and at bedtime. All subjects subsequently rinsed their mouths with water and expectorated the oral contents. The same procedure was followed on Days 2 and 3. On Day 4, all subjects provided a first morning void urine specimen, gargled after breakfast, and provided urine specimens at 2, 4, and 6 h after this final gargle. All urine specimens were frozen within 30 min of collection. Analytical methods The analytical methods used in this study have been described in a previous publication (6). Briefly, ethanol in urine was measured using the Microgenics DRI Ethyl Alcohol Assay (Microgenics, Fremont, CA) with a lower limit of quantitation of 20 mg/dl; the method is linear to 350 mg/dl. All specimens were screened for EtG using an enzyme immunoassay (DRI EtG, Microgenics); however, all specimens were subjected to confirmatory analysis regardless of the screening result. Creatinine in urine was measured by the DRI Creatinine Specimen Validity Detect Test (Thermo Scientific, Waltham, MA) with a linear range of 0.21 to 350 mg/dl. Creatinine-adjusted EtG and EtS concentrations were based on the conversion of nanogram EtG or EtS per milliliter urine to microgram EtG or EtS per gram of creatinine. EtG and EtS were measured by a liquid chromatography tandem mass spectrometry (LC MS MS) method developed by Millennium Research Institute (San Diego, CA), on an Aria Transcend multiplexing LC system paired with a Quantum Ultra triple-stage quadrupole MS and Xcalibur software (Thermo Scientific). The details of the LC MS MS method have been previously published (7). The selectivity of the method was assessed against a library of approximately 100 drugs and metabolites, and no interference was observed. Day-to-day coefficients of variation of the EtG assay were 8% and 3% at 625 and 1250 ng/ml, respectively, and 7% at 200 ng/ml for the EtS assay, using quantitative values from three independent batches per day obtained over five days. Day-to-day coefficients of variation were 7% and 4% for the EtG and EtS assays, respectively, at their lower limits of quantitation. The analytical performance characteristics for the EtG and EtS confirmatory assays are summarized in Table I. Results Urine EtG, EtS, and creatinine results are presented in Table II. None of the subjects had detectable EtG in their urine at the beginning of the study, although two had detectable concentrations of EtS (6 and 82 ng/ml; 16 and 83 µg/g creatinine). Ethanol concentrations in all specimens were below the quantitation threshold of 20 mg/dl. Among all specimens, only one had a detectable concentration of EtG (173 ng/ml; 117 µg/g creatinine); this single specimen was a 2-h post-gargle specimen on the final day of the study. EtS was detected in the urine of 7 of the 10 participants, but it was not detected in the single specimen with detectable EtG. The maximum EtS concentration detected was 104 ng/ml (or 112 µg/g creatinine, in another subject). Three of the 10 subjects produced a total of 8 urinary EtS concentrations above 50 ng/ml, and 10 specimens exceeded 50 µg/g creatinine. Three specimens had EtS concentrations exceeding 100 ng/ml. Screening immunoassays were imperfect predictors of urinary EtG concentrations in this study. At a screening threshold of 100 ng/ml, one of the specimens screened negative for EtG, but yielded an EtG concentration of 173 ng/ml measured by LC MS MS. Two specimens produced screening results above the screening threshold (221 and 13,622 ng/ml) but did not yield detectable EtG concentrations by LC MS MS. Table I. Performance of the EtG and EtS Methods* EtG EtS Method LC MS MS LC MS MS Linear range ,000 ng/ml ,000 ng/ml Interday precision (% CV) 7% 4% Interday accuracy (% Bias) 11% 7% * The accuracy of the assays were determined by quantitation of EtG and EtS in quality control samples prepared in synthetic urine at concentrations of 750 ng/ml for EtG and 200 ng/ml for EtS. Interday precision and bias were determined by repeating the assay (n = 20). EtS linearity and bias were also assessed between 10 and 100 ng/ml. The average bias for urine specimens containing 10 ng/ml EtS was 23%, and it was less than 20% at 50, 70, and 100 ng/ml. Discussion There are innumerable environmental sources of incidental ethanol exposure, many of which have been shown to produce appreciable quantities of urinary ethanol metabolites. Several investigators have reported urinary concentrations of EtG and EtS after intensive use of ethanol-containing hand sanitizers. Reisfield et al. (6) recently demonstrated peak urinary EtG and EtS concentrations of 2001 ng/ml (1998 µg/g creatinine) and 83 ng/ml (94 µg/g creatinine), respectively, following intensive use of ethanol (62%)-containing hand sanitizer. Musshoff et al. (1) recently reported measurable quantities of ethanol in a variety of foods and beverages. This German study found peak urinary EtG concentrations of 512 ng/ml following 265

3 Table II. Summary of Data Obtained for Urinary EtG, EtS, and Creatinine EtG EtS Creatinine EtG EtS (ng/ml) (ng/ml) (mg/dl) µg/g creatinine µg/g creatinine Subject 1 A* B C D E F G Subject 2 A B C D E F G Subject 3 A B C D E F G Subject 4 A B C D E F G Subject 5 A B C D E F G Subject 6 A B C D E F G * A: pre-exposure morning void, day 1; B: first void, study day 2; C: first void, study day 3; D: first void, study day 4; E: day 4, 2 h after final mouthwash; F: day 4, 4 h after final mouthwash; and G: day 4, 6 h after final mouthwash. (See Study Protocol in Materials and Methods.) the consumption of 2 3 L of nonalcoholic beer (ethanol content: 3.6 g/l), peak EtS concentrations of 648 ng/ml following consumption of up to 2 L of grape juice (ethanol content: 1.25 g/l), peak EtG concentrations of 200 ng/ml following consumption of 750 g of sauerkraut (ethanol content: 2 g/kg), and peak EtG concentrations of 120 ng/ml following consumption of g of ripe bananas (ethanol content: 5 g/kg). In the case of the nonalcoholic beer, EtG was detectable for up to 7 and 13 h at cutoffs of 500 and 100 ng/ml, respectively. Current EtG and EtS cutoffs for distinguishing ethanol consumption from incidental exposures are non-uniform, empirically derived, and have been vigorously debated (8,9). We identified two peer-reviewed articles that examined urinary ethanol and metabolites after use of ethanol-containing mouthwash. In the first, Costantino et al. (10) performed a twopart study. In the first part, nine subjects provided a baseline (pre-exposure) urine specimen, which was required to be negative for ethanol and EtG. Subjects then gargled with a mouthful of Cepacol (12% ethanol) mouthwash for 30 s and repeated this until the entire 4-oz. bottle had been used. All gargling was completed over a 15-min period. Multiple urine specimens (two to seven) were collected over the following 24 h, and all specimens were refrigerated within 1 h of collection. Eight of the nine subjects produced at least one urine specimen with detectable EtG. The maximum EtG was 345 ng/ml (227 µg/g creatinine), obtained 90 min after gargling. Omitted from the data and discussion was the fact that the maximum corrected EtG was 790 µg/g creatinine, obtained 74 min after exposure. In the second part of the study, 11 subjects gargled with Cepacol mouthwash three times daily for five days. The first morning void urine was collected each day. Eight of the 11 subjects produced at least one EtG-positive urine. Sixteen of the 66 specimens (and of the 55 non-baseline specimens) had detectable EtG at a cutoff of 50 ng/ml. The maximum EtG concentration was 108 ng/ml on the final morning void of one subject. Urine creatinine concentrations for this part of the study were not reported. In another study, Høiseth et al. (11) ex- 266

4 amined urinary, blood, and oral fluid concentrations of ethanol, EtG, and EtS following the use of mouthwash, nonalcoholic wine, or 3.75 ml of vodka. Four subjects rinsed their mouths with 15 ml of Listerine mouthwash (21.6% ethanol) for eight cycles of rinsing for 1 min followed by resting for 30 s. Urine samples were collected at 1.5, 3.5, 5.5, and 7.5 h after use of mouthwash. Ethanol, tested by an enzymatic method, was undetectable in all urine samples. EtG and EtS, measured by LC MS MS, were undetectable in all samples at the LOD and LOQ of 170 and 370 ng/ml, respectively. A non-peer-reviewed publication included a study involving two subjects given intensive exposure to mouthwash (12). Subjects used 20 ml of Target Springmint Antiseptic Mouth Table II (continued). Summary of Data Obtained for Urinary EtG, EtS, and Creatinine EtG EtS Creatinine EtG EtS (ng/ml) (ng/ml) (mg/dl) µg/g creatinine µg/g creatinine Subject 7 A* B C D E F G Subject 8 A B C D E F G Subject 9 A B C D E F G Subject 10 A B C D E F G * A: pre-exposure morning void, day 1; B: first void, study day 2; C: first void, study day 3; D: first void, study day 4; E: day 4, 2 h after final mouthwash; F: day 4, 4 h after final mouthwash; and G: day 4, 6 h after final mouthwash. (See Study Protocol in Materials and Methods.) Rinse (21.6% ethanol), swished between the teeth for 30 s, hourly for 8 h. Urine specimens were collected at 2, 4, 6, 8, and 16 h (first next-morning void). EtG and EtS measurements were made by LC MS MS at the detection limits of 37.8 and 7.2 ng/ml, respectively. A maximum urinary EtG concentration of 366 ng/ml (194 µg/g creatinine) was measured in one of the subjects 6 h after exposure. The maximum urinary EtS concentration was 73 ng/ml (41 µg/g creatinine) in the same subject 8 h following exposure. Our work differs from previous studies in several respects. First, we used mouthwash with the highest available ethanol content (26.9%). Second, subjects gargled with the mouthwash rather than merely swishing it between their teeth. Third, four times daily gargling (twice the recommended frequency), rather than gargling a large volume over a short period of time, simulates realistic use of mouthwash. Notwithstanding these differences, our study mostly confirms and extends the findings of others. Specifically, intensive but plausible mouthwash use does not yield urinary ethanol at the quantifiable limit of 20 mg/dl, and does not produce raw or corrected urinary EtG or EtS concentrations in excess of 200 ng/ml or 200 µg/g creatinine. As in our previous study of dermal exposure to ethanol-containing hand sanitizer (6), small quantities of ethanol were detected below the quantifiable limit of 20 mg/dl. Precision studies with ethanolfree controls, however, revealed that some of the measured urinary ethanol concentrations were slightly above a 99% statistical threshold for background noise using the enzymatic ethanol assay. We suspect that the very low detectable concentrations of urinary ethanol were likely due to oropharyngeal mucosal absorption, inadvertent swallowing with subsequent gastrointestinal uptake, and/or endogenous production of ethanol. In agreement with the studies performed by Costantino et al. (10), Høiseth et al. (11), and Jones et al. (12), our results do not demonstrate any apparent accumulation of EtG. Likewise, in agreement with the data in Jones and coworkers study (12), we found no evidence of daily accumulation of EtS. An EtG and EtS threshold of at least 500 ng/ml is adequate to distinguish between intentional ethanol use and incidental exposure caused by intensive use of high alcohol content mouthwash. Use of a threshold at 100 or 200 ng/ml has a potential for false-positive results in patients who regularly use mouthwash. 267

5 Conclusions Four-times-daily exposure to high ethanol content mouthwash produced maximum urinary EtG concentrations of 173 ng/ml (117 µg/g creatinine) and maximum EtS concentrations of 104 ng/ml (112 µg/g creatinine). Ethanol was not detected in any specimen at the quantifiable limit of 20 mg/dl. The urinary concentrations of EtG and EtS measured in this study were well below the commonly accepted (United States) threshold of 500 ng/ml for evidence of deliberate ethanol ingestion. This confirms and extends previous work that ethanolcontaining mouthwash does not result in metabolite concentrations that exceed 500 ng/ml or 500 µg/g creatinine. These data, which reflect urinary ethanol metabolite concentrations under circumstances that simulate reasonable scenarios for intensive use of mouthwash, demonstrate that such use is unlikely to produce urinary ethanol metabolite concentrations that, by current standards, may be interpreted as ethanol consumption. References 1. F. Musshoff, E. Albermann, and B. Madea. Ethyl glucuronide and ethyl sulfate in urine after consumption of various beverages and foods misleading results? Int. J. Legal Med. 124: (2010). 2. A Thierauf, A. Wohlfarth, V. Auwärter, M.G. Perdekamp, F.M. Wurst, and W. Weinmann. Urine tested positive for ethyl glucuronide and ethyl sulfate after the consumption of yeast and sugar. Forensic Sci. Int. 202: e45 e47 (2010). 3. A. Thierauf, C.C. Halter, S. Rana, V. Auwaerter, A. Wohlfarth, F.M. Wurst, and W. Weinmann. Urine tested positive for ethyl glucuronide after trace amounts of ethanol. Addiction 104: (2009). 4. A. Thierauf, H. Gnann, A. Wohlfarth, V. Auwärter, M.G. Perdekamp, K.J. Buttler, F.M. Wurst, and W. Weinmann. Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of non-alcoholic beer. Forensic Sci. Int. 202: (2010). 5. G.E. Skipper, W. Weinmann, A. Thierauf, P. Schaefer, G. Wiesbeck, J.P. Allen, M. Miller, and F.M. Wurst. Ethyl glucuronide: a biomarker to identify alcohol use by health professionals recovering from substance use disorders. Alcohol Alcohol. 39: (2004). 6. G.M. Reisfield, B.A. Goldberger, B.O. Crews, A.J. Pesce, G.R. Wilson, S.A. Teitelbaum, and R.L. Bertholf. Ethyl glucuronide, ethyl sulfate, and ethanol in urine after sustained exposure to an ethanol-based hand sanitizer. J. Anal. Toxicol. 35: (2011). 7. B. Crews, S. Latyshev, C. Mikel, P. Almazan, R. West, A. Pesce, and C. West. Improved detection of ethyl glucuronide and ethyl sulfate in a pain management population using high-throughput LC MS/MS. J. Opioid Manag. 6(6): (2010). 8. K. Helliker. A test for alcohol and its flaws. Wall Street Journal, August 12, S.B. Karch. Ethanol-based hand cleansers. J. Forensic Leg. Med. 16: (2009). 10. A. Costantino, E.J. Digregorio, W. Korn, S. Spayd, and F. Rieders. The effect of the use of mouthwash on ethylglucuronide concentrations in urine. J. Anal. Toxicol. 30: (2006). 11. G. Høiseth, B. Yttredal, R. Karinen, H. Gjerde, and A. Christophersen. Levels of ethyl glucuronide and ethyl sulfate in oral fluid, blood, and urine after use of mouthwash and ingestion of nonalcoholic wine. J. Anal. Toxicol. 34: (2010). 12. J.T. Jones, M.R. Jones, C.A. Plate, and D. Lewis. Ethyl glucuronide and ethyl sulfate concentrations following use of ethanol containing mouthwash. whitepapers_etgmouthwash_fd_ pdf (accessed November 2010). Manuscript received November 22, 2010; revision received January 16,