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Excessive urinary excretion of isopropyl glucuronide after isopropanol abuse
Forensic Science International 266 (2016) 250–253
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Excessive urinary excretion of isopropyl glucuronide after isopropanol abuse Torsten Arndt a,*, Reinhild Beyreiß a, Werner Hartmann b, Stefanie Schro¨fel a, Karsten Stemmerich a a b
Bioscientia Institut fu¨r Medizinische Diagnostik GmbH, Konrad-Adenauer-Straße 17, D-55218 Ingelheim, Germany Klinik fu¨r Forensische Psychiatrie und Psychotherapie, Zentrum fu¨r Psychiatrie Reichenau, Feursteinstraße 55, D-78479 Reichenau, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 February 2016 Received in revised form 18 May 2016 Accepted 27 May 2016 Available online 3 June 2016
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. 860 mg/L or approx. 1540 mg/g creatinine (forensic cut-off 0.1 mg/L, clinical cut-off 0.5 mg/L). LC-MS/MS confirmatory analysis was negative for EtG (<0.05 mg/L; forensic cut-off 0.1 mg/L), but strongly positive for 2-propyl glucuronide (approx. 1100 mg/L or 2000 mg/g creatinine; cut-off 0.1 mg/L). 1-propyl, 1-butyl, and tert-butyl glucuronide were negative (<0.05 mg/L; cut-off 0.1 mg/L), 2-butyl glucuronide was 0.1 mg/L (cut-off 0.1 mg/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. ß 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Disinfectant Ethyl glucuronide Propyl glucuronide Intoxication Isopropanol
1. Introduction Ethyl glucuronide (EtG) in urine is considered a specific marker of recent alcohol consumption. EtG screening is frequently done by immunoassay and confirmatory analysis by mass spectrometry [1]. False-positive EtG screenings regarding ethanol consumption were found after medication with chloral hydrate [2] or sevoflurane [3], and after inhalative exposition to propyl alcohols
Abbreviations: EtG, ethyl glucuronide. * Corresponding author. Tel.: +49 6132 781 349; fax: +49 6132 781 428. E-mail address: [email protected] (T. Arndt). http://dx.doi.org/10.1016/j.forsciint.2016.05.027 0379-0738/ß 2016 Elsevier Ireland Ltd. All rights reserved.
[4] or ethanol [5] from hand disinfectants. We report on a falsepositive EtG immunoassay result caused by excessive urinary isopropyl glucuronide excretion most probably due to hand or surface disinfectant abuse. 2. Materials and methods 2.1. Patient We describe an alcohol-dependent patient (male, 53 years, normal body weight) who committed an offence under the influence of alcohol. He was hospitalized in a forensic-psychiatric clinic from October 2014 until November 2015. Since then, he has
T. Arndt et al. / Forensic Science International 266 (2016) 250–253
251
2.3.5. Chromatography MassChrom EtG column (Chromsystems, Munich, Germany) at 40 8C, mobile phase 0.1% (v/v) formic acid (A) and 2 mM ammonium formate in methanol (B), gradient from 90/10 A/B to 100% B in 5 min, flow rate 500 mL/min, total analysis time including washing and column reconditioning 9 min, samples stored at 10 8C until injection.
been treated in a rehabilitation centre for patients with addiction diseases. During hospitalization in situations of conflict (stress), the patient showed repeatedly a withdrawn behaviour with periods of hunger and thirst. Diabetes or diabetic metabolic situations were suspected but ruled out by repeated analyses of ketone bodies in urine, fasting serum blood glucose and HbA1c in EDTA blood (data not shown). Alcohol consumption was checked e.g. by the analysis of EtG in urine. Immunological EtG screenings were positive. A value of 110 mg/L (72 mg/g creatinine) was obtained during an episode of suspected intoxication presenting with metabolic acidosis and respiratory compensation and ketonuria. However, confirmatory EtG analyses by LC-MS/MS were repeatedly negative. The patient’s wife reported episodes of industrial cleaning fluid abuse, but this could not be confirmed and was not admitted to by the patient. The patient was considered open-minded and communicative by the staff of the rehabilitation centre. On the day of interest in January 2016, he appeared confused and almost unable to speak. He showed miosis and an increased heart rate. Blood pressure was normal. Breath alcohol analysis with a Dra¨ger Alkomat Alcotest 6810 (Dra¨ger, Lu¨beck, Germany) yielded 0.24 mg/L. A urine sample was taken and sent to our laboratory for EtG analysis. This was the first time that we became involved in this case.
2.3.6. Mass spectrometry/DMS Source in negative electrospray mode, temperature 600 8C, curtain gas 40 psi, ion spray voltage 2500 V, nebulizer and turbo gas 60 psi each. MRM-transitions for quantification and identification with unit resolution in Q1 and Q3: EtG 221-75/85 (declustering potential (DP) 50 V, collision energy (CE) 20/ 22 V), D5-EtG 226-75/85 (DP 75 V, CE 20/ 22 V), propyl glucuronides 235-75/85 (DP 75 V, CE 20/ 22 V) and butyl glucuronides 249-75/85 (DP 69 V, CE 21/ 23 V). Dwell time 100 ms for each transition, except D5-EtG (50 ms). SelexION separation voltage (SV) 4000 V, DMS offset (DMO) 3 V and compensation voltages (COV) 4.5 V for EtG and D5-EtG, 5.5 V for propyl glucuronides and 7.0 V for butyl glucuronides. Autoanalyzer assays and LC-MS/MS were validated according to the Guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) [6], using the Valistat software.
2.2. Material
3. Results
2.2.1. Reference substances 1-Propyl b-D-glucuronide (AthenaES, Baltimore, USA), 2-propyl b-D-glucuronide, 1-butyl b-D-glucuronide, 2-butyl b-D-glucuronide, and tert-butyl b-D-glucuronide (custom syntheses from Aurora Analytics, Baltimore, USA delivered by AthenaES), ethyl bD-glucuronide (EtG) and internal standard D5-EtG (Lipomed, Weil am Rhein, Germany).
EtG screening by immunoassay was positive, approx. 860 mg/L or approx. 1540 mg/g creatinine (forensic cut-off 0.1 mg/L, clinical cut-off 0.5 mg/L). Subsequent LC-MS/MS confirmatory analysis was negative for EtG, but strongly positive for 2-propyl glucuronide (Fig. 1). In detail: ethyl glucuronide was <0.05 mg/L as the lower limit of quantification (forensic cut-off 0.1 mg/L) and 2-propyl glucuronide was approx. 1100 mg/L or 2000 mg/g creatinine (cut-off 0.1 mg/L). 1-propyl, 1-butyl, and tert-butyl glucuronide were <0.05 mg/L (negative, cut-off 0.1 mg/L) and 2-butyl glucuronide was 0.1 mg/L (cut-off 0.1 mg/L). Creatinine, used as a measure of urine dilution, was 557 mg/L (reference range for men 390–2590 mg/L [7]).
2.2.2. Reagents Formic acid and ammonium formate (both ‘‘eluent additive for LC-MS’’ grade) (Sigma-Aldrich, Steinheim, Germany), water and methanol (both ‘‘Ultra LC-MS’’ grade) (Carl Roth, Karlsruhe, Germany). DRI ethyl glururonide immunoassay and creatinine assay (Jaffe, kinetic) (Thermo Fisher Scientific Microgenics, Passau, Germany). 2.3. Methods 2.3.1. Assays The ethyl glururonide and the creatinine assays were run on an AU680 analyzer (Beckman Coulter, Krefeld, Germany) according to the testkit instructions. 2.3.2. LC-MS/MS 1290 Infinity LC (Agilent, Waldbronn, Germany) coupled to a MistraSwitch column oven (MayLab Analytical Instruments, Vienna, Austria), a QTRAP 5500 mass spectrometer (Sciex, Darmstadt, Germany) equipped with a differential ion mobility spectrometry (DMS) cell (SelexION) and an autosampler PAL HTCxt (CTC Analytics, Zwingen, Switzerland). 2.3.3. Sample preparation To 50 mL urine, calibrator, or control 500 mL of internal standard solution (1 mg/L D5-EtG in 0.1% (v/v) formic acid) were added. Injection volume was 10 mL. 2.3.4. Calibration/quantification A six point calibration (0.05/0.1/0.5/1/5/10 mg/L of each analyte) in blank urine was used.
4. Discussion To our knowledge, such an excessive urinary isopropyl glucuronide concentration has not been described so far. The treating physician was immediately notified and provided the above given information. Consequently, the suspicion of recurrent industrial cleaning fluid abuse was raised and potential sources of such chemicals were assessed. During hospitalization, the patient had access to the hand disinfectant Sterillium classic pure (30 g 1-propanol, 45 g 2propanol, 0.2 g mecetronium etilsulfate per 100 g; Bode Chemie, Berlin, Germany) and the window cleaning fluid Vitrex (5–15% 2propanol and 2.5–5% 2-butoxy ethanol; Pramol-Chemie, Bazenheid, Switzerland). If not brought from the clinic to the rehabilitation centre, the patient had no access to these products in January 2016, the time of presentation. Furthermore, 1-propyl glucuronide was not found in the urine (Fig. 1) but it should have been detectable after Sterillium classic pure abuse as deduced from our data regarding 1-propyl glucuronide excretion in urine after Sterillium classic pure vapor inhalation by hand disinfection [4]. During his stay in the rehabilitation centre and thus at the time of presentation, the patient had free access to the following industrial chemicals, being all disinfectants delivered from Wachendorff Chemie (Troisdorf-Spich, Germany): ‘‘RheoseptWD plus’’–a disinfectant without any alcohol content and thus of secondary interest for our case. ‘‘Rheosol-Alkoholreiniger’’–an
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excretion when compared with our previous data [4,5]. In fact, we consider 2-propyl glucuronide concentrations of approx. 1100 mg/ L or approx. 2000 mg/g creatinine as indicative of a 2-propanol intoxication. Data regarding the excretion of 2-propyl glucuronide for patients with 2-propanol abuse are lacking. Indeed, laboratory diagnosis and follow-up of 2-propanol intoxication (usually referred to as isopropanol intoxication) is frequently done by measurement of 2-propanol (isopropanol) and/or acetone in blood and/or urine [8,9]. To our knowledge, this is the first report on urinary 2-propyl glucuronide measurement and its use as a marker of a 2-propanol intoxication. Spiking urine with pure reference substance, 2-propyl glucuronide showed a cross-reactivity of 69–84% with the DRI EtG immunoassay [10]. This is confirmed by our case, yielding a crossreactivity of approx. 71%. Thus, we consider the false-positive EtG immunoassay screening to be caused by the cross-reaction of the large amount of 2-propyl glucuronide in the urine of the patient. Medication of the patient has been ruled out as a reason for the false-positive immunoassay result (data not shown). The breath ethanol result of 0.24 mg/L was most propably due to the crossreaction of the analyzer with 2-propanol (personal information by the manufacturer of the alcohol breath analyzer). After submitting this report to Forensic Science International and in the time of the Reviewing process, the patient admitted to have taken diluted ‘‘Rheosept-Ha¨nde- und Hautdesinfektion’’ disinfactant with originally 63% (v/v) 2-propanol several times during his stay in the rehabilitation centre and thus at the time of presentation. This is in agreement with our finding of only 2-propyl glucuronide in the urine. As a result, it has been proven that disinfectant abuse caused the excessive excretion of 2-propyl glucuronide in the urine of our acutely intoxicated and chronically alcohol-dependent patient with a history of industrial cleaning fluid abuse. The treating physician considered the detection of 2-propyl glucuronide the landmark finding for the final diagnosis. 5. Conclusions
Fig. 1. LC-MS/MS ion chromatograms (quantifier MRM of EtG and propyl glucuronides): (A) standard mixture (0.1 mg/L each) in blank urine. (B) Typical urine sample after ethanol consumption (EtG 1.9 mg/L), (C) urine from the patient with most probably disinfectant abuse: EtG and 1-propyl glucuronide (1-PpG) negative (<0.05 mg/L as the lower limit of quantification; cut-off 0.1 mg/L), 2propyl glucuronide (2-PpG; syn. isopropyl glucuronide) approx. 1100 mg/L or approx. 2000 mg/g creatinine (cut-off 0.1 mg/L).
all purpose cleaner with 15–20% (v/v) ethanol, ruled out by the negative LC-MS/MS EtG result. ‘‘Rheosept-Ha¨nde- und Hautdesinfektion’’ a hand and skin disinfectant with 63% (v/v) 2-propanol and ‘‘Rheosept-FD plus’’ – a surface disinfectant with 5–10% (v/v) 2-propanol, both potential sources of 2-propanol intoxication. Inhalative exposition to the vapor of these 2-propanol-containing disinfectants can not explain the excessive 2-propyl glucuronide
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 1propanol (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. References [1] N.E. Walsham, R.A. Sherwood, Ethyl glucuronide, Ann. Clin. Biochem. 49 (2012) 110–117. [2] T. Arndt, B. Gierten, B. Gu¨ssregen, A. Werle, J. Gru¨ner, False-positive ethyl glucuronide immunoassay screening associated with chloral hydrate medication as confirmed by LC-MS/MS and self-medication, Forensic Sci. Int. 184 (2009) e27–e29. [3] M. Bo¨ttcher, D. Ku¨hne, False-positive ethyl glucuronide immunoassay after anestesia with sevoflurane, Toxichem. Krimtech. 82 (2015) 112 [Posterabstract]. [4] T. Arndt, J. Gru¨ner, S. Schro¨fel, K. Stemmerich, False-positive ethyl glucuronide immunoassay screening caused by a propyl alcohol-based hand sanitizer, Forensic Sci. Int. 223 (2012) 359–363. [5] T. Arndt, S. Schro¨fel, B. Gu¨ssregen, K. Stemmerich, Inhalation but not transdermal resorption of hand sanitizer ethanol causes positive ethyl glucuronide findings in urine, Forensic Sci. Int. 237 (2014) 126–130.
T. Arndt et al. / Forensic Science International 266 (2016) 250–253 [6] Guidelines and Recommendations of the GTFCh, Appendix B. Requirements for the Validation of Analytical Methods. Version 01, June 01, 2009 http://gtfch.org/cms/images/ stories/files/Appendix%20B%20GTFCh%2020090601.pdf, last cited May 18, 2016. [7] T. Arndt, Urine-creatinine concentration as a marker of urine dilution: reflections using a cohort of 45,000 samples, Forensic Sci. Int. 186 (2009) 48–51. [8] World Health Organization Task Group on Environmental Health Criteria for 2-Propanol, International Program on Chemical Safety, Environmental Health
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Criteria 103: 2-Propanol. Geneva, 1990 www.inchem.org/documents/ehc/ehc/ ehc103.htm; last cited May 18, 2016. [9] R.C. Baselt, Disposition of Toxic Drugs and Chemicals in Man, 10th ed., Biomedical Publications, Seal Beach, California, USA, 2014. [10] T. Arndt, R. Beyreiß, S. Schro¨fel, K. Stemmerich, Cross-reaction of propyl and butyl alcohol glucuronides with an ethyl glucuronide enzyme immunoassay, Forensic Sci. Int. 241 (2014) 84–86.
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Excessive urinary excretion of isopropyl glucuronide after isopropanol abuse Torsten Arndt a,*, Reinhild Beyreiß a, Werner Hartmann b, Stefanie Schro¨fel a, Karsten Stemmerich a a b
Bioscientia Institut fu¨r Medizinische Diagnostik GmbH, Konrad-Adenauer-Straße 17, D-55218 Ingelheim, Germany Klinik fu¨r Forensische Psychiatrie und Psychotherapie, Zentrum fu¨r Psychiatrie Reichenau, Feursteinstraße 55, D-78479 Reichenau, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 February 2016 Received in revised form 18 May 2016 Accepted 27 May 2016 Available online 3 June 2016
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. 860 mg/L or approx. 1540 mg/g creatinine (forensic cut-off 0.1 mg/L, clinical cut-off 0.5 mg/L). LC-MS/MS confirmatory analysis was negative for EtG (<0.05 mg/L; forensic cut-off 0.1 mg/L), but strongly positive for 2-propyl glucuronide (approx. 1100 mg/L or 2000 mg/g creatinine; cut-off 0.1 mg/L). 1-propyl, 1-butyl, and tert-butyl glucuronide were negative (<0.05 mg/L; cut-off 0.1 mg/L), 2-butyl glucuronide was 0.1 mg/L (cut-off 0.1 mg/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. ß 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Disinfectant Ethyl glucuronide Propyl glucuronide Intoxication Isopropanol
1. Introduction Ethyl glucuronide (EtG) in urine is considered a specific marker of recent alcohol consumption. EtG screening is frequently done by immunoassay and confirmatory analysis by mass spectrometry [1]. False-positive EtG screenings regarding ethanol consumption were found after medication with chloral hydrate [2] or sevoflurane [3], and after inhalative exposition to propyl alcohols
Abbreviations: EtG, ethyl glucuronide. * Corresponding author. Tel.: +49 6132 781 349; fax: +49 6132 781 428. E-mail address: [email protected] (T. Arndt). http://dx.doi.org/10.1016/j.forsciint.2016.05.027 0379-0738/ß 2016 Elsevier Ireland Ltd. All rights reserved.
[4] or ethanol [5] from hand disinfectants. We report on a falsepositive EtG immunoassay result caused by excessive urinary isopropyl glucuronide excretion most probably due to hand or surface disinfectant abuse. 2. Materials and methods 2.1. Patient We describe an alcohol-dependent patient (male, 53 years, normal body weight) who committed an offence under the influence of alcohol. He was hospitalized in a forensic-psychiatric clinic from October 2014 until November 2015. Since then, he has
T. Arndt et al. / Forensic Science International 266 (2016) 250–253
251
2.3.5. Chromatography MassChrom EtG column (Chromsystems, Munich, Germany) at 40 8C, mobile phase 0.1% (v/v) formic acid (A) and 2 mM ammonium formate in methanol (B), gradient from 90/10 A/B to 100% B in 5 min, flow rate 500 mL/min, total analysis time including washing and column reconditioning 9 min, samples stored at 10 8C until injection.
been treated in a rehabilitation centre for patients with addiction diseases. During hospitalization in situations of conflict (stress), the patient showed repeatedly a withdrawn behaviour with periods of hunger and thirst. Diabetes or diabetic metabolic situations were suspected but ruled out by repeated analyses of ketone bodies in urine, fasting serum blood glucose and HbA1c in EDTA blood (data not shown). Alcohol consumption was checked e.g. by the analysis of EtG in urine. Immunological EtG screenings were positive. A value of 110 mg/L (72 mg/g creatinine) was obtained during an episode of suspected intoxication presenting with metabolic acidosis and respiratory compensation and ketonuria. However, confirmatory EtG analyses by LC-MS/MS were repeatedly negative. The patient’s wife reported episodes of industrial cleaning fluid abuse, but this could not be confirmed and was not admitted to by the patient. The patient was considered open-minded and communicative by the staff of the rehabilitation centre. On the day of interest in January 2016, he appeared confused and almost unable to speak. He showed miosis and an increased heart rate. Blood pressure was normal. Breath alcohol analysis with a Dra¨ger Alkomat Alcotest 6810 (Dra¨ger, Lu¨beck, Germany) yielded 0.24 mg/L. A urine sample was taken and sent to our laboratory for EtG analysis. This was the first time that we became involved in this case.
2.3.6. Mass spectrometry/DMS Source in negative electrospray mode, temperature 600 8C, curtain gas 40 psi, ion spray voltage 2500 V, nebulizer and turbo gas 60 psi each. MRM-transitions for quantification and identification with unit resolution in Q1 and Q3: EtG 221-75/85 (declustering potential (DP) 50 V, collision energy (CE) 20/ 22 V), D5-EtG 226-75/85 (DP 75 V, CE 20/ 22 V), propyl glucuronides 235-75/85 (DP 75 V, CE 20/ 22 V) and butyl glucuronides 249-75/85 (DP 69 V, CE 21/ 23 V). Dwell time 100 ms for each transition, except D5-EtG (50 ms). SelexION separation voltage (SV) 4000 V, DMS offset (DMO) 3 V and compensation voltages (COV) 4.5 V for EtG and D5-EtG, 5.5 V for propyl glucuronides and 7.0 V for butyl glucuronides. Autoanalyzer assays and LC-MS/MS were validated according to the Guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) [6], using the Valistat software.
2.2. Material
3. Results
2.2.1. Reference substances 1-Propyl b-D-glucuronide (AthenaES, Baltimore, USA), 2-propyl b-D-glucuronide, 1-butyl b-D-glucuronide, 2-butyl b-D-glucuronide, and tert-butyl b-D-glucuronide (custom syntheses from Aurora Analytics, Baltimore, USA delivered by AthenaES), ethyl bD-glucuronide (EtG) and internal standard D5-EtG (Lipomed, Weil am Rhein, Germany).
EtG screening by immunoassay was positive, approx. 860 mg/L or approx. 1540 mg/g creatinine (forensic cut-off 0.1 mg/L, clinical cut-off 0.5 mg/L). Subsequent LC-MS/MS confirmatory analysis was negative for EtG, but strongly positive for 2-propyl glucuronide (Fig. 1). In detail: ethyl glucuronide was <0.05 mg/L as the lower limit of quantification (forensic cut-off 0.1 mg/L) and 2-propyl glucuronide was approx. 1100 mg/L or 2000 mg/g creatinine (cut-off 0.1 mg/L). 1-propyl, 1-butyl, and tert-butyl glucuronide were <0.05 mg/L (negative, cut-off 0.1 mg/L) and 2-butyl glucuronide was 0.1 mg/L (cut-off 0.1 mg/L). Creatinine, used as a measure of urine dilution, was 557 mg/L (reference range for men 390–2590 mg/L [7]).
2.2.2. Reagents Formic acid and ammonium formate (both ‘‘eluent additive for LC-MS’’ grade) (Sigma-Aldrich, Steinheim, Germany), water and methanol (both ‘‘Ultra LC-MS’’ grade) (Carl Roth, Karlsruhe, Germany). DRI ethyl glururonide immunoassay and creatinine assay (Jaffe, kinetic) (Thermo Fisher Scientific Microgenics, Passau, Germany). 2.3. Methods 2.3.1. Assays The ethyl glururonide and the creatinine assays were run on an AU680 analyzer (Beckman Coulter, Krefeld, Germany) according to the testkit instructions. 2.3.2. LC-MS/MS 1290 Infinity LC (Agilent, Waldbronn, Germany) coupled to a MistraSwitch column oven (MayLab Analytical Instruments, Vienna, Austria), a QTRAP 5500 mass spectrometer (Sciex, Darmstadt, Germany) equipped with a differential ion mobility spectrometry (DMS) cell (SelexION) and an autosampler PAL HTCxt (CTC Analytics, Zwingen, Switzerland). 2.3.3. Sample preparation To 50 mL urine, calibrator, or control 500 mL of internal standard solution (1 mg/L D5-EtG in 0.1% (v/v) formic acid) were added. Injection volume was 10 mL. 2.3.4. Calibration/quantification A six point calibration (0.05/0.1/0.5/1/5/10 mg/L of each analyte) in blank urine was used.
4. Discussion To our knowledge, such an excessive urinary isopropyl glucuronide concentration has not been described so far. The treating physician was immediately notified and provided the above given information. Consequently, the suspicion of recurrent industrial cleaning fluid abuse was raised and potential sources of such chemicals were assessed. During hospitalization, the patient had access to the hand disinfectant Sterillium classic pure (30 g 1-propanol, 45 g 2propanol, 0.2 g mecetronium etilsulfate per 100 g; Bode Chemie, Berlin, Germany) and the window cleaning fluid Vitrex (5–15% 2propanol and 2.5–5% 2-butoxy ethanol; Pramol-Chemie, Bazenheid, Switzerland). If not brought from the clinic to the rehabilitation centre, the patient had no access to these products in January 2016, the time of presentation. Furthermore, 1-propyl glucuronide was not found in the urine (Fig. 1) but it should have been detectable after Sterillium classic pure abuse as deduced from our data regarding 1-propyl glucuronide excretion in urine after Sterillium classic pure vapor inhalation by hand disinfection [4]. During his stay in the rehabilitation centre and thus at the time of presentation, the patient had free access to the following industrial chemicals, being all disinfectants delivered from Wachendorff Chemie (Troisdorf-Spich, Germany): ‘‘RheoseptWD plus’’–a disinfectant without any alcohol content and thus of secondary interest for our case. ‘‘Rheosol-Alkoholreiniger’’–an
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T. Arndt et al. / Forensic Science International 266 (2016) 250–253
excretion when compared with our previous data [4,5]. In fact, we consider 2-propyl glucuronide concentrations of approx. 1100 mg/ L or approx. 2000 mg/g creatinine as indicative of a 2-propanol intoxication. Data regarding the excretion of 2-propyl glucuronide for patients with 2-propanol abuse are lacking. Indeed, laboratory diagnosis and follow-up of 2-propanol intoxication (usually referred to as isopropanol intoxication) is frequently done by measurement of 2-propanol (isopropanol) and/or acetone in blood and/or urine [8,9]. To our knowledge, this is the first report on urinary 2-propyl glucuronide measurement and its use as a marker of a 2-propanol intoxication. Spiking urine with pure reference substance, 2-propyl glucuronide showed a cross-reactivity of 69–84% with the DRI EtG immunoassay [10]. This is confirmed by our case, yielding a crossreactivity of approx. 71%. Thus, we consider the false-positive EtG immunoassay screening to be caused by the cross-reaction of the large amount of 2-propyl glucuronide in the urine of the patient. Medication of the patient has been ruled out as a reason for the false-positive immunoassay result (data not shown). The breath ethanol result of 0.24 mg/L was most propably due to the crossreaction of the analyzer with 2-propanol (personal information by the manufacturer of the alcohol breath analyzer). After submitting this report to Forensic Science International and in the time of the Reviewing process, the patient admitted to have taken diluted ‘‘Rheosept-Ha¨nde- und Hautdesinfektion’’ disinfactant with originally 63% (v/v) 2-propanol several times during his stay in the rehabilitation centre and thus at the time of presentation. This is in agreement with our finding of only 2-propyl glucuronide in the urine. As a result, it has been proven that disinfectant abuse caused the excessive excretion of 2-propyl glucuronide in the urine of our acutely intoxicated and chronically alcohol-dependent patient with a history of industrial cleaning fluid abuse. The treating physician considered the detection of 2-propyl glucuronide the landmark finding for the final diagnosis. 5. Conclusions
Fig. 1. LC-MS/MS ion chromatograms (quantifier MRM of EtG and propyl glucuronides): (A) standard mixture (0.1 mg/L each) in blank urine. (B) Typical urine sample after ethanol consumption (EtG 1.9 mg/L), (C) urine from the patient with most probably disinfectant abuse: EtG and 1-propyl glucuronide (1-PpG) negative (<0.05 mg/L as the lower limit of quantification; cut-off 0.1 mg/L), 2propyl glucuronide (2-PpG; syn. isopropyl glucuronide) approx. 1100 mg/L or approx. 2000 mg/g creatinine (cut-off 0.1 mg/L).
all purpose cleaner with 15–20% (v/v) ethanol, ruled out by the negative LC-MS/MS EtG result. ‘‘Rheosept-Ha¨nde- und Hautdesinfektion’’ a hand and skin disinfectant with 63% (v/v) 2-propanol and ‘‘Rheosept-FD plus’’ – a surface disinfectant with 5–10% (v/v) 2-propanol, both potential sources of 2-propanol intoxication. Inhalative exposition to the vapor of these 2-propanol-containing disinfectants can not explain the excessive 2-propyl glucuronide
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 1propanol (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. References [1] N.E. Walsham, R.A. Sherwood, Ethyl glucuronide, Ann. Clin. Biochem. 49 (2012) 110–117. [2] T. Arndt, B. Gierten, B. Gu¨ssregen, A. Werle, J. Gru¨ner, False-positive ethyl glucuronide immunoassay screening associated with chloral hydrate medication as confirmed by LC-MS/MS and self-medication, Forensic Sci. Int. 184 (2009) e27–e29. [3] M. Bo¨ttcher, D. Ku¨hne, False-positive ethyl glucuronide immunoassay after anestesia with sevoflurane, Toxichem. Krimtech. 82 (2015) 112 [Posterabstract]. [4] T. Arndt, J. Gru¨ner, S. Schro¨fel, K. Stemmerich, False-positive ethyl glucuronide immunoassay screening caused by a propyl alcohol-based hand sanitizer, Forensic Sci. Int. 223 (2012) 359–363. [5] T. Arndt, S. Schro¨fel, B. Gu¨ssregen, K. Stemmerich, Inhalation but not transdermal resorption of hand sanitizer ethanol causes positive ethyl glucuronide findings in urine, Forensic Sci. Int. 237 (2014) 126–130.
T. Arndt et al. / Forensic Science International 266 (2016) 250–253 [6] Guidelines and Recommendations of the GTFCh, Appendix B. Requirements for the Validation of Analytical Methods. Version 01, June 01, 2009 http://gtfch.org/cms/images/ stories/files/Appendix%20B%20GTFCh%2020090601.pdf, last cited May 18, 2016. [7] T. Arndt, Urine-creatinine concentration as a marker of urine dilution: reflections using a cohort of 45,000 samples, Forensic Sci. Int. 186 (2009) 48–51. [8] World Health Organization Task Group on Environmental Health Criteria for 2-Propanol, International Program on Chemical Safety, Environmental Health
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Criteria 103: 2-Propanol. Geneva, 1990 www.inchem.org/documents/ehc/ehc/ ehc103.htm; last cited May 18, 2016. [9] R.C. Baselt, Disposition of Toxic Drugs and Chemicals in Man, 10th ed., Biomedical Publications, Seal Beach, California, USA, 2014. [10] T. Arndt, R. Beyreiß, S. Schro¨fel, K. Stemmerich, Cross-reaction of propyl and butyl alcohol glucuronides with an ethyl glucuronide enzyme immunoassay, Forensic Sci. Int. 241 (2014) 84–86.