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Positive EtG findings in hair as a result of a cosmetic treatment
Forensic Science International 218 (2012) 97–100
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Positive EtG findings in hair as a result of a cosmetic treatment§ Frank Sporkert a,*, Hicham Kharbouche a,1, Marc P. Augsburger a,2, Clementine Klemm b,3, Markus R. Baumgartner c,4 a
University Centre of Legal Medicine Lausanne-Geneva, Rue du Bugnon 21, CH-1011 Lausanne, Switzerland Institute of Legal Medicine, Kantonsspital, Rorschacherstr. 93, CH-9007 St.Gallen, Switzerland c Institute of Legal Medicine, Klinisch-Forensisches Labor, Kurvenstrasse 17, CH-8006 Zu¨rich, Switzerland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2011 Accepted 21 April 2011 Available online 20 October 2011
In a case of a driving ability assessment, hair analysis for ethyl glucuronide (EtG) was requested by the authorities. The person concerned denied alcohol consumption and did not present any clinical sign of alcoholism. However, EtG was found in concentrations of up to 910 pg/mg in hair from different sampling dates suggesting an excessive drinking behavior. The person declared to use a hair lotion on a regularly base. To evaluate a possible effect of the hair lotion, prospective blood and urine controls as well as hair sampling of scalp and pubic hair were performed. The traditional clinical biomarkers of ethanol consumption, CDT and GGT, were inconspicuous in three blood samples taken. EtG was not detected in all collected urine samples. The hair lotion was transmitted to our laboratory. The ethanol concentration in this lotion was determined with 35 g/L. The EtG immunoassay gave a positive result indicating EtG, which could be confirmed by GC–MS/MS-NCI. In a follow-up experiment the lotion was applied to the hair of a volunteer over a period of six weeks. After this treatment, EtG could be measured in the hair at a concentration of 72 pg/mg suggesting chronic and excessive alcohol consumption. Overnight incubation of EtG free hair in the lotion yielded an EtG concentration of 140 pg/mg. In the present case, the positive EtG hair findings could be interpreted as the result of an EtG containing hair care product. To our knowledge, the existence of such a product has not yet been reported, and it is exceptionally unusual to find EtG in cosmetics. Therefore, external sources for hair contamination should always be taken into account when unusual cosmetic treatment is mentioned. In those cases, it is recommended to analyze the hair product for a possible contamination with EtG. The analysis of body hair can help to reveal problems occurring from cosmetic treatment of head hair. As a consequence, the assessment of drinking behavior should be based on more than one diagnostic parameter. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ethyl glucuronide Hair Hair-care Cosmetic treatment
1. Introduction Hair is steadily exposed to hair care, sunshine, rain, smoke and hair. These external sources can influence the concentrations of substances detected in hair. Cannabinoids in hair are known to be sensible to sunlight [1] but their presence in hair may also be the result of external incorporation [2]. Opiates and
§ This paper is part of the special issue entitled: Selected papers from the Chamonix 2011 Society of Hair Testing Meeting, Guest-edited by Pascal Kintz. * Corresponding author. Tel.: +41 21 314 56 26, fax: +41 21 314 73 29. E-mail addresses: [email protected] (F. Sporkert), [email protected] (H. Kharbouche), [email protected] (M.P. Augsburger), [email protected] (C. Klemm), [email protected] (M.R. Baumgartner). 1 Tel.: +41 21 314 56 26. 2 Tel.: +41 21 314 70 85. 3 Tel.: +41 71 494 30 15. 4 Tel.: +41 44 360 23 71.
0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.10.009
cocaine concentrations are decreased by oxidative bleaching [3– 5]. On the other hand, cosmetic treatment can also lead to an increase of concentrations as reported in the case of fatty acid ethyl esters (FAEE) which are formed directly on the head when ethanol containing hair care products are applied [6,7]. Despite the wide use of hair EtG in forensic routine work, only few has been reported in the literature about the influence of hair-care products on the EtG concentration in hair. As for other substances, the use of bleaching products can lead to a significant decrease of the EtG concentration [8]. The question if cosmetic treatment can also yield to false positive results in the case of ethyl glucuronide (EtG) has generally been refused by scientists as ethyl glucuronide is basically formed in the liver, lung and intestine by conjugation of ethanol with UDP glucuronic acid catalyzed by UDP-glucuronosyltransferases (UGT) in the presence of ethanol [9]. Since several UGT subtypes are able to catalyze this reaction, the EtG formation should not depend on genetic polymorphism [10].
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F. Sporkert et al. / Forensic Science International 218 (2012) 97–100
In this paper, the authors present a case, where EtG was found in high concentrations in hair as a result of a cosmetic treatment with a hair lotion containing high amounts of EtG. A self experiment was carried out in order to evaluate the possibility of incorporating EtG from external sources into hair in a concentration suggesting excessive alcohol consumption.
10 g/day at the end of the experiment. The hair was cut at three occasions with an electrical shaver as short as possible (residual hair length < 1 mm): (1) before, (2) one day after the first treatment, and (3) four weeks after having used the lotion on a daily base. In another experiment, an EtG negative hair strand was incubated in vitro for three days in the concerned hair lotion. The hair samples were analyzed for EtG according to a validated protocol as described above with the difference of three washing steps with water instead of one.
2. Case 4. Results In a case of an evaluation of driving ability after a car accident in February 2008, a 40-year-old woman had to prove her abstinence from alcohol. The mandated department of traffic medicine requested, among others, hair testing for EtG. Over a two-year period between 2008 and 2010, several hair sampling were carried out and yielded to contradicting results from high EtG concentrations to negative results. As a result, the woman was declared inapt for driving. The woman denied any excessive alcohol consumption, claimed the regular use of an ethanol containing hair lotion and asked for a contra expertise. Chronic and excessive alcohol consumption, which was suggested by some of the test results, should be also detectable by the measurement of the traditional and less sensitive biomarkers CDT and GGT. Therefore, three blood and hair sampling were carried out in the following almost two years. However, low amounts of alcohol intake may still not be detectable in hair which implicates that hair analysis cannot be used to prove total abstinence. In contrast to the long term monitoring by EtG hair testing, in urine, EtG can be detected usually up to two days after a drinking event, in case of excessive drinking up to maximal five days [11,12]. In the present case, we therefore recommended also urine testing for EtG which was performed weekly over a time period of four months. Finally, the hair lotion was carefully investigated and also analyzed for EtG. 3. Materials and methods 3.1. Samples Hair samples from the person concerned were taken between April 2008 and August 2010. The sampling was performed according to a standard protocol during medical assessments. In addition, 16 urine samples were collected on a weekly base between the 10.11.2009 and the 08.03.2010. In August 2010, the administered hair lotion was transmitted to one of the labs. The authors obtained written consent from the person concerned for all additional analyses as well as for the publication of the results. 3.2. Methods The hair samples were analyzed for EtG according to our fully validated method [13]. Briefly, a hair segment was washed for 5 min, first with water and secondly with acetone. The dried hair was powdered in a ball mill. About 30 mg were taken for analysis. The sample was incubated for 2 h in an ultrasonic bath with 1 ml of distilled water in presence of the internal standard EtG-D5 (Lipomed, Arlesheim, CH). The extract was purified at a 60 mg OASIS-Max SPE cartridge (Waters, MA, USA). The eluate was evaporated and derivatized with PFPA for GC–MS/MS-NCI measurement on a Varian 300 MS. Additional measurements for EtG in hair using the same extraction procedures were carried out in the other institutes either on a Thermo TSQ Quantum XLS Triple Quadruple GC–MS/MS (Thermo Fisher Scientific, CA, USA) or by LC–MS/MS on a QTrap 4000 (AB Sciex, Darmstadt, Germany) with their own validated methods. EtG in urine and in the lotion as well as and creatinine were measured with the DRI EtG enzyme immunoassay on the CDx90 Analyzer (Thermo Scientific Microgenics, Passau, Germany). CDT was determined using Ceofix CDT-Kit on an Agilent capillary electrophoresis according to our published method [14]. The liver enzyme GGT was measured by an immunological test on a Dimension XPand plus (Siemens Healthcare Diagnostics, Deerfield, IL, USA). The ethanol content in the hair lotion was determined after 1/ 100 dilution of the lotion with distilled water by our routinely used HS-GC-FID (Agilent 1888 HS and Agilent 6850 GC-FID). 3.3. Self experiment The hair lotion was applied every second day to one of the author’s hair on the right head side over a six week period. The author’s daily ethanol intake was estimated with 15 g/day in the beginning of the treatment decreasing to less than
Altogether, four hair samples including head hair and pubic hair were taken from the woman between April 2008 and August 2010. The resulting EtG concentrations are presented in Table 1. Between June 2008 and August 2010, three blood sampling were carried out in order to control the liver enzyme GGT and CDT. The results are shown in Table 2. In order to control abstinence effectively, urine samplings were carried out weekly during a time period of four months (November 2009–March 2010). All of the samples were tested negative for EtG (<100 ng/ml). At last, the hair lotion which had been used by the person concerned was transmitted to our laboratory. Corresponding to the manufacture’s data, the product is an alcoholic plant extract. In contrast to manufacture’s recommendation the lotion was not washed out after the application. The ethanol concentration of the lotion was determined at 35 g/L or 44 vol.%. EtG was first measured by immunoassay suggesting a concentration of >2.0 mg/ml. The subsequent determination of EtG by means of GC–MS/MS-NCI yielded a concentration of 2.7 mg/ml. These results were confirmed in two independent measurements in the laboratories of Zurich and St. Gallen. A second hair lotion from a different lot was achieved independently. The above mentioned results could be confirmed as well. In order to elucidate the possibility of EtG incorporation by the hair lotion, a self experiment was performed. The application of the Table 1 EtG concentrations in the woman’s hair. Date of sampling
Type of hair
Segment (cm)
EtG concentration
11.04.2008 20.06.2008
Head hair Head hair
20.09.2009 30.10.2009
Head hair Head hair
06.08.2010
Pubic hair Head hair
0–6 0–2 2–4 4–6 6–12 0–5 0–3 3–6 2 (in total) 0–3 3–6 6–9 9–12
444a 47 130 460 910 32 NDb ND ND ND ND ND ND
[pg/mg]
a b
Result of a private laboratory (analytical method unknown). ND: not detected (LOD: 3 pg/mg).
Table 2 GGT and CDT concentrations measured in blood of the person concerned. Date of sampling
Marker
Concentration
Reference valuesa
20.06.2008
CDT-CE GGT CDT-CE GGT CDT-CE GGT
0.7% 22 U/L 0.5% 29 U/L 0.7% 16 U/L
<1.7% 5–55 U/L <1.7% 5–55 U/L <1.7% 5–55 U/L
30.10.2009 06.08.2010
a Base for the reference values: specificity for CDT >99% (data non-published), GGT: 95% prediction interval.
F. Sporkert et al. / Forensic Science International 218 (2012) 97–100 Table 3 EtG concentrations in treated (right side) and untreated (left side) parts of scalp hair after administration of the hair lotion. Sampling date
Head side
Hair length [cm]
EtG concentration [pg/mg]
1 2
31.10.2010 12.12.2010
3
10.01.2011
Right Right Left Right Left
1.5 1.5 1.5 1.0 1.0
14a 72a 11 6.9 NDb
Sampling
a Confirmed in independent measurements in the laboratories of Zurich and St. Gallen. b ND: not detected (LOD: 3 pg/mg).
lotion to the author’s hair led to an increase of the concentration of EtG compared to the untreated hair on the left head side. Noteworthy, measurements for treated and untreated hair were always performed in parallel. The results of the measured EtG concentrations before, directly after and four weeks after the treatment are summarized in Table 3. In a parallel experiment, a negative hair strand was incubated in the lotion for 3 days. EtG analysis according to the validated method yielded a concentration of 140 pg/mg. 5. Discussion Hair analyses carried out between April 2008 and September 2009 yielded concentrations of >30 pg/mg indicating a chronic and excessive alcohol consumption according to the consensus of the Society of Hair Testing (SoHT) [15] and our own results [16]. An excessive drinking behavior seemed to be even more evident because the EtG concentrations were not only close above the cutoff of 30 pg/mg but also highly elevated values from 130 up to 910 pg/mg of EtG were measured in the client’s head hair. In contrast to these findings, the concentrations for the blood markers CDT and GGT as well as for the EtG urine did not indicate excessive alcohol consumption even if the sampling was carried out at different occasions which did not totally correspond to the period covered by the hair tests. But at least, the first blood sampling from June 2008 covers parts of the proximal 3 cm hair segment. The EtG concentrations measured in the hair samples from April 2008 and September 2009 decrease significantly from the distal to the proximal segment. This may indicate a break or a less frequent administration of the lotion or a strong decline of the alcohol consumption. The absence of EtG in the hair taken at the 30.10.2009 suggests that the EtG was completely washed out after abandonment or a change of the hair treatment. Otherwise, EtG should still be detectable since the hair segments from the 20.09.2009 and the 30.10.2009 represent overlapping time periods. The person concerned declared that she finally followed the manufacturer’s instructions and started to wash out carefully the hair some minutes after the administration what she had never done in the earlier treatment. Another strong hint against chronic and excessive alcohol consumption was the absence of EtG in the pubic hair. EtG concentrations in pubic hair are often strongly elevated compared to scalp hair [17] probably due to contact with urine. Another explanation might be the wider time window due to the higher portion of telogene hair of about 50% [18]. The remarkable difference in the EtG hair concentrations measured in the hair of the client in April and June 2008 (up to 910 pg/mg) compared to those found after self application (72 pg/ mg) can be explained by differences in the experimental design of the self experiment. The hair of the client had been treated for already more than one year before the first sampling according to
99
the person concerned. In contrast, the self experiment started with cut hair. Only 1 mm remained on the scalp. When the lotion was applied, a big part flew down, so that the contact time with the hair was shorter than with longer hair. Since the hair of the client was longer than 20 cm, the number of applications to the analyzed parts of the hair was greater than in the self experiment. This hypothesis appeared to be confirmed by the increasing EtG concentrations towards the distal segments (from 47 up to 910 pg/ mg) of the person concerned in a period where constant application was claimed. However, from the data of this study some alcohol consumption of the client cannot be completely excluded and may potentially have contributed to some extent to the highly elevated EtG concentrations of up to 910 pg/mg, The EtG concentration of 140 pg/mg which was obtained for the in vitro incubated blank hair sample cannot be compared in toto with the highest concentrations found in the real case since a more extensive washing procedure was applied including 3 aqueous wash steps instead of only one as in the standard protocol. In conclusion, the data indicate that highly elevated EtG concentrations can be found in hair due to the regular use of an EtG contaminated cosmetic which was not washed out after use. The assessment of hair care is therefore crucial for the evaluation of results from hair analysis and should be attentively regarded as an influencing factor. In the present case, all claims of the client had been rejected by the driving license authorities and the traffic medicine department even though other clinical, medical or psychological parameters did not confirm the suspicion of excessive drinking. As a consequence the driving license was revoked twice for a total of 24 months. To our knowledge, this is the first reported case where it is shown that a cosmetic hair product contains EtG. As a consequence, falsely elevated EtG values due to external contamination from this EtG-containing hair product were observed. It has been demonstrated that high ethanol concentration alone of 44 vol.% should not produce highly elevated EtG concentrations such as found in the present case [19]. The present work shows that the EtG from the lotion could be readily incorporated into the hair and could not be removed after applying two washing steps. Furthermore, we could demonstrate that the EtG concentration rapidly decreases when the administration is abandoned as shown in the hair sample of the author taken four weeks after the last application. A small portion of the decrease in the EtG concentration in the case of the self experiment is certainly related to a diminution of the author’s daily alcohol intake from 15 to less than 10 g/day. So far, the herein presented product is up to now the only known hair care containing ethyl glucuronide. According to our experience hair care products typically do not contain EtG. A number of over 15 commercially available hair care products from Germany and Switzerland were tested negative for EtG (data not published). The question of the origin of the EtG found in the lotion is currently under further investigation. Since UDP-glucuronyltransferases are also present in plants [20,21] an extraction of these enzymes which produce EtG in the presence of ethanol cannot be excluded. Another possibility for the presence of EtG in the lotion is a direct extraction of already formed EtG from one of the plants used for the production of the lotion. 6. Conclusion The unusual case of an external contamination of hair with EtG by an EtG-containing hair lotion was presented. In conclusion, we strongly recommend to assess the use of hair care products and the hair treatment very carefully at the hair sampling. In critical cases, hair products should be analyzed for the
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presence of EtG. Furthermore, body hair can help to reveal problems occurring from cosmetic treatment of head hair. As a consequence, the expertise of the drinking behavior should be based on more than one diagnostic parameter. References [1] G. Skopp, L. Potsch, M. Mauden, Stability of cannabinoids in hair samples exposed to sunlight, Clin. Chem. 46 (2000) 1846–1848. [2] V. Auwa¨rter, A. Wohlfarth, J. Traber, D. Thieme, W. Weinmann, Hair analysis for delta9-tetrahydrocannabinolic acid A – new insights into the mechanism of drug incorporation of cannabinoids into hair, Forensic Sci. Int. 196 (2010) 10–13. [3] L. Potsch, G. Skopp, Stability of opiates in hair fibers after exposure to cosmetic treatment, Forensic Sci. Int. 81 (1996) 95–102. [4] G. Skopp, L. Potsch, M.R. Moeller, On cosmetically treated hair – aspects and pitfalls of interpretation, Forensic Sci. Int. 84 (1997) 43–52. [5] V. Cirimele, P. Kintz, P. Mangin, Drug concentrations in human hair after bleaching, J. Anal. Toxicol. 19 (1995) 331–332. [6] S. Hartwig, V. Auwarter, F. Pragst, Effect of hair care and hair cosmetics on the concentrations of fatty acid ethyl esters in hair as markers of chronically elevated alcohol consumption, Forensic Sci. Int. 131 (2003) 90–97. [7] J. Gareri, B. Appenzeller, P. Walasek, G. Koren, Impact of hair-care products on FAEE hair concentrations in substance abuse monitoring, Anal. Bioanal. Chem. (2011). [8] L. Morini, A. Zucchella, A. Polettini, L. Politi, A. Groppi, Effect of bleaching on ethyl glucuronide in hair: an in vitro experiment, Forensic Sci. Int. 198 (2010) 23–27. [9] J.E. Manautou, G.P. Carlson, Comparison of pulmonary and hepatic glucuronidation and sulphation of ethanol in rat and rabbit in vitro, Xenobiotica 22 (1992) 1309–1319. [10] R.S. Foti, M.B. Fisher, Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs, Forensic Sci. Int. 153 (2005) 109–116.
[11] F.M. Wurst, C. Kempter, S. Seidl, A. Alt, Ethyl glucuronide – a marker of alcohol consumption and a relapse marker with clinical and forensic implications, Alcohol Alcohol. 34 (1999) 71–77. [12] A. Helander, M. Bottcher, C. Fehr, N. Dahmen, O. Beck, Detection times for urinary ethyl glucuronide and ethyl sulfate in heavy drinkers during alcohol detoxification, Alcohol Alcohol. 44 (2009) 55–61. [13] H. Kharbouche, F. Sporkert, S. Troxler, M. Augsburger, P. Mangin, C. Staub, Development and validation of a gas chromatography–negative chemical ionization tandem mass spectrometry method for the determination of ethyl glucuronide in hair and its application to forensic toxicology, J Chromatogr. B 877 (2009) 2337–2343. [14] J.B. Daeppen, F. Anex, B. Favrat, A. Bissery, J. Leutwyler, R. Gammeter, P. Mangin, M. Augsburger, Carbohydrate-deficient transferrin measured by capillary zone electrophoresis and by turbidimetric immunoassay for identification of young heavy drinkers, Clin. Chem. 51 (2005) 1046–1048. [15] P. Kintz, Consensus of the Society of Hair Testing on hair testing for chronic excessive alcohol consumption 2009, Forensic Sci. Int. 196 (2010) 2. [16] H. Kharbouche, Inte´reˆt de l’e´thylglucuronide comme marqueur d’abus d’alcool, aspects analytiques, diagnostiques et me´dico-le´gaux, Thesis, University of Lausanne, Faculty of Biology and Medicine, 7-2-2011. [17] P. Kintz, M. Villain, E. Vallet, M. Etter, G. Salquebre, V. Cirimele, Ethyl glucuronide: unusual distribution between head hair and pubic hair, Forensic Sci. Int. (2007). [18] F. Pragst, M.A. Balikova, State of the art in hair analysis for detection of drug and alcohol abuse, Clin. Chim. Acta (2006). [19] L. Martins, T. Binz, M. Yegles, Influence of ethanol containing cosmetics on ethyl glucuronide concentration in hair, Ann Toxicol Anal. 23 (2011) 10. [20] C.M.M. Gachon, M. Langlois-Meurinne, P. Saindrenan, Plant secondary metabolism glycosyltransferases: the emerging functional analysis, Trends Plant Sci. 10 (2005) 542–549. [21] J. Ross, Y. Li, E. Lim, D.J. Bowles, Higher plant glycosyltransferases, Genome Biol. 2 (2001), Reviews 3004.
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Positive EtG findings in hair as a result of a cosmetic treatment§ Frank Sporkert a,*, Hicham Kharbouche a,1, Marc P. Augsburger a,2, Clementine Klemm b,3, Markus R. Baumgartner c,4 a
University Centre of Legal Medicine Lausanne-Geneva, Rue du Bugnon 21, CH-1011 Lausanne, Switzerland Institute of Legal Medicine, Kantonsspital, Rorschacherstr. 93, CH-9007 St.Gallen, Switzerland c Institute of Legal Medicine, Klinisch-Forensisches Labor, Kurvenstrasse 17, CH-8006 Zu¨rich, Switzerland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2011 Accepted 21 April 2011 Available online 20 October 2011
In a case of a driving ability assessment, hair analysis for ethyl glucuronide (EtG) was requested by the authorities. The person concerned denied alcohol consumption and did not present any clinical sign of alcoholism. However, EtG was found in concentrations of up to 910 pg/mg in hair from different sampling dates suggesting an excessive drinking behavior. The person declared to use a hair lotion on a regularly base. To evaluate a possible effect of the hair lotion, prospective blood and urine controls as well as hair sampling of scalp and pubic hair were performed. The traditional clinical biomarkers of ethanol consumption, CDT and GGT, were inconspicuous in three blood samples taken. EtG was not detected in all collected urine samples. The hair lotion was transmitted to our laboratory. The ethanol concentration in this lotion was determined with 35 g/L. The EtG immunoassay gave a positive result indicating EtG, which could be confirmed by GC–MS/MS-NCI. In a follow-up experiment the lotion was applied to the hair of a volunteer over a period of six weeks. After this treatment, EtG could be measured in the hair at a concentration of 72 pg/mg suggesting chronic and excessive alcohol consumption. Overnight incubation of EtG free hair in the lotion yielded an EtG concentration of 140 pg/mg. In the present case, the positive EtG hair findings could be interpreted as the result of an EtG containing hair care product. To our knowledge, the existence of such a product has not yet been reported, and it is exceptionally unusual to find EtG in cosmetics. Therefore, external sources for hair contamination should always be taken into account when unusual cosmetic treatment is mentioned. In those cases, it is recommended to analyze the hair product for a possible contamination with EtG. The analysis of body hair can help to reveal problems occurring from cosmetic treatment of head hair. As a consequence, the assessment of drinking behavior should be based on more than one diagnostic parameter. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ethyl glucuronide Hair Hair-care Cosmetic treatment
1. Introduction Hair is steadily exposed to hair care, sunshine, rain, smoke and hair. These external sources can influence the concentrations of substances detected in hair. Cannabinoids in hair are known to be sensible to sunlight [1] but their presence in hair may also be the result of external incorporation [2]. Opiates and
§ This paper is part of the special issue entitled: Selected papers from the Chamonix 2011 Society of Hair Testing Meeting, Guest-edited by Pascal Kintz. * Corresponding author. Tel.: +41 21 314 56 26, fax: +41 21 314 73 29. E-mail addresses: [email protected] (F. Sporkert), [email protected] (H. Kharbouche), [email protected] (M.P. Augsburger), [email protected] (C. Klemm), [email protected] (M.R. Baumgartner). 1 Tel.: +41 21 314 56 26. 2 Tel.: +41 21 314 70 85. 3 Tel.: +41 71 494 30 15. 4 Tel.: +41 44 360 23 71.
0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.10.009
cocaine concentrations are decreased by oxidative bleaching [3– 5]. On the other hand, cosmetic treatment can also lead to an increase of concentrations as reported in the case of fatty acid ethyl esters (FAEE) which are formed directly on the head when ethanol containing hair care products are applied [6,7]. Despite the wide use of hair EtG in forensic routine work, only few has been reported in the literature about the influence of hair-care products on the EtG concentration in hair. As for other substances, the use of bleaching products can lead to a significant decrease of the EtG concentration [8]. The question if cosmetic treatment can also yield to false positive results in the case of ethyl glucuronide (EtG) has generally been refused by scientists as ethyl glucuronide is basically formed in the liver, lung and intestine by conjugation of ethanol with UDP glucuronic acid catalyzed by UDP-glucuronosyltransferases (UGT) in the presence of ethanol [9]. Since several UGT subtypes are able to catalyze this reaction, the EtG formation should not depend on genetic polymorphism [10].
98
F. Sporkert et al. / Forensic Science International 218 (2012) 97–100
In this paper, the authors present a case, where EtG was found in high concentrations in hair as a result of a cosmetic treatment with a hair lotion containing high amounts of EtG. A self experiment was carried out in order to evaluate the possibility of incorporating EtG from external sources into hair in a concentration suggesting excessive alcohol consumption.
10 g/day at the end of the experiment. The hair was cut at three occasions with an electrical shaver as short as possible (residual hair length < 1 mm): (1) before, (2) one day after the first treatment, and (3) four weeks after having used the lotion on a daily base. In another experiment, an EtG negative hair strand was incubated in vitro for three days in the concerned hair lotion. The hair samples were analyzed for EtG according to a validated protocol as described above with the difference of three washing steps with water instead of one.
2. Case 4. Results In a case of an evaluation of driving ability after a car accident in February 2008, a 40-year-old woman had to prove her abstinence from alcohol. The mandated department of traffic medicine requested, among others, hair testing for EtG. Over a two-year period between 2008 and 2010, several hair sampling were carried out and yielded to contradicting results from high EtG concentrations to negative results. As a result, the woman was declared inapt for driving. The woman denied any excessive alcohol consumption, claimed the regular use of an ethanol containing hair lotion and asked for a contra expertise. Chronic and excessive alcohol consumption, which was suggested by some of the test results, should be also detectable by the measurement of the traditional and less sensitive biomarkers CDT and GGT. Therefore, three blood and hair sampling were carried out in the following almost two years. However, low amounts of alcohol intake may still not be detectable in hair which implicates that hair analysis cannot be used to prove total abstinence. In contrast to the long term monitoring by EtG hair testing, in urine, EtG can be detected usually up to two days after a drinking event, in case of excessive drinking up to maximal five days [11,12]. In the present case, we therefore recommended also urine testing for EtG which was performed weekly over a time period of four months. Finally, the hair lotion was carefully investigated and also analyzed for EtG. 3. Materials and methods 3.1. Samples Hair samples from the person concerned were taken between April 2008 and August 2010. The sampling was performed according to a standard protocol during medical assessments. In addition, 16 urine samples were collected on a weekly base between the 10.11.2009 and the 08.03.2010. In August 2010, the administered hair lotion was transmitted to one of the labs. The authors obtained written consent from the person concerned for all additional analyses as well as for the publication of the results. 3.2. Methods The hair samples were analyzed for EtG according to our fully validated method [13]. Briefly, a hair segment was washed for 5 min, first with water and secondly with acetone. The dried hair was powdered in a ball mill. About 30 mg were taken for analysis. The sample was incubated for 2 h in an ultrasonic bath with 1 ml of distilled water in presence of the internal standard EtG-D5 (Lipomed, Arlesheim, CH). The extract was purified at a 60 mg OASIS-Max SPE cartridge (Waters, MA, USA). The eluate was evaporated and derivatized with PFPA for GC–MS/MS-NCI measurement on a Varian 300 MS. Additional measurements for EtG in hair using the same extraction procedures were carried out in the other institutes either on a Thermo TSQ Quantum XLS Triple Quadruple GC–MS/MS (Thermo Fisher Scientific, CA, USA) or by LC–MS/MS on a QTrap 4000 (AB Sciex, Darmstadt, Germany) with their own validated methods. EtG in urine and in the lotion as well as and creatinine were measured with the DRI EtG enzyme immunoassay on the CDx90 Analyzer (Thermo Scientific Microgenics, Passau, Germany). CDT was determined using Ceofix CDT-Kit on an Agilent capillary electrophoresis according to our published method [14]. The liver enzyme GGT was measured by an immunological test on a Dimension XPand plus (Siemens Healthcare Diagnostics, Deerfield, IL, USA). The ethanol content in the hair lotion was determined after 1/ 100 dilution of the lotion with distilled water by our routinely used HS-GC-FID (Agilent 1888 HS and Agilent 6850 GC-FID). 3.3. Self experiment The hair lotion was applied every second day to one of the author’s hair on the right head side over a six week period. The author’s daily ethanol intake was estimated with 15 g/day in the beginning of the treatment decreasing to less than
Altogether, four hair samples including head hair and pubic hair were taken from the woman between April 2008 and August 2010. The resulting EtG concentrations are presented in Table 1. Between June 2008 and August 2010, three blood sampling were carried out in order to control the liver enzyme GGT and CDT. The results are shown in Table 2. In order to control abstinence effectively, urine samplings were carried out weekly during a time period of four months (November 2009–March 2010). All of the samples were tested negative for EtG (<100 ng/ml). At last, the hair lotion which had been used by the person concerned was transmitted to our laboratory. Corresponding to the manufacture’s data, the product is an alcoholic plant extract. In contrast to manufacture’s recommendation the lotion was not washed out after the application. The ethanol concentration of the lotion was determined at 35 g/L or 44 vol.%. EtG was first measured by immunoassay suggesting a concentration of >2.0 mg/ml. The subsequent determination of EtG by means of GC–MS/MS-NCI yielded a concentration of 2.7 mg/ml. These results were confirmed in two independent measurements in the laboratories of Zurich and St. Gallen. A second hair lotion from a different lot was achieved independently. The above mentioned results could be confirmed as well. In order to elucidate the possibility of EtG incorporation by the hair lotion, a self experiment was performed. The application of the Table 1 EtG concentrations in the woman’s hair. Date of sampling
Type of hair
Segment (cm)
EtG concentration
11.04.2008 20.06.2008
Head hair Head hair
20.09.2009 30.10.2009
Head hair Head hair
06.08.2010
Pubic hair Head hair
0–6 0–2 2–4 4–6 6–12 0–5 0–3 3–6 2 (in total) 0–3 3–6 6–9 9–12
444a 47 130 460 910 32 NDb ND ND ND ND ND ND
[pg/mg]
a b
Result of a private laboratory (analytical method unknown). ND: not detected (LOD: 3 pg/mg).
Table 2 GGT and CDT concentrations measured in blood of the person concerned. Date of sampling
Marker
Concentration
Reference valuesa
20.06.2008
CDT-CE GGT CDT-CE GGT CDT-CE GGT
0.7% 22 U/L 0.5% 29 U/L 0.7% 16 U/L
<1.7% 5–55 U/L <1.7% 5–55 U/L <1.7% 5–55 U/L
30.10.2009 06.08.2010
a Base for the reference values: specificity for CDT >99% (data non-published), GGT: 95% prediction interval.
F. Sporkert et al. / Forensic Science International 218 (2012) 97–100 Table 3 EtG concentrations in treated (right side) and untreated (left side) parts of scalp hair after administration of the hair lotion. Sampling date
Head side
Hair length [cm]
EtG concentration [pg/mg]
1 2
31.10.2010 12.12.2010
3
10.01.2011
Right Right Left Right Left
1.5 1.5 1.5 1.0 1.0
14a 72a 11 6.9 NDb
Sampling
a Confirmed in independent measurements in the laboratories of Zurich and St. Gallen. b ND: not detected (LOD: 3 pg/mg).
lotion to the author’s hair led to an increase of the concentration of EtG compared to the untreated hair on the left head side. Noteworthy, measurements for treated and untreated hair were always performed in parallel. The results of the measured EtG concentrations before, directly after and four weeks after the treatment are summarized in Table 3. In a parallel experiment, a negative hair strand was incubated in the lotion for 3 days. EtG analysis according to the validated method yielded a concentration of 140 pg/mg. 5. Discussion Hair analyses carried out between April 2008 and September 2009 yielded concentrations of >30 pg/mg indicating a chronic and excessive alcohol consumption according to the consensus of the Society of Hair Testing (SoHT) [15] and our own results [16]. An excessive drinking behavior seemed to be even more evident because the EtG concentrations were not only close above the cutoff of 30 pg/mg but also highly elevated values from 130 up to 910 pg/mg of EtG were measured in the client’s head hair. In contrast to these findings, the concentrations for the blood markers CDT and GGT as well as for the EtG urine did not indicate excessive alcohol consumption even if the sampling was carried out at different occasions which did not totally correspond to the period covered by the hair tests. But at least, the first blood sampling from June 2008 covers parts of the proximal 3 cm hair segment. The EtG concentrations measured in the hair samples from April 2008 and September 2009 decrease significantly from the distal to the proximal segment. This may indicate a break or a less frequent administration of the lotion or a strong decline of the alcohol consumption. The absence of EtG in the hair taken at the 30.10.2009 suggests that the EtG was completely washed out after abandonment or a change of the hair treatment. Otherwise, EtG should still be detectable since the hair segments from the 20.09.2009 and the 30.10.2009 represent overlapping time periods. The person concerned declared that she finally followed the manufacturer’s instructions and started to wash out carefully the hair some minutes after the administration what she had never done in the earlier treatment. Another strong hint against chronic and excessive alcohol consumption was the absence of EtG in the pubic hair. EtG concentrations in pubic hair are often strongly elevated compared to scalp hair [17] probably due to contact with urine. Another explanation might be the wider time window due to the higher portion of telogene hair of about 50% [18]. The remarkable difference in the EtG hair concentrations measured in the hair of the client in April and June 2008 (up to 910 pg/mg) compared to those found after self application (72 pg/ mg) can be explained by differences in the experimental design of the self experiment. The hair of the client had been treated for already more than one year before the first sampling according to
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the person concerned. In contrast, the self experiment started with cut hair. Only 1 mm remained on the scalp. When the lotion was applied, a big part flew down, so that the contact time with the hair was shorter than with longer hair. Since the hair of the client was longer than 20 cm, the number of applications to the analyzed parts of the hair was greater than in the self experiment. This hypothesis appeared to be confirmed by the increasing EtG concentrations towards the distal segments (from 47 up to 910 pg/ mg) of the person concerned in a period where constant application was claimed. However, from the data of this study some alcohol consumption of the client cannot be completely excluded and may potentially have contributed to some extent to the highly elevated EtG concentrations of up to 910 pg/mg, The EtG concentration of 140 pg/mg which was obtained for the in vitro incubated blank hair sample cannot be compared in toto with the highest concentrations found in the real case since a more extensive washing procedure was applied including 3 aqueous wash steps instead of only one as in the standard protocol. In conclusion, the data indicate that highly elevated EtG concentrations can be found in hair due to the regular use of an EtG contaminated cosmetic which was not washed out after use. The assessment of hair care is therefore crucial for the evaluation of results from hair analysis and should be attentively regarded as an influencing factor. In the present case, all claims of the client had been rejected by the driving license authorities and the traffic medicine department even though other clinical, medical or psychological parameters did not confirm the suspicion of excessive drinking. As a consequence the driving license was revoked twice for a total of 24 months. To our knowledge, this is the first reported case where it is shown that a cosmetic hair product contains EtG. As a consequence, falsely elevated EtG values due to external contamination from this EtG-containing hair product were observed. It has been demonstrated that high ethanol concentration alone of 44 vol.% should not produce highly elevated EtG concentrations such as found in the present case [19]. The present work shows that the EtG from the lotion could be readily incorporated into the hair and could not be removed after applying two washing steps. Furthermore, we could demonstrate that the EtG concentration rapidly decreases when the administration is abandoned as shown in the hair sample of the author taken four weeks after the last application. A small portion of the decrease in the EtG concentration in the case of the self experiment is certainly related to a diminution of the author’s daily alcohol intake from 15 to less than 10 g/day. So far, the herein presented product is up to now the only known hair care containing ethyl glucuronide. According to our experience hair care products typically do not contain EtG. A number of over 15 commercially available hair care products from Germany and Switzerland were tested negative for EtG (data not published). The question of the origin of the EtG found in the lotion is currently under further investigation. Since UDP-glucuronyltransferases are also present in plants [20,21] an extraction of these enzymes which produce EtG in the presence of ethanol cannot be excluded. Another possibility for the presence of EtG in the lotion is a direct extraction of already formed EtG from one of the plants used for the production of the lotion. 6. Conclusion The unusual case of an external contamination of hair with EtG by an EtG-containing hair lotion was presented. In conclusion, we strongly recommend to assess the use of hair care products and the hair treatment very carefully at the hair sampling. In critical cases, hair products should be analyzed for the
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