Forensic Science International 244 (2014) 30–35

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Degradation of the ethyl glucuronide content in hair by hydrogen peroxide and a non-destructive assay for oxidative hair treatment using infra-red spectroscopy Dominic Ammann, Roland Becker *, Anka Kohl, Jessica Ha¨nisch, Irene Nehls Federal Institute for Materials Research and Testing (BAM), Berlin, Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 May 2014 Received in revised form 25 July 2014 Accepted 31 July 2014 Available online 13 August 2014

The assessment of quantification results of the alcohol abuse marker ethyl glucuronide (EtG) in hair in comparison to the cut-off values for the drinking behavior may be complicated by cosmetic hair bleaching. Thus, the impact of increasing exposure to hydrogen peroxide on the EtG content of hair was investigated. Simultaneously, the change of absorbance in the range of 1000–1100 cm1 indicative for the oxidation of cystine was investigated non-destructively by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) using pulverized portions of the respective hair samples. Hair samples treated with hydrogen peroxide consistently displayed a significantly increased absorbance at 1040 cm1 associated with the formation of cysteic acid. The EtG content decreased significantly if the hair was treated with alkaline hydrogen peroxide as during cosmetic bleaching. It could be shown that ATR-FTIR is capable of detecting an exposure to hydrogen peroxide when still no brightening was visible and already before the EtG content deteriorated significantly. Thus, hair samples suspected of having been exposed to oxidative treatment may be checked non-destructively by a readily available technique. This assay is also possible retrospectively after EtG extraction and using archived samples. ß 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Hair testing Oxidation Bleaching Cysteic acid HPLC–MS/MS

1. Introduction In recent years, the determination of ethyl glucuronide (EtG), a minor metabolite of ethanol, in human hair gained increasing importance for the assessment of the overall drinking behavior [1–5]. Correct quantification of the actual EtG content in a person’s hair sample is prerequisite for its comparison with generally accepted cut-off values [6] especially in legal cases and further scenarios with serious consequences for the involved parties. Hair bleaching is a possible way to dissemble lower EtG assays or generate false negative EtG results. It has been reported that treatment of hair involving cosmetic bleaching leads to complete loss of EtG [7]. However, it could not be decided, to which extent the EtG losses were induced by detergent mediated washing-off or by oxidative degradation. Meanwhile, there is evidence that EtG is chemically degraded by hydrogen peroxide in aqueous solution

* Corresponding author at: Federal Institute for Materials Research and Testing (BAM), Richard-Willsta¨tter-Strasse 11, D-12489 Berlin, Germany. Tel.: +49 30 81041171; fax: +49 30 81041177. E-mail address: [email protected] (R. Becker). http://dx.doi.org/10.1016/j.forsciint.2014.07.029 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.

[8]. However, oxidative treatment of hair and potential EtG loss may not be obvious to the analyst and an assay to detect manipulated samples would increase the reliability of EtG results. This investigation aimed at clarifying two questions: Under which conditions does oxidative treatment of hair comparable to cosmetic bleaching lead to a significant decrease of the EtG content pretending an underestimation of the drinking behavior? Is the exposure of a given hair sample to oxidative agents detectable by technical means with practical relevance? Therefore, no commercial bleach was applied but the effects of detergent-free hydrogen peroxide alone and in combination with a buffer and ammonia were investigated in detail. Concentrations and exposure periods matched the conditions of cosmetic bleaching. Fourier transform infrared spectroscopy (FTIR) was used to investigate significant changes in the hair matrix induced by oxidative treatment because of its availability in forensic laboratories, its sensitivity and its non-destructive operation principle. Furthermore, FTIR has successfully been employed to detect the oxidation induced alteration of hair [9–11] and wool [12–14]. The ATR technique allowed recording of IR spectra directly from powdered hair samples without further sample preparation.

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2. Materials and methods

2.5. Quantification of ethyl glucuronide with HPLC–MS/MS

2.1. Chemicals and reagents

50 mg of the hair (snippet size: 1– 2 mm) were washed with dichloromethane and methanol as described above. After airdrying, the sample was submitted to a combined grinding and extraction procedure known as micropulverization and described elsewhere in detail [16]. In brief, two stainless steel balls (Ø = 5 mm) were added to the Eppendorf tube followed by 550 mL of water and 50 mL of an aqueous EtG-d5 solution (100 ng/mL) as internal standard. Up to ten closed tubes were placed equally into each of the two PTFE-adaptors with a capacity of ten conical plastic tubes (Retsch, Haan, Germany). The samples were milled for 30 min with a frequency of 30 s1 using the mixer mill MM400. Thereafter, the extract was filtered through a 0.2 mm regenerated cellulose Phenex1 filter (Phenomenex, Aschaffenburg, Germany) and 150 mL of the filtrate allowing three injections were submitted to HPLC analysis. Quantification of EtG was performed on an Agilent 1200 series HPLC binary pump system (Agilent Technologies, Waldbronn, Germany) equipped with an autosampler and coupled to an API 4000 Q-Trap1 high performance hybrid triple quadrupole/ linear ion trap mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, California/Concord, Ontario, Canada). Chromatographic separation was carried out using a combination of a 10 mm  2.1 mm Hypercarb guard column (3 mm particle size) with a 100 mm  2.1 mm Hypercarb column (Thermo Scientific, Waltham, USA) with 3 mm particle size. A mixture of 93% water, 7% acetonitrile and 0.1% formic acid was used as mobile phase at a flow rate of 0.3 mL/min. The injection volume was 50 mL. The separation was carried out isocratically over a total run time of 10 min and the analytes displayed retention times of 3.5 min (native EtG) and 3.4 min (EtG-d5). The oven temperature was held at 30 8C. The mass spectrometer was run in the multiple reaction mode (MRM, dwell time: 100 msec) with negative ionization. Transitions monitored were m/z 220.8 ! 74.9 (quantifier) for native EtG and m/z 226.0 ! 74.9 for EtG-d5 as well as m/z 220.8 ! 84.9 (qualifier) for native EtG and m/z 226.0 ! 84.9 for EtG-d5. The first and third quadrupole were set to unit resolution. Source parameters were: ion spray voltage, 4000 V; desolvation temperature, 500 8C; ion source gas 1, 80 (arbitrary units) a.u.; ion source gas 2, 90 a.u.; curtain gas, 25 a.u.; collision gas 12 a.u.; declustering potential, 28 V for both transitions of native EtG and 27 V for both transitions of EtG-d5; collision energy, 27 eV for the transition m/z 220.8 ! 74.9 and 26 eV for the other ones; entrance potential, 9 V for the quantifier transitions and 10 V for the qualifier transitions; and collision cell exit potential, 7 V for all four transitions. Data were collected with the Analyst 1.5.2 and processed with the Analyst 1.6.2 software packages (Applied Biosystems/MDS SCIEX).

Native EtG and labeled EtG-d5 were obtained from Medichem (Steinenbronn, Germany). Hydrogen peroxide (trace select, 30%), Trizma1 base (99.9%), hydrochloric acid (37%), ammonium hydroxide (trace select, 25%), and formic acid (for mass spectrometry, 98%) were purchased from Sigma–Aldrich (Steinheim, Germany). Dichloromethane and methanol were obtained in picograde quality from LGC Standards GmbH (Wesel, Germany). Acetonitrile (HPLC grade) was from VWR International (Leuven, Belgium). Deionized water was prepared with a Milli-Q system (Millipore, Billerica, MA, USA). 2.2. Origin of hair samples and sample pre-treatment Hair samples were anonymously received from male volunteers with known age and abstinence from hair dying or bleaching but suspected or known alcohol consumption. Each hair strand as received from an individual was humidified with a small amount of water and gently pressed between paper filters to remove excess water. Then the strand was aligned on a plastic board and manually cut with a ceramic household knife to pieces of 1–2 mm of length. The moist cuttings were cooled to 28 8C and lyophilized for 24 h using a LYOVAC GT 2/GT 2-E lyophilizer (FINN-AQUA, Hu¨rth, Germany). After equilibration with the laboratory atmosphere the cut hair material was homogenized by overhead shaking for 48 h using an REAX 20 end over end mixer (Heidolph, Schwabach, Germany). The hair pool was obtained from a local barber and contained hair without oxidative treatment from a number of male donors and was cut and homogenized as reported elsewhere [15]. 2.3. Bleaching procedure Three different bleaching solutions were prepared: Solution A: 10% neutral H2O2 in water; solution B: 10% H2O2 in 50 mM Trizma1-HCl buffer at pH 8.0; solution C: 10% H2O2 in 50 mM Trizma1-HCl buffer adjusted to pH 10.0 with ammonium hydroxide. 50 mg of the hair sample under investigation (snippet size: 1– 2 mm) were weighed into a 2 mL conical plastic self-lock tube with attached lid (Eppendorf AG, Hamburg, Germany) and submerged in 1 mL of dichloromethane for 15 min. After pipetting the dichloromethane off, 1 mL of methanol was added and allowed just to perfuse to hair and then removed immediately. After airdrying over night the sample was submerge in the respective solution A, B or C for 10, 30, 45 or 60 min. Thereafter, the solution was removed and the hair sample was washed three times with 1 mL of water. The moist sample was cooled to 28 8C and then lyophilized for 24 h.

2.6. Calibration and quality control 2.4. Infrared spectroscopy 50 mg of the designated sample were filled in 5 mL stainless steel milling flask together with two stainless steel milling balls of (Ø = 5 mm) and pulverized in a Retsch Mixer mill MM400 (Retsch, Haan, Germany) for 10 min at frequency of 30 s1. 10 mg of the powdered hair was applied to an ATR Diamond Golden Gate accessory mounted on an IFS66v FTIR vacuum spectrometer (Bruker, Ettlingen, Germany). Every measurement included 2000 background and samples scans covering the range between 4000 and 600 cm1 with a resolution of 2 cm1. IR spectra were processed using the Origin 9.0 software package (OriginLab Corp., Northampton, MA, USA).

Fourteen aqueous calibration solutions in the concentration range between 100 pg/mL and 80,000 pg/mL, corresponding to a concentration range between 1 and 800 pg EtG per mg hair were prepared gravimetrically. At least six calibration points were used for each measurement sequence depending on the concentration of the samples. Coefficients of determination R2 were in all cases better than 0.998. Procedural blanks were analyzed with each sequence of 30 injections in duplicate. Procedural background levels of EtG were carefully controlled and throughout insignificant. Additionally, duplicate injections of two quality control materials (QM) were done after each sequence of 20 injections to monitor the quantitative reproducibility of the analytical procedure. No significant drift was observed. These QM materials

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consisted of aqueous solutions containing 10.8 and 35.7 pg EtG per mg water. LOD and LOQ of EtG in hair was estimated as 1 pg/mg and 3 pg/mg on basis of a signal to noise ratio of three and nine, respectively. 3. Results and discussion 3.1. Oxidative treatment of hair and EtG content Fig. 1 depicts the change of the EtG content in the hair pool after stepwise prolonged treatment with hydrogen peroxide and with increasing pH. It is seen that neutral hydrogen peroxide does not decrease the EtG content significantly while at pH 8 a significant degradation is observed after a 60 min exposure period (Welch test, p < 0.001). The fact that the EtG content remains virtually unaffected by treatment with solution A up to 1 h proves that aqueous EtG extraction is irrelevant for the exposure periods investigated in this work. At pH 10 in the presence of ammonia the degradation of the original EtG content is already significant after 10 min (p < 0.01) and becomes more conspicuous with exposure duration (30 min: p < 0.001; 45 and 60 min: p < 1013). It is very likely that EtG is indeed chemically degraded on treatment of hair samples with alkaline hydrogen peroxide. This is supported by the observation of Kerekes and Yegles with aqueous EtG solutions treated with hydrogen peroxide [8]. It is further well documented that alkaline hydrogen peroxide oxidizes the aldehyde function of monosaccharides [17,18]. In case of aldose acetales similar to EtG the secondary alcoholic functions are oxidized followed by ring cleavage [17]. It should be noted that only the application of solution C, which resembles the conditions of commercial bleaching, leads to a visible brightening of the treated hair sample. An example is given in the supplementary Fig. S1. This decrease of the EtG content upon bleaching was further investigated with the hair strands obtained from different individual displaying a range of authentic EtG contents. Fig. 2 depicts the results obtained after treatment with solutions B and C. An exposure period of 45 min comes close to what is recommended for the application of commercially available cosmetic bleach and was considered to result in a significant effect from the initial experiments with the hair pool.

It is seen that the original EtG contents in the hair strands are significantly reduced by exposure to both alkaline bleaching solutions (Fig. 2). In case of hair samples 1, 3, 4, and 5 EtG levels obviously indicating elevated or even excessive alcohol consumption over the period represented by the respective hair sample are reduced to social drinker or even teetotaler levels. Hair sample 2 with a low EtG content generally considered typical for nondrinkers and close to the EtG detection limit displays the least reduction. It should be noted that hair samples 1–5 were strands of different length and taken at different distances from the scalp of individual donors with potentially irregular drinking habit. Hair age, thickness, swelling characteristics and washing habits vary among the donors and are assumed to affect the different extent of EtG degradation in the samples 1–5. In case of commercial bleaching products with optimized detergent composition as employed by Morini et al. [7] personal hair characteristics become obsolete and EtG becomes undetectable due to an increased washing-off effect or matrix effects caused by the detergents during electrospray ionization. 3.2. Infrared spectroscopic detection of hair bleaching Fig. 3 depicts the infrared spectra of hair samples from the hair pool before and after oxidative treatment with hydrogen between 10 and 60 min. The spectra are dominated by the amide I (1640– 1650 cm1), amide II (1510–1515 cm1) and amide III (1227 cm1) absorption bands along with the absorption of CH2 and CH3 bending modes at 1445 cm1 as to be expected from the literature [19]. After oxidative treatment a new band at 1040 cm1 is observed as the sole significant change. This band has been identified as the symmetric S–O stretch mode n(S–O) of the sulfonic group of cysteic acid formed by cysteine oxidation [10,11,20]. Cysteine is abundant in human hair and typically makes up between 17% and 18% of the hair keratin [11,21–23]. With continued exposure to H2O2 the intensity of the n(S–O) band at 1040 cm1 tends to increase. The absorption bands of further oxidized sulfur species reported in the connection with oxidation of cystine [20], human hair [10,11,20,24,25] or wool [12–14,26] under various conditions were not observed. This is in accordance with [27] where treatment of hair with alkaline hydrogen peroxide led nearly

Fig. 1. Ethyl glucuronide content in the hair pool after oxidative treatment (means and standard deviations; n = number of runs with the respective exposure; *p < 0.05, **p < 0.01).

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Fig. 2. Deterioration of the EtG content in authentic hair samples 1–5 after treatment with alkaline hydrogen peroxide (means and standard deviations; n = number of runs with the respective exposure; *p < 0.05, **p < 0.01).

exclusively to cysteic acid while acid hydrogen peroxide also displayed significant amounts of further oxidation products which might be regarded as intermediate in the oxidation of cystine to cysteic acid. A collection of these compounds, their infrared bands and the respective literature can be found in the supplementary Table S1. It should be noted that hair specimens from different donors do not seem to display any significant differences with regard to their IR spectra before oxidative treatment. This is in accordance with studies including a large number of individuals of different sex, age and hair color [9,28]. The possible occurrence of minor cysteic acid portions in untreated hair [11,21] suggests the definition of a cut-off for

Fig. 3. IR spectra of the hair pool before and after treatment with alkaline hydrogen peroxide (ATR-FTIR).

significantly increased cysteic acid content. For that purpose the cysteic acid content needs to be determined semi-quantitatively. This is most conveniently done by using an IR absorption band that remains unaltered by hair treatment as internal standard. For this purpose the amide I [10,11,13,28], amide II [28] or the amide III bands [10,29] as well as the CH2-deformation mode [29] have been used after normalization of the respective spectra. In this work all spectra were normalized to a base line defined by two reference points. These were the absorbance minimum between the amide I and amide II band (1580 cm1) and the absorbance at 903 cm1. The peak heights above this baseline were expressed as ratios of the n(S–O) mode to the amide I, amide II, and amide III bands. Details on the normalization are given in the supplementary Fig. S2 while Fig. 4 depicts the distributions of the ratios in all tested hair samples. It is already seen by the naked eye that the n(S–O)/amide absorption ratio is significantly higher in all three cases after oxidative treatment, most conspicuously in case of the amide III band. A Welch-test confirmed significantly different means before and after treatment (in all cases: p < 1015) and an F-test revealed significantly different variances (in all cases: p < 0.01). A more detailed presentation of absorbance ratios observed after treatment with solutions A–C is given in the supplementary Fig. S3. It was seen that even a short treatment period of 10 min with any solution resulted in significant formation of cysteic acid. Thus, ATR-FTIR is capable of clearly detecting oxidative treatment before EtG content degradation becomes relevant. A further tool to visualize the significance of the change after oxidative treatment is provided by derivative spectroscopy. Second order derivatives of FTIR spectra have been employed to study surface properties of hair [24]. Fig. 5 depicts typical first and second derivatives of IR spectra from a hair specimen before and after oxidative treatment. After oxidative treatment, the n(S–O) absorption region is clearly and throughout distinguished from untreated specimen by the contrarily oriented maxima after first order derivatization and a strong band after second order derivatization. A suggestion how to construct a cut-off that defines the derivatives as being significantly indicative for an increased cysteic acid content is given in the supplementary Figs. S4–S5. The spectra and derivatives of untreated hair samples do not display these characteristics (see supplementary Fig. S6).

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Fig. 4. Box-plots of ratios of n(S–O) absorption band to the amide I, II, and III bands before and after oxidative treatment.

Fig. 5. FTIR absorbance and derivatives of a treated hair sample (hair sample 1, H2O2, pH 8, 45 min).

3.3. Practical application of the procedure A given hair specimen may be assessed with regard to a potential bleaching history by visual screening of the absorbance around 1040 cm1. Original FTIR spectra and the first and second order derivatives display characteristic patterns only present after deliberate oxidation of the hair matrix. Experienced-based

cut-off limits may be developed from compiled FTIR measurements along the principles outlined in the supplementary material. Blond or pigment-free hair cannot be assessed regarding a potential oxidative treatment by the naked eye and grinding of any hair specimen results in a gray powder. It should also be noted that the treatment of hair with hydrogen peroxide does not necessarily

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result in visually obvious brightening. Thus, the runs with neutral hydrogen peroxide and at pH 8 led to distinct n(S–O) absorption bands without any visible change of the originally brown hair (supplementary Fig. S1). It was seen that a significant formation of cysteic acid occurs with any treatment with hydrogen peroxide before the EtG content is severely deteriorated. Therefore, the detection of cysteic acid may be regarded as a safe way to identify suspicious specimens. It has been shown that untreated hair contains minor amounts of cysteic acid [11,22] as well as traces of cysteine-S-monoxide and cysteine-S-dioxide [11]. Photo-oxidation is regarded a major reason for this phenomenon [12,26] closely related to the photoyellowing of wool [26]. However, this effect is only expected to reach observable extent at the distal end and with increasing hair length and weathering. Scalp hair collection for reasonable EtG quantification as recommended by the SoHT should be narrowed to the 0–6 cm of the proximal end [6]. Therefore, it is not likely to sample hair significantly affected by photo-oxidation and a significant observation of cysteic acid is highly indicative of recent oxidative manipulation of a given hair specimen under investigation. It has been demonstrated that dry milling of the hair sample is advantageous to maximize EtG extraction yields [15,30] and this work revealed that it does not affect the detection of cysteic acid. It is recommended to use a pulverized hair sample for FTIR analysis though single hair analysis has been reported [31]. As in the quantification of EtG a certain minimum sample amount appears prerequisite to be sufficiently representative. The few mg required for FTIR analysis may even be used for EtG analysis or archival storage of specimen copies. According to a literature report the cysteic acid content remains stable in hair samples archived at ambient temperatures [9]. The ATR-FTIR assay for cysteic was also successful applied to hair samples after aqueous extraction of EtG using mircopulverization [16] and subsequent drying (data not shown). Thus, suspicious samples may be archived even after being extracted and tested later for oxidative manipulation if doubts might arise. 4. Conclusion The EtG content in hair degrades when treated with alkaline hydrogen peroxide under condition similar to cosmetic bleaching. The effect becomes more distinct in the presence of ammonia. Even before significant degradation of the EtG content, cysteic acid is formed in amounts detectable by routine ATR-FTIR spectroscopy. Thus, in doubtful cases the oxidative treatment of a hair sample can be tested non-destructively and without loss of sample using widely available equipment. This test is applicable to archived samples and even to samples already having been extracted and dried. Acknowledgement Financial support from the German Federal Ministry for Economic Affairs and Energy via ‘‘Zentrales Innovationsprogramm Mittelstand’’ (FKZ KF2201048SK2) is gratefully acknowledged. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.forsciint. 2014.07.029.

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Degradation of the ethyl glucuronide content in hair by hydrogen peroxide and a non-destructive assay for oxidative hair treatment using infra-red spectroscopy.

The assessment of quantification results of the alcohol abuse marker ethyl glucuronide (EtG) in hair in comparison to the cut-off values for the drink...
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