Int J Legal Med (2014) 128:69–72 DOI 10.1007/s00414-013-0939-z
TECHNICAL NOTE
Determination of ethyl glucuronide in hair: a rapid sample pretreatment involving simultaneous milling and extraction Bettina Mönch & Roland Becker & Irene Nehls
Received: 13 September 2013 / Accepted: 29 October 2013 / Published online: 13 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Keywords Ethyl glucuronide . Hair . Micropulverized extraction . GC-MS
importance for the assessment of the long-term drinking habits [1–4]. The quantification of EtG follows a straightforward scheme: Particle size reduction of the hair sample is followed by washing and extraction, optionally an extract cleanup, in case of GC, a derivatization, and finally the quantification using LC-MS/MS [5–7] or GC-MS(/MS) [8–11]. Options for the particle size reduction of the hair sample are manual cutting to snippets or grinding to a powder. Recent studies showed that the grinding of hair samples leads to a higher detectable amount of EtG compared to millimeter-size snippets [12, 13]. The combination of hair grinding with the analyte extraction in a one-step procedure is a rapid and straightforward sample pretreatment called micropulverized extraction and was so far applied for the determination of amphetamines and methamphetamines [14, 15], ketamine [16], cocaine, and other drugs of abuse [17] and for therapeutic drugs [18–20]. However, to our knowledge, it has not been reported for the quantification of EtG in hair. Therefore, the aim of this study was to develop a procedure that combines grinding of hair samples with the extraction of EtG and to compare it with conventional sample pretreatment methods.
Introduction
Materials and methods
In recent years, the determination of ethyl glucuronide (EtG), a minor metabolite of ethanol, in human hair gained
Origin of the hair samples
Abstract A combination of simultaneous milling and extraction known as micropulverized extraction was developed for the quantification of the alcohol marker ethyl glucuronide (EtG) in hair samples using a homogeneous reference material and a mixer mill. Best extraction results from 50 mg of hair were obtained with 2-mL plastic tubes containing two steel balls (∅ = 5 mm), 0.5 mL of water and with an oscillating frequency of 30 s−1 over a period of 30 min. EtG was quantified employing a validated GC-MS procedure involving derivatization with pentafluoropropionic acid anhydride. This micropulverization procedure was compared with dry milling followed by separate aqueous extraction and with aqueous extraction after manual cutting to millimeter-size snippets. Micropulverization yielded 28.0±1.70 pg/mg and was seen to be superior to manually cutting (23.0±0.83 pg/mg) and equivalent to dry grinding (27.7±1.71 pg/mg) with regard to completeness of EtG extraction. The option to process up to 20 samples simultaneously makes micropulverization especially valuable for the high throughput of urgent samples.
Electronic supplementary material The online version of this article (doi:10.1007/s00414-013-0939-z) contains supplementary material, which is available to authorized users. B. Mönch : R. Becker (*) : I. Nehls Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Strasse 11, 12489 Berlin, Germany e-mail: [email protected]
A hair material containing EtG in the range considered as being indicative for moderate drinking behavior was obtained from Medichem (Steinenbronn, Germany). This material consisted of cut hair (2–3 mm) and was produced from authentic hair samples obtained from different persons in order to adjust the desired EtG level. The homogeneity of this material was established, and the results are described elsewhere [11].
70
Chemicals and reagents Native EtG and labeled EtG-d5 were obtained from Medichem (Steinenbronn, Germany). Stock solutions (2 mg/mL each) were prepared in high-purity water prepared with a Milli-Q system (Millipore, USA). All other chemicals were of highest analytical grade. Dichloromethane and methanol were supplied by Promochem (Wesel, Germany). Methane 5.5, hydrogen (99.999 %), and carbon dioxide AligalTM2 for PTV cooling were purchased from Air Liquide (Düsseldorf, Germany). Washing and extraction of cut hair samples The manually cut hair samples were processed following a modification of the procedure described by Pragst et al. [2]. About 50 mg of hair were weighed exactly into a 2-mL conical plastic tube with attached lid (safe-lock tubes, Eppendorf AG, Hamburg, Germany). Therein, the hair was submersed 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 air-drying, 0.45 mL of water was added, followed by 0.05 mL of aqueous EtG-d5 solution (115 ng/g) as internal standard (IS). After standing for 2 days at room temperature, the samples were filtered. Washing and extraction of powdered hair samples Milling was performed with a Pulverisette 23-ball mill (Fritsch, Idar-Oberstein, Germany), as described elsewhere [13]. About 60 mg of the manually cut hair were filled into a 5-mL grinding bowl and two stainless steel balls (Ø=1 cm) were added. Samples were ground for 2 min at a frequency of 30 s−1 to obtain a crude powder. For each EtG determination, 50 mg of hair were weighed exactly into a 2-mL conical plastic tube with attached lid. Therein, the hair was washed and extracted as described for the manually cut hair samples. One-step procedure for milling and extraction About 50 mg of the manually cut hair sample were weighted exactly into a 2-mL conical plastic self-lock tube (see above) with attached lid and washed with the method described above. After air-drying, 0.45 mL of water was added, followed by 0.05 mL of aqueous EtG-d5 solution (115 ng/g) as IS. Then, two stainless steel balls (Ø=0.5 cm) were added and 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 at a frequency of 30 s−1 using a mixer mill MM 400 (Retsch, Haan, Germany). Then, the samples were centrifugalized for 10 min at a frequency of 13,400 rpm with
Int J Legal Med (2014) 128:69–72
a MiniSpin® centrifuge (Eppendorf AG, Hamburg, Germany). The clear supernatant was pipetted of and filtered. The extract temperature was measured by holding an IRt h e r m o m e t e r ( G T- 1 0 1 , G e r a t h e r m M e d i c a l A G , Geschwenda, Germany) 3 cm above the extract surface. Ethyl glucuronide quantification by GC-MS The aqueous hair extracts were lyophilized, and EtG was quantified using a validated procedure involving PFPA derivatization [11]. Details of the procedure using negative chemical ionization and the quality assurance are collected in the Supplementary information. Each extraction during method development was performed in double (n =2) and each extract was quantified in double.
Results and discussion Development of the combined milling and extraction procedure Though milling and extraction were combined in one step, the whole sample pretreatment prior to instrumental involves several steps: Manual cutting into snippets, washing of the hair to remove potential contamination, addition of solvent, internal standard and milling balls, followed by the use of the mixer mill, the centrifugation of the obtained suspension, and the filtration of the clear supernatant. The work flow of the whole procedure is presented in Supplemental Fig. S1. Completeness and rapidity of the newly developed extraction method depend on various parameters to be optimized. These are the oscillating frequency and duration of the milling procedure as well as the number, the size, and the composition of the balls. Water was the extractant of choice due to the excellent solubility of EtG and in accordance with nearly the entire literature. The mixer mill was run at the highest possible frequency for an optimal rapidity of the process, and stainless steel balls were selected. It was seen that the employed tubes withstand a loading of 20 balls (Ø = 3 mm) and 5 balls (Ø = 5 mm), respectively, for 60 min at maximum frequency without signs of damage. The number and the size of the balls were optimized simultaneously. The volume of the 2-mL tubes allowed the use of up to eight 3-mm balls and up to six 5-mm balls. Firstly, the effect of the number of milling balls was investigated with a fixed milling period of 30 min. Figure 1 reveals that the 5-mm balls led to a significantly greater EtG extraction compared to the 3-mm balls. It is further obvious that the best results were obtained with two 5-mm balls. A further increase of balls led to decreasing EtG extraction. This observation was confirmed in an independent reproducibility experiment.
Int J Legal Med (2014) 128:69–72
Fig. 1 Optimization of number and size of the milling balls and presentation of the mean detectable EtG contents with standard deviations (n =2)
Secondly, the optimal milling period for the use of two 5-mm balls was evaluated. Figure 2 depicts the results obtained after extraction times between 5 and 60 min. Obviously, the variability of extractable EtG with extraction time is hardly significant and tends to reach an optimum at 30 min. The slightly higher values from 20 min onward may be due to the moderate temperature increase with extraction time. Thus, a period of 30 min and the use of two 5-mm balls were regarded as the optimal conditions for the determination of EtG in hair. Throughout the investigation, the closed tubes (maximum of ten) were always equally distributed between the two PTFEadaptors, with a capacity of ten conical plastic tubes each. That way, no leakage of the tube lids occurred. Verification of the combined milling and extraction procedure The described procedure needs to be tested regarding the equivalence with dry milling followed by extraction and regarding its superiority over manual cutting followed by extraction.
71
Therefore, EtG was quantified in ten samples each that were either manually cut or ground to a dry crude powder and then extracted with water for 2 days at room temperature [11] and in ten samples that were submitted to the combined pulverization/extraction method (two 5-mm balls, 30 min). The EtG contents observed in the respective extracts are shown in Table 1. The three data sets were normally distributed, free of outliers, trends, and were tested for significant differences between the measurements series (F-test) and the respective means (t-test). It is obvious that both measurement series involving milling resulted in higher amounts of detectable EtG than manual cutting in accordance with literature reports [12, 13]. It is also seen that there is no significant difference between dry milling, followed by extraction and the combination of milling and extraction.
Conclusion Micropulverization, the combined milling and extraction, was shown to be applicable for the quantification of EtG in hair and equivalent to dry grinding with consecutive extraction. This method displays excellent efficiency in terms of completeness and rapidity of extraction. As 20 samples can be Table 1 Ethyl glucuronide content in picograms per milligram extracted from hair using different options for particle size reduction Replicate extraction
Manual cutting
Dry milling
Combined milling and extraction (micropulverisation)
1 2 3 4 5 6 7 8
23.3 23.2 24.6 23.4 22.3 21.9 22.7 22.4
28.1 30.4 29.7 25.2 26.4 28.4 26.9 27.0
27.8 30.2 29.8 25.6 26.7 29.5 26.3 26.8
9 10 Average Standard deviation Difference from manual cutting
22.6 23.5 23.0 0.83
26.0 28.6 27.7 1.71 4.7 (20.4 %)
27.2 29.5 28.0 1.70 5.0 (21.7 %)
p-values F-test 9.96×10−8
t-test
Manual cutting–dry milling
Fig. 2 Optimization of the milling/extraction time, presentation of mean detectable EtG contents, and standard deviations (n =2)
Manual cutting–combined milling and extraction Dry milling–combined milling and extraction
2.08×10
1.97×10−6 1.12×10−6
0.721
0.721
−7
72
processed simultaneously, this method opens prospects for automatization and allows a high throughput of urgent samples.
Int J Legal Med (2014) 128:69–72
11.
12.
References 1. Morini L, Politi L, Polettini A (2009) Ethyl glucuronide in hair. A sensitive and specific marker of chronic heavy drinking. Addiction 104:915–920 2. Pragst F, Rothe M, Moench B, Hastedt M, Herre S, Simmert D (2010) Combined use of fatty acid ethyl esters and ethyl glucuronide in hair for diagnosis of alcohol abuse: interpretation and advantages. Forensic Sci Int 196:101–110 3. Agius R, Nadulski T, Kahl H-G, Dufax B (2012) Ethyl glucuronide in hair—a highly effective test for monitoring of alcohol consumption. Forensic Sci Int 218:10–14 4. Hastedt M, Herre S, Pragst F, Rothe M, Hartwig S (2012) Workplace alcohol testing program by combined use of ethyl glucuronide and fatty acid ethyl esters in hair. Alcohol Alcohol 47:127–132 5. Morini L, Politi L, Groppi A, Stramesi C, Polettini A (2006) Determination of ethyl glucuronide in hair samples by liquid chromatography/electrospray tandem mass spectrometry. J Mass Spectrom 41:34–42 6. Lamoureux F, Gaulier JM, Sauvage FL, Mercerolle M, Vallejo C, Lachâtre G (2009) Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction. Anal Bioanal Chem 394:1895–1901 7. Albermann ME, Musshoff F, Madea B (2010) A fully validated highperformance liquid chromatography-tandem mass spectrometry method for the determination of ethyl glucuronide in hair for the proof of strict alcohol abstinence. Anal Bioanal Chem 396:2441–2447 8. Kharbouche H, Sporkert F, Troxler S, Augsburger M, Mangin P, Staub C (2009) 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 877:2337–2343 9. Alvarez I, Bermejo AM, Tabernero MJ, Fernandez P, Cabarcos P, Lopez P (2009) Microwave-assisted extraction: a simpler and faster method for the determination of ethyl glucuronide in hair by gas chromatography– mass spectrometry. Anal Bioanal Chem 393:1345–1350 10. Shi Y, Shen B, Xiang P, Yan H, Shen M (2010) Determination of ethyl glucuronide in hair samples of Chinese people by protein
13.
14.
15.
16.
17.
18.
19.
20.
precipitation (PPT) and large volume injection–gas chromatography–tandem mass spectrometry (LVI–GC/MS/MS). J Chromatogr B 878:3161–3166 Mönch B, Becker R, Jung C, Nehls I (2013) The homogeneity testing of EtG in hair reference materials: a high-throughput procedure using GC–NCI–MS. Forensic Sci Int 226:202–207 Albermann ME, Musshoff F, Aengenheister L, Madea B (2012) Investigations on the influence of different grinding procedures on measured ethyl glucuronide concentrations in hair determined with an optimized and validated LC-MS/MS method. Anal Bioanal Chem 403:769–776 Mönch B, Becker R, Nehls I (2013) Quantification of ethyl glucuronide in hair: effect of milling on extraction efficiency. Alcohol Alcohol 48:558–563 Miyaguchi H, Kakuta M, Iwata YT, Matsuda H, Tazawa H, Kimura H, Inoue H (2007) Development of a micropulverized extraction method for rapid toxicological analysis of methamphetamine in hair. J Chromatogr A 1163:43–48 Miyaguchi H, Iwata YT, Kanamori T, Tsujikawa K, Kuwayama K, Inoue H (2009) Rapid identification and quantification of methamphetamine and amphetamine in hair by gas chromatography/mass spectrometry coupled with micropulverized extraction, aqueous acetylation and microextraction by packed sorbent. J Chromatogr A 1216:4063–4070 Inagaki S, Makino H, Fukushima T, Min JZ, Toshimasa Toyooka T (2009) Rapid detection of ketamine and norketamine in rat hair using micropulverized extraction and ultra-performance liquid chromatography–electrospray ionization mass spectrometry. Biomed Chromatogr 23:1245–1250 Favretto D, Vogliardi S, Stocchero G, Nalesso A, Tucci M, Ferrara SD (2011) High performance liquid chromatography–high resolution mass spectrometry and micropulverized extraction for the quantification of amphetamines, cocaine, opioids, benzodiazepines, antidepressants and hallucinogens in 2.5 mg hair samples. J Chromatogr A 1218:6583–6595 Miyaguchi H (2013) Determination of zolpidem in human hair by micropulverized extraction based on the evaluation of relative extraction efficiency of seven psychoactive drugs from an incurred human hair specimen. J Chromatogr A 1293:28–35 Favretto D, Stocchero G, Vogliardi S, Frison G, Trevisanuto G-D, Castagna F, Ferrara SD (2010) Neonatal hair analysis by liquid chromatography-high-resolution mass spectrometry to reveal gestational exposure to venlafaxine. Ther Drug Monit 32:30–39 Favretto D, Stocchero G, Nalesso A, Vogliardi S, Boscolo-Berto R, Montisci M, Ferrara D (2013) Monitoring haloperidol exposure in body fluids and hair of children by liquid chromatography–highresolution mass spectrometry. Ther Drug Monit 35:493–501
TECHNICAL NOTE
Determination of ethyl glucuronide in hair: a rapid sample pretreatment involving simultaneous milling and extraction Bettina Mönch & Roland Becker & Irene Nehls
Received: 13 September 2013 / Accepted: 29 October 2013 / Published online: 13 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Keywords Ethyl glucuronide . Hair . Micropulverized extraction . GC-MS
importance for the assessment of the long-term drinking habits [1–4]. The quantification of EtG follows a straightforward scheme: Particle size reduction of the hair sample is followed by washing and extraction, optionally an extract cleanup, in case of GC, a derivatization, and finally the quantification using LC-MS/MS [5–7] or GC-MS(/MS) [8–11]. Options for the particle size reduction of the hair sample are manual cutting to snippets or grinding to a powder. Recent studies showed that the grinding of hair samples leads to a higher detectable amount of EtG compared to millimeter-size snippets [12, 13]. The combination of hair grinding with the analyte extraction in a one-step procedure is a rapid and straightforward sample pretreatment called micropulverized extraction and was so far applied for the determination of amphetamines and methamphetamines [14, 15], ketamine [16], cocaine, and other drugs of abuse [17] and for therapeutic drugs [18–20]. However, to our knowledge, it has not been reported for the quantification of EtG in hair. Therefore, the aim of this study was to develop a procedure that combines grinding of hair samples with the extraction of EtG and to compare it with conventional sample pretreatment methods.
Introduction
Materials and methods
In recent years, the determination of ethyl glucuronide (EtG), a minor metabolite of ethanol, in human hair gained
Origin of the hair samples
Abstract A combination of simultaneous milling and extraction known as micropulverized extraction was developed for the quantification of the alcohol marker ethyl glucuronide (EtG) in hair samples using a homogeneous reference material and a mixer mill. Best extraction results from 50 mg of hair were obtained with 2-mL plastic tubes containing two steel balls (∅ = 5 mm), 0.5 mL of water and with an oscillating frequency of 30 s−1 over a period of 30 min. EtG was quantified employing a validated GC-MS procedure involving derivatization with pentafluoropropionic acid anhydride. This micropulverization procedure was compared with dry milling followed by separate aqueous extraction and with aqueous extraction after manual cutting to millimeter-size snippets. Micropulverization yielded 28.0±1.70 pg/mg and was seen to be superior to manually cutting (23.0±0.83 pg/mg) and equivalent to dry grinding (27.7±1.71 pg/mg) with regard to completeness of EtG extraction. The option to process up to 20 samples simultaneously makes micropulverization especially valuable for the high throughput of urgent samples.
Electronic supplementary material The online version of this article (doi:10.1007/s00414-013-0939-z) contains supplementary material, which is available to authorized users. B. Mönch : R. Becker (*) : I. Nehls Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Strasse 11, 12489 Berlin, Germany e-mail: [email protected]
A hair material containing EtG in the range considered as being indicative for moderate drinking behavior was obtained from Medichem (Steinenbronn, Germany). This material consisted of cut hair (2–3 mm) and was produced from authentic hair samples obtained from different persons in order to adjust the desired EtG level. The homogeneity of this material was established, and the results are described elsewhere [11].
70
Chemicals and reagents Native EtG and labeled EtG-d5 were obtained from Medichem (Steinenbronn, Germany). Stock solutions (2 mg/mL each) were prepared in high-purity water prepared with a Milli-Q system (Millipore, USA). All other chemicals were of highest analytical grade. Dichloromethane and methanol were supplied by Promochem (Wesel, Germany). Methane 5.5, hydrogen (99.999 %), and carbon dioxide AligalTM2 for PTV cooling were purchased from Air Liquide (Düsseldorf, Germany). Washing and extraction of cut hair samples The manually cut hair samples were processed following a modification of the procedure described by Pragst et al. [2]. About 50 mg of hair were weighed exactly into a 2-mL conical plastic tube with attached lid (safe-lock tubes, Eppendorf AG, Hamburg, Germany). Therein, the hair was submersed 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 air-drying, 0.45 mL of water was added, followed by 0.05 mL of aqueous EtG-d5 solution (115 ng/g) as internal standard (IS). After standing for 2 days at room temperature, the samples were filtered. Washing and extraction of powdered hair samples Milling was performed with a Pulverisette 23-ball mill (Fritsch, Idar-Oberstein, Germany), as described elsewhere [13]. About 60 mg of the manually cut hair were filled into a 5-mL grinding bowl and two stainless steel balls (Ø=1 cm) were added. Samples were ground for 2 min at a frequency of 30 s−1 to obtain a crude powder. For each EtG determination, 50 mg of hair were weighed exactly into a 2-mL conical plastic tube with attached lid. Therein, the hair was washed and extracted as described for the manually cut hair samples. One-step procedure for milling and extraction About 50 mg of the manually cut hair sample were weighted exactly into a 2-mL conical plastic self-lock tube (see above) with attached lid and washed with the method described above. After air-drying, 0.45 mL of water was added, followed by 0.05 mL of aqueous EtG-d5 solution (115 ng/g) as IS. Then, two stainless steel balls (Ø=0.5 cm) were added and 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 at a frequency of 30 s−1 using a mixer mill MM 400 (Retsch, Haan, Germany). Then, the samples were centrifugalized for 10 min at a frequency of 13,400 rpm with
Int J Legal Med (2014) 128:69–72
a MiniSpin® centrifuge (Eppendorf AG, Hamburg, Germany). The clear supernatant was pipetted of and filtered. The extract temperature was measured by holding an IRt h e r m o m e t e r ( G T- 1 0 1 , G e r a t h e r m M e d i c a l A G , Geschwenda, Germany) 3 cm above the extract surface. Ethyl glucuronide quantification by GC-MS The aqueous hair extracts were lyophilized, and EtG was quantified using a validated procedure involving PFPA derivatization [11]. Details of the procedure using negative chemical ionization and the quality assurance are collected in the Supplementary information. Each extraction during method development was performed in double (n =2) and each extract was quantified in double.
Results and discussion Development of the combined milling and extraction procedure Though milling and extraction were combined in one step, the whole sample pretreatment prior to instrumental involves several steps: Manual cutting into snippets, washing of the hair to remove potential contamination, addition of solvent, internal standard and milling balls, followed by the use of the mixer mill, the centrifugation of the obtained suspension, and the filtration of the clear supernatant. The work flow of the whole procedure is presented in Supplemental Fig. S1. Completeness and rapidity of the newly developed extraction method depend on various parameters to be optimized. These are the oscillating frequency and duration of the milling procedure as well as the number, the size, and the composition of the balls. Water was the extractant of choice due to the excellent solubility of EtG and in accordance with nearly the entire literature. The mixer mill was run at the highest possible frequency for an optimal rapidity of the process, and stainless steel balls were selected. It was seen that the employed tubes withstand a loading of 20 balls (Ø = 3 mm) and 5 balls (Ø = 5 mm), respectively, for 60 min at maximum frequency without signs of damage. The number and the size of the balls were optimized simultaneously. The volume of the 2-mL tubes allowed the use of up to eight 3-mm balls and up to six 5-mm balls. Firstly, the effect of the number of milling balls was investigated with a fixed milling period of 30 min. Figure 1 reveals that the 5-mm balls led to a significantly greater EtG extraction compared to the 3-mm balls. It is further obvious that the best results were obtained with two 5-mm balls. A further increase of balls led to decreasing EtG extraction. This observation was confirmed in an independent reproducibility experiment.
Int J Legal Med (2014) 128:69–72
Fig. 1 Optimization of number and size of the milling balls and presentation of the mean detectable EtG contents with standard deviations (n =2)
Secondly, the optimal milling period for the use of two 5-mm balls was evaluated. Figure 2 depicts the results obtained after extraction times between 5 and 60 min. Obviously, the variability of extractable EtG with extraction time is hardly significant and tends to reach an optimum at 30 min. The slightly higher values from 20 min onward may be due to the moderate temperature increase with extraction time. Thus, a period of 30 min and the use of two 5-mm balls were regarded as the optimal conditions for the determination of EtG in hair. Throughout the investigation, the closed tubes (maximum of ten) were always equally distributed between the two PTFEadaptors, with a capacity of ten conical plastic tubes each. That way, no leakage of the tube lids occurred. Verification of the combined milling and extraction procedure The described procedure needs to be tested regarding the equivalence with dry milling followed by extraction and regarding its superiority over manual cutting followed by extraction.
71
Therefore, EtG was quantified in ten samples each that were either manually cut or ground to a dry crude powder and then extracted with water for 2 days at room temperature [11] and in ten samples that were submitted to the combined pulverization/extraction method (two 5-mm balls, 30 min). The EtG contents observed in the respective extracts are shown in Table 1. The three data sets were normally distributed, free of outliers, trends, and were tested for significant differences between the measurements series (F-test) and the respective means (t-test). It is obvious that both measurement series involving milling resulted in higher amounts of detectable EtG than manual cutting in accordance with literature reports [12, 13]. It is also seen that there is no significant difference between dry milling, followed by extraction and the combination of milling and extraction.
Conclusion Micropulverization, the combined milling and extraction, was shown to be applicable for the quantification of EtG in hair and equivalent to dry grinding with consecutive extraction. This method displays excellent efficiency in terms of completeness and rapidity of extraction. As 20 samples can be Table 1 Ethyl glucuronide content in picograms per milligram extracted from hair using different options for particle size reduction Replicate extraction
Manual cutting
Dry milling
Combined milling and extraction (micropulverisation)
1 2 3 4 5 6 7 8
23.3 23.2 24.6 23.4 22.3 21.9 22.7 22.4
28.1 30.4 29.7 25.2 26.4 28.4 26.9 27.0
27.8 30.2 29.8 25.6 26.7 29.5 26.3 26.8
9 10 Average Standard deviation Difference from manual cutting
22.6 23.5 23.0 0.83
26.0 28.6 27.7 1.71 4.7 (20.4 %)
27.2 29.5 28.0 1.70 5.0 (21.7 %)
p-values F-test 9.96×10−8
t-test
Manual cutting–dry milling
Fig. 2 Optimization of the milling/extraction time, presentation of mean detectable EtG contents, and standard deviations (n =2)
Manual cutting–combined milling and extraction Dry milling–combined milling and extraction
2.08×10
1.97×10−6 1.12×10−6
0.721
0.721
−7
72
processed simultaneously, this method opens prospects for automatization and allows a high throughput of urgent samples.
Int J Legal Med (2014) 128:69–72
11.
12.
References 1. Morini L, Politi L, Polettini A (2009) Ethyl glucuronide in hair. A sensitive and specific marker of chronic heavy drinking. Addiction 104:915–920 2. Pragst F, Rothe M, Moench B, Hastedt M, Herre S, Simmert D (2010) Combined use of fatty acid ethyl esters and ethyl glucuronide in hair for diagnosis of alcohol abuse: interpretation and advantages. Forensic Sci Int 196:101–110 3. Agius R, Nadulski T, Kahl H-G, Dufax B (2012) Ethyl glucuronide in hair—a highly effective test for monitoring of alcohol consumption. Forensic Sci Int 218:10–14 4. Hastedt M, Herre S, Pragst F, Rothe M, Hartwig S (2012) Workplace alcohol testing program by combined use of ethyl glucuronide and fatty acid ethyl esters in hair. Alcohol Alcohol 47:127–132 5. Morini L, Politi L, Groppi A, Stramesi C, Polettini A (2006) Determination of ethyl glucuronide in hair samples by liquid chromatography/electrospray tandem mass spectrometry. J Mass Spectrom 41:34–42 6. Lamoureux F, Gaulier JM, Sauvage FL, Mercerolle M, Vallejo C, Lachâtre G (2009) Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction. Anal Bioanal Chem 394:1895–1901 7. Albermann ME, Musshoff F, Madea B (2010) A fully validated highperformance liquid chromatography-tandem mass spectrometry method for the determination of ethyl glucuronide in hair for the proof of strict alcohol abstinence. Anal Bioanal Chem 396:2441–2447 8. Kharbouche H, Sporkert F, Troxler S, Augsburger M, Mangin P, Staub C (2009) 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 877:2337–2343 9. Alvarez I, Bermejo AM, Tabernero MJ, Fernandez P, Cabarcos P, Lopez P (2009) Microwave-assisted extraction: a simpler and faster method for the determination of ethyl glucuronide in hair by gas chromatography– mass spectrometry. Anal Bioanal Chem 393:1345–1350 10. Shi Y, Shen B, Xiang P, Yan H, Shen M (2010) Determination of ethyl glucuronide in hair samples of Chinese people by protein
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