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Alcohol consumption or contamination: A preliminary study on the determination of the ethanol origin by stable carbon isotope analysis
Accepted Manuscript Title: Alcohol Consumption or Contamination: A Preliminary Study on the Determination of the Ethanol Origin by Stable Carbon Isotope Analysis Authors: Hang Chen, Baohua Shen, Sujing Zhang, Ping Xiang, Xianyi Zhuo, Min Shen PII: DOI: Reference:
S0379-0738(18)30333-5 https://doi.org/10.1016/j.forsciint.2018.06.013 FSI 9360
To appear in:
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Received date: Revised date: Accepted date:
9-1-2018 4-6-2018 10-6-2018
Please cite this article as: Hang Chen, Baohua Shen, Sujing Zhang, Ping Xiang, Xianyi Zhuo, Min Shen, Alcohol Consumption or Contamination: A Preliminary Study on the Determination of the Ethanol Origin by Stable Carbon Isotope Analysis, Forensic Science International https://doi.org/10.1016/j.forsciint.2018.06.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Alcohol Consumption or Contamination: A Preliminary Study on the Determination of the Ethanol Origin by Stable Carbon Isotope Analysis Hang Chen, Baohua Shen, Sujing Zhang, Ping Xiang, Xianyi Zhuo, Min Shen*1
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Academy of Forensic Science, P.R.China, Shanghai 200063, China
Highlights
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The stable isotope analysis technique was applied to the analysis of alcohol in body fluids in medico-legal cases. There was a significant difference in the carbon isotopic characteristics between edible alcohol and non-edible alcohol.
The 13C values of ethanol may be useful indicators to determine origin determination of alcohol in biological samples
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First author: Hang Chen: [email protected]
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1CONTACT: Min Shen: [email protected]
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Abstract
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The origin of ethanol detected in bio-samples whether it be from the consumption of alcoholic beverages or contamination with disinfectants has been questioned in court cases in China recently. The stable carbon isotope naturally occurs in carbon-containing compounds and can help determine the origin of the compound in question. In total, 42 types of beers and 11 types of disinfectants were analyzed by gas chromatography-isotope ratio mass spectrometry.
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Consumption and contamination experiments were carried out with 6 volunteers. The 13C values of ethanol ranged from -29.51 ‰ to -18.36 ‰ for the beer samples, which reflected the botanical
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features of C3 plants or mixtures of C3 and C4 plants. The 13C values of ethanol ranged from -17.7 ‰ to -14.4 ‰ for disinfectants, which reflected the different origins of ethanol in
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disinfectants from those in beer. The 13C value did not change in vivo after being consumed within the time limit used in this study. These characteristics of the 13C values will facilitate to interpret whether the ethanol detected in bio-samples originated from consumption or contamination.
Keywords 13C values; Ethanol; Origin; Consumption; Contamination; Isotope ratio mass spectrometry
1.Introduction The origin of ethanol may be the key piece of information to define an alcohol-related case. Ethanol content is the most commonly requested analysis in forensic toxicology laboratories [1], including in cases of violence, sexual assault, motor vehicle accidents and other alcohol-related 1 / 13
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cases [2, 3]. The single results of an ethanol content analysis have become increasingly likely to be questioned in court. Chinese media have recently reported many cases as follows: when lawyers were questioned, investigators could not exclude the possibility of contamination during sampling. Thus, the traditional evidence of alcohol consumption is rebutted by lawyers. Specific ethanol metabolites, such as ethyl glucuronide [4, 5] and ethyl sulfate [6], are used to reflect alcohol consumption. However, in recent cases, lawyers have pointed out that the detected metabolites may be derived from alcohol consumption a long time before the incident due to the longer half-lives of these metabolites [7, 8]. Novel markers such as iso-α-acids for alcohol consumption have been studied recently [9, 10]. Using the logical relationship between ethanol-related compounds and ethanol itself, lawyers are still able to find new arguments. The stable carbon isotope naturally occurs in carbon-containing compounds, and it is a new indicator of the origin of ethanol. The stable carbon isotope is expressed as a 13C value and is related to an international standard (Vienna Pee Dee Belemnite, VPDB); this value has been used
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to determine the geographical origin of wine [11-14]. In forensic studies, the feasibility of the 13C value is also being studied, such as in the analysis of explosives [15-17] and illegal drugs [18]. The aim of the present study was to explore the potential of stable carbon isotope analysis in the determination of ethanol origins for forensic purposes. As a preliminary study, we chose beer as a typical example of alcohol consumption because it represents the largest market in China [19]. At the same time, disinfectants are chosen as the typical example of alcohol contamination, which has been a controversial issue in recent years. According to the approval of the ethics committee,
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non-invasive samples (urine and oral fluids) were used in this study. The 13C values of ethanol in consumption bio-samples and in contamination bio-samples were compared.
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2. Material and methods 2.1. Reagents
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The n-alkane isotopic standard (Type A6) was purchased from Biogeochemical Laboratories, Indiana University (Bloomington, IN, USA). Wine ethanol certified reference material (BCR-660) was purchased from the Directorate F (health, consumer and reference materials), European Commission’s Joint Research Centre (Geel, Belgium). A series of ethanol standard solutions (0.1, 0.2, 0.5, 0.8, 1.0, 2.0, and 3.0 mg/mL) were prepared with pure ethanol (analytical reagent, Linfeng, SH, China) in water. Acetonitrile (HPLC, gradient grade) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Water (LC/MS grade) was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Tert-butanol was purchased from Chem Service (Westchester, PA, USA) and diluted to 0.04 mg/mL in water.
2.2. HS-GC-FID measurements
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Headspace-Gas Chromatography-Flame Ionization Detector (HS-GC-FID) analysis was performed following a previously described procedure [20] in which 0.1 mL of urine or oral fluid was placed directly into a capped and sealed vial with 0.5 mL of 0.04 mg/mL tert-butanol (as an internal standard). After vortexing the sample for 30 s, the vial was put on a 7697A headspace sampler (Agilent Technologies, Santa Clara, CA, USA). The equilibration time and temperature were 10 min and 65 °C, respectively. Headspace vapor was injected into a 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) and separated in double columns (DB-ALC1: 30 m × 0.32 mm i.d., 1.8 μm film thickness; DB-ALC2: 30 m × 0.32 mm i.d., 1.2 μm film thickness; Agilent Technologies, Santa Clara, CA, USA) at 40°C. Ethanol was detected with 2 / 13
double FIDs. The standard curve for ethanol was obtained by plotting the peak area ratio of ethanol to tert-butanol versus the standard ethanol series solution concentrations (0.1, 0.2, 0.5, 0.8, 1.0, 2.0 and 3.0 mg/mL). The correlation coefficient of the method was greater than 0.999, the precision and accuracy of the method were less than 5%, the detection limit was 0.01mg/mL, and the limit of quantification was 0.1 mg/mL.
2.3. GC-IRMS measurements
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Before Gas Chromatography–Stable Isotope Ratio Mass Spectrometry (GC-IRMS) analysis, samples were prepared by two different methods according to the ethanol content to remove the interference of endogenous substances, such as protein in the sample. Beer and disinfectant samples with high ethanol contents were diluted with acetonitrile to an ethanol concentration of approximately 1.0 mg/mL. The urine and oral fluid samples with low ethanol contents were centrifugally separated at 12000 rpm for 5 min, and then the supernatants were filtered by 0.22-mm nylon membranes. The GC-IRMS analysis was performed using a Trace GC Ultra gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) connected to an MAT 253 isotope ratio mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with a GC Isolink combustion reactor interface (Thermo Fisher Scientific, Waltham, MA, USA). An HPINNOWAX column (30 m × 0.32 mm i.d., 0.25-μm film thickness, Agilent Technologies, Santa Clara, CA, USA) was used for chromatographic separation. The injection temperature was 200 °C, the injection volume was 2 μL, and the post-injection dwell time was 5 s. The oven was programmed as follows: an initial temperature of 45 °C for 3 min, which was increased at a rate of 1 °C/min to 52 °C, then increased at a rate of 20 °C/min to 200 °C and kept at the final temperature for 2 min. Helium was used as the carrier with an injection rate of 1 mL/min. The combustion interface temperature was set to 1020 °C. The CO2 reference gas pressure was 2 bars, and the gas was introduced into the IRMS at 30 s, 850 s and 900 s for 20 s each time. The back flush was switched off at 90 s and switched on at 800 s.
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The 13C values were expressed against VPDB standard and were calculated using the formula (13C = [(13C/12C)sample / (13C/12C)VPDB] - 1). This calculation was performed using CO2 as a reference gas that was calibrated by an n-alkane isotopic standard (Type A6). BCR-660 was analyzed at the beginning of each sequence to test the accuracy of the instrument. Isodat 3.0 (Thermo Fisher Scientific, Bremen, Germany) software was used for data acquisition and isotope value calculation. SPSS 19.0 (IBM, USA) was used for statistical calculations.
2.4. Validation of the GC-IRMS method
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Selectivity was measured by pure ethanol, diluent (acetonitrile), wort (a simulated beer matrix), blank urine and blank oral fluid. The linear range was measured by a series of ethanol standard solutions (0.1, 0.2, 0.5, 0.8, 1.0, 2.0, and 3.0 mg/mL). The method’s precision (represented by the standard deviation) and accuracy (represented by bias) were measured together on 7 different days using BCR-660.
2.5. Samples Beer samples (Table 1) and alcohol-containing disinfectant samples were purchased from supermarkets or online shops. Two types of wort were purchased from supermarkets.
2.6. Consumption and contamination experiments Six volunteers (3 males and 3 females, ages 21~49 years), who provided oral informed 3 / 13
consent, were involved in the experiments. Urine samples (20 mL) were collected in a plastic collector at each sampling point. To remove the residual beer in the mouth after consumption, all volunteers gargled with 100 mL of drinking water after beer consumption. Then, oral fluid samples (5 mL) were collected in a clean corning tube at each sampling point. All bio-samples were analyzed by HS-GC-MS and GC-IRMS within 6 h after being collected.
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Consumption experiment 1 Before the experiment, urine and oral fluid samples were collected from all 6 volunteers for ethanol-negative confirmation. The pasteurized Lager beer imports from Denmark (No. 40 in this study, with a wort concentration of 10.5 °P, and fermented by barley) from the same barrel (net volume: 2000 mL) were consumed by all the volunteers (each volunteer consumed approximately 330 mL). Urine and oral fluid samples were collected 1 h later.
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Consumption experiment 2 Before the experiment, urine and oral fluid samples were collected from volunteer #1 for ethanol-negative confirmation. The pasteurized Lager beer imports from Germany (No. 41 in this study, with a wort concentration of 17.0 °P fermented by barley and with a net volume of 500 mL) were consumed by volunteer #1. Urine and oral fluid samples were collected at 1 h, 2 h, 3 h, 4 h, and 6 h after consumption. After 2 days, urine and oral fluid samples were collected again from volunteer #1 for ethanol-negative confirmation. Then, the pasteurized ale beer imports from Germany (No. 42 in this study with a wort concentration of 11.2 °P fermented by barley and cane sugar and with a net volume of 500 mL) were consumed as described previously. Similarly, urine and oral fluid samples were collected at 1 h, 2 h, 3 h, 4 h, and 6 h after consumption.
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Contamination experiment The ethanol-negative urine and oral fluid samples were used as blank samples. Then, 20 μL of alcohol-containing disinfectant was added to 10 mL of the blank sample one by one to prepare the contaminated samples.
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3. Results and discussion 3.1. Method validation
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The selectivity of the GC-IRMS method was assessed by absolute ethanol, diluent (acetonitrile), wort, blank urine and blank oral fluid. The retention time of ethanol was obtained via absolute ethanol analysis with a small sample volume (0.2 μL) and a high split ratio (200:1). Figure 1A shows a typical GC-IRMS chromatogram of ethanol. There were no interfering peaks in the retention window of ethanol (Figures 1B, 1C, 1D and 1E). Figures 1F, 1G and 1H show typical GC-IRMS chromatograms of a beer sample, urine sample and oral fluid sample, respectively. In a previous study by Ai, acetonitrile was used instead of water as the solvent for the δ13C analysis of ethanol in alcoholic beverages by GC/IRMS [21]. In this study, we used acetonitrile as the diluent because it can eliminate protein in beer and avoid the blocking of chromatographic columns. Figure 2 contains a plot of the mean intensities for the m/z 44 peak amplitude (mV) at ethanol concentrations from 0.1 to 3.0 mg/mL and a plot of 13C values from ethanol at 4 / 13
concentrations from 0.1 to 3.0 mg/mL. As an integrated result, accurate 13C values could be obtained when the sample signal for the m/z 44 peak was near 1000~10000 mV. Therefore, it was better to control the concentration of ethanol in the sample to be within 0.5 to 2.0 mg/mL. Because of this limitation, we have adopted different pretreatment methods for two kinds of samples with different ethanol concentrations.
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The observed 13C values of ethanol in BCR-660 over a 7-day period were -27.2 ‰ to -26.7 ‰. The nominal 13C value of ethanol in BCR-660 was -26.7 ‰. The standard deviation (precision) observed over 7 days was 0.2 ‰, and the biases (accuracy) of the observed date from the certified date were less than 1.8 %. To avoid the isotopic fractionation caused by heating during the headspace process, we selected another processing method different from GC-FID analysis. The verification data showed that direct dilution and filtration led to good precision and accuracy.
3.2. Reference 13C values of ethanol in beer
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First,10 bottles of No. 1 beer and 10 cans of No. 2 beer with different production dates were
analyzed to determine whether the different production dates (batches) affect the 13C value of ethanol in beer. According to the standard deviation test, the standard deviation was less than
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0.5 ‰. Production dates do not affect the 13C value. Next, 13C values of ethanol in 42 types of beer were analyzed (Table 1). Similar to the research by Brooks [22], the distribution of the 13C value of ethanol in beer was bimodal, with one peak ranging from -29.5 ‰ to -25.4 ‰ and the other ranging from -20.1 ‰ to -18.4 ‰. The dates grouped by the fermentative method showed significant differences (P< 0.01) according to the t-tests. In fact, this difference was related to the botanical origin (Figure 3). All lager beer samples analyzed in this study used C3 ingredients, such as barley, wheat and rice. The ale beer samples analyzed in this study had a higher isotopic ratio because C4 plants (such as cane and corn) were mixed in the ingredients. Due to the different carbon dioxide absorption pathways, the
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13C values of C3 plants ranged from approximately -34 ‰ to -22 ‰, while the 13C values of C4 plants ranged from -17 ‰ to -11 ‰ [23]. Similar to Brooks’ results [22], the median 13C value of ethanol in beer samples with all C3 ingredients was -26.9 ‰. After adding C4 plants as ingredients,
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the median 13C values of ethanol increased to -18.6 ‰. Further, the distribution of 13C values of the beer samples with C3 ingredients was bimodal. The 13C values of ethanol in unpasteurized beer samples ranged from -27.0 ‰ to -25.4 ‰ and ranged from -29.5 ‰ to -26.8 ‰ for pasteurized beers. This difference may have originated from the temperature difference during the process, as pasteurized beer is heated and disinfected at approximately 60°C [24]. Different producers or wort concentrations did not significantly affect the 13C values of ethanol in beer with C3 ingredients (P>0.05).
3.3. Reference 13C values of ethanol in disinfectants
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The 13C values of ethanol in 11 types of alcohol-containing disinfectants ranged from -17.7 ‰ to -14.4 ‰ (Table 2). In China, commercialized ethanol is mainly derived from corn, a typical C4 plant [25, 26]. In this study, 11 types of best-selling alcohol-containing disinfectants from Chinese online shops were bought as samples. As expected, all of these disinfectants had higher 13C values than those in beer samples.
3.4. The difference between the 13C values in consumption and contamination samples In consumption experiment 1, 6 volunteers consumed No. 40 beer (13C = -28.0 ‰) from the 5 / 13
same barrel, and urine and oral fluids were collected 1 h later and analyzed. The 13C values of ethanol ranged from -27.3 ‰ to -26.7 ‰ in urine and from -27.3 ‰ to -26.3 ‰ in oral fluids (Table 3). The 13C values of ethanol in all bio-samples were compared with the No. 40 beer. The differences were less than 1.3 ‰ for urine and less than 1.7 ‰ for oral fluid. In consumption experiment 2, volunteer #1 consumed No. 41 beer (13C = -27.9 ‰) and No. 42 beer (13C = -18.4 ‰) on two different days. Urine and oral fluids were collected at 0 (before beer consumed), 1, 2, 3, 4, and 6 h after consumption. The 13C values of ethanol ranged from -27.9 ‰ to -27.5 ‰ in urine and ranged from -27.7 ‰to -27.3 ‰ in oral fluids after consuming No. 41 beer (Table 4). The 13C values of ethanol in urine and oral fluids after consuming No. 42 beer ranged from -18.2 ‰ to -18.0 ‰ and from -18.8 ‰ to -18.2 ‰, respectively (Table 5).
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Similar to Experiment 1, differences in the 13C values between the bio-samples and beer were calculated one by one. The differences were less than 0.6 ‰. In the contamination experiment, contaminated urine and oral fluid samples were analyzed
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(Table 2). The 13C values of ethanol ranged from -17.8 ‰ to -14.2 ‰ in urine and from -17.9 ‰ to -14.5 ‰ in oral fluids after being contaminated. The differences in 13C values between contaminated bio-samples and disinfectants were less than 0.4 ‰.
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The results of these experiments show that the isotopic characteristics (13C value) of ethanol can be transferred to the bio-samples, regardless of consumption or contamination. The 13C value of ethanol with a pure C3 origin in urine and oral fluid was lower than -25.0 ‰, and the 13C value of ethanol with a pure C4 origin in urine and oral fluid was higher than -18.0 ‰ (Figure 3). This obvious data difference shows the potential of stable carbon isotope analysis to determine the origin of ethanol in vivo. Because most alcoholic beverages are from C3 plants [27] and most non-edible alcohol is produced from C4 plants [28], stable carbon isotope analysis can provide data on consumption and contamination. However, some alcoholic beverages are brewed with C4 plants (such as vodka, bourbon whisky and rum), and the production of ethanol from residues of C3 plants (such as wheat [29]) has also become a recent research direction. The origin of ethanol is not clear when it comes from a mixture of C3 and C4 plants. The inadequate differentiation of the ethanol origin by 13C values may require stable oxygen isotope values and stable hydrogen isotope values.
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3.5. Analysis of a sample in an authentic case
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In a traffic accident, the blood alcohol content of the driver was detected to be 0.82 mg/mL. According to Chinese law, an individual with a blood alcohol content greater than 0.80 mg/mL should be prosecuted for criminal liabilities. The driver argued that he did not drink alcohol before the accident and that his blood was contaminated by alcohol swabs when drawing the blood. He said that he saw the nurse use a tincture for skin disinfection. The police investigated the hospital where the blood draws were performed. They found that the hospital purchased two kinds of disinfectants, a tincture (red bottle) and an alcohol-free sterile swab (blue bottle). In the absence of more information, the driver was going to be acquitted. The driver's urine was analyzed, and the 13C value of ethanol in the urine was -28.4 ‰. This value indicates that the origin of ethanol detected in the urine originated from C3 plants. For further confirmation, we analyzed the tincture provided by the hospital. The 13C value of ethanol in the tincture was -15.0 ‰, which was obviously different from the ethanol present in the urine of the driver. We speculate that the driver had consumed alcohol. According to our analysis, the police believed that the driver was suspected of lying. By replaying the surveillance video, the police found two important points. First, the hospital used a 6 / 13
sterile swab from a blue bottle when drawing blood from the driver. Second, half an hour before the traffic accident, the driver exited a restaurant. By comparing the driver's credit card records and restaurant bill records, it was proven that the driver consumed 2 bottles of beer in the restaurant before the accident. According to the police investigation, the type of beer consumed by the driver was the same as the No. 19 beer sample (13C = -28.4 ‰) in this study. This finding was consistent with our analytical data (the 13C value of ethanol in the urine was also -28.4 ‰). In a comprehensive survey, the police uncovered the truth and forced the driver to assume the criminal responsibility he deserved.
4. Conclusion
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The 13C values of ethanol in beer and disinfectant samples were measured. The stable carbon isotope characteristics of beer have the botanical features of C3 plants or a mixture of C3 and C4 plants. The isotopic characteristics of commercial alcohol are relatively simple, only showing the botanical features of C4 plants. These characteristics were transferred to bio-samples without changes in consumption and contamination experiments. Through the application of a case, the potential and value of stable isotope analysis technology in forensic analysis was explained. More studies need to be conducted on how to describe the origin of mixed botanical ethanol.
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Funding
Acknowledgment
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This study was sponsored by Shanghai Sailing Program (17YF1420100) and the supporting from Academy of Forensic Science (GY2016Z-1).
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This study thanks for 13th National Key R&D Program of China (2016YFC0800704), Shanghai Key Laboratory of Forensic Medicine (17DZ2273200) and Shanghai Forensic Service Platform (16DZ2290900).
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Figure 1. GC-IRMS chromatograms (m/z 44 ion current as a function of time) for 13C analysis of pure
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ethanol (A), acetonitrile (B), wort (C), blank urine (D), blank oral fluid (E), beer sample (F), urine
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sample (G) and oral fluid sample (H).
Figure 2. (A) Plot of the linear range of the experiments of the mean intensity utilizing ethanol. (B) Plot of the linear range of the experiments of the mean 13C values utilizing ethanol.
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Figure 3. Plot of the 13C values of ethanol from different origins and the relationship of 13C with
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botanical origins.
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Table. 1 Reference 13C values of ethanol [‰] obtained from the studied beer samples and its basic information Sample Producer
Fermentative
Wort
Botanical
Modes
Concentration (°P)
Origin
Un-pasteurized
Lager
9.0
Barley
-26.7±0.5
China
2**
China
Pasteurized
Lager
8.0
Barley, Rice
-28.2±0.2
3
China
Un-pasteurized
Lager
9.0
Barley
-26.6
4
China
Un-pasteurized
Lager
8.0
Barley
-26.4
5
China
Un-pasteurized
Lager
8.0
Barley
-26.3
6
China
Un-pasteurized
Lager
8.0
Barley
-26.5
7
China
Un-pasteurized
Lager
9.7
Barley
-25.9
8
China
Un-pasteurized
Lager
8.0
Barley
9
China
Un-pasteurized
Lager
11.0
Barley, Wheat
10
China
Un-pasteurized
Lager
11.0
Barley, Rice
11
China
Un-pasteurized
Lager
9.0
12
China
Un-pasteurized
Lager
9.0
13
China
Un-pasteurized
Lager
8.0
14
China
Un-pasteurized
Lager
8.0
15
China
Un-pasteurized
Lager
8.0
16
China
Un-pasteurized
Lager
8.0
17
China
Un-pasteurized
Lager
13.5
18
China
Pasteurized
Lager
8.0
19
China
Pasteurized
Lager
20
China
Pasteurized
Lager
21
China
Pasteurized
Lager Lager Lager
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Barley, Rice
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1**
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No. *
-26.2 -26.6 -26.4 -26.2 -26.4
Barley, Rice
-27.0
Wheat, Rice
-26.3
Barley, Wheat
-26.5
Barley, Wheat
-25.4
Barley, Wheat
-26.6
Barley, Rice
-28.5
Barley, Rice
-28.4
11.4
Barley
-27.8
9.0
Barley, Rice
-28.3
9.0
Barley, Rice
-28.8
16.3
Barley
-26.2
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Barley, Wheat
9.0
China
Pasteurized
23
Germany
Un-pasteurized
24
Germany
Un-pasteurized
Lager
12.4
Wheat
-26.1
25
Germany
Pasteurized
Lager
11.5
Barley
-28.2
26
Germany
Pasteurized
Lager
12.2
Barley
-27.5
27
Germany
Pasteurized
Lager
12.4
Wheat
-27.2
28
Germany
Pasteurized
Lager
12.5
Barley
-27.8
29
Germany
Pasteurized
Lager
12.4
Barley
-26.8
30
America
Pasteurized
Ale
11.8
Barley, Cane sugar
-20.1
31
America
Pasteurized
Ale
8.1
Barley, Corn sugar
-19.8
32
America
Pasteurized
Lager
10.3
Barley, Rice
-29.5
33
America
Pasteurized
Lager
9.7
Wheat
-27.2
34
Belgium
Pasteurized
Ale
11.7
Barley, Wheat, Cane sugar
-18.8
35
Belgium
Pasteurized
Lager
15.5
Barley, Wheat
-28.3
36
Belgium
Pasteurized
Lager
11.7
Barley, Wheat
-28.7
37
Britain
Pasteurized
Ale
10.5
Barley, Rice, Cane sugar
-18.4
38
Denmark
Pasteurized
Ale
10.5
Barley, Corn
-18.5
39
Thailand
Pasteurized
Lager
12.3
Barley, Wheat
-28.1
40
Denmark
Pasteurized
Lager
10.5
Barley
-28.0
41
Germany
Pasteurized
Lager
17.0
Barley
-27.9
42
Germany
Pasteurized
Ale
11.2
Barley, Cane sugar
-18.4
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13C (‰)
Processing
* Each beer was purchased 1 bottle/tin as a random test sample in addition to samples No. 1 and 2. ** For No. 1 and No. 2 beer samples, 10 bottles/tins with different production dates were purchased from different supermarkets (or online stores). 12 / 13
Table. 2 Reference 13C values of ethanol in disinfectants and contaminated bio-samples Sample No. 43 44 45 46 47 48 49 50 51 52 53
13C
13C
(‰)
-16.6 -16.9 -17.7 -15.7 -16.8 -14.4 -16.8 -16.8 -16.6 -17.5 -15.8
Urine Difference with disinfectants (‰) 0.0 0.1 0.1 0.2 0.1 0.2 0.1 0.1 0.4 0.1 0.1
(‰)
-16.6 -17.1 -17.8 -15.6 -16.6 -14.2 -16.7 -16.7 -17.0 -17.4 -15.9
13C
Oral fluid Difference with disinfectants (‰) 0.3 0.1 0.2 0.3 0.0 0.1 0.2 0.0 0.4 0.2 0.4
(‰)
-17.0 -16.8 -17.9 -15.5 -16.8 -14.5 -16.5 -16.8 -16.2 -17.3 -15.4
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Disinfectants
Table. 3 Reference 13C values of ethanol in urine and oral fluid after the consumption of No. 40 beer Urine 13C (‰)
0.5 0.5 0.6 0.6 0.5 0.7
-27.0 -26.7 -27.2 -27.3 -27.3 -27.3
1# 2# 3# 4# 5# 6#
Conc.*(mg/mL)
13C (‰)
0.5 0.5 0.4 0.5 0.5 0.5
-26.9 -26.3 -27.0 -26.5 -27.3 -26.9
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* Concentrations of ethanol in corresponding bio-samples
Difference from No. 40 beer (‰) 1.1 1.7 0.9 1.4 0.7 1.1
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Conc.*(mg/mL)
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Volunteer
Oral fluid Difference from No. 40 beer (‰) 1.0 1.3 0.8 0.7 0.6 0.7
Table. 4 Reference 13C values of ethanol in urine and oral fluid after the consumption of No. 41 beer
-*** -27.5 -27.9 -
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0.0 0.9 0.5 0.4 0.2 0.1
(‰)
Difference from No. 41 beer (‰) 0.4 0.0
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0h** 1h 2h 3h 4h 6h
Conc.*(mg/mL)
13C
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Urine Time
Conc.(mg/mL)
Oral fluid 13C
0.0 0.8 0.6 0.4 0.2 0.1
(‰)
-27.3 -27.7 -
Difference from No. 41 beer (‰) 0.6 0.2
* Concentrations of ethanol in corresponding bio-samples ** Before beer consumed
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*** Lower than linear range of method
Table. 5 Reference 13C values of ethanol in urine and oral fluid after the consumption of No. 42 beer
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Urine
Time
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0h** 1h 2h 3h 4h 6h
Conc.*(mg/mL) 0.0 1.0 0.8 0.6 0.4 0.3
13C
(‰)
-*** -18.0 -18.1 -18.2 -
Oral fluid Difference from No. 42 beer (‰) 0.3 0.3 0.1
* Concentrations of ethanol in corresponding bio-samples ** Before beer was consumed *** Lower than the linear range of the method
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Conc.(mg/mL)
13C (‰)
0.0 0.9 0.7 0.6 0.4 0.2
-18.2 -18.2 -18.8 -
Difference from No. 42 beer (‰) 0.2 0.2 0.5
S0379-0738(18)30333-5 https://doi.org/10.1016/j.forsciint.2018.06.013 FSI 9360
To appear in:
FSI
Received date: Revised date: Accepted date:
9-1-2018 4-6-2018 10-6-2018
Please cite this article as: Hang Chen, Baohua Shen, Sujing Zhang, Ping Xiang, Xianyi Zhuo, Min Shen, Alcohol Consumption or Contamination: A Preliminary Study on the Determination of the Ethanol Origin by Stable Carbon Isotope Analysis, Forensic Science International https://doi.org/10.1016/j.forsciint.2018.06.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Alcohol Consumption or Contamination: A Preliminary Study on the Determination of the Ethanol Origin by Stable Carbon Isotope Analysis Hang Chen, Baohua Shen, Sujing Zhang, Ping Xiang, Xianyi Zhuo, Min Shen*1
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Academy of Forensic Science, P.R.China, Shanghai 200063, China
Highlights
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The stable isotope analysis technique was applied to the analysis of alcohol in body fluids in medico-legal cases. There was a significant difference in the carbon isotopic characteristics between edible alcohol and non-edible alcohol.
The 13C values of ethanol may be useful indicators to determine origin determination of alcohol in biological samples
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First author: Hang Chen: [email protected]
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1CONTACT: Min Shen: [email protected]
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Abstract
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The origin of ethanol detected in bio-samples whether it be from the consumption of alcoholic beverages or contamination with disinfectants has been questioned in court cases in China recently. The stable carbon isotope naturally occurs in carbon-containing compounds and can help determine the origin of the compound in question. In total, 42 types of beers and 11 types of disinfectants were analyzed by gas chromatography-isotope ratio mass spectrometry.
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Consumption and contamination experiments were carried out with 6 volunteers. The 13C values of ethanol ranged from -29.51 ‰ to -18.36 ‰ for the beer samples, which reflected the botanical
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features of C3 plants or mixtures of C3 and C4 plants. The 13C values of ethanol ranged from -17.7 ‰ to -14.4 ‰ for disinfectants, which reflected the different origins of ethanol in
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disinfectants from those in beer. The 13C value did not change in vivo after being consumed within the time limit used in this study. These characteristics of the 13C values will facilitate to interpret whether the ethanol detected in bio-samples originated from consumption or contamination.
Keywords 13C values; Ethanol; Origin; Consumption; Contamination; Isotope ratio mass spectrometry
1.Introduction The origin of ethanol may be the key piece of information to define an alcohol-related case. Ethanol content is the most commonly requested analysis in forensic toxicology laboratories [1], including in cases of violence, sexual assault, motor vehicle accidents and other alcohol-related 1 / 13
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cases [2, 3]. The single results of an ethanol content analysis have become increasingly likely to be questioned in court. Chinese media have recently reported many cases as follows: when lawyers were questioned, investigators could not exclude the possibility of contamination during sampling. Thus, the traditional evidence of alcohol consumption is rebutted by lawyers. Specific ethanol metabolites, such as ethyl glucuronide [4, 5] and ethyl sulfate [6], are used to reflect alcohol consumption. However, in recent cases, lawyers have pointed out that the detected metabolites may be derived from alcohol consumption a long time before the incident due to the longer half-lives of these metabolites [7, 8]. Novel markers such as iso-α-acids for alcohol consumption have been studied recently [9, 10]. Using the logical relationship between ethanol-related compounds and ethanol itself, lawyers are still able to find new arguments. The stable carbon isotope naturally occurs in carbon-containing compounds, and it is a new indicator of the origin of ethanol. The stable carbon isotope is expressed as a 13C value and is related to an international standard (Vienna Pee Dee Belemnite, VPDB); this value has been used
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to determine the geographical origin of wine [11-14]. In forensic studies, the feasibility of the 13C value is also being studied, such as in the analysis of explosives [15-17] and illegal drugs [18]. The aim of the present study was to explore the potential of stable carbon isotope analysis in the determination of ethanol origins for forensic purposes. As a preliminary study, we chose beer as a typical example of alcohol consumption because it represents the largest market in China [19]. At the same time, disinfectants are chosen as the typical example of alcohol contamination, which has been a controversial issue in recent years. According to the approval of the ethics committee,
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non-invasive samples (urine and oral fluids) were used in this study. The 13C values of ethanol in consumption bio-samples and in contamination bio-samples were compared.
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2. Material and methods 2.1. Reagents
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The n-alkane isotopic standard (Type A6) was purchased from Biogeochemical Laboratories, Indiana University (Bloomington, IN, USA). Wine ethanol certified reference material (BCR-660) was purchased from the Directorate F (health, consumer and reference materials), European Commission’s Joint Research Centre (Geel, Belgium). A series of ethanol standard solutions (0.1, 0.2, 0.5, 0.8, 1.0, 2.0, and 3.0 mg/mL) were prepared with pure ethanol (analytical reagent, Linfeng, SH, China) in water. Acetonitrile (HPLC, gradient grade) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Water (LC/MS grade) was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Tert-butanol was purchased from Chem Service (Westchester, PA, USA) and diluted to 0.04 mg/mL in water.
2.2. HS-GC-FID measurements
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Headspace-Gas Chromatography-Flame Ionization Detector (HS-GC-FID) analysis was performed following a previously described procedure [20] in which 0.1 mL of urine or oral fluid was placed directly into a capped and sealed vial with 0.5 mL of 0.04 mg/mL tert-butanol (as an internal standard). After vortexing the sample for 30 s, the vial was put on a 7697A headspace sampler (Agilent Technologies, Santa Clara, CA, USA). The equilibration time and temperature were 10 min and 65 °C, respectively. Headspace vapor was injected into a 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) and separated in double columns (DB-ALC1: 30 m × 0.32 mm i.d., 1.8 μm film thickness; DB-ALC2: 30 m × 0.32 mm i.d., 1.2 μm film thickness; Agilent Technologies, Santa Clara, CA, USA) at 40°C. Ethanol was detected with 2 / 13
double FIDs. The standard curve for ethanol was obtained by plotting the peak area ratio of ethanol to tert-butanol versus the standard ethanol series solution concentrations (0.1, 0.2, 0.5, 0.8, 1.0, 2.0 and 3.0 mg/mL). The correlation coefficient of the method was greater than 0.999, the precision and accuracy of the method were less than 5%, the detection limit was 0.01mg/mL, and the limit of quantification was 0.1 mg/mL.
2.3. GC-IRMS measurements
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Before Gas Chromatography–Stable Isotope Ratio Mass Spectrometry (GC-IRMS) analysis, samples were prepared by two different methods according to the ethanol content to remove the interference of endogenous substances, such as protein in the sample. Beer and disinfectant samples with high ethanol contents were diluted with acetonitrile to an ethanol concentration of approximately 1.0 mg/mL. The urine and oral fluid samples with low ethanol contents were centrifugally separated at 12000 rpm for 5 min, and then the supernatants were filtered by 0.22-mm nylon membranes. The GC-IRMS analysis was performed using a Trace GC Ultra gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) connected to an MAT 253 isotope ratio mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with a GC Isolink combustion reactor interface (Thermo Fisher Scientific, Waltham, MA, USA). An HPINNOWAX column (30 m × 0.32 mm i.d., 0.25-μm film thickness, Agilent Technologies, Santa Clara, CA, USA) was used for chromatographic separation. The injection temperature was 200 °C, the injection volume was 2 μL, and the post-injection dwell time was 5 s. The oven was programmed as follows: an initial temperature of 45 °C for 3 min, which was increased at a rate of 1 °C/min to 52 °C, then increased at a rate of 20 °C/min to 200 °C and kept at the final temperature for 2 min. Helium was used as the carrier with an injection rate of 1 mL/min. The combustion interface temperature was set to 1020 °C. The CO2 reference gas pressure was 2 bars, and the gas was introduced into the IRMS at 30 s, 850 s and 900 s for 20 s each time. The back flush was switched off at 90 s and switched on at 800 s.
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The 13C values were expressed against VPDB standard and were calculated using the formula (13C = [(13C/12C)sample / (13C/12C)VPDB] - 1). This calculation was performed using CO2 as a reference gas that was calibrated by an n-alkane isotopic standard (Type A6). BCR-660 was analyzed at the beginning of each sequence to test the accuracy of the instrument. Isodat 3.0 (Thermo Fisher Scientific, Bremen, Germany) software was used for data acquisition and isotope value calculation. SPSS 19.0 (IBM, USA) was used for statistical calculations.
2.4. Validation of the GC-IRMS method
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Selectivity was measured by pure ethanol, diluent (acetonitrile), wort (a simulated beer matrix), blank urine and blank oral fluid. The linear range was measured by a series of ethanol standard solutions (0.1, 0.2, 0.5, 0.8, 1.0, 2.0, and 3.0 mg/mL). The method’s precision (represented by the standard deviation) and accuracy (represented by bias) were measured together on 7 different days using BCR-660.
2.5. Samples Beer samples (Table 1) and alcohol-containing disinfectant samples were purchased from supermarkets or online shops. Two types of wort were purchased from supermarkets.
2.6. Consumption and contamination experiments Six volunteers (3 males and 3 females, ages 21~49 years), who provided oral informed 3 / 13
consent, were involved in the experiments. Urine samples (20 mL) were collected in a plastic collector at each sampling point. To remove the residual beer in the mouth after consumption, all volunteers gargled with 100 mL of drinking water after beer consumption. Then, oral fluid samples (5 mL) were collected in a clean corning tube at each sampling point. All bio-samples were analyzed by HS-GC-MS and GC-IRMS within 6 h after being collected.
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Consumption experiment 1 Before the experiment, urine and oral fluid samples were collected from all 6 volunteers for ethanol-negative confirmation. The pasteurized Lager beer imports from Denmark (No. 40 in this study, with a wort concentration of 10.5 °P, and fermented by barley) from the same barrel (net volume: 2000 mL) were consumed by all the volunteers (each volunteer consumed approximately 330 mL). Urine and oral fluid samples were collected 1 h later.
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Consumption experiment 2 Before the experiment, urine and oral fluid samples were collected from volunteer #1 for ethanol-negative confirmation. The pasteurized Lager beer imports from Germany (No. 41 in this study, with a wort concentration of 17.0 °P fermented by barley and with a net volume of 500 mL) were consumed by volunteer #1. Urine and oral fluid samples were collected at 1 h, 2 h, 3 h, 4 h, and 6 h after consumption. After 2 days, urine and oral fluid samples were collected again from volunteer #1 for ethanol-negative confirmation. Then, the pasteurized ale beer imports from Germany (No. 42 in this study with a wort concentration of 11.2 °P fermented by barley and cane sugar and with a net volume of 500 mL) were consumed as described previously. Similarly, urine and oral fluid samples were collected at 1 h, 2 h, 3 h, 4 h, and 6 h after consumption.
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Contamination experiment The ethanol-negative urine and oral fluid samples were used as blank samples. Then, 20 μL of alcohol-containing disinfectant was added to 10 mL of the blank sample one by one to prepare the contaminated samples.
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3. Results and discussion 3.1. Method validation
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The selectivity of the GC-IRMS method was assessed by absolute ethanol, diluent (acetonitrile), wort, blank urine and blank oral fluid. The retention time of ethanol was obtained via absolute ethanol analysis with a small sample volume (0.2 μL) and a high split ratio (200:1). Figure 1A shows a typical GC-IRMS chromatogram of ethanol. There were no interfering peaks in the retention window of ethanol (Figures 1B, 1C, 1D and 1E). Figures 1F, 1G and 1H show typical GC-IRMS chromatograms of a beer sample, urine sample and oral fluid sample, respectively. In a previous study by Ai, acetonitrile was used instead of water as the solvent for the δ13C analysis of ethanol in alcoholic beverages by GC/IRMS [21]. In this study, we used acetonitrile as the diluent because it can eliminate protein in beer and avoid the blocking of chromatographic columns. Figure 2 contains a plot of the mean intensities for the m/z 44 peak amplitude (mV) at ethanol concentrations from 0.1 to 3.0 mg/mL and a plot of 13C values from ethanol at 4 / 13
concentrations from 0.1 to 3.0 mg/mL. As an integrated result, accurate 13C values could be obtained when the sample signal for the m/z 44 peak was near 1000~10000 mV. Therefore, it was better to control the concentration of ethanol in the sample to be within 0.5 to 2.0 mg/mL. Because of this limitation, we have adopted different pretreatment methods for two kinds of samples with different ethanol concentrations.
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The observed 13C values of ethanol in BCR-660 over a 7-day period were -27.2 ‰ to -26.7 ‰. The nominal 13C value of ethanol in BCR-660 was -26.7 ‰. The standard deviation (precision) observed over 7 days was 0.2 ‰, and the biases (accuracy) of the observed date from the certified date were less than 1.8 %. To avoid the isotopic fractionation caused by heating during the headspace process, we selected another processing method different from GC-FID analysis. The verification data showed that direct dilution and filtration led to good precision and accuracy.
3.2. Reference 13C values of ethanol in beer
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First,10 bottles of No. 1 beer and 10 cans of No. 2 beer with different production dates were
analyzed to determine whether the different production dates (batches) affect the 13C value of ethanol in beer. According to the standard deviation test, the standard deviation was less than
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0.5 ‰. Production dates do not affect the 13C value. Next, 13C values of ethanol in 42 types of beer were analyzed (Table 1). Similar to the research by Brooks [22], the distribution of the 13C value of ethanol in beer was bimodal, with one peak ranging from -29.5 ‰ to -25.4 ‰ and the other ranging from -20.1 ‰ to -18.4 ‰. The dates grouped by the fermentative method showed significant differences (P< 0.01) according to the t-tests. In fact, this difference was related to the botanical origin (Figure 3). All lager beer samples analyzed in this study used C3 ingredients, such as barley, wheat and rice. The ale beer samples analyzed in this study had a higher isotopic ratio because C4 plants (such as cane and corn) were mixed in the ingredients. Due to the different carbon dioxide absorption pathways, the
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13C values of C3 plants ranged from approximately -34 ‰ to -22 ‰, while the 13C values of C4 plants ranged from -17 ‰ to -11 ‰ [23]. Similar to Brooks’ results [22], the median 13C value of ethanol in beer samples with all C3 ingredients was -26.9 ‰. After adding C4 plants as ingredients,
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the median 13C values of ethanol increased to -18.6 ‰. Further, the distribution of 13C values of the beer samples with C3 ingredients was bimodal. The 13C values of ethanol in unpasteurized beer samples ranged from -27.0 ‰ to -25.4 ‰ and ranged from -29.5 ‰ to -26.8 ‰ for pasteurized beers. This difference may have originated from the temperature difference during the process, as pasteurized beer is heated and disinfected at approximately 60°C [24]. Different producers or wort concentrations did not significantly affect the 13C values of ethanol in beer with C3 ingredients (P>0.05).
3.3. Reference 13C values of ethanol in disinfectants
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The 13C values of ethanol in 11 types of alcohol-containing disinfectants ranged from -17.7 ‰ to -14.4 ‰ (Table 2). In China, commercialized ethanol is mainly derived from corn, a typical C4 plant [25, 26]. In this study, 11 types of best-selling alcohol-containing disinfectants from Chinese online shops were bought as samples. As expected, all of these disinfectants had higher 13C values than those in beer samples.
3.4. The difference between the 13C values in consumption and contamination samples In consumption experiment 1, 6 volunteers consumed No. 40 beer (13C = -28.0 ‰) from the 5 / 13
same barrel, and urine and oral fluids were collected 1 h later and analyzed. The 13C values of ethanol ranged from -27.3 ‰ to -26.7 ‰ in urine and from -27.3 ‰ to -26.3 ‰ in oral fluids (Table 3). The 13C values of ethanol in all bio-samples were compared with the No. 40 beer. The differences were less than 1.3 ‰ for urine and less than 1.7 ‰ for oral fluid. In consumption experiment 2, volunteer #1 consumed No. 41 beer (13C = -27.9 ‰) and No. 42 beer (13C = -18.4 ‰) on two different days. Urine and oral fluids were collected at 0 (before beer consumed), 1, 2, 3, 4, and 6 h after consumption. The 13C values of ethanol ranged from -27.9 ‰ to -27.5 ‰ in urine and ranged from -27.7 ‰to -27.3 ‰ in oral fluids after consuming No. 41 beer (Table 4). The 13C values of ethanol in urine and oral fluids after consuming No. 42 beer ranged from -18.2 ‰ to -18.0 ‰ and from -18.8 ‰ to -18.2 ‰, respectively (Table 5).
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Similar to Experiment 1, differences in the 13C values between the bio-samples and beer were calculated one by one. The differences were less than 0.6 ‰. In the contamination experiment, contaminated urine and oral fluid samples were analyzed
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(Table 2). The 13C values of ethanol ranged from -17.8 ‰ to -14.2 ‰ in urine and from -17.9 ‰ to -14.5 ‰ in oral fluids after being contaminated. The differences in 13C values between contaminated bio-samples and disinfectants were less than 0.4 ‰.
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The results of these experiments show that the isotopic characteristics (13C value) of ethanol can be transferred to the bio-samples, regardless of consumption or contamination. The 13C value of ethanol with a pure C3 origin in urine and oral fluid was lower than -25.0 ‰, and the 13C value of ethanol with a pure C4 origin in urine and oral fluid was higher than -18.0 ‰ (Figure 3). This obvious data difference shows the potential of stable carbon isotope analysis to determine the origin of ethanol in vivo. Because most alcoholic beverages are from C3 plants [27] and most non-edible alcohol is produced from C4 plants [28], stable carbon isotope analysis can provide data on consumption and contamination. However, some alcoholic beverages are brewed with C4 plants (such as vodka, bourbon whisky and rum), and the production of ethanol from residues of C3 plants (such as wheat [29]) has also become a recent research direction. The origin of ethanol is not clear when it comes from a mixture of C3 and C4 plants. The inadequate differentiation of the ethanol origin by 13C values may require stable oxygen isotope values and stable hydrogen isotope values.
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3.5. Analysis of a sample in an authentic case
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In a traffic accident, the blood alcohol content of the driver was detected to be 0.82 mg/mL. According to Chinese law, an individual with a blood alcohol content greater than 0.80 mg/mL should be prosecuted for criminal liabilities. The driver argued that he did not drink alcohol before the accident and that his blood was contaminated by alcohol swabs when drawing the blood. He said that he saw the nurse use a tincture for skin disinfection. The police investigated the hospital where the blood draws were performed. They found that the hospital purchased two kinds of disinfectants, a tincture (red bottle) and an alcohol-free sterile swab (blue bottle). In the absence of more information, the driver was going to be acquitted. The driver's urine was analyzed, and the 13C value of ethanol in the urine was -28.4 ‰. This value indicates that the origin of ethanol detected in the urine originated from C3 plants. For further confirmation, we analyzed the tincture provided by the hospital. The 13C value of ethanol in the tincture was -15.0 ‰, which was obviously different from the ethanol present in the urine of the driver. We speculate that the driver had consumed alcohol. According to our analysis, the police believed that the driver was suspected of lying. By replaying the surveillance video, the police found two important points. First, the hospital used a 6 / 13
sterile swab from a blue bottle when drawing blood from the driver. Second, half an hour before the traffic accident, the driver exited a restaurant. By comparing the driver's credit card records and restaurant bill records, it was proven that the driver consumed 2 bottles of beer in the restaurant before the accident. According to the police investigation, the type of beer consumed by the driver was the same as the No. 19 beer sample (13C = -28.4 ‰) in this study. This finding was consistent with our analytical data (the 13C value of ethanol in the urine was also -28.4 ‰). In a comprehensive survey, the police uncovered the truth and forced the driver to assume the criminal responsibility he deserved.
4. Conclusion
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The 13C values of ethanol in beer and disinfectant samples were measured. The stable carbon isotope characteristics of beer have the botanical features of C3 plants or a mixture of C3 and C4 plants. The isotopic characteristics of commercial alcohol are relatively simple, only showing the botanical features of C4 plants. These characteristics were transferred to bio-samples without changes in consumption and contamination experiments. Through the application of a case, the potential and value of stable isotope analysis technology in forensic analysis was explained. More studies need to be conducted on how to describe the origin of mixed botanical ethanol.
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Funding
Acknowledgment
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This study was sponsored by Shanghai Sailing Program (17YF1420100) and the supporting from Academy of Forensic Science (GY2016Z-1).
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This study thanks for 13th National Key R&D Program of China (2016YFC0800704), Shanghai Key Laboratory of Forensic Medicine (17DZ2273200) and Shanghai Forensic Service Platform (16DZ2290900).
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Figure 1. GC-IRMS chromatograms (m/z 44 ion current as a function of time) for 13C analysis of pure
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ethanol (A), acetonitrile (B), wort (C), blank urine (D), blank oral fluid (E), beer sample (F), urine
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sample (G) and oral fluid sample (H).
Figure 2. (A) Plot of the linear range of the experiments of the mean intensity utilizing ethanol. (B) Plot of the linear range of the experiments of the mean 13C values utilizing ethanol.
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Figure 3. Plot of the 13C values of ethanol from different origins and the relationship of 13C with
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botanical origins.
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Table. 1 Reference 13C values of ethanol [‰] obtained from the studied beer samples and its basic information Sample Producer
Fermentative
Wort
Botanical
Modes
Concentration (°P)
Origin
Un-pasteurized
Lager
9.0
Barley
-26.7±0.5
China
2**
China
Pasteurized
Lager
8.0
Barley, Rice
-28.2±0.2
3
China
Un-pasteurized
Lager
9.0
Barley
-26.6
4
China
Un-pasteurized
Lager
8.0
Barley
-26.4
5
China
Un-pasteurized
Lager
8.0
Barley
-26.3
6
China
Un-pasteurized
Lager
8.0
Barley
-26.5
7
China
Un-pasteurized
Lager
9.7
Barley
-25.9
8
China
Un-pasteurized
Lager
8.0
Barley
9
China
Un-pasteurized
Lager
11.0
Barley, Wheat
10
China
Un-pasteurized
Lager
11.0
Barley, Rice
11
China
Un-pasteurized
Lager
9.0
12
China
Un-pasteurized
Lager
9.0
13
China
Un-pasteurized
Lager
8.0
14
China
Un-pasteurized
Lager
8.0
15
China
Un-pasteurized
Lager
8.0
16
China
Un-pasteurized
Lager
8.0
17
China
Un-pasteurized
Lager
13.5
18
China
Pasteurized
Lager
8.0
19
China
Pasteurized
Lager
20
China
Pasteurized
Lager
21
China
Pasteurized
Lager Lager Lager
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Barley, Rice
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1**
A
No. *
-26.2 -26.6 -26.4 -26.2 -26.4
Barley, Rice
-27.0
Wheat, Rice
-26.3
Barley, Wheat
-26.5
Barley, Wheat
-25.4
Barley, Wheat
-26.6
Barley, Rice
-28.5
Barley, Rice
-28.4
11.4
Barley
-27.8
9.0
Barley, Rice
-28.3
9.0
Barley, Rice
-28.8
16.3
Barley
-26.2
N
U
Barley, Wheat
9.0
China
Pasteurized
23
Germany
Un-pasteurized
24
Germany
Un-pasteurized
Lager
12.4
Wheat
-26.1
25
Germany
Pasteurized
Lager
11.5
Barley
-28.2
26
Germany
Pasteurized
Lager
12.2
Barley
-27.5
27
Germany
Pasteurized
Lager
12.4
Wheat
-27.2
28
Germany
Pasteurized
Lager
12.5
Barley
-27.8
29
Germany
Pasteurized
Lager
12.4
Barley
-26.8
30
America
Pasteurized
Ale
11.8
Barley, Cane sugar
-20.1
31
America
Pasteurized
Ale
8.1
Barley, Corn sugar
-19.8
32
America
Pasteurized
Lager
10.3
Barley, Rice
-29.5
33
America
Pasteurized
Lager
9.7
Wheat
-27.2
34
Belgium
Pasteurized
Ale
11.7
Barley, Wheat, Cane sugar
-18.8
35
Belgium
Pasteurized
Lager
15.5
Barley, Wheat
-28.3
36
Belgium
Pasteurized
Lager
11.7
Barley, Wheat
-28.7
37
Britain
Pasteurized
Ale
10.5
Barley, Rice, Cane sugar
-18.4
38
Denmark
Pasteurized
Ale
10.5
Barley, Corn
-18.5
39
Thailand
Pasteurized
Lager
12.3
Barley, Wheat
-28.1
40
Denmark
Pasteurized
Lager
10.5
Barley
-28.0
41
Germany
Pasteurized
Lager
17.0
Barley
-27.9
42
Germany
Pasteurized
Ale
11.2
Barley, Cane sugar
-18.4
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M
22
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13C (‰)
Processing
* Each beer was purchased 1 bottle/tin as a random test sample in addition to samples No. 1 and 2. ** For No. 1 and No. 2 beer samples, 10 bottles/tins with different production dates were purchased from different supermarkets (or online stores). 12 / 13
Table. 2 Reference 13C values of ethanol in disinfectants and contaminated bio-samples Sample No. 43 44 45 46 47 48 49 50 51 52 53
13C
13C
(‰)
-16.6 -16.9 -17.7 -15.7 -16.8 -14.4 -16.8 -16.8 -16.6 -17.5 -15.8
Urine Difference with disinfectants (‰) 0.0 0.1 0.1 0.2 0.1 0.2 0.1 0.1 0.4 0.1 0.1
(‰)
-16.6 -17.1 -17.8 -15.6 -16.6 -14.2 -16.7 -16.7 -17.0 -17.4 -15.9
13C
Oral fluid Difference with disinfectants (‰) 0.3 0.1 0.2 0.3 0.0 0.1 0.2 0.0 0.4 0.2 0.4
(‰)
-17.0 -16.8 -17.9 -15.5 -16.8 -14.5 -16.5 -16.8 -16.2 -17.3 -15.4
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Disinfectants
Table. 3 Reference 13C values of ethanol in urine and oral fluid after the consumption of No. 40 beer Urine 13C (‰)
0.5 0.5 0.6 0.6 0.5 0.7
-27.0 -26.7 -27.2 -27.3 -27.3 -27.3
1# 2# 3# 4# 5# 6#
Conc.*(mg/mL)
13C (‰)
0.5 0.5 0.4 0.5 0.5 0.5
-26.9 -26.3 -27.0 -26.5 -27.3 -26.9
N
* Concentrations of ethanol in corresponding bio-samples
Difference from No. 40 beer (‰) 1.1 1.7 0.9 1.4 0.7 1.1
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Conc.*(mg/mL)
U
Volunteer
Oral fluid Difference from No. 40 beer (‰) 1.0 1.3 0.8 0.7 0.6 0.7
Table. 4 Reference 13C values of ethanol in urine and oral fluid after the consumption of No. 41 beer
-*** -27.5 -27.9 -
M
0.0 0.9 0.5 0.4 0.2 0.1
(‰)
Difference from No. 41 beer (‰) 0.4 0.0
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0h** 1h 2h 3h 4h 6h
Conc.*(mg/mL)
13C
A
Urine Time
Conc.(mg/mL)
Oral fluid 13C
0.0 0.8 0.6 0.4 0.2 0.1
(‰)
-27.3 -27.7 -
Difference from No. 41 beer (‰) 0.6 0.2
* Concentrations of ethanol in corresponding bio-samples ** Before beer consumed
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*** Lower than linear range of method
Table. 5 Reference 13C values of ethanol in urine and oral fluid after the consumption of No. 42 beer
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Urine
Time
A
0h** 1h 2h 3h 4h 6h
Conc.*(mg/mL) 0.0 1.0 0.8 0.6 0.4 0.3
13C
(‰)
-*** -18.0 -18.1 -18.2 -
Oral fluid Difference from No. 42 beer (‰) 0.3 0.3 0.1
* Concentrations of ethanol in corresponding bio-samples ** Before beer was consumed *** Lower than the linear range of the method
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Conc.(mg/mL)
13C (‰)
0.0 0.9 0.7 0.6 0.4 0.2
-18.2 -18.2 -18.8 -
Difference from No. 42 beer (‰) 0.2 0.2 0.5