Ethanol stability in unpreserved refrigerated antemortem blood

Ethanol stability in preserved antemortem blood has been widely studied since it is a common practice in cases involving suspected impaired driving to collect antemortem blood in evacuated blood tubes containing sodium fluoride. In some situations, antemortem blood is submitted to a forensic laboratory for ethanol analysis in evacuated blood tubes that contain only an anticoagulant. There has been limited research on ethanol stability in antemortem blood stored without a preservative. On two occasions, antemortem blood was collected from five ethanol-free individuals into 6-ml Vacutainer® tubes containing only 10.8 mg potassium EDTA. The blood tubes were spiked with ethanol to approximately either 0.08 or 0.15 g/dl. Dual-FID headspace gas chromatography was used to analyze 58 blood tubes, 29 from each session, for ethanol 1 day after sample collection and again after 1 year of refrigerated storage (~4°C). Statistically significant decreases in ethanol were detected at the 0.05 level of significance. Mean decreases in ethanol after 1 year of storage for the 0.08 and 0.15 g/dl samples were 0.013 and 0.010 g/dl, respectively. The mean ethanol decrease across all tubes was 0.012 g/dl. The range of decreases for the 58 blood tubes was 0.003–0.018 g/dl. The mean ethanol decreases measured in this unpreserved antemortem blood are comparable in magnitude to those previously observed in antemortem blood containing sodium fluoride after 1 year of refrigerated storage. Ethanol did not increase in the antemortem blood samples despite the absence of sodium fluoride.     

Evaluation of the Analytical Performance of a Fuel Cell Breath Alcohol Testing Instrument: A Seven‐Year Comprehensive Study

Abstract: Between 2003 and 2009, 54,255 breath test sequences were performed on 129 AlcoSensor IV–XL evidential instruments in Orange County, CA. The overall mean breath alcohol concentration and standard deviation from these tests was 0.141 ± 0.051 g/210 L. Of these test sequences, 38,580 successfully resulted in two valid breath alcohol results, with 97.5% of these results agreeing within ±0.020 g/210 L of each other and 86.3% within ±0.010 g/210 L. The mean absolute difference between duplicate tests was 0.006 g/210 L with a median of 0.004 g/210 L. Of the 2.5% of duplicate test results that did not agree within ±0.020 g/210 L, 95% of these had a breath alcohol concentration of 0.10 g/210 L or greater and 77% had an alcohol concentration of 0.15 g/210 L or greater. The data indicate that the AlcoSensor IV–XL can measure a breath sample for alcohol concentration with adequate precision even amid the effects of biological variations.     

Variability in the Blood/Breath Alcohol Ratio and Implications for Evidentiary Purposes

The breath analyzer is an indispensable tool for identifying alcohol levels among drivers. While numerous studies have shown high correlations between blood and breath alcohol concentrations, most are limited by the study design. This study seeks to assess this relationship by minimizing potential measurement bias, document time from alcohol consumption to testing, and adjusting for potential confounders. A blinded study was performed using conditions closely resembling those in the field. The Draeger 7110 MKIII IL breath analyzer was used to assess breath alcohol concentrations (BrAC). Participants were 61 healthy volunteers aged 21–37 years with body mass index ≤30 and no history of alcoholism. A total of 242 valid blood/breath tests were performed in four test sets. The study results showed a high correlation coefficient between BrAC and blood alcohol concentration (BAC) levels (r = 0.983) with high sensitivity (97%) and specificity (93%). This strong association between the breath analyzer and BAC persisted even after adjustment for various stages of alcohol absorption. These results illustrate the high diagnostic sensitivity of the breath analyzer in field‐tested conditions.     

The Accuracy of Handheld Pre‐Arrest Breath Test Instruments as a Predictor of the Evidential Breath Alcohol Test Results

Drunk driving is a serious threat to public safety. All available and appropriate tools for curbing this threat should be employed to their full extent. The handheld pre‐arrest breath test instrument (PBT) is one tool for identifying the alcohol‐impaired driver and enforcing drunk driving legislation. A set of data was evaluated (n = 1779) where the PBT instrument was employed in drunk driving arrests to develop a multivariate predictive model. When maintained and operated by trained personnel, the PBT provides a reasonable estimate of the evidential test result within the relevant forensic range (95% prediction interval:  ± 0.003 g/210 L). ROC analysis shows that a multivariate model for PBT prediction of the evidentiary alcohol concentration above versus below the legal limit of 0.08 g/210 L has excellent performance with an AUC of 0.96. These results would be of value in evidential hearings seeking to admit the PBT results in drunk driving trials.     

顶空气相色谱法测定血液中乙醇不确定度的评估

目的 评估血液中乙醇测定结果的不确定度。方法 用顶空气相色谱法测定血液样本中乙醇质量浓度,从测定程序分析测量不确定度的来源,计算测定结果的不确定度。结果 血液样本中乙醇质量浓度两次测定平均值为1.00mg/mL,扩展不确定度为0.02 mg/mL。结论 血液中乙醇测定结果的不确定度主要来源于平行测定的误差。     

Reliability of breath-alcohol analysis in individuals with gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) is widespread in the population among all age groups and in both sexes. The reliability of breath alcohol analysis in subjects suffering from GERD is unknown. We investigated the relationship between breath-alcohol concentration (BrAC) and blood-alcohol concentration (BAC) in 5 male and 5 female subjects all suffering from severe gastroesophageal reflux disease and scheduled for antireflux surgery. Each subject served in two experiments in random order about 1-2 weeks apart. Both times they drank the same dose of ethanol (approximately 0.3 g/kg) as either beer, white wine, or vodka mixed with orange juice before venous blood and end-expired breath samples were obtained at 5-10 min intervals for 4 h. An attempt was made to provoke gastroesophageal reflux in one of the drinking experiments by applying an abdominal compression belt. Blood-ethanol concentration was determined by headspace gas chromatography and breath-ethanol was measured with an electrochemical instrument (Alcolmeter SD-400) or a quantitative infrared analyzer (Data-Master). During the absorption of alcohol, which occurred during the first 90 min after the start of drinking, BrAC (mg/210 L) tended to be the same or higher than venous BAC (mg/dL). In the post-peak phase, the BAC always exceeded BrAC. Four of the 10 subjects definitely experienced gastric reflux during the study although this did not result in widely deviant BrAC readings compared with BAC when sampling occurred at 5-min intervals. We conclude that the risk of alcohol erupting from the stomach into the mouth owing to gastric reflux and falsely increasing the result of an evidential breath-alcohol test is highly improbable.     

New Zealand's breath and blood alcohol testing programs: further data analysis and forensic implications

Paired blood and breath alcohol concentrations (BAC, in g/dL, and BrAC, in g/210 L), were determined for 11,837 drivers apprehended by the New Zealand Police. For each driver, duplicate BAC measurements were made using headspace gas chromatography and duplicate BrAC measurements were made with either Intoxilyzer 5000, Seres 679T or Seres 679ENZ Ethylometre infrared analysers. The variability of differences between duplicate results is described in detail, as well as the variability of differences between the paired BrAC and BAC results. The mean delay between breath and blood sampling was 0.73 h, ranging from 0.17 to 3.1 8h. BAC values at the time of breath testing were estimated by adjusting BAC results using an assumed blood alcohol clearance rate. The paired BrAC and time-adjusted BAC results were analysed with the aim of estimating the proportion of false-positive BrAC results, using the time-adjusted BAC results as references. When BAC results were not time-adjusted, the false-positive rate (BrAC>BAC) was 31.3% but after time-adjustment using 0.019 g/dL/h as the blood alcohol clearance rate, the false-positive rate was only 2.8%. However, harmful false-positives (defined as cases where BrAC>0.1 g/210L, while BAC< or =0.1g/dL) occurred at a rate of only 0.14%. When the lower of duplicate breath test results were used as the evidential results instead of the means, the harmful false-positive rate dropped to 0.04%.     

Comparison of ethanol concentrations in venous blood and end-expired breath during a controlled drinking study

Concentration-time profiles of ethanol were determined for venous whole blood and end-expired breath during a controlled drinking experiment in which healthy men (n=9) and women (n=9) drank 0.40-0.65 g ethanol per kg body weight in 20-30 min. Specimens of blood and breath were obtained for analysis of ethanol starting at 50-60 min post-dosing and then every 30-60 min for 3-6 h. This protocol furnished 130 blood-breath pairs for statistical evaluation. Blood-ethanol concentration (BAC, mg/g) was determined by headspace gas chromatography and breath-ethanol concentration (BrAC, mg/2l) was determined with a quantitative infrared analyzer (Intoxilyzer 5000S), which is the instrument currently used in Sweden for legal purposes. In 18 instances the Intoxilyzer 5000S gave readings of 0.00 mg/2l whereas the actual BAC was 0.08 mg/g on average (range 0.04-0.15 mg/g). The remaining 112 blood- and breath-alcohol measurements were highly correlated (r=0.97) and the regression relationship was BAC=0.10+0.91BrAC and the residual standard deviation (S.D.) was 0.042 mg/g (8.4%). The slope (0.91+/-0.0217) differed significantly from unity being 9% low and the intercept (0.10+/-0.0101) deviated from zero (t=10.2, P<0.001), indicating the presence of both proportional and constant bias, respectively. The mean bias (BAC - BrAC) was 0.068 mg/g and the 95% limits of agreement were -0.021 and 0.156 mg/g. The average BAC/BrAC ratio was 2448+/-540 (+/-S.D.) with a median of 2351 and 2.5th and 97.5th percentiles of 1836 and 4082. We found no significant gender-related differences in BAC/BrAC ratios, being 2553+/-576 for men and 2417+/-494 for women (t=1.34, P>0.05). The mean rate of ethanol disappearance from blood was 0.157+/-0.021 mg/(g per hour), which was very close to the elimination rate from breath of 0.161+/-0.021 mg/(2l per hour) (P>0.05). Breath-test results obtained with Intoxilyzer 5000S (mg/2l) were generally less than the coexisting concentrations of ethanol in venous blood (mg/g), which gives an advantage to the suspect who provides breath compared with blood in cases close to a threshold alcohol limit.     

The Effects of Gastroesophageal Reflux Disease on Forensic Breath Alcohol Testing

Fifteen test subjects, 10 of whom were diagnosed with gastroesophageal reflux disease (GERD), were dosed with alcohol to BACs above 0.150 g/dL. Blood and breath assays taken at 20-min intervals for 8 h after dosing demonstrated close agreement between postabsorptive BAC and BrAC values. Three subjects exhibited elevated breath alcohol concentrations up to 0.105 g/dL during the absorptive phase that were apparently due to the passage of gastric alcohol through the lower esophageal sphincter not attributable to eruction or regurgitation. The effect of gastric alcohol was not consistently proportional to the amount of unabsorbed gastric alcohol. Absorption of alcohol in the esophagus explains the nonproportionality. Breath samples contaminated by GERD-related alcohol leakage from the stomach into a breath sample were found only when there was a high concentration of alcohol in the stomach. When contaminated breath samples were encountered, they were irreproducible in magnitude.     

Results of a proposed breath alcohol proficiency test program

Although proficiency test programs have long been used in both clinical and forensic laboratories, they have not found uniform application in forensic breath alcohol programs. An initial effort to develop a proficiency test program appropriate to forensic breath alcohol analysis is described herein. A total of 11 jurisdictions participated in which 27 modern instruments were evaluated. Five wet bath simulator solutions with ethanol vapor concentrations ranging from 0.0254 to 0.2659 g/210 L were sent to participating programs, instructing them to perform n = 10 measurements on each solution using the same instrument. Four of the solutions contained ethanol only and one contained ethanol mixed with acetone. The systematic errors for all instruments ranged from -11.3% to +11.4% while the coefficient of variations ranged from zero to 6.1%. A components-of-variance analysis revealed at least 79% of the total variance as being due to the between-instrument component for all concentrations. Improving proficiency test program development should consider: (1) clear protocol instructions, (2) frequency of proficiency testing, (3) use lower concentrations for determining limits-of-detection and -quantitation, etc. Despite the lack of a biological component, proficiency test participation should enhance the credibility of forensic breath test programs.     

腐败血液中乙醇的顶空气相色谱分析

目的分析血液腐败后产生的乙醇及其他物质并探讨腐败血液中乙醇的检测及计算方法。方法以正常人空白血液制作腐败血样,采用1,4-二氧六环为内标物,通过顶空气相色谱进行定性及定量分析。结果血中乙醇在0.0625～1mg/mL范围内线性关系良好（r^2=0.9996）,各质量浓度组的变异系数（CV%）〈2%,血中乙醇的最低检出限为1μg/mL（S/N≥3）。腐败血样所产生乙醇与正丙醇的比例大致为25：1。结论检验方法简便、准确。为法医毒化检验相关工作提供了依据。     

Headspace gas chromatographic determination of ethanol: the use of factorial design to study effects of blood storage and headspace conditions on ethanol stability and acetaldehyde formation in whole blood and plasma

Our headspace gas chromatographic flame ionization detection (HS-GC-FID) method for ethanol determination showed slightly, but consistently, low ethanol concentrations in whole blood (blood) in proficiency testing programs (QC-samples). Ethanol and acetaldehyde were determined using HS-GC-FID with capillary columns, headspace equilibration temperature (HS-T degrees ) of 70 degrees C and 20 min equilibration time (HS-EqT). Full factorial designs were used to study the variables HS-T degrees (50 degrees -70 degrees C), HS-EqT (15-25 min), ethanol concentration (0.20-1.20 g/kg) and storage at room temperature (0-6 days) with three sample-sets; plasma, hemolyzed blood and non-hemolyzed blood. A decrease in the ethanol concentration in blood was seen as a nearly equivalent increase in the acetaldehyde concentration. This effect was not observed in plasma, indicating chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells. The variables showed different magnitude of effects in hemolyzed and non-hemolyzed blood. A decrease in ethanol concentration was seen even after a few days of storage and also when changing the HS-T degrees from 50 to 70 degrees C. The formation of acetaldehyde was dependent on all the variables and combinations of these (interactions) and HS-T degrees was involved in all the significant interaction effects. Favorable instrumental conditions were found to be HS-T degrees of 50 degrees C and HS-EqT of 15-25 min. The ethanol concentrations obtained for the range 0.04-2.5 g/kg after analyzing authentic forensic blood samples with a HS-T degrees of 50 degrees C were statistically significantly higher than at 70 degrees C (+0.0154 g/kg, p < 0.0001, n = 180). In conclusion, chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells has been shown to contribute to lowered ethanol concentrations in blood samples. Storage conditions before analysis and the headspace equilibration temperature during analysis were important for the determination of blood ethanol concentrations.     

Effects of Homeopathic Mother Tinctures on Breath Alcohol Testing

In some countries, it is illegal to drive with any detectable amount of alcohol in blood; in others, the legal limit is 0.5 g/L or lower. Recently, some defendants charged with driving under the influence of alcohol and have claimed that positive breath alcohol test results were due to the ingestion of homeopathic mother tinctures. These preparations are obtained by maceration, digestion, infusion, or decoction of herbal material in hydroalcoholic solvent. A series of tests were conducted to evaluate the alcoholic content of three homeopathic mother tinctures and their ability to produce inaccurate breath alcohol results. Nine of 30 subjects gave positive results (0.11–0.82 g/L) when tests were taken within 1 min after drinking mother tincture. All tests taken at least 15 min after the mother tincture consumption and resulted in alcohol-free readings. An observation period of 15–20 min prior to breath alcohol testing eliminates the possibility of false-positive results.     

The Stability of 4-Chloromethcathinone in Blood and Vitreous Humor

We present results of our study on the stability of 4-chloromethcathinone (4-CMC) in authentic postmortem peripheral blood and vitreous humor samples. The stability of 4-CMC was determined in postmortem blood samples (for a period of 90 days) and vitreous humor (30 days) at three different temperatures: −15°C, +4°C, and + 23°C. The analyses were carried out using ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). In both materials, the lowest 4-CMC stability was demonstrated at room temperature. The blood samples stored in a freezer (−15°C) showed stability for the entire study period (90 days), while in the case of the vitreous humor sample stored at the same temperature the concentration of the substance decreased by 53% after 30 days. The study carried out in authentic postmortem blood and vitreous humor samples confirms the previous reports of 4-CMC instability in biological material. Authors suggest that the biological material should be stored frozen until analyses are carried out as soon as possible after collection of the material.     

Evaluation of breath alcohol instruments II. In vivo experiments with Alcolmeter Pocket Model

The precision and accuracy of an Alcolmeter Pocket Model breath alcohol instrument have been investigated in experiments with human subjects under controlled conditions. The instrument response was zero in all tests with breath samples from alcohol-free subjects. The standard deviations of ethanol determinations in breath were ±0.0722 mg/ml during ethanol absorption and ±0.0416 mg/ml during ethanol elimination. The standard deviation during the elimination phase increased with ethanol concentration in the sample, being ±0.0416 mg/ml on average at a mean concentration of 0.420 mg/ml, corresponding to a coefficient of variation of 9.9%.The blood alcohol estimates using the Alcolmeter were somewhat too high during active absorption of ethanol, and too low during elimination, when a constant blood-breath alcohol ratio of 2100:1 was used to calibrate the instrument. During the elimination phase of ethanol kinetics and at a mean blood alcohol concentration of 0.50 mg/ml, the mean Alcolmeter result was 0.456 ± 0.169 mg/ml with 95% confidence, i.e. varying between 0.287 and 0.625 mg/ml 95 times out of 100 tests at this critical blood alcohol level.     

Duplicate breath testing: some statistical analyses

Duplicate breath alcohol testing from each individual provides confidence in the results when reasonable agreement (i.e. +/- 0.02 g/210 L) is achieved. For this reason many jurisdictions require duplicate testing. The State of Washington has recently implemented an infrared breath testing program and now requires two breath samples from each individual. Statistical analysis of 1847 duplicate breath tests is presented. Three variables are analyzed: first alcohol result (ALC1), the absolute difference between the two breath samples (DIFFA), and the signed difference between the two breath samples (DIFFS). The first breath alcohol result ranged from 0.021 to 0.338 g/210 L with a mean of 0.157 g/210 L. The absolute difference ranged from 0.00 to 0.05 g/210 L. The signed difference ranged from -0.05 g/210 L to 0.05 g/210 L. The absolute difference was regressed upon the first alcohol result and resulted in poor linear correlation of r = 0.212. Duplicate breath test differences do not appear to be a function of subject's alcohol level, but rather of sample provision.     

血液乙醇同源化合物分析及其法医学意义

酒精饮料申除含多量乙醇外,还含有微量甲醇、杂醇等乙醇同源化合物,且具有和乙醇类似的代谢及药代动力学特征,均参与肝ADH代谢,并与乙醇存在条件性竞争抑制。利用其代谢特性,在某些复杂醉酒驾车案件的法医学调查中,可根据血液中乙醇同源化合物的定量检测结果,结合对应血液乙醇质量浓度,帮助核实或推算饮酒者申诉的饮酒时间的真实性。     

The rate of dissipation of mouth alcohol in alcohol positive subjects

Seven subjects participated in a two-part study to evaluate mouth alcohol dissipation in alcohol positive subjects. In part one, subjects rinsed their mouths with a vodka solution and were breath tested after 1, 2, 3, 4, and 5 min intervals. On average, breath alcohol concentration (BrAC) decreased 20.4% (range 3.2-47.9%) between 1 and 2 min after rinsing. In part two of the study, multiple breath tests were administered after rinsing once with the vodka solution. The BrAC decreased more than 0.020 g/210 L between the first and second tests for all subjects (average 0.095 g/210 L, range 0.021-0.162 g/210 L). The average time for subjects to reach their unbiased BrAC was 9.35 min (range 4-13 min) after rinsing. This study reaffirms the need for duplicate breath testing and confirms that the minimum of a 15-min observation period is sufficient for mouth alcohol to dissipate in alcohol positive subjects.     

A large-scale study of the relationship between blood and breath alcohol concentrations in New Zealand drinking drivers

Blood alcohol concentrations (BAC) and corresponding breath alcohol concentrations (BrAC) were determined for 21,582 drivers apprehended by New Zealand police. BAC was measured using headspace gas chromatography, and BrAC was determined with Intoxilyzer 5000 or Seres Ethylometre infrared analysers. The delay (DEL) between breath testing and blood sampling ranged from 0.03 to 5.4 h. BAC/BrAC ratios were calculated before and after BAC values were corrected for DEL using 19 mg/dL/h as an estimate of the blood alcohol clearance rate. Calculations were performed for single and duplicate breath samples obtained using the Intoxilyzer (groups I-1 and I-2) and Seres devices (groups S-1 and S-2). Before correction for DEL, BAC/BrAC ratios for groups I-1, I-2, S-1, and S-2 were (mean+/-SD) 2320+/-260, 2180+/-242, 2330+/-276, and 2250+/-259, respectively. After BAC values were adjusted for DEL, BAC/BrAC ratios for these groups were (mean+/-SD) 2510+/-256, 2370+/-240, 2520+/-280, and 2440+/-260, respectively. Our results indicate that in New Zealand the mean BAC/BrAC ratio is 19-26% higher than the ratio of the respective legal limits (2000).     

Delayed ethanol analysis of breath specimens: long-term field experience with commercial silica gel tubes and breathalyzer collection

Delayed ethanol analysis was performed on breath specimens collected with commercial silica gel tubes using multiple Breathalyzer instruments. Eleven hundred and nine results were obtained from an ethanol testing program over a five-year period. Only 2.5% of the specimens had apparent collection errors. For the valid specimens, the most frequent result was 0.11 g/210 L and the mean result was 0.14 g/210 L. For 642 specimens, delayed results were compared with direct results. Direct results were greater than delayed results for 55%, less than for 27%, and equal to for 18% of the pairs. When fixed tolerance limits of +/- 0.03 were used, 81% of the direct results were confirmed. The confirmation percentage was best in the critical range of direct results, 0.05 to 0.15 g/210 L. The collection tubes showed no substantial variability in retaining ethanol during storage and releasing ethanol for analysis.