Driving under the influence of zopiclone: Elimination between two consecutive blood samples.

AIM: Zopiclone is a widely used hypnotic drug which is frequently detected in apprehended drivers. For assessments in forensic cases, the elimination half-life (t1/2) of a drug is sometimes important. A t1/2 of 3.5-6.5 h for zopiclone is previously reported in healthy individuals, but different factors like age and drug-interactions can affect the t1/2 of zopiclone. The aim of this study was to describe concentrations of zopiclone and co-ingestion of additional drugs in apprehended drivers, and to investigate the t1/2 of zopiclone based on two consecutive blood samples. METHODS: Data was collected from apprehended drivers in Norway between 2003 and 2021. All cases where zopiclone was detected were included. In a subset of the material, two consecutive whole blood samples were collected ≥ 20 and < 60 min apart. Concentrations of zopiclone in blood were determined by LC-MS or UHPLC-MS/MS. The elimination and t1/2 of zopiclone was estimated from the concentration change of zopiclone and the time interval between the two consecutive blood samples, under the assumption of first order kinetics. RESULTS: The median concentration among all zopiclone positive cases was 0.044 mg/L (IQR 0.070 mg/L) (n = 2401). The most frequent additional drugs detected were ethanol (36%), diazepam (22%), amphetamine (14%) and THC (14%). In zopiclone-only cases (n = 364), the median concentration of zopiclone was 0.066 mg/L (IQR 0.115 mg/L). In 112 cases, two consecutive blood samples were collected. Of these, 28 cases showed increasing concentrations of zopiclone between the two sampling time points. Among the cases in which the concentration decreased (n = 84), the median C1 was 0.048 mg/L (IQR 0.062 mg/L) and the median C2 was 0.043 mg/L (IQR 0.056 mg/L). A Bayesian statistical model was used to obtain the posterior distribution of t1/2. The posterior median of t1/2 was estimated to 3.1 h (IQR=0.39 h) when including only the cases showing decreasing concentrations, and this increased to 3.8 h (IQR=0.52 h) when also including samples showing non outlying increase in concentrations. There was no statistically significant gender difference in the calculated half-lives (two-sided Mann-Whitney U test, p = .525). CONCLUSIONS: This study showed that zopiclone is frequently detected in apprehended drivers in supra therapeutic concentrations and poly drug cases. The elimination of zopiclone in blood from two consecutive blood samples indicated an apparent t1/2 of between 3.1 and 3.8 h, which is within the lower range of what previous experimental studies on healthy individuals have reported.

Comparative Evaluation of Carboxyhemoglobin Quantification in Postmortem Whole Blood by CO-Oximetry and Headspace Gas Chromatography with Flame Ionization Detection and Atom Absorption Spectrophotometry.

A comparative evaluation of two methods used for carboxyhemoglobin (COHb) determination in postmortem whole blood was performed: carbon monoxide (CO)-oximetry measuring at 128 wavelengths and headspace gas chromatography with flame ionization detection (HS-GC--FID) where CO was determined after catalytic reduction of CO to CH4 and Fe was determined by atom absorption spectrophotometry (AAS, 248.3 nm). An aliquot of 100 µL whole blood was loaded into the CO-oximetry module. In the HS-GC--FID analysis, to 1.0 mL of whole blood, 3.0 mL of saponin solution was added, mixed and centrifuged. To 20 mL HS vials, 400 µL of the supernatant was added and the vials were immediately sealed. One milliliter of potassium hexacyanoferrat (III) solution was added through the HS septum and mixed. The samples were incubated at 70°C for 5 min. CO was separated using He as carrier gas and a CP-Molsieve 5 Å PLOT capillary column. Fe was determined using 400 µL of the saponin supernatant diluted to 10 mL by water. During a period of ∼3 years, 124 postmortem whole blood samples were analyzed. Bland-Altman method comparison showed satisfactory agreement and no significant bias between the methods for the whole saturation range (5 to 85% COHb). Five samples, all with %COHb >40, showed deviations of more than 10% COHb in absolute terms. One sample, in the lower COHb range <10%, was false negative on the CO-oximetry method. The between-assay accuracy, reported as bias, at 60% COHb was -0.8% and -9.0%, and precision, reported as relative standard deviation, was 1.6% and 7.7%, for the CO-oximetry and HS-GC--FID-AAS methods, respectively. Both methods obtained satisfactory results in proficiency testing rounds, with z-scores <±2 (n = 11). This study showed that the CO-oximetry method based on the 128-wavelength principle and the HS-GC--FID-AAS method are comparable and satisfactory for %COHb determination in postmortem whole blood.

Frequency of postmortem ethanol formation in blood, urine and vitreous humor - Improving diagnostic accuracy with the use of ethylsulphate and putrefactive alcohols.

PURPOSE: This study aimed to compare the frequency of postmortem ethanol formation in blood, urine and vitreous humor according to negative ethylsulphate (EtS) in blood or positive putrefactive alcohols (PA's) in either medium. Furthermore, it aimed to evaluate the interpretational value of calculated ethanol ratios in relation to EtS and PA results. METHODS: Blood ethanol positive forensic cases were included; one dataset consisting of 2504 cases with EtS analysed in blood and another dataset with 8001 cases where PA's were analysed. RESULTS: PA's were found in 24.4% of cases. EtS was negative in 15.3%, 9.4% and 7.4% of cases that were positive for ethanol in blood, urine and vitreous humor, respectively. In EtS negative cases, the concentrations of ethanol in blood, urine and vitreous humor were lower than 0.20 g/kg in 51.3%, 67.4% and 77.8%, respectively. It was 1.0 g/kg or higher in blood in 4.2% of cases. More EtS negative and PA positive cases were seen in central compared to peripheral blood. Ethanol ratios between urine or vitreous humor and blood were significantly lower in both EtS negative and PA positive cases, but large variations were observed. CONCLUSION: EtS and PA analysis improve the diagnostic accuracy of ethanol in postmortem cases. Postmortem ethanol formation in vitreous humor and urine were both more frequent than expected and we recommend the analysis of ethanol primarily in peripheral blood if available.

Relationship between betahydroxybutyrate (BHB) and acetone concentrations in postmortem blood and cause of death.

Unexpected death caused by diabetic or alcoholic ketoacidosis is easily overlooked due to the non-specific symptoms. Although the acid betahydroxybutyrate (BHB) is the most abundant ketone body formed in conditions with ketoacidosis, routine analysis in postmortem investigations often only includes the neutral ketone body acetone. This study aims to evaluate the usefulness of implementing routine BHB analysis in postmortem cases, by investigating the relationship between BHB and acetone concentrations in postmortem blood and the main cause of death. From our database of forensic autopsy cases examined from 2012 to 2015, there were 376 cases with BHB and/or acetone detected in postmortem blood that could be paired with data from the Norwegian Cause of Death Registry. Cases were categorized into three groups based on cause of death: "Diabetes-related" (n = 38), "Alcohol-related" (n = 35) and "Other" (n = 303). Analysis of BHB in blood was performed using UHPLC-MS/MS (limit of quantification (LOQ) 52 mg/L) and of acetone using HS-GC-FID (LOQ 87 mg/L). For the purpose of the study, the acetone method was also validated for a LOQ of 23 mg/L. The median BHB concentration was significantly higher in the group of diabetes-related deaths (671 mg/L, range 68-1311 mg/L) compared to the group of alcohol-related (304 mg/L, range 65-1555 mg/L, p <0.001) and other causes of deaths (113 mg/L, range 0-1402 mg/L, p <0.001). In seven deaths (1.9%), the BHB blood concentration was above the suggested pathological threshold of 250 mg/L, without detection of acetone in blood above 23 mg/L. In 15% of deaths by other causes than diabetes or alcohol, a pathologically significant BHB blood concentration was detected. Our results indicate that BHB is a more reliable marker of pathologically significant ketoacidosis than acetone, and we suggest that BHB should be routinely analyzed in postmortem investigations.

Quantitative Determination of Ethyl Glucuronide and Ethyl Sulfate in Postmortem and Antemortem Whole Blood Using Phospholipid Removal 96-Well Plate and UHPLC-MS-MS.

Postmortem ethanol formation is a well-known problem in forensic toxicology. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are ethanol metabolites that can be used to distinguish antemortem alcohol intake from postmortem formation of ethanol and in addition can be a helpful tool in assessment of the hip-flask defense. To an aliquot of 100 µL whole blood, internal standard (IS) and water was added before protein precipitation treatment (PPT) with ice-cold acetonitrile (ACN). The supernatants were filtered through a 96-well phospholipid removal plate, evaporated to dryness and reconstituted in 150 µL water/ACN/formic acid (FA). Identification of compounds was performed using multiple reaction monitoring (MRM) in negative mode. Gradient elution was performed on a C18 column with methanol (MeOH) and 0.1% FA. The run time was 4.5 min, and 0.5 µL was injected on an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) instrument. Linearity was achieved (coefficient of determination (R2) ≥ 0.999) for EtG in the range of 0.089 to 22 mg/L (0.40-100 µM) and EtS 0.025 to 6.3 mg/L (0.20-50 µM). The limit of quantification (LOQ) was 0.067 mg/L (0.30 µM) for EtG and 0.019 mg/L (0.15 µM) for EtS. Between assay accuracy was -15% to 8% and precision reported as relative standard deviation (RSD) was ≤ 4.5%. Precision, estimated as the RSD of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤ 6.7%. Recovery was ≥ 61% for EtG and ≥ 77% for EtS and matrix effects (ME) were 99% to 103%. Method comparison was carried out with a previously used UHPLC-MS-MS method, and satisfactory agreement was achieved, and external proficiency testing control samples had z-score < ± 1. The method has been used in routine work for more than 4 years analyzing about 6,000 antemortem and postmortem whole blood samples and has proven to be robust and reliable.

Comparative Study of Postmortem Concentrations of Antidepressants in Several Different Matrices.

Peripheral blood (PB) is considered to be the golden standard for measuring postmortem drug concentrations. In several cases, PB is however not available, but information regarding drug findings might still be crucial in order to determine the cause of death. Antidepressants are frequently detected in postmortem samples from forensic toxicology cases, but the literature investigating concentrations in other matrices than peripheral and heart blood is limited.We here describe a study for comparison of concentrations for a large number of different drugs in six different matrices. A total of 173 postmortem cases were included in the study material. The results from 44 cases with findings of antidepressants (amitriptyline/nortriptyline, citalopram, mianserin, mirtazapine, paroxetine, sertraline, trimipramine and venlafaxine) are presented in this article. Concentrations in peripheral and cardiac blood (CB), pericardial fluid (PF), two muscle samples and vitreous humour (VH) are compared. Ratios between concentrations in different matrices have also been compiled from available literature.All the investigated antidepressants were detected in all different matrices, and comparable concentration levels were found in the different matrices with a few exceptions. Concentrations in VH were generally lower than in the other matrices, and in a few cases with low concentrations in blood the antidepressants were not detected in VH. For most of the cases, ratios of 0.5-2 were found between concentration in PB and that in the other matrices. Some deviant concentrations where however found.This study shows that CB, PF, muscle and VH can provide important indications of the corresponding concentrations in PB when PB is not available.

EtG and EtS in Autopsy Blood Samples With and Without Putrefaction Using UPLC-MS-MS.

Analytical challenges related to postmortem specimens are well known. The degree of putrefaction of the corpse will influence the quality of the blood samples, and both the efficiency of sample preparation and the subsequent chromatographic performance can be affected. An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed and validated for the determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in postmortem whole blood. Sample preparation prior to UPLC-MS-MS analysis consisted of protein precipitation and filtration through a phospholipid removal plate. Chromatography was achieved using an HSS T3 column and gradient elution with formic acid in water in combination with methanol. The injection volume was 0.5 µL. Negative electrospray ionization was performed in the multiple reaction monitoring mode. Two transitions were monitored for the analytes and one for the internal standards. The between-assay relative standard deviations were in the range of 1.7-7.0% and the limits of quantification were 0.025 and 0.009 mg/L for EtG and EtS, respectively. Recovery was 51-55% and matrix effects ranged from 98% to 106% (corrected with internal standard). Blood samples from nine autopsy cases with various extents of putrefaction were analyzed. The sample preparation efficiently removed the phospholipids from the blood specimens. The samples were clean and the analytical quality of the chromatographic performance was satisfactory for both analytes irrespective of the degree of putrefaction.

Ethanol elimination rates at low concentrations based on two consecutive blood samples.

INTRODUCTION: For ethanol, the elimination curve change from apparent zero to apparent first order kinetics at low blood alcohol concentrations (BACs). This is less studied than elimination at higher BACs, and knowledge about this low BAC elimination is especially missing in drunk drivers representing a population with a high frequency of heavy drinkers with increased rate of ethanol metabolism. The aim of this study was to investigate the point at which elimination rates turns from zero to first order kinetics and the exact elimination rates at the very low BAC intervals in drunk drivers. METHODS: Two consecutively collected samples from suspected drunk drivers were used. All samples were analyzed by two headspace gas chromatography flame ionization detector methods (limit of quantification=0.04g/kg). The elimination rates at BACs below 0.25g/kg (study group, n=175) was studied in detail, and compared to the elimination rates in a moderate BAC reference group (n=789) as well as a high BAC reference group (n=4435). RESULTS: There were no differences in age, gender and drivings occurring during night-time between the study group and the reference groups. The mean elimination rates were stable at 0.18-0.19g/kg/h from a BAC of 4.0g/kg and until BAC in the first blood sample fell below 0.19g/kg. At BACs below 0.19g/kg, the mean elimination rate gradually declined from 0.163g/kg/h to the lowest elimination rate of 0.083g/kg/h. There was no relation between the concentration of ethanol and elimination rate at BACs above 0.19g/kg (Pearson's r=0.035, p=0.3), but there was a strong relation between concentration of ethanol and elimination rate at BACs below 0.19g/kg (Pearson's r=0.56, p<0.001). CONCLUSION: In conclusion, the present study showed that in a population of drunk drivers, the shift from zero order to first order kinetics occurs when BAC falls below 0.19g/kg. Below this points, the present study indicate that drunk drivers show elimination rates comparable to the normal population. These results could assist in back-calculations in cases of drunk driving involving low BACs.

Determination of safety margins for whole blood concentrations of alcohol and nineteen drugs in driving under the influence cases.

Legislative limits for driving under the influence of 20 non-alcohol drugs were introduced in Norway in February 2012. Per se limits corresponding to blood alcohol concentrations (BAC) of 0.2g/kg were established for 20 psychoactive drugs, and limits for graded sanctions corresponding to BACs of 0.5 and 1.2g/kg were determined for 13 of these drugs. This new legislation made it possible for the courts to make sentences based on the analytical results, similar to the situation for alcohol. To ensure that the reported concentration is as least as high as the true concentration, with a 99% safety level, safety margins had to be calculated for each of the substances. Diazepam, tetrahydrocannabinol (THC) and alcohol were used as model substances to establish a new model for estimating the safety margins. The model was compared with a previous used model established several years ago, by a similar yet much simpler model, and they were found to be in agreement. The measurement uncertainties depend on the standard batch used, the work list and the measurements' replicate. A Bayesian modelling approach was used to determine the parameters in the model, using a dataset of 4700 diazepam positive specimens and 5400 THC positive specimens. Different safety margins were considered for low and high concentration levels of diazepam (≤2µM (0.6mg/L) and >2µM) and THC (≤0.01µM (0.003mg/L) and >0.01µM). The safety margins were for diazepam 19.5% (≤2µM) and 34% (>2µM), for THC 19.5% (≤0.01µM) and 24.9% (>0.01µM). Concentration dependent safety margins for BAC were based on a dataset of 29500 alcohol positive specimens, and were in the range 10.4% (0.1g/kg) to 4.0% (4.0g/kg) at a 99% safety level. A simplified approach was used to establish safety margins for the compounds amphetamine, MDMA, methamphetamine, alprazolam, phenazepam, flunitrazepam, clonazepam, nitrazepam, oxazepam, buprenorphine, GHB, methadone, ketamine, cocaine, morphine, zolpidem and zopiclone. The safety margins for these drugs were in the range 34-41%.

Has the intake of THC by cannabis users changed over the last decade? Evidence of increased exposure by analysis of blood THC concentrations in impaired drivers.

The main psychoactive substance, Δ9-tetrahydrocannabinol (THC) can be present in highly variable amounts in different cannabis preparations. An increase in THC content in cannabis products has been suggested, and reported from several countries. However, it has not yet been investigated if products with high potency lead to increased human exposure, and thus to higher risk of adverse effects. In this study, we examined the mean concentrations of THC in whole blood samples from drivers apprehended in Norway in the period between 2000 and 2010 suspected of driving under the influence of drugs. Cases with only THC (n=1747) have been compared to cases with only ethanol (n=38796) or amphetamines (n=2493). The increase in mean THC concentration measured from 2000 to 2010 was from 4.0 ± 0.3 to 6.6 ± 0.4 ng/ml (58%), compared to 3% for ethanol and 16% for the amphetamines. This increase in THC concentrations was to some extent paralleled by an increase in the percentage of drivers which were judged as lightly impaired by a physician. Monitoring concentrations of drugs of abuse in blood from apprehended drivers indicated an increasing exposure to THC in Norway. If similar trends are observed globally, it should be further explored if this type of information could be used to elucidate the drug consumption patterns in a population and accordingly the consequences with regard to adverse effects of cannabis from a public health perspective.

Prevalence of alcohol and drugs among Norwegian motor vehicle drivers: a roadside survey.

The objective of the study was to determine the prevalence of alcohol, psychoactive medicinal drugs and illegal drugs among drivers in Norwegian road traffic. Drivers of motor vehicles were selected from April 2005 to April 2006 in the south-eastern part of Norway, surrounding, but not including the capital, Oslo. A stratified two-stage cluster sampling procedure was used. In the first stage, random road sites and time intervals were selected, and in the second stage, drivers were stopped by random at those sites and time intervals. Altogether about 12,000 drivers were asked to provide a sample of oral fluid (saliva) and answer a few questions. Samples of oral fluid were obtained from 88% of the drivers, of whom 30% were females and 70% males. The prevalence of each drug was estimated by a weighted average using weights adjusted for under- or over-sampling compared to traffic statistics. Alcohol or drugs were found in oral fluid samples of 4.5% of the drivers; alcohol in 0.4%, psychoactive medicinal drugs in 3.4%, and illegal drugs in 1.0%. Illegal drugs were found more frequently in samples from younger drivers, while psychoactive medicinal drugs were more frequently found in samples from older drivers. Psychoactive medicinal drugs were more prevalent among females than males, among drivers stopped on working days rather than weekends, and among those who reported annual driving distances less than 16,000 km. The drugs found most frequently were zopiclone (1.4%), benzodiazepines (1.4%), codeine (0.8%), tetrahydrocannabinol (0.6%) and amphetamines (0.3%). Two or more drugs were found in 0.6% of the samples, corresponding to 15% of the drug-positive drivers.

In vitro formation of ethanol in autopsy samples containing fluoride ions.

We present a case of a death of a diabetic man where the concentration of ethanol in post-mortem blood rose from 0.4 g/l 2 days after autopsy to 3.5 g/l 10 days after autopsy. The presence of fluoride ions in this blood sample was determined with ion chromatography and verified that fluoride ions were added to the vials. The concentrations of free fluoride, corresponding to 0.21 and 0.25% w/v potassium fluoride in blood and urine, respectively, were somewhat lower than the recommended 1% w/v. However, the amount of fluoride ions bound to calcium, proteins and other compounds in the samples is unknown. The blood sample was also subject to microbiological examination, which revealed growth of bacteria. In addition, a very high concentration of glucose was found in vitreous humour from the deceased. To determine whether the ethanol detected at the first analysis was of ante-mortem origin, ethyl glucuronide was analysed. Its absence, in the blood as well as the urine sample, strongly supported the theory that, in this case, all the ethanol detected was formed post-mortem. This case showed that ethanol may be formed in vitro at a very high concentration, despite the verified presence of fluoride ions. Possible reasons for this unusual formation of ethanol were the abundant presence of bacteria, a high level of glucose and, possibly, an insufficient amount of fluoride added to the vials.

Simultaneous determination of 6 beta-blockers, 3 calcium-channel antagonists, 4 angiotensin-II antagonists and 1 antiarrhythmic drug in post-mortem whole blood by automated solid phase extraction and liquid chromatography mass spectrometry. Method development and robustness testing by experimental design.

A method for the simultaneous determination of the beta-blockers atenolol, sotalol, metoprolol, bisoprolol, propranolol and carvedilol, the calcium-channel antagonists diltiazem, amlodipine and verapamil, the angiotensin-II antagonists losartan, irbesartan, valsartan and telmisartan, and the antiarrhythmic drug flecainide, in whole blood samples from forensic autopsies was developed. Sample clean-up was achieved by precipitation and solid phase extraction (SPE) with a mixed-mode column. Quantification was performed by reversed phase high performance liquid chromatography with positive electrospray ionization mass spectrometric detection (HPLC-MS). The method has been developed and robustness tested by systematically searching for satisfactory conditions using experimental designs including factorial and response surface designs. With the exception of amlodipine, the concentration limit of quantification (cLOQ) covered low therapeutic concentration levels for all the compounds. Within assay precisions and accuracies (bias) were 3.4-21% RSD and from -24 to 21% for the concentration range 1.00-5.00 microM, respectively. Between assay precisions were 4.4-28% RSD for the concentration range from 0.1 to 5 microM and recoveries varied from 9 to 103%. The method is used for determination of cardiovascular drugs in post-mortem whole blood samples from forensic autopsy cases.

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.

An automated alcohol dehydrogenase method for ethanol quantification in urine and whole blood.

In a previous work, an automated alcohol dehydrogenase method for the quantification of ethanol in whole blood (blood) specimens was presented. In the present work the application of the method to urine specimens has been investigated. Also, method robustness to routine analysis of urine and blood specimens during a period of eight months is shown. The limits of detection and quantification for urine were 0.0012 g/dL and 0.0042 g/dL, respectively. Relative standard deviations for the repeatability and within-laboratory reproducibility were in the ranges 1.4-4.1% and 1.8-4.6%, respectively. The method was compared with two headspace gas chromatography-flame ionization detection methods using authentic forensic urine specimens (n = 305) and blood specimens (n = 3186). Passing-Bablok regression for the concentration range 0.01-0.48 g/dL (urine) and 0.002-0.40 g/dL (blood) showed a statistically significant difference, for urine y = 0.9313 (0.9250 - 0.9377)x + 0.0038 (0.0029-0.0044) and for blood y = 0.9493 (0.9491 - 0.9495)x + 0.0032 (0.00318-0.00323), at 95% confidence level. The results of the external quality control specimens were in accordance with the reported theoretical concentrations.

Fast quantification of ethanol in whole blood specimens by the enzymatic alcohol dehydrogenase method. Optimization by experimental design.

A sensitive, fast, simple, and high-throughput enzymatic method for the quantification of ethanol in whole blood (blood) on Hitachi 917 is presented. Alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde using the coenzyme nicotinamide adenine dinucleotide (NAD), which is concurrently reduced to form NADH. Method development was performed with the aid of factorial design, varying pH, and concentrations of NAD+ and ADH. The linear range increased and reaction end point decreased with increasing NAD+ concentration and pH. The method was linear in the concentration range 0.0024-0.4220 g/dL. The limits of detection and quantification were 0.0007 g/dL and 0.0024 g/dL, respectively. Relative standard deviations for the repeatability and within-laboratory reproducibility were in the ranges 0.7-5.7% and 1.6-8.9%, respectively. The correlation coefficient when compared with headspace gas chromatography-flame ionization detection methods was 0.9903. Analysis of authentic positive blood specimens gave results that were slightly lower than those of the reference method.