Stability of diazepam in blood samples at different storage conditions and in the presence of alcohol

Diazepam is one of the mostly used benzodiazepines and it is frequently analyzed in different biological samples, especially blood samples. The diazepam stability in the sample matrices is an important factor regarding reliable data obtaining. The storage is the main factor determining the stability of diazepam in blood samples and it is the object of the study presented. Remaining diazepam amount in spiked whole blood and plasma samples were tested at different storage temperatures, in the absence or presence of sodium fluoride as stabilizer as well as the influence of ethanol on diazepam stability was evaluated. The results of the study indicated that the temperature is the main storage factor affecting diazepam stability. In the fluoride stabilized blood samples the amount of diazepam decreases up to 85% of initial level when stored at -20° C for the period of testing (12 weeks). The presence of low (0.5 g/L) or high (3g/L) ethanol concentrations influences the stability of diazepam at -20 °C. In whole blood samples, the combination of sodium fluoride and ethanol decreases additionally (15-25%) the concentration of the analyte. Freeze-thaw experiments of whole blood samples show about 5-9% decrease in diazepam concentration after the first cycle. The freeze-thaw experiments on plasma samples, containing ethanol and/or fluoride show insignificant decreases of analyte concentration. Further experiments on benzodiazepines stability at different storage conditions or in combination of different factors should be undertaken in forensic toxicology to ensure the data quality, their reliability and reproducibility.     

The unreliability of using a urine ethanol concentration to predict a blood ethanol concentration

Of approximately 5,000 forensic cases with a positive ethanol result, over 1,000 were available in which both blood and urine were present for comparison of ethanol content. Data were examined for calculation of the urine to blood ethanol concentration ratio, with the intent of evaluating the validity of predicting a blood ethanol level given a urine ethanol level. The overall urine to blood ethanol concentration ratio was 1.57:1 with a range of 0.7 to 21.0:1. The extremely wide range of values implies that a large degree of error would be introduced if a mean ratio was used when predicting a blood ethanol level from a urine ethanol level.     

A study on the combined action of CO and HCN in terms of concentration-time products

Acute toxicity at single and combined exposures of CO and HCN was studied on rats in terms of concentration-time product (ppm . min) necessary to kill animals (lethal CT). The animal was exposed individually to test gas in an animal chamber made of transparent plastics, and test gas was made in gas chamber connected to the animal chamber by a wide and short piece of plastic tube. HCN was produced by addition of NaCN solution to H2SO4 and in case of CO exposure, various amounts of pure CO were introduced. During exposure, gas samples were frequently taken. After exposure, blood sample was withdrawn from the right side of the heart. CO concentrations in the gas and blood were determined gas chromatographically. HCN in the gas sample was measured spectrophotometrically, after being absorbed into NaOH solution in a glass vessel devised by our laboratory. At single exposures, mean lethal CT for CO was 78,000 +/- 22,000 and for HCN was 4,700 +/- 940. In combined exposure, various combinations of CO and HCN were used. A fractional CT, defined as a ratio of CT to lethal CT, multiplied by 100, was calculated for each gas. A linear relationship between fractional CTs of HCN and CO was considered to show a simple additive action between the two gases. The sum of both fractional CTs averaged 100 +/- 26. On the other hand, linear relation was not observed between blood levels of the two toxicants at death.     

Physiological variations in blood ethanol measurements during the post-absorptive state

Specimens of arterial plasma and venous whole blood were obtained at 3-10 min intervals during the post-peak phase of ethanol metabolism in healthy volunteers. The concentrations of ethanol in blood and plasma were determined by headspace gas chromatography. This method had a standard deviation of 0.28 mg/dl for whole blood and 0.26 mg/dl for plasma and the coefficients of variation were 0.43% and 0.79% respectively. The physiological variation from time-to-time, expressed as the residual standard deviation after fitting the ethanol concentration-time regression relationships, ranged from 0.43-3.7 mg/dl (0.65-16%). The time-to-time variations in concentrations of ethanol were maximum when there were problems in getting an unimpeded flow of blood through the indwelling catheters. The results do not support the existence of sporadic fluctuations or spiking in the blood alcohol concentration-time profile during the post-absorptive state. Instead, this study underscores the need to control carefully the method of sampling blood and in this way keep pre-analytical sources of variation to a minimum.     

血液中乙醇保存稳定性研究

目的确定血液中乙醇最佳保存条件,探讨影响血液中乙醇含量稳定性的主要因素。方法对血液保存的温度(-20、4、20℃)、防腐剂(NaF、无防腐剂、Na2O2)、储存容器中空气所占比例(0%、25%、50%)和血醇质量浓度(0.2、0.8、2.0mg/mL)四个因素采用正交试验L9(34)方法分组,样本采用顶空气相色谱法进行测定,测定结果采用方差分析进行讨论。结果在20℃保存且不加入防腐剂的两组样本中血醇浓度变化明显,其余变化不明显。结论血液样本在4℃、储存容器中空气比例为50%和加防腐剂(NaF)的条件下保存,稳定性最佳;四个影响因素中温度为影响血液中乙醇含量稳定性的主要因素。     

Validation of an ion-trap gas chromatographic-mass spectrometric method for the determination of cocaine and metabolites and cocaethylene in post mortem whole blood

The coingestion of cocaine (COC) and ethanol is a very frequent occurrence and is known to increase the risk of morbidity and mortality. The formation occurs of a transesterification product, the cocaethylene (CE), which is even more toxic than cocaine. In order to study the role of ethanol as an agent of interaction in lethal cocaine intoxication, and to establish its influence in post mortem cocaine concentrations, an ion-trap gas chromatographic-mass spectrometric method (GC-MS) was validated to quantify simultaneously the agent and its biotransformation products, benzoylecgonine (BE), ecgoninemethylester (EME) and the 'biomarker' of the interaction, the CE present in whole blood. Deuterated internal standards were added to 2 ml of post mortem whole blood and extracted in Bond Elut Certify columns. The residues were evaporated and derivatized with N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (MTBSTFA). Detection was performed by electron impact ionization. The monitored ions were m/z 82/85 for EME-tert-butyldimethylsilyl (TBDMS)/EME-d3-TBDMS; m/z 182/185 for COC/COC-d3; m/z 196/199 for CE/CE-d3 and m/z 282/285 for BE-TBDMS/BE-d3-TBDMS. The limits of detection and quantification were found to be 25 ng and 50 ng ml(-1), respectively, for COC and CE, and 50 and 100 ng ml(-1) for BE and EME. Accuracy was different for each of the compounds, varying from 65 to 98%. The dynamic range of the assay was 50-2000 ng ml(-1).     

Medicolegal studies on alcohol detected in dead bodies — alcohol levels in skeletal muscle

An experiment was carried out on rats to determine whether or not a skeletal muscle sample was suitable for the determination of ethanol concentration in a carcass. Gas chromatography was used to estimate the ethanol and n-propanol concentrations in the femoral muscle and intracardial blood. The ethanol concentration of each sample was corrected according to the moisture ratio of circulating blood, viz., 78.5%.The ethanol concentration ratio of blood to muscle was 1.03 two hours after ethanol administration. When the carcasses of rats pre-treated with ethanol were stored at 15 °C and 25 °C, respectively, the ethanol concentrations in muscle and blood increased with time. At all times the concentration was higher in blood than in muscle, and also higher in samples collected from the carcass stored at 25 °C than at 15 °C.When the control carcass was stored in the same manner, the postmortem production of ethanol was noticed in both blood and muscle. As in the experimental rats, the control rats exhibited a higher blood ethanol than muscle ethanol level. Again, the ethanol concentration was higher in samples collected from the carcass stored at 25 °C than at 15 °C. The ratio of ethanol to n-propanol was less than 20:1 in blood and less than 10.1 in muscle.These results suggest that skeletal muscle may be a suitable tissue for the postmortem detection of ethanol.     

A pharmacokinetic study of ethyl glucuronide in blood and urine: applications to forensic toxicology

This pharmacokinetic study investigated the kinetics of ethanol and its metabolite ethyl glucuronide (EtG) in blood and urine during the whole time course of absorption and elimination. There are few previous studies on the kinetics of EtG in blood, and we wanted to evaluate whether such knowledge could yield valuable information regarding the time of ethanol ingestion in forensic cases, such as, for instance, drunk driving. Ten male volunteers consumed ethanol at a fixed dose of 0.5 g/kg body weight in a fasted state. Blood samples were collected for 14 h and urine samples were collected for 45-50 h after the start of drinking. EtG reached its maximum concentration (C(max)) in blood after a median of 4 h (range 3.5-5), a median of 3 h (range 2-4.5) after C(max) for ethanol. The ethanol-to-EtG ratios in blood (ethanol in g/L, EtG in mg/L) were >1 only for the first median 3.5 h (range 2.5-3.5) after drinking. EtG elimination occurred with a median half-life of 2.2 h (range 1.7-3.1 h), and the renal clearance was 8.32 L/h (median, range 5.25-20.86). The concentrations of EtG were always much higher in urine than in blood. The total amount of EtG excreted in the urine was median 30 mg (range 21.5-39.7), representing 0.017% (median, range 0.013-0.022) of the ethanol given, on a molar basis. The information from the present study may be a valuable supplement to determine the time of ethanol ingestion. For this purpose, two subsequent increasing EtG values and a high ethanol-to-EtG ratio in blood would support information of recent drinking.     

Kinetics of ethanol degradation in forensic blood samples

To determine ethanol in human post-mortem blood samples is problematic, largely due to the inappropriate and variable methods of preserving and storing, which can cause decomposition and loss of alcohol concentration. In this study, four crucial parameters of sample conservation were studied: temperature (T), percentage of air chamber in container (%CA), ethanol concentration in blood and post-mortem time. Blood samples from post-mortem cases were stored under different conditions (ethanol levels were known in all cases); factorial design variables: (%CA) 0, 5, 20, 35, 65%; storage temperature: 25, 4 and -10 degrees C; in a total of 15 experiments. No preserving agent was used in samples. Quantification of ethanol in blood was carried out by gas chromatography with head-space FID detector. Initial ethanol concentration ranged from 0.50 to 4.30 g/L. The kinetics of degradation observed was pseudo-first-order. The parameter that characterised the kinetics of ethanol degradation (k(0)) ranged from (4 x 10(-4) and 5.0 x 10(-1) day(-1)), depending on storage conditions. A strong dependence between ethanol degradation and the content of the air chamber was observed and this dependence was found to be stronger than that between degradation and temperature; there was an experimental relation between (k(0)) and (%CA). Activation energy for different conditions, i.e. 0, 5, 20, 35 and 65 (%CA), were calculated and contour plots were made. A mathematical equation relating air chamber, temperature and ethanol concentration at a certain time was determined. This equation allowed estimation of initial concentrations of ethanol with minimal error. A good correlation between experimental data and data calculated with the equation was obtained (r(2) = 0.9998). The best storage conditions were: 0% CA and storage at -10 degrees C, obtaining an ethanol degradation of 0.01% after 15 days. However, 33% of ethanol degradation was obtained with 35% CA at 25 degrees C after 15 days. This equation is useful in forensic cases in which original concentration of ethanol has to be estimated under different sample storage conditions.     

Forensic chemical testing of cadaveric material for acetonitrile

Gas chromatographic conditions for qualitative and quantitative evaluation of acetonitrile in biological material were determined, including those for reactive gas chromatography. Absolute and relative time of acetonitrile and concomitant substances retention in three columns of different polarity was determined. Study of the time of acetonitrile retention in biological material showed that acetonitrile concentration in the blood virtually did not change in cadaveric material stored in a hermetically closed flask for 2 weeks at 20 +/- 3 degrees C, while its concentration in the stomach decreased by 10-15%. Distribution of acetonitrile in human viscera in lethal poisoning was studied; the agent was evenly distributed in the gastric wall, intestine, liver, and kidney, while its concentrations in the lung and brain were 2-3 times higher. Forensic chemical expert analyses of the blood, urine, and viscera from corpses of humans dead from lethal acetonitrile poisoning showed that lethal concentration in the blood was 28.3-57.0 mg and in the urine 23.2-40.6 mg/100 ml.     

Hydrogen cyanide and carbon monoxide in blood of convicted dead in a polyurethane combustion: a proposition for the data analysis

Carbon monoxide is a well-known toxic component in fire atmospheres. However, the importance of hydrogen cyanide as a toxic agent in fire causalities is under discussion. A tragic polyurethane mattress fire provoked death of 35 convicts in a prison (Unit I, Olmos, Penitenciary Service of Buenos Aires Province, Argentina), in 1990. There is no report of any investigation carried out with such a large amount of victims in Argentina. Carboxihemoglobin (COHb) and hydrogen cyanide (HCN) were quantified in victims blood to elucidate the cause of the death. Saturation of COHb ranged between 4 and 18%, and HCN 2.0-7.2mg/l. These latter values were higher than the lethal levels reported in literature. Other toxic components routinely measured (ethanol, methanol, aldehydes and other volatile compounds) gave negative results on the 35 cases. Neither drugs of abuse nor psychotropics were detected. Statistical chi(2) analysis was applied to find differences between HCN and COHb concentrations. Saturation of COHb and HCN in blood were not independent variables (chi(2)=8.25). Moreover, the ratio COHb/HCN was constant (0.47+/-0.04). In order to evaluate the contribution of each toxic to the diagnosis, a lethal index was defined for each toxic (LI(CO) and LI(HCN)). The most probable cause of death could be inferred by a suitable plot of both indexes. The results indicated that death in the 35 fire victims was probably caused by HCN, generated during the extensive polyurethane decomposition provoked by a rapid increase of temperature.     

The influence of physical properties and lipid content of bile on the human blood/bile ethanol ratio

The relationship between ethanol levels in blood and bile was determined in human postmortem specimens. The influences of several physical properties--surface tension, specific gravity and viscosity--and bile lipid content on the blood/bile ethanol ratio were evaluated. A gas chromatographic direct injection technique was employed to determine the ethanol concentrations in postmortem blood and bile specimens. A positive correlation was established between the levels in the two fluids. No correlation could be found between the blood/bile ethanol ratios and the aforementioned physical properties of bile. Correction of the observed bile ethanol for lipid content had an insignificant effect on the ratio. The average blood/bile ethanol ratio was 1.03 +/- 0.29 (range: 0.32-2.91). The wide range observed makes it undesirable to use bile ethanol concentrations to predict specific blood ethanol concentrations. However, under certain conditions, bile ethanol levels may be used to estimate blood concentrations within a range of values.     

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.     

Comparative study of ethanol levels in blood versus bone marrow,vitreous humor,bile and urine

Post-mortem ethanol levels in blood were compared to corresponding levels in rib bone marrow, vitreous humor, urine and bile. In forensic toxicology, a good correlation between blood and a tissue or body fluid is needed to estimate a blood alcohol concentration when blood is unavailable or contaminated. In this study, direct injection and headspace gas-chromatographic techniques were employed to quantitate the ethanol concentrations. Comparable findings by these two techniques showed a reproducibility of results. When the determined bone marrow ethanol levels were corrected for the lipid fraction, a consistent correlation could be established between ethanol levels in blood and bone marrow. The relationship (linearity and ratio range) between ethanol levels in blood and corrected levels in bone marrow was better than that between blood and vitreous humor, bile or urine. This study showed that blood ethanol levels can be predicted by extrapolating the corrected rib bone marrow ethanol level.     

Excretion of alcohol in urine and diuresis in healthy men in relation to their age, the dose administered and the time after drinking

Healthy male volunteers drank neat whisky in amounts corresponding to 0.51, 0.68, or 0.85 g ethanol/kg body weight in 15-25 min after an overnight (10 h) fast. Urine was collected immediately before drinking and then at 60 min intervals for 7-8 h after intake. The volumes of urine voided were measured and the concentrations of alcohol (UAC) were determined by an enzymatic method. Ethanol-induced diuresis showed large inter-subject variations. The flow of urine was maximum between 60 and 120 min post-drinking when the median rates of production were 117 ml/h (range 55-335), 113 ml/h (range 41-453) and 373 ml/h (range 215-485) for 0.51, 0.68, and 0.85 g ethanol/kg respectively. The output of urine returned to normal (30-60 ml/h) after the peak UAC had passed despite an elevated blood alcohol concentration (BAC). The average amount of alcohol excreted in urine was 0.29 g (S.D. 0.119), 0.44 g (S.D. 0.246), and 1.00 g (S.D. 0.427) after the consumption of 0.51, 0.68 and 0.85 g ethanol/kg respectively. Neither peak diuresis nor the amount of alcohol excreted depended on a subject's age between 20 and 60 years. This work shows that after drinking a moderate dose of alcohol, only 0.7-1.5% of the amount consumed is excreted unchanged in urine. Ethanol-induced diuresis is most pronounced for the first 1-2 h after drinking (rising BAC). The production of urine returns to normal during the post-absorptive state.     

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.     

Forensic medicine and toxicologic aspects of 2-propanol poisoning

Two cases of poisoning with 2-propanol (isopropylalcohol) are reported. In one case, nail polish remover was drunk by a 2-year-old child. The concentration of 2-propanol and its metabolite acetone in the blood could be observed over a period of approximately 50 h. The highest concentration of 2-propanol determined was 4.22 g/l. Acetone reached a maximum value of 2.27 g/l 12 h after ingestion. The child survived without any observable after-effects. In the second case, a 35-year-old man drank ethanol in addition to 2-propanol. The poisoning was lethal. The possible time of intake before death is discussed in relation to the estimated levels of ethanol, 2-propanol and acetone found in the blood and urine. The histomorphological findings are often important as well with regard to time of intake.     

The diagnostic significance of the quantitative glycogen content in cadaveric tissues in different types of death

The author developed a method for glycogen content determination in histological sections of the liver, heart and the skeletal muscles, stained according to Best, in case of death from ischaemic heart disease, acute ethanol intoxication, overcooling. Different variation limits of glycogen level in these kinds of death as compared to data in case of death from lethal mechanical injuries ("normal value") were determined.     

Kinetics of ethanol in biological sections

Ethanol concentration in alveolocapillary blood (ACB), venous blood (VB), capillary blood (CB), saliva and urine was measured in healthy men and women aged 19-45 years 20, 40, 60, 90, 120, 180, 240 and 300 min after a single intake of 20% ethanol solution in soda water in a dose 0.8 g/kg body mass. Two types of kinetic curves were established. Calculations with Vidmark equation for different biomedia were made. Ethanol levels in all BM studied coincided in the resorption phase. In the elimination phase, ethanol concentration forms a sequence: ACB < saliva < VB < urine. Correlations and correlation coefficients of ethanol concentrations in different BM were estimated. The ethanol concentration correlation urine/ACB 1.71 +/- 0.15 and VB/ACB 1.45 +/- 0.07 is proposed for use in tests for alcohol intoxication.     

Handling of inspired vaporized ethanol in the airways and lungs (with comments on forensic aspects)

Collections of expired air and chemical determinations of ethanol concentrations in inspired and expired air showed that during prolonged inspiration of ethanol (vapour)-containing air about 55% was absorbed by adult human subjects. The fractional absorption was not detectably affected by variations in tidal volume (0.7-2.1 liters), nor was it significantly reduced in experiments where, due to preceding oral intake, the ethanol concentration of systemic blood was up to 50 times higher than that of inspired air. In these experiments the difference between the rates of change in blood alcohol concentration (beta 60) during and before ethanol inhalation agreed well with values calculated from measured respiratory absorptions. Mass spectrometric recordings of ethanol concentration in expired air vs. expired volume, taken in a state of steady uptake, also gave absorption fractions of about 0.55, and showed that the concentration in end-expiratory air did not fall below some 30% of that of the inspired air. These and other findings show that a large part of ethanol being inspired is deposited in the airway linings to be released again to ethanol-free alveolar air expired through the airways. It is concluded that inspired ethanol deserves consideration as a source of elevations of blood alcohol concentrations.