Comparison of Different Analytical Methods for the Determination of Carbon Monoxide in Postmortem Blood

The determination of carbon monoxide (CO) and carboxyhemoglobin (COHb) is of utmost importance in forensic toxicology to determine the cause of death in cases of CO poisoning, fire, and explosions. To this end, reliable and updated analytical methods are required. In this paper, four different methods for the determination of carbon monoxide in postmortem blood samples were compared: (i) the spectrophotometric determination of COHb applying the method proposed by Rodkey and modified by Beutler–West, (ii) the spectrophotometric determination of CO using a micro-diffusion-based method, (iii) the determination of CO by gas chromatography coupled to a TCD detector, and (iv) the determination of COHb by blood gas analysis. Three postmortem blood samples were analyzed with all methods, and the results were comparable. The applied methodologies showed different features depending on the sensitivity, sample preparation, and volume. The HS-GC/TCD method in our hand was the most appropriate, on postmortem samples, and versatile to apply. Unfortunately, only a limited number of postmortem blood samples were available for this study due to the rarity of that kind of intoxication in our jurisdiction.     

Determination of total hemoglobin in forensic blood samples with special reference to carboxyhemoglobin analysis

For the determination of total hemoglobin (Hb) in blood containing elevated carboxyhemoglobin (COHb), a newly developed reagent containing a 100-fold concentration of ferricyanide (20 g/l) and a 2-fold concentration of Sterox SE was compared with a standard reagent (0.2 g/l ferricyanide), the reagent of van Kampen and Zijlstra, using forensic blood samples and experimentally heated blood samples. There were no significant differences between the spectra of hemiglobincyanide (HiCN) solution produced with our reagent and the van Kampen and Zijlstra reagent using experimentally heated blood samples. Although the spectra of HiCN changed gradually with increased heating time and with the passage of time after mixing, the absorbance at 540 nm (A540) did not change until at least 120 min for both the reagents. When forensic blood samples containing elevated COHb were mixed with the van Kampen and Zijlstra reagent, total-Hb concentrations determined 5 min after mixing were 10-20% higher than those determined after 180 min. The overestimates of total Hb determined after 5 min resulted in comparable underestimates of percentage saturation of COHb (COHb%) when COHb% was obtained from the ratio of COHb content, determined by gas chromatogrpahy, to total-Hb concentration in blood. However, there was an extremely good correlation between the values of total Hb in forensic blood samples determined with the van Kampen and Zijlstra reagent after 180 min and those determined with our reagent after 5 min. From the results obtained, our reagent proved to be suitable for the determination of total Hb in forensic science practice.     

Performance of immunoassays in screening for opiates,cannabinoids and amphetamines in post-mortem blood

Several immunoassay methods for screening of abused drugs in whole blood were evaluated in post-mortem forensic toxicology. Blood samples known to be positive or negative for opiates, cannabinoids or amphetamines by gas chromatography-mass spectrometry (GC-MS) were analysed by EMIT II Plus and EMIT d.a.u., Syva RapidTest and Triage 8 after acetone precipitation. In these experiments, the EMIT immunoassay method was modified by using the Dade Behring VIVA analyser to detect substances more sensitively. Low concentrations of abused drugs were detected in blood samples. The sensitivities of the modified EMIT method for opiates, cannabinoids and amphetamines were 100, 86 and 98%, respectively, whereas the values were below 86% with the other methods. The specificities of all immunoassay methods for opiates and cannabinoids were 83% or above but 51-85% for amphetamines. Sample rejection occurred in a few cases with the EMIT amphetamine assays. The modified EMIT immunoassay system presented here seems to be useful for screening of drugs of abuse in post-mortem blood samples, especially when urine is not available.     

Blood carbon monoxide and hydrogen cyanide concentrations in the fatalities of fire and non-fire associated civil aviation accidents, 1991-1998

To evaluate pathophysiological significance of post-mortem urinary myoglobin levels in determining the cause of death, we investigated 210 forensic autopsy cases, partially in comparison with serum levels. Post-mortem serum myoglobin levels were extraordinary high in most cases possibly due to post-mortem change. Urinary myoglobin levels did not correlate with the serum levels, showing possible post-mortem elevation in cases of a prolonged post-mortem period over 48h. A high (>1000 ng/ml), moderate (100-1000 ng/ml), slight (50-100 ng/ml) and not significant (<50 ng/ml) elevation of urinary myoglobin were observed in 26, 43, 31 and 110 cases, respectively. Half the highly elevated cases were those with a survival time over 24h. In cases of minor muscle injury such as head trauma, elevation of urinary myoglobin level was closely related to longer survival. In acute/subacute deaths with a post-mortem interval within 48h, a significant difference was observed in relation to the blood carboxyhemoglobin (COHb) levels of fire victims: myoglobinuria over 100 ng/ml was more frequently and markedly observed in cases with COHb below 60% than over 60%, suggesting muscle damage in fatal burns. Similar elevation was observed in heat stroke victims, and also in some cases of acute and subacute death from polytrauma, asphyxiation, drowning, electricity and spontaneous cerebral bleeding, but not in myocardial infarction. Thus, it was suggested that high post-mortem urinary myoglobin levels in acute and subacute death cases may be a possible indicator of antemortem massive skeletal muscle damage as well as exertional muscle hyperactivity or convulsive disorders associated with hypoxia.     

Post mortem kinetics of secobarbital

Post-mortem changes in barbiturate concentrations were evidenced using a rat-secobarbital model. The method used for the extraction and HPLC assay of barbiturates is suitable for all biologic fluids and post-mortem tissues. Kinetic data obtained is of excellent quality. Various modelization constants were defined. This experimental work emphasizes the difficulty of post-mortem toxicology, as concentrations found at the time of autopsy may be different from concentrations at the time of death, in blood as well as in tissues.     

顶空气质联用法测定腐败血、肝检材中CO

目的研究CO中毒腐败血、肝组织检材中CO的HS/GC/MS检测。方法用HS/GC/MS法分析碳氧血红蛋白(COHb)血的线性范围。配制10%、30%、50%、70%浓度COHb血样,分别在室温、冷藏、冷冻条件下保存,分别在当日、第4、14、45d进行测定,比较实验结果。腐败肝组织由雄性健康家兔通CO气体致死,当天解剖,家兔肝常温隔绝空气保存并放35d至腐败,期间进行不定期顶空测定分析。结果制备的COHb血在0-100%之间有良好的线性关系Y=2.4X+2.2(r=0.9995)。以此方法测定家兔CO中毒致死的COHb新鲜血的浓度和4℃下放置45dCOHb腐败血,结果表明温度对血样中COHb%的测定影响最大。采用HS/GC/MS法检测,每次只需0.25ml血样或1g肝脏,分析一次时间只需3min,均可检测出新鲜检材和常温放置45d的腐败肝组织检材CO的含量。结论HS/GC/MS法能检出CO中毒的腐败生物检材中CO。     

Carbon monoxide concentrations in the 2009 Victorian Bushfire disaster victims

Blood was available for the estimation of carboxyhemoglobin saturation (COHb) in 30 of the 173 persons who died in the Victorian bushfires in February 2009. The ages of these 30 deaths ranged from 3 to 80 years and there were 8 females. 13 cases (43%) were considered negative (less than 5% COHb), 12 (40%) were between 5 and 40% COHb, 2 (6.7%) between 40 and 50% and 3 (10%) were greater than 50% COHb. There were 6 persons either found within a building or a car and the COHb in these cases ranged up to 69% (mean 50%). There were 5 cases where the location was unable to be determined as either indoor or outdoor due to the extensive nature of the fire. The remaining 19 deceased persons were all located outside in the open and the concentration of COHb in these cases ranged up to 30% (mean 19%). Hydrogen cyanide was only detected in two deceased persons at concentrations of 0.5 and 2.7 mg/L, respectively. 13 deceased were found to have soot in the airways following necropsy but this did not correlate with the COHb levels.     

Headspace solid phase microextraction for the gas chromatographic analysis of methyl-parathion in post-mortem human samples. Application in a suicide case by intravenous injection

A simple and rapid procedure for the determination of methyl-parathion (m-p) in post-mortem biological samples was developed using headspace solid phase microextraction (SPME) and gas chromatography (GC) with nitrogen-phosphorous detection (NPD). Methyl-parathion was extracted on 85 microm polyacrylate SPME fiber. Salt addition, extraction temperature, and extraction time were optimized to enhance the sensitivity of the method. The linearity (y = 0.0473x - 0.0113, R2 = 0.9992) and the dynamic range (0.1-40 microg/ml) were found very satisfactory. The recoveries of methyl-parathion were found to be 46% in spiked human whole blood, 53% in spiked homogenized liver tissue, and 54% in spiked homogenized kidney tissue compared with samples prepared in water. The coefficients of variations for 2, 4, and 20 microg/ml of methyl-parathion in blood ranged from 0.9 to 5.1%, whereas the detection limit of the method was satisfactory (1 ng/ml in aqueous samples, 50 ng/ml in whole blood). The developed procedure was applied to post-mortem biological samples from a 21-year-old woman fatally poisoned (suicide) by intravenous injection of methyl-parathion. The intact insecticide was found in the post-mortem blood at a concentration of 24 microg/ml. No methyl-parathion was detected in the liver, kidneys, and gastric contents.     

Failure to detect elevated levels of carboxyhemoglobin in infants dying from SIDS

Carboxyhemoglobin (COHb) levels were determined in stored blood samples from 91 infants diagnosed to have died from the sudden infant death syndrome (SIDS) (0.59+/-0.41%, excluding one outlying value of 10.83%); 48 age-matched controls (0.53+/-0.38%); and three individuals who died from fire related causes (41+/-20%). No statistical differences in COHb levels were detected between blood from SIDS and control infants (p = 0.43).     

The routine use of C-reactive protein in forensic investigations

In clinical medicine, C-reactive protein (CRP) is extensively used as a general marker for immune system activation, and post-mortem applicability has been established [M.Q. Fujita, B.L. Zhu, K. Ishida, L. Quan, S. Oritani, H. Maeda, Serum C-reactive protein levels in postmortem blood-an analysis with special reference to the cause of death and survival time, Forensic Sci. Int. 130 (2002) 160-166; L. Uhlin-Hansen, C-reactive protein (CRP), a comparison of pre- and post-mortem blood levels, Forensic Sci. Int. 124 (2001) 32-35]. We have analysed the routine use of CRP in non-selected cases. Scarcity of blood available for analysis is a common problem in forensic investigation, and in response to this we have developed a method using liver as a source. In 50 consecutive autopsy cases, we have evaluated method, validated results and discussed their interpretation. In three cases the analysis was not possible. For each of the remaining cases (n=47) we have analysed whole blood, serum and/or liver samples. 57% (n=25) had serum CRP > 10 mg/L. Serum levels were higher than in whole blood or liver. CRP levels in serum and whole blood samples were stable in more than one month after death, making storage for later analysis possible. Liver levels peaked at one week, but after one month putrefaction was obvious. CRP levels were independent of the post-mortem interval. The use of liver as a source has not yet been described in literature. Our results in liver samples correlate well with plasma results, and liver is a good post-mortem alternative when blood is not available. We conclude that CRP measurements are easy, viable and inexpensive in a forensic setting, and that the number of cases with CRP elevation is high in a non-selected forensic material. In cases of doubt, marked elevation of CRP is an indicator of natural mode of death, and in cases of trauma, it indicates vital reaction. It can be used as a pre-autopsy screening, leading to a more extensive search for diseases not easily diagnosed, such as sepsis or ketoacidosis.     

二阶导数光谱双波长法测定血中碳氧血红蛋白饱和度的研究

本文将双波长法用于二阶导数光谱中,提出一种新的COHb饱和度测定方法。用本法测定了已知COHb饱和度的标准血样和未知COHb饱和度的检血,都得到了比较满意的结果。     

Two fatal intoxication cases with imidacloprid: LC/MS analysis

Imidacloprid [1-(6-chloro-3pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a new and potent nitromethylene insecticide with high insecticidal activity at very low application rates. It is the first highly effective insecticide that, like nicotine, acts on the nervous system, causing blockage of postsynaptic nicotinergic acetylcholine receptors. Two fatal cases with this insecticide in two male individuals, of 33 and 66 years old, are presented. An LC/MS with electrospray method for measuring imidacloprid and its metabolites in post-mortem samples is described. In the chromatographic separation, a reverse-phase column XTerra MS C18 (2.1mm i.d.x 150 mm, 5 microm) was used and the mobile phase composed with acetonitrile and 0.1% formic acid (15:85), at a 0.25 mL/min flow rate. Samples were prepared with a liquid-liquid extraction procedure with dichloromethane. Calibration curves for imidacloprid in blood and urine samples were linear from 0.2 to 15 microg/mL. The mean recovery was 86% with a coefficient of variation of +/-5.9%. The detection limit was 0.002 microg/mL. Quantitative results were obtained for all post-mortem matrices available of the two fatal cases: blood, urine, stomach contents, lung, liver and kidney. The imidacloprid blood concentrations found in two-cases were 12.5 and 2.05 microg/mL. The authors validated a method to detect and quantify imidacloprid in post-mortem samples, and to our knowledge for the first time a post-mortem tissue distribution was performed on various samples for this insecticide.     

Threshold values in toxicology - useful or not?

In many fields of toxicology, numbers are used as threshold values, e.g. as "acceptable daily intake values" resulting in maximum permissible concentrations in food or in animal feed by using "safety factors"; maximal admissible concentrations of toxic substances in the air at the workplace; cut-off values in analytical toxicology; limit values for biological specimens in the case of driving under the influence of drugs, guidance values for environmental specimens, etc. The philosophy behind these values must be well understood and they should only be applied to real cases by persons with enough toxicological background. The bad use of these numbers in toxicology can have dramatic consequences. Especially in regulatory toxicology their use should be made with great care. Moreover, tremendous improvements in analytical methodology, e.g. the decreasing of the limits of detection for many potentially toxic substances in recent years, should not end up in an overestimation of risks to humans. To avoid these abuses careful interpretations of analytical findings by qualified toxicologists are of paramount importance. The use and abuse of some of these threshold values will be outlined in several applications from analytical toxicology, risk assessment issues, forensic toxicology in post-mortem cases, as well as from the drugs and driving cases. Generally, if threshold values are considered as guidance values and not as the "absolute truth" in toxicology, they may be very useful in the interpretation of toxicology data.     

Cyanide, carboxyhemoglobin and blood acid-base state in animals exposed to combustion products of various combinations of acrylic fiber and gauze

In order to examine the usefulness of blood cyanide concentrations as an indicator of whether or not a victim was alive at the start of a fire, blood cyanide concentrations were measured in the bodies that we autopsied in our institute between January 1986 and March, 1987. In the present study, bodies with advanced decomposition were excluded. Thirty-six bodies were included: cyanide as well as carboxyhemoglobin (COHb) were detected in four charred bodies found at the scene of a fire. On the other hand, cyanide was not detected in any of the remaining 30 bodies except in two cases suspected of having ingested a cyanide compound. Rats and rabbits were made to inhale the combustion products of various combinations of acrylic fiber (hydrogen cyanide generating material when heated) and gauze (carbon monoxide generating material when heated). The exposure to the combustion products was continued until death in the rat and until apnea in the rabbit. The concentration of hydrogen cyanide in the exposure chamber and that of blood cyanide, at the time the animal died, correlated with the amount of acrylic fiber heated. In addition to differences in blood COHb and cyanide concentrations, there were also differences in blood gas concentrations between the acrylic fiber and the gauze groups. When the rabbits were switched to room air after the occurrence of apnea, the blood gas value began to normalize.     

Gamma-hydroxybutyric acid stability and formation in blood and urine

Gamma-hydroxybutyric acid (GHB) can cause problems in interpretation of toxicological findings due to its endogenous nature, significant production in tissues after death and potential formation in stored samples. Our study was designed to determine the influence of storage conditions on GHB levels and its possible in vitro formation in blood and urine in cases where no exogenous use of GHB or its precursors was suspected. The samples were prepared by validated method based on liquid-liquid reextraction with adipic acid internal standard and MSTFA derivatization and assayed on a GC-MS operating in EI SIM mode. The first part of the study was performed with pooled blood and urine samples obtained from living and deceased subjects stored with and without NaF (1% w/v) at 4 and -20 degrees C over 8 months. In ante-mortem samples (both blood and urine) no significant GHB production was found. After 4 months of storage, the substantial GHB rise up to 100 mg/Lwas observed in post-mortem blood stored at 4 degrees C without NaF with subsequent gradual decrease in following months. The inhibition of GHB production was apparent during storage in NaF treated frozen blood samples. In post-mortem urine only slight temporary GHB levels were ascertained (up to 8 mg/L). The second part of our study was aimed to analyse 20 individual post-mortem blood samples stored at 4 degrees C for 16-27 days between autopsy and analysis without preservation followed by storage at 4 degrees C with NaF for 4 months. The temporary GHB production with maximum of 28 mg/Lwas detected in some samples.     

一氧化碳（CO）中毒检测方法

作者查阅近20年国内外相关文献的基础上,对一氧化碳(CO)中毒的检验方法进行了全面的阐述,同时对每种方法的优缺点和适用条件作了比较,并对适用于我国国情的顶空气质联用法进行了改良.     

Determination of venlafaxine in post-mortem whole blood by HS-SPME and GC-NPD

Venlafaxine is a phenethylamine derivative widely prescribed for the treatment of depression which inhibits both serotonin and norepinephrine reuptake (SNRI). In treatment with antidepressants of patient with depression and other psychiatric disorders there is also increased risk of suicidal thought and behaviour. Several lethal intoxications involving venlafaxine usually among psychotic patients have been reported in the literature. Sample preparation is of the greatest significance for a successful toxicological analysis. The development of simple, effective and rapid extraction procedures of drugs from post-mortem biological samples is a challenge. Headspace-solid phase microextraction (HS-SPME) offers significant advantages such as simplicity, low cost, compatibility with analytical systems, automation and solvent-free extraction. The aim of our work was the optimization of a HS-SPME procedure for the determination of venlafaxine in post-mortem biological samples by gas chromatography (GC) with nitrogen-phosphorous detection (NPD). Venlafaxine was extracted on 100 μm Polydimethylsiloxone Coating-Red (PDMS) SPME fiber and determined by GC-NPD. Salt addition, extraction temperature, preheating and extraction time were optimized to enhance the recovery of the extraction from aqueous solution spiked with venlafaxine. Finally the developed procedure was applied to post-mortem biological samples of a fatally poisoned woman by venlafaxine. The drug was quantified in post-mortem blood gastric and oesophagus contents of the deceased woman. A simple and rapid procedure using HS-SPME was developed for sample preparation of venlafaxine in post-mortem biological samples prior to GC-NPD determination. Validation data was satisfactory, thus enabling application in the toxicological analysis of forensic samples.     

Serum alcohol concentrations in trauma patients determined by immunoassay versus gas chromatography

There has been an extensive discussion in the forensic toxicology community regarding the effect of trauma on the enzymatic method for ethanol analysis. There is a paucity of information in the literature that addresses this question. This study was designed to compare the Dade Behring Dimension enzymatic method with the reliable gas chromatographic method. The blood samples collected at the same time from trauma patients were analyzed by both methods. The result of the study shows no significant quantitative difference between the enzymatic and gas liquid chromatographic (GLC) methods. Also, the enzymatic method did not show any false positive ethanol in cases where the GLC method showed a negative finding.     

Massive acute arsenic poisonings

Arsenic poisonings are still important in the field of toxicology, though they are not as frequent as about 20-30 years ago. In this paper, the arsenic concentrations in ante- and post-mortem materials, and also forensic and anatomo-pathological aspects in three cases of massive acute poisoning with arsenic(III) oxide (two of them with unexplained criminalistic background, in which arsenic was taken for amphetamine and one suicide), are presented. Ante-mortem blood and urine arsenic concentrations ranged from 2.3 to 6.7 microg/ml, respectively. Post-mortem tissue total arsenic concentrations were also detected in large concentrations. In case 3, the contents of the duodenum contained as much as 30.1% arsenic(III) oxide. The high concentrations of arsenic detected in blood and tissues in all presented cases are particularly noteworthy in that they are very rarely detected at these concentrations in fatal arsenic poisonings.     

Validity of CO-oximetric determination of carboxyhaemoglobin in putrefying blood and body cavity fluid

Most commercial CO-oximeters pose warning flags in their measured carboxyhaemoglobin (COHb) levels when total haemoglobin (tHb) concentrations in the samples are below a certain threshold, typically about 3-4gdl. The warning flags acknowledge the fact that the haemoglobin levels are outside the ranges these instruments are designed (or validated) for. By using a parallel GC method and special sample treatment methods prior to CO-oximetric measurements, this study demonstrates that CO-oximeters could actually produce valid COHb results for samples with tHb as low as 1g/dl, and that the warning flags have no bearing to the accuracy and precision of the measurements. This study also indicates that warning flags due to high methaemoglobin (MetHb) and sulphaemoglobin (SHb), and often turbidity did not affect the validity of the CO-oximetric measurements.