Simple determination of carboxyhemoglobin by double wavelength spectrophotometry of absorbance difference and the comparison with gas chromatographic method

This paper describes the spectrophotometric determination of carboxyhemoglobin (CO-Hb) in blood on the basis of double wavelength spectrophotometry of absorbance difference. Absorbance measurements are made in the 500–600 nm region at a blood dilution of 100–200-fold. Blood is diluted with a solution containing Na₂S₂O₄ to provide two components of CO-Hb and deoxyhemoglobin (deoxy-Hb). Absorbance difference at the two wavelengths at which deoxy-Hb has the same absorbance reflects only the CO-Hb component because the opposite component is nulled out of the mixture. After measurement of the absorbance difference, the measuring solution is saturated with CO gas to make all Hb derivative CO-Hb and remeasured at the same wavelengths. The percent of CO-Hb is considered the absorbance difference ratio. Results obtained by the present method was in satisfactory agreement with gas chromatographic data in blood not containing methemoglobin (Met-Hb). Comparative experiments using the gas chromatographic method and the present method were performed with samples containing Met-Hb. However, while there is a deficiency in the gas chromatographic method when the samples contain Met-Hb, the results of the present method were in close agreement with theoretical values when samples are mixed with CO-Hb, O₂-Hb and Met-Hb. Advantages of this method are that it is simple and accurate, standard curve or equation for calculation and accurate dilution are not necessary.     

Experimental studies on death by fire in automobiles and exhaust gas poisoning

Studies were made on the acid-base balance, blood gases, and carbon monoxide (CO), cyanide, and sulfur dioxide concentrations in the blood of albino rabbits that died from automobile exhaust gas poisoning (group I) or fires in cars (complete combustion, group II; incomplete combustion, group III). In group I, the temperature and CO concentration increased gradually to 35 degrees C and 5.2% in 70 min. The animals died after 9 min, when the values were 20 degrees C and 5.2%, respectively. In group II the animals died after 9 min, when the values were 55 degrees C and 1.95%, respectively. In group III, the temperature was very high (870 degrees C), but the CO concentration was not (0.6-1.3%) after 4 min. The animals died after 5 min. In all experimental groups, marked acidosis and hypoxemia were seen, but the CO2 tension (PCO2) was high, in contrast to previous studies on pure CO poisoning. In group I, the level of carboxyhemoglobin (CO-Hb) was significantly higher (91.2 +/- 3.4% in arterial blood, 87.5 +/- 8.1% in venous blood; p less than 0.01) than in groups II and III. Although the O2 tensions of venous and arterial blood (PvO2, PaO2) were very low, that of arterial blood was higher, suggesting that O2 was still being utilized in the tissues at the time of death. In group II, CO-Hb was high (57.7 +/- 16.0% in arterial blood, 61.2 +/- 20.6% in venous blood) and the acid-base balance indicated marked acidosis. In group III, the CO-Hb, PCO2 and cyanide levels in the blood were very high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term storage of blood samples as whole blood at extremely low temperatures for methemoglobin determination

Changes in methemoglobin (Met-Hb) concentrations during storage of whole blood and a hemolysate at refrigerated or various freezing temperatures were examined using experimentally prepared blood samples. When whole blood was stored at 3 degrees C, rapid reduction of Met-Hb was observed in the nitrite-treated blood whereas neither reduction nor formation of Met-Hb was observed in the untreated and heated blood within 7 days. When hemolysate was stored at 3 degrees C, Met-Hb concentrations were stable within a few days regardless of the initial values. However, slight autoxidation was observed 7 days after storage in the untreated and heated blood. When whole blood was stored at various freezing temperatures, Met-Hb concentrations were practically stable until at least 30 days at -80 degrees C or -196 degrees C regardless of the initial values, although considerable autoxidation was observed at -30 degrees C especially in the blood containing small amounts of Met-Hb. Based on the results obtained, a new method was devised for long-term storage of whole blood at extremely low temperatures for Met-Hb determinations.     

Storage of blood for methemoglobin determination: comparison of storage with a cryoprotectant at -30 degrees C and without any additions at -80 degrees C or -196 degrees C

Changes in methemoglobin (Met-Hb) concentrations during storage of whole blood or mixtures of blood and a cryoprotectant at refrigerated or various freezing temperatures were examined using blood samples from nitrite-administered rats and from autopsy cadavers. When whole blood was stored at 3 degrees C, Met-Hb reduction was observed in blood samples from nitrite-administered rats and in the blood from a victim poisoned by a weed killer containing some oxidant. When samples were stored at -30 degrees C, Met-Hb formation by autoxidation was inevitably observed in blood samples stored as whole blood, whereas addition of a cryoprotectant to whole blood could prevent Met-Hb formation in all the blood samples. When whole blood was stored at -80 degrees C or -196 degrees C, Met-Hb concentrations were practically stable until at least 30 days regardless of the initial values except in the control rat blood samples stored at -80 degrees C which showed slight formation of Met-Hb. From the results obtained, both the storage with a cryoprotectant at -30 degrees C and that without any additions at -80 degrees C or -196 degrees C proved to be suitable for long-term storage of blood samples from autopsy cadavers for Met-Hb determination.     

HS-GC内标曲线法测定血中乙醇含量的不确定度评估

目的建立自动顶空-气相色谱(HS-GC)内标曲线法测定血中乙醇含量的不确定评估方法。方法从分析测定程序着手,依据不确定度评定的指导性文件,分析不确定度来源,量化不确定度分量,计算检测结果的合成标准不确定度和扩展不确定度。结果各相对不确定度来自于检材重复性检测为3.4%,乙醇标准溶液为0.71%,检材为0.61%,叔丁醇内标溶液为0.41%,标准曲线为1.1%,气相色谱仪为1.3%,血液中乙醇的相对扩展不确定度为3.9%。结论血液中乙醇含量的不确定度主要来源于检材重复性检测、气相色谱仪、乙醇标准曲线。     

Postmortem formation of carbon monoxide

Since carbon monoxide (CO) production after death was suggested in a drowned body, CO and carboxyhemoglobin (HbCO) levels in blood and body cavity fluids of cadavers which were not exposed to fire and CO have been analyzed. CO released from the tissues was determined by gas chromatography and gas chromatography-mass spectrometry, and the total concentration of hemoglobin (Hb) was measured as cyanmethemoglobin (CNmHb). The HbCO level was calculated by the ratio of CO content and CO-binding capacity. CO levels (ml/100 g at STP) of the seven cases in which blood and body cavity fluids could be collected ranged from 0.13 to 0.87 in blood and 0.02 to 0.80 in body cavity fluids. HbCO levels in blood and body cavity fluids were from 0.3 to 6.0% and from 2.3 to 44.1%, respectively. In a typical case showing postmortem formation of CO, the CO levels in body cavity fluids were higher than that in blood. It is suggested that CO in a putrefied body is due to CO in blood prior to death and the CO formed by the decomposition of Hb, myoglobin and other substances during putrefaction. The significance of HbCO levels in body cavity fluids of cases with marked postmortem decomposition seems difficult to interpret without the value of HbCO in blood.     

Quantitative evaluation of volatile hydrocarbons in post-mortem blood in forensic autopsy cases of fire-related deaths

Volatile hydrocarbons in post-mortem blood from victims of fires were analyzed quantitatively by headspace gas chromatography mass spectrometry. The benzene and styrene concentrations in the blood were positively correlated with the carboxyhemoglobin (CO-Hb) concentration, which is evidence that the deceased inhaled the hydrocarbons and carbon monoxide simultaneously. By contrast, the concentrations of toluene and CO-Hb in the blood were not significantly correlated. This lack of correlation could be explained by two different sources of toluene, with low blood concentrations of toluene arising when the deceased inhaled smoke and high blood concentrations of toluene arising when the deceased inhaled petroleum vapor or other unknown vapors. The quantity of soot deposited in the respiratory tract was classified into four grades (-, 1+, 2+, 3+). The mean CO-Hb concentration in the 1+ soot group was significantly lower than those in the 2+ (p<0.05) and 3+ (p<0.01) soot groups. The blood CO-Hb concentrations in the 1+ soot group were all below 30%. Those indicated that the deceased aspirated smoke that contained both soot and carbon monoxide. The wide variation in CO-Hb concentrations for each soot classification could be caused by the different types of smoke produced by different materials. For example, petroleum combustion with a limited supply of oxygen, like in a compartment fire, may produce a large volume of dense black smoke that contains a large quantity of soot. Soot deposits in the airways and the blood CO-Hb concentration are basic and essential autopsy findings that are used to investigate fire-related deaths. The quantitative GC-MS analysis of blood volatile hydrocarbons can provide additional useful information on the cause of the fire and the circumstances surrounding the death. In combination, these three findings are useful for the reconstruction of cases.     

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.     

Blood cyanide determination in two cases of fatal intoxication: comparison between headspace gas chromatography and a spectrophotometric method

Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automated headspace gas chromatographic method with nitrogen-phosphorus detector (HS-GC/NPD). In the former, hydrogen cyanide (HCN) was quantitatively determined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS-GC/NPD method, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liberated as HCN. The spectrophotometric (VIS) and HS-GC/NPD methods were validated on postmortem blood samples fortified with potassium cyanide in the ranges 0.5-10 and 0.05-5 mug/mL, respectively. Detection limits were 0.2 mug/mL for VIS and 0.05 mug/mL for HS-GC/NPD. This work shows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable for rapid diagnosis of cyanide in postmortem cases.     

Fatality due to methyl acetylene-propadiene (MAPP) inhalation

A 33-year-old man died after intentionally inhaling a gaseous mix of methyl acetylene (propyne) and propadiene (allene) commonly known as MAPP, which is used for soldering and welding. He was found with a plastic bag securely placed over his head and a cylinder of MAPP alongside his head. The cylinder had been vented into the bag using a flexible hose. A comprehensive toxicological analysis revealed only a trace of diphenhydramine in the liver and 0.02 mg/L of morphine in the urine. Analysis of blood by headspace gas chromatography (HS-GC) detected two unknown peaks. These were determined to be the components of MAPP gas. MAPP was quantitated in femoral blood (59.6 mg/L) and brain (43.6 mg/kg) using a HS-GC method. The cause of death was attributed to acute MAPP intoxication, and the manner was determined to be suicide. A discussion on the analytical and interpretive considerations commonly encountered when analyzing volatile compounds is also presented.     

Intratracheal gas analysis for volatile substances by gas chromatography/mass spectrometry--application to forensic autopsies

Intratracheal gas analysis was carried out by gas chromatography/mass spectrometry (GC/MS) in 20 burned body cases (13 males and 7 females). Volatile aromatic and aliphatic hydrocarbons were detected by GC/MS using a GS-Q column with the intratracheal gas as well as the blood in 19 cases. The characteristic patterns of mass chromatograms for gasoline, kerosene (gas oil), and liquid petroleum gas could be differentiated from each other using the intratracheal gas. The burned body in one case showed no presence of volatile substances in the intratracheal gas, nor intratracheal soot, although high concentrations (1 microg/g and more) of volatile substances were detected on the clothes. The victim also had normal CO-Hb concentrations (0.1 to 0.2%) in the heart blood. The results of intratracheal gas analysis were consistent with signs of the vital reaction. In conclusion, intratracheal gas analysis provides a supportive method for diagnosing the cause of death in burned bodies, and yields for at least 48 hours valuable information on volatile hydrocarbons (being detected in deliberate or accidental fire cases) to which the body had been exposed just before death.     

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.     

Accuracy and Usefulness of the AVOXimeter 4000 as Routine Analysis of Carboxyhemoglobin

The measurement of blood carboxyhemoglobin (CO‐Hb) is important to determine the cause of death. The AVOXimeter 4000 (AVOX), a portable CO‐oximeter, has the advantages of a low purchase price and operating cost, ease of operation, and rapid results. Little information is available on the usefulness of AVOX in the forensic sample, and the previous study investigated only six samples. Therefore, in this study, we confirmed the usefulness of the AVOX through a comparison of its re s ults with data previously obtained using the double wavelength spectrophotometric method in autopsies. Regression analysis was performed between CO‐Hb levels measured by the AVOX and those measured by the conventional double wavelength spectrophotometric method in postmortem blood samples: a significant correlation was observed. This study suggests the usefulness of the AVOX to analyze postmortem blood, and the AVOX is suitable for routine forensic analysis and can be applied at the crime scene.     

Production of carbon monoxide in cadavers

Carbon monoxide (CO), total hemoglobin (Hb) and carboxyhemoglobin (HbCO) in the blood and reddish discolored body cavity fluids of cadavers which had not been exposed to fire and CO were analyzed. In 13 cadavers found on land, the maximum saturation of HbCO in the blood was 3.6%, and was 10.1% in the body cavity fluids. There was only one case in which the HbCO saturations in the body cavity fluids were more than 10%. In seven drowned bodies found in fresh water, the highest HbCO saturation in the blood was 6.1%, and was 44.1% in the body cavity fluids. There were three cases in which the HbCO saturations in the body cavity fluids were more than 10%. In 12 drowned bodies found in sea water, the HbCO saturations in the blood were not more than 6.2%, and the maximum saturation of HbCO in the body cavity fluids was 83.7%. There were eight cases in which the HbCO saturations in the body cavity fluids were more than 10%. The results seem to indicate that the interpretation of HbCO saturation in the blood would not be affected significantly by the postmortem formation of CO, and that body cavity fluids should not be used for CO determination.     

Computer-aided headspace gas chromatography applied to blood-alcohol analysis: importance of online process control

This paper describes the analysis of ethanol in blood specimens from suspect drunk drivers and the associated quality assurance procedures currently used in Sweden for legal purposes. Aliquots of whole blood from two separate Vacutainer tubes are diluted with 1-propanol as internal standard before analysis by headspace gas chromatography (HS-GC) with three different stationary phases: Carbopak B, Carbopak C, and 15% Carbowax 20 M. The actual HS-GC analysis, the integration of chromatographic peaks, the collection and processing of results, as well as the quality control tests involve the use of computer-aided techniques. The standard deviation of analysis(y) increased with concentration of ethanol in the blood specimen(x), and above 0.50 mg/g the regression equation was y = 0.0033 + 0.0153x. The prosecution blood-alcohol concentration (BAC) is the mean of three separate determinations made by different laboratory technicians working independently with different sets of equipment. A deduction is made from the mean analytical result to compensate for random and systematic errors inherent in the method. At BACs of 0.5 and 1.5 mg/g, which are the statutory limits in Sweden, the allowances currently made are 0.06 and 0.09 mg/g, respectively. Accordingly, the reduced prosecution BAC is less than the actual BAC with a statistical confidence of 99.9%.     

Accidental poisonings involving carbon monoxide, heating systems, and confined spaces

Eleven incidents of carbon monoxide (CO) intoxication resulting in sixteen fatalities are reported. All of the cases involved heating systems as either the source or the means of distributing the CO. Blood samples were analyzed for ethanol and CO. Elevated blood CO saturations were found in 14 of the 16 victims while none of the victims had a blood ethanol concentration greater than 0.10% (w/v).     

血液样品中乙醇稳定性的实验研究

目的考察在不同存放条件下含乙醇的血液样品中乙醇浓度的变化情况。方法采用顶空-气相色谱法,以异丙醇为内标,对存放条件不同的血液样品中乙醇进行检测。结果冷冻(-10℃)条件存放1至30天,血液样品中乙醇含量无显著变化;冷藏(4℃)条件存放1至30天,血液样品中乙醇含量变化不显著;室温(28℃~33℃)条件存放1至30天,血液样品中乙醇含量显著改变。结论含有乙醇的血液样品,在冷冻、冷藏条件下可较稳定的存放30天;在室温条件下存放30天血液样品中乙醇浓度发生显著变化,不可在此条件下存放。     

直接进样气相色谱法测定全血中乙醇的含量

目的建立一种直接进样气相色谱法检测全血中的乙醇。方法全血经硫酸铝钾沉淀蛋白,加入内标异丙醇后,用直接进样气相色谱法进行检测,FID为检测器,用保留时间定性,内标标准曲线法定量。结果该方法线性范围为0.2-1.4mg/m L,相关系数R=0.999 3,总分析时间不超过5min,最低检测限为0.01mg/m L,相对标准偏差（RSD）〈5%,平均回收率为92.3%;所建立的方法与顶空气相色谱（HS-GC）法比较相对偏差（RD）〈10%。结论该方法可用于全血中乙醇的检测。     

A simple spectrophotometric method for the determination of carboxyhemoglobin in blood

The spectrophotometric method for the determination of carboxyhemoglobin (HbCO) in blood reported by Fretwurst and Meinecke was modified so as to give the same values of percentage HbCO (HbCO%) as those determined by the oxygen electrode method. Values of HbCO% of nine practical samples determined by both the oxygen electrode method and the present method were nearly identical regardless of the presence of methemoglobin (Met-Hb) in blood. The present method is suitable for forensic practice.     

Suicidal and accidental carbon monoxide poisonings due to charcoal fires in closed spaces

The number of fatal carbon monoxide poisonings has significantly decreased since the introduction of natural gas. Apart from their occurrence during house fires, accidental and suicidal carbon monoxide poisonings are only rarely seen nowadays. We report on 7 cases of unintentional and intentional fatal carbon monoxide poisoning (CO-Hb concentrations between 56.4% and 74%) due to improper handling of charcoal fires in closed spaces. Autopsy findings and results of the toxicological analysis are presented and discussed.