Direct carboxyhemoglobin determination by derivative spectroscopy

A fast and sensitive method for the determination of carboxyhemoglobin (COHb) in blood using derivative spectroscopy is described. The addition of sodium dithionite as a reducing agent is not required. The concentration interval studied was from 0.5% to 100% COHb.     

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.     

血中碳氧血红蛋白饱和度测定影响因素的研究

目的 考察血中碳氧血红蛋白饱和度(HbCO％)测定的影响因素,为其结果评定和所需样品保存条件提供实验依据。方法 利用三种分光光度法,测定30d内不同条件下保存的CO阳性血的HbCO％的变化。结果 还原双波长法、双波长法测定结果比较稳定,单波长法抗干扰能力较差;尸检所取血样的保存条件包括温度、保存时间及与空气接触程度对HbCO％的测定均有影响,其中温度影响较为显著。结论 利用还原双波长法与双波长法,并结合光谱扫描观察峰形变化可得到比较可靠的结果。30d内4℃条件下,密闭容器中血样接触少量空气不影响其HbCO％的测定。     

A practical method for the accurate determination of methemoglobin in blood containing carboxyhemoglobin

Kinetics of the oxidation of carboxyhemoglobin (HbCO) by potassium ferricyanide was studied photometrically in a weakly acid solution. An increase in the absorbance at 630 nm reached a maximum within 10 min when over a 100-fold excess of ferricyanide to hemoglobin iron was used. A slight decrease in the absorbance was observed after completion of the reaction when over a 500-fold excess of the reagent was used. In the presence of 0.4% Sterox SE, the absorbance began to decrease without complete oxidation. From these findings, a simple, rapid and accurate method for the determination of methemoglobin (Met-Hb) in blood was devised. The method was compared with two other methods, using 11 blood samples containing various amounts of HbCO, and proved to be suitable for blood containing elevated HbCO as well as for ordinary blood.     

False positive reaction for carboxyhemoglobin in blood from liver tissue

On spectrophotometric determination of carboxyhemoglobin in blood collected from the liver of three bodies at three days post-mortem, false positive results were found (5 – 15 per cent saturation), since samples of heart blood collected a few hours after death did not contain carboxyhemoglobin.     

A comparison of carboxyhemoglobin saturation values in postmortem heart blood and peripheral blood specimens

The following is a study conducted to determine whether there was any significant difference in carboxyhemoglobin (COHb) saturation levels between the heart blood and blood collected from a peripheral site. The average heart blood to peripheral blood COHb saturation level ratio in the 42 cases studied was 1.09. Sixty-two percent (26 of 42) of the cases had a heart blood to peripheral blood ratio between 0.9 and 1.1; 74% (31 of 42) had a ratio between 0.8 and 1.2. Eighty-three percent (35 of 42) had a ratio between 0.7 and 1.3. There were four cases where the heart blood to peripheral blood ratio was either below 0.6 or greater than 1.4. The differences between the two sites were not statistically significant.     

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.     

Rapid detection of caffeine in blood by freeze-out extraction

A new method for the detection of caffeine in blood has been proposed based on the combination of extraction and freezing-out to eliminate the influence of sample matrix. Metrological characteristics of the method are presented. Selectivity of detection is achieved by optimal conditions of analysis by high performance liquid chromatography. The method is technically simple and cost-efficient, it ensures rapid performance of the studies.     

Automated determination of carboxyhemoglobin contents in autopsy materials using head-space gas chromatography/mass spectrometry

To establish a method for the routine analysis of carboxyhemoglobin (COHb) in autopsy materials including those which have undergone postmortem changes, e.g. thermo-coagulation, putrifaction and contamination, an automated head-space gas chromatography/mass spectrometry (GC/MS) analysis was utilized. The procedure consisted of preparation of the sample in a vial and a carbon monoxide (CO) saturated sample, for estimation of hemoglobin content, in another vial, the addition of n-octanol, potassium ferricyanide and an internal standard (t-butanol), GC separation and determination of CO using a GC/MS system equipped with an automated head-space gas sampler. The method was practical not only with the blood and bone marrow aspirates to confirm the findings on the CO-oximeter system, but also with the thermo-coagulated and putrified blood.     

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.     

Changes in the concentration of carboxyhemoglobin in the blood under the action of high temperatures

Changes in carboxyhemoglobin concentrations are studied in 18 specimens of cadaveric blood 1-2 h and 3 days after heating to 50, 65, 70, 75, 80, and 90 degrees C for 5, 10, and 20 min. The concentration of carboxyhemoglobin decreased to 15% after heating at 70-75 degrees C for 10 and 20 min. Heating to 80-90 degrees makes the measurements impossible. Five-min heating at 50-65 degrees C did not change the concentration of carboxyhemoglobin in the blood.     

血液样品中防腐剂对碳氧血红蛋白稳定性的影响

目的研究临床上常用8种试剂对血液样品中碳氧血红蛋白(HbCO)稳定性的影响。方法将血液样品分为高、低两个HbCO浓度组,选用临床常用的甲醛、氟化钠、乙二胺四乙酸二钠、亚硝酸钠、草酸钾、肝素钠、柠檬酸钾及氟化钠与草酸钾混合物(1:3)8种试剂,按常用浓度分别添加到血样品中,并于添加后0h、2h、8h、24h、3d、7d用紫外可见光分光光度法检测其中HbCO饱和度,用统计学方法进行结果分析。结果本实验选用的8种试剂只有甲醛和亚硝酸钠对HbCO的稳定性影响较为显著,而其余6种对HbCO稳定性的影响无统计学意义。结论在检验疑似CO中毒并经甲醛或亚硝酸钠防腐的检材时应慎重,以免导致错误的鉴定结论。     

Rapid determination of flunitrazepam in alcoholic beverages by desorption electrospray ionization-mass spectrometry

Desorption electrospray ionization-mass spectrometry (DESI-MS), a novel ambient ionization technique, was used in this study for determining flunitrazepam in various alcoholic beverages. Using this technique, no pretreatment of the samples was necessary and identification of the drug was accomplished in individual samples in minutes. In addition, the acquired mass spectra provide the information of the identity of the drink based on the detected characteristic ions from the matrices. This study also demonstrates the capability of DESI-MS to perform quantitative analysis of simulated evidence samples with a limit of quantification of 3μg/mL. Furthermore it has been shown that this method can be used for high-throughput analysis whereby six samples were analyzed in a row within 6 minutes and no observable sample carry-over was noted. DESI-MS shows potential as a rapid, sensitive, and selective technique for forensic analysis of spiked beverages which are typical evidence of drug facilitated sexual assault and robbery cases.     

Analysis of carboxyhemoglobin and cyanide in blood from victims of the Dupont Plaza Hotel fire in Puerto Rico

Ninety-seven people died from a fire that occurred in the Dupont Plaza Hotel in Puerto Rico on 31 Dec. 1986. All, except four who died later in the hospital, were found dead at the scene. All of the fatalities at the hotel (except for eight) were burned beyond recognition. Blood from seventy-eight of the victims was screened for carboxyhemoglobin at the Institute for Forensic Sciences in Puerto Rico and was then sent to the National Institute of Standards and Technology, Gaithersburg, Maryland, for analysis of carboxyhemoglobin and cyanide concentrations. The blood data indicated that carbon monoxide and hydrogen cyanide, singly or combined, were probably not responsible for the majority of the deaths that occurred in the badly burned victims. On the other hand, the significantly higher carboxyhemoglobin in the nonburned victims indicated that carbon monoxide alone or combined with hydrogen cyanide probably played a major role in the cause of their deaths.     

Rapid determination of ethylene glycol in biological material

A rapid and simple modification of a gas chromatographic (AFID) method of ethylene glycol determination, based on derivation with n-butylboronic acid, was developed.