Keiko Kudo

Determination of cyanide and thiocyanate in blood by gas chromatography and gas chromatography-mass spectrometry

We devised a sensitive and simple method for determining cyanide and its major metabolite, thiocyanate, in blood using an extractive alkylation technique. Pentafluorobenzyl bromide was used as the alkylating agent, and tetradecyldimethylbenzylammonium chloride was used as the phase-transfer catalyst. The derivatives obtained were analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with an electron-capture detection. The detection limits of cyanide and thiocyanate were 0.01 and 0.003 mumol/ml, respectively, while the gross recovery of both compounds was 80%. The calibration curve was linear over the concentration range from 0.02 to 1.0 mumol/ml for cyanide and from 0.01 to 1.0 mumol/ml for thiocyanate. The accuracy and precision of the method were evaluated, and the coefficients of variation were found to be within 10%. Using the method, the blood levels of two victims who had died from cyanide poisoning were determined.

Carbonization of pitches—IV: Carbonization of polycyclic aromatic hydrocarbons under the presence of aluminum chloride catalyst

Abstract Using aluminum chloride as a catalyst, polycyclic aromatic hydrocarbons were converted into carbons of two different structures, although at the early stage all carbonization processes proceeded via molten phase. The structures were examined with an optical microscope under crossed nicois. The first type of structure was a flow pattern with large oriented needle like domains observed for carbons obtained from naphthalene, chrysene, and anthracene, whereas the second one was a mosaic structure for carbon from pyrene. A significant subsequent graphitization at 2500°C (as expressed by Lc) took place for both types regardless of the different size of oriented domains. The orientation of domains depends on the rate of carbonization controlled by the holding temperature, time and quantity of the catalyst. Appropriate conditions of carbonization suppressing too fast generation of nuclei so as to permit the formation of ordered arrangement of condensed molecules were found to yield a needle-like coke also from pyrene, thus showing that the passing through a molten phase is not a sufficient condition for formation of a needle-like structure.

The usefulness of thiosulfate as an indicator of hydrogen sulfide poisoning: three cases

Abstract We examined the usefulness of thiosulfate as an indicator of hydrogen sulfide poisoning by analysing sulfide and thiosulfate in three cases. In the first (non-fatal) case sulfide and thiosulfate were not detected in the blood samples from any of the four workers involved in the accident. In the urine samples, only thiosulfate was detected in three out of the four workers at a concentration of 0.12–0.43 μmol/ml, which was 4–14 times higher than the level in a healthy person. In the second (fatal) case sulfide and thiosulfate were detected in the blood sample at concentrations of 0.007 μmol/ml for sulfide, and 0.025 μmol/ml for thiosulfate. The thiosulfate concentration was at least 8 times higher than the level in a healthy person. In the third (fatal) case sulfide and thiosulfate were detected in the blood sample at concentrations of 0.95 μmol/ml for sulfide, and 0.12 μmol/ml for thiosulfate. Based on the above results, we concluded that thiosulfate in urine is the only indicator to prove hydrogen sulfide poisoning in non-fatal cases, while the analysis of sulfide in fatal cases should be accompanied by the measurement of thiosulfate in blood.

Sulfide concentrations in postmortem mammalian tissues.

Postmortem changes in sulfide concentrations in body tissues were examined in autopsied rats exposed to hydrogen sulfide concentrations of 550 to 650 ppm, and in nonexposed rats and humans. Analyses were made by gas chromatography, following an extractive alkylation. Sulfide concentrations in the blood, liver, and kidneys of rats increased in both the exposed and nonexposed groups, depending on the lapse of time after death. On the other hand, the lung, brain, and muscle showed little or no change in sulfide concentration with elapse of time after death. The data obtained from human tissues were almost the same as those for rats, except data for blood, in which no or little increase of sulfide was observed.

Determination of nitrate in blood by gas chromatography and gas chromatography–mass spectrometry

We devised a sensitive and simple method for determining nitrate in whole blood, using an extractive alkylation technique. Nitrate in whole blood was reduced to nitrite by hydrazine sulfate in the presence of Cu2+ and Zn2+ as catalysts, and alkylated with pentafluorobenzyl bromide using tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst. The obtained derivative was analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with electron-capture detection. The detection limit of nitrate in whole blood was 0.01 mM. The calibration curve was linear over the concentration range from 0.02 to 1.0 mM for nitrate in whole blood. The accuracy and precision of the method were evaluated and the relative standard deviations were found to be within 10%. Using this method, the blood nitrate levels of two victims who committed suicide by inhaling automobile exhaust gas were determined.

Pattern of poisoning in Japan: selection of drugs and poisons for systematic toxicological analysis

Patterns of poisoning are known to be different in different countries, because of the local environmental, cultural, and religious situations. Therefore, in Japan, it is important to know the pattern of poisoning in our own country and to prepare for every poisoning case by establishing an efficient systematic toxicological analysis system in forensic practice. We conducted a retrospective study of the kinds of compounds causing poisonings and the frequency of their use based on two series of reports dealing with poisoning cases in Japan prepared by the National Research Institute of Police Science and the Japanese Society of Legal Medicine for 2003 to 2006. From these reports, 459 and 177 compounds, respectively, were extracted as poisonous compounds over the study period. After data analysis, we selected 314 drugs and poisons as important target compounds for systematic drug analysis in Japan; they included 36 volatile compounds, 14 abused drugs, 170 medical drugs, 60 pesticides, 13 natural toxins, and 21 others. This is the first study to show the toxic drugs and poisons to be analyzed in Japan based on frequency of use, and as such the list will be useful in establishing the most efficient screening system in forensic practice.

Methamphetamine and amphetamine concentrations in postmortem rabbit tissues

The feasibility of detecting methamphetamine and its major metabolite, amphetamine, in postmortem tissues over a 2-year period was examined. It is important to determine if the abuse and toxic effects of drugs can be proved from evidence found in decayed, submerged, or stained tissue materials. The blood, urine, liver, skeletal muscle, skin and extremity bones from rabbits given methamphetamine intravenously were kept at room temperature, under 4 different conditions: sealed in a test tube, dried in the open air, submerged in tap water and stained on gauze. Methamphetamine was present in all the samples, with slight change in concentration in case of sealed and air dried tissues. Changes varied in bones kept in water. There were considerable decreases in methamphetamine in blood and urine stains. Despite long term storage, drug abuse and/or toxicity could be determined, in all tissues examined.

Extractive Alkylation and Gas Chromatographic Analysis of Sulfide

A sensitive analysis of sulfide in blood was established, using an extractive alkylation technique. Pentafluorobenzyl bromide was used as the alkylating agent, tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst, and potassium dihydrogenphosphate as the buffer to suppress the formation of sulfide. Mass fragmentography was used to identify the sulfide derivative and gas chromatography with an electron capture detector was used for quantitative determination, with the lowest limit of detection being about 0.01 microgram/g. The blood level of rats exposed to hydrogen sulfide was also determined.

Construction of calibration-locking databases for rapid and reliable drug screening by gas chromatography-mass spectrometry

Unique calibration-locking databases were constructed for rapid and semiquantitative drug screening by gas chromatography-mass spectrometry (GCMS). In addition to the free-drug database of 127 drugs, a drug database with acetylating reagents was constructed to increase the number of detectable compounds in the analysis by GC-MS; 156 drugs, including 30 drugs of abuse, 42 hypnotics and their metabolites, 18 antipsychotic drugs, 15 antidepressants, and 12 antipyretic analgesic agents, were registered with parameters, such as the mass spectrum, retention time, qualifier ion/target ion percentage, and calibration curve using the novel GC-MS software NAGINATA. Diazepam-d5 was used as internal standard for construction of each calibration curve in the range of 0.01–5.0 μg/ml for most drugs. We examined the applicability of the constructed database to analyzing whole blood samples spiked with 40 drugs most commonly encountered in toxicological cases in Japan. The drugs in blood were extracted using enhanced polymer columns (Focus), subjected to GC-MS after incubation with acetylating reagents, and screened by the drug database. Among the 40 drugs examined, 38 and 30 drugs were successfully identifi ed at the level of 1 and 0.1 μg/ml, respectively, without using standard compounds. The time required for data analysis was less than 1 min, and semiquantitative data were also obtained simultaneously. Because new drugs and metabolites can easily be added to the databases, we can recommend them as useful tools in clinical and forensic toxicological screening.

Fatal and nonfatal poisoning by hydrogen sulfide at an industrial waste site.

An adult man (A) entered a pit to collect seepage at an industrial waste site in Japan. As he suddenly lost consciousness, three colleagues (B, C, D) entered the pit to rescue him. All of these men lost consciousness in the pit. Two workers (A and B) died soon after the accident, one worker (C) died 22 days after the accident, and one worker (D) survived. Since hydrogen sulfide gas was detected in the atmosphere of the pit, gas poisoning was suspected. Toxicological analyses of sulfide and thiosulfate, a metabolite of sulfide, in blood and urine of the victims were made using the extractive alkylation technique combined with gas chromatography/mass spectrometry (GC/MS). Sulfide was detected in the blood of A and B at levels of 0.13 and 0.11 mg/L, respectively, somewhat higher than in healthy persons. Thiosulfate was detected in whole blood of deceased victims A and B, in the plasma of deceased victim C, at concentrations of 10.53, 4.59, and 4.14 mg/L, respectively. These values were similar to those found in fatal cases of hydrogen sulfide poisoning. Thiosulfate was not detected in the plasma of survivor D. With respect to urine samples, thiosulfate was the highest in the non-acute death victim C (137.20 mg/L), followed by that in the survivor D (29.34 mg/L), and low (0.90 mg/L) and not detected in the acute death victims, A and B, respectively. Based on these results, all four patients were victims of hydrogen sulfide poisoning. The concentrations of thiosulfate in blood and urine were more useful than that for sulfide for determining hydrogen sulfide poisoning. Thiosulfate in urine was the only indicator of hydrogen sulfide poisoning in the non-fatal victim.