| Co-Investigator(Kenkyū-buntansha) |
HIRATA Yukari Fujita Health University, School of Medicine, Research Associate, 医学部, 助手 (50156676)
KATSUMATA Yoshinao Nagoya University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (30109326)
SUZUKI Osamu Hamamatsu University, School of Medicine, Professor, 医学部, 教授 (70093044)
KANEKO Rina Fujita Health University, School of Medicine, Research Associate, 医学部, 助手 (70367697)
HAMAJIMA Makoto Fujita Health University, School of Medicine, Research Associate, 医学部, 助手 (20189608)
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| Research Abstract |
In 1998, an extraordinary murder incidence took place in Wakayama ; 4 victims were killed. After the incidence, a lot of imitative poisoning cases occurred. Several inorganic compounds, such as arsenic, cyanide or azides, were used in the poisoning cases. In the case of sodium azide, it was then regulated as a poison by the Poisonous and Deleterious Substances Control Law in 2000. It is thus important to screen these inorganic compounds rapidly with less labor. Ion chromatography (IC) has been widely used for the separation of inorganic cations and anions ; IC is easy to use and highly quantitative, although IC cannot identify and specify these inorganic ions. So far, IC has been applied mainly for environmental samples, such as surface water samples. In this project, we have tried to develop simple, rapid and robust methods for the analyses of inorganic ions in the biological samples by IC.
Sodium azide is widely used as preservative for laboratory reagents. However, it is highly toxic
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and the intake of about I g sodium azide would be fatal. In rat, its oral LD_<50> value is estimated to be 45 mg/kg, which is only 5 times greater than that potassium cyanide. Thus we have developed a simple method for analysis of sodium azide. We tried several methods for purification of azide from biological samples ; it was revealed that Conway diffusion method would be simple and fast. Azide ion in various samples was extracted using a Conway microdiffusion cell ; hydrazoic acid was vaporized from 1 mL sample (plasma or beverages) by adding 1 mL of 5% sulfuric acid in the outer groove, and absorbed in 250 μL of 0.1 M NaOH solution in the central round basin. Incubation for 30 min at 37℃ was sufficient for the purification of azide from biological samples. For IC analysis, a 20-μL aliquot was subjected to IC system equipped with a suppressor and a conductivity detector (Dionex, DX 500 system). The precolumn and separation column used were an AG 15 (20 mm × 2 mm i.d.) and an AS 15 (250 mm × 2 mm i.d.) semimicrocolumns. The mobile phase was 38 mM NaOH and the flow rate was set at 0.4 mL/min. The retention time of azide ion was about 10 mm. Combination of the reduction of NaOH solution in the Conway diffusion method and the use of semimicrocolums, has enabled 4 to 5 times higher sensitivity compared to methods previously reported ; the detection limit for serum samples was about 30 ng/mL Its calibration curve showed good linearity in the range of 50 ng/mL and 10 μg/mL serum We have validated this method; the CV values for intra-day and day-to-day measurements were less than 6.8 % and 93 %, respectively. In the case of beverages, Comway diffusion method was needed for accurate quantitaion because of the interference of some impurity peaks. Azide ion was detected from rat serum samples 1 or 4 hours after its oral administration (10 mg/kg). Thus the present method can be applicable for clinical and forensic toxicology, because of its simplicity and sensitivity. We are now preparing a paper for submitting. In the next research, we are going to obtain data about the decay of azide ion of different samples under several environmental conditions. Less
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