[Simplified rapid test for poisons in emergency and critical care medicine].

In emergency and critical care medicine, it is important to guess which poisons that patients have taken or been exposed to. The assumption and identification of save lives. Therefore an accurate screening system is required to treat acute poisoning patients in clinical toxicology. However, the ability of a medical center is not sufficient to analyze poisonous substances using analytical equipment. Moreover, the handling and maintenance of the equipment are tedious and costly. To improve these problems, a simple detection method should be established to identify poisons and to treat acute patients in emergency and critical care medicine. In our laboratory, various supports have been attempted for the training of analysts who cope with poisoning incidents and accidents due to toxic substances. Moreover, a simple detection method for toxic substances utilized in the medical center was developed without using expensive analysis apparatus. However, it is impossible to detect and identify chemical warfare agents in a clinical laboratory, because of possible secondary exposure to such dangerous substances in insufficient analytical laboratory equipment. Therefore it is necessary to contact related organizations possessing the proper facilities.

Single hair analysis of methamphetamine and amphetamine by solid phase microextraction coupled with in matrix derivatization.

A sensitive method for detection of methamphetamine (MA) and amphetamine (AP) in human hair was developed using solid phase microextraction (SPME) and one-pot derivatization. MA and AP were directly derivatized to N-propoxycarbonyl derivatives in an aqueous solution by propylchloroformate in a one-pot reaction before extraction by SPME. The derivatives were extracted to a coating of SPME from a headspace of the vial. The adsorbed derivatives were thermally desorbed in the injection port of a gas chromatograph. Pentadeuterated MA was used as an internal standard. The absolute recoveries of MA and AP from the spiked hair were 2.80-17.5%, respectively. The calibration curves showed linearity in the range of 0.05-20 ng/0.08 mg/vial for MA and 0.1-20 ng/0.08 mg/vial for AP in hair. Detection limits (S/N = 3) of MA and AP were 0.02 and 0.05 ng/0.08 mg/vial. The coefficients of variation of intraday were 1.04-26.4%. Additionally, this proposed method was applied to segmental analysis in clinical and medico-legal cases of MA intoxication.

Kinetic characteristics and toxic effects of benzalkonium chloride following intravascular and oral administration in rats.

Kinetic characteristics and toxic effects of benzalkonium chloride (BZK) following injection via jugular vein (JV), femoral artery (FA) and oral administration (PO) were experimentally investigated using rats. The BZK concentrations in blood and tissues (lung, liver and kidney) were determined by high-performance liquid chromatography with solid phase extraction. Toxic doses of 15 and 250 mg/kg of BZK were used for intravascular (JV and FA) and PO administration, respectively. The fatal effects appeared soon after the dose in JV-rats, while delayed in FA- or PO-rats. The blood BZK concentrations and the elimination half-lives were similar between JV- and FA-rats, while the distribution of BZK in tissues was slightly different. In PO administration, the rats that aspirated BZK into their lungs had some symptoms, while the rats that did not aspirate BZK appeared to be normal. The BZK concentrations in blood and tissues were significantly higher in the aspirated PO-rats. The toxic degree of BZK was correlated with the BZK concentration in orally dosed rats. Lung and kidney had higher BZK concentrations compared to blood or liver, and they could be the target organs of BZK.Keyword: Benzalkonium chloride

[On-site screening of toxic substances in clinical laboratory and poisoning information system].

An accurate screening system is required to treat acute poisoning patients in clinical toxicology. However, the medical center analysis of poisonous substances using machines is not sufficient. Moreover, the handling and maintenance of such machines are tedious and costly. To improve these problems and employ effective information, we have developed a simple detection method and constructed a support system using the Internet. Various support systems have been attempted for the training of analysts who can cope with a poisoning incident (accident) involving toxic substances. Our simple detection method for toxic substances in the medical center was developed without using expensive analysis apparati. As technical support for the analysts of medical laboratories, the following items were completed: 1) training for analysts, 2) research of analytical techniques in the medical centers (accuracy management), 3) creation of an analysis manual, 4) construction of on-line analysis manuals, 5) construction of the poisoning information system on the Internet, 6) construction of a system for requesting analysis of poisoning, 7) a quick-detection method for toxic substances and 8) examination of the insurance application.

Miniaturized sample preparation method for determination of amphetamines in urine.

A simple and miniaturized sample preparation method for determination of amphetamines in urine was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). Urine was directly applied to the extraction column that was pre-packed with Extrelut and sodium carbonate. Amphetamine (AP) and methamphetamine (MA) in urine were adsorbed on the surface of Extrelut. AP and MA were then converted to a free base and derivatized to N-propoxycarbonyl derivatives using propylchloroformate on the column. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 100 and 102%, respectively. The calibration curves showed linearity in the range of 0.50-50 microg/mL for AP and MA in urine. When urine samples containing two different concentrations (0.50 and 5.0 microg/mL) of AP and MA were determined, the intra-day and inter-day coefficients of variation were 1.4-7.7%. This method was applied to 14 medico-legal cases of MA intoxication. The results were compared and a good agreement was obtained with a HPLC method.

[Development of salicylic acid detector tube].

A detector tube was successfully devised for the screening of salicylic acid in urine. It, named "salicylic acid detector tube", consists of glass tube in which silica gel coated with 5% (w/w) of ferric chloride is enclosed. A pipette rubber cap was attached to an end of the tube, and another end was inserted into urine sample. The sample was then introduced into the tube, the color of the reagent immediately turned purple under the condition of more than 50 microg/ml of salicylic acid in urine. This device was useful for the emergency screening of salicylic acid in acute poisoning cases with aspirin.

The levels of kerosene components in biological samples after repeated dermal exposure to kerosene in rats.

The current study was experimentally investigated using rats whether or not kerosene components are accumulated from daily repeated dermal exposure. Rats received daily 1h-exposure to kerosene for 5 days (5K), daily 1h-exposure for 4 days and left for 1 day (4KL), a single 1h-exposure (1K), a single 1h-exposure and left for 1 day (1KL), or a single 1h-exposure, sacrificed and left dead for 1 day (1KLD). Kerosene components, trimethylbenzenes (TMBs) and aliphatic hydrocarbons (AHCs) in blood and tissues were determined by GC-MS. In blood, almost the same concentrations of TMBs were detected in the rats sacrificed immediately after exposure (5K, 1K and 1KLD), and only trace levels were detected in the rats sacrificed 1 day after exposure (4 and 1KL). Almost the same levels of AHCs in blood were detected among groups except for the rats sacrificed 1 day after a single exposure (1KL), in which AHCs were slightly lower. These results suggest that (1) AHCs tend to be accumulated from daily exposure, while TMBs do not, (2) the proportions of detected kerosene components in blood can be an indicator of whether the last exposure occurred just before death or not, (3) the kerosene levels last at least 1 day without blood circulation.

Distribution and disposition of benzalkonium chloride following various routes of administration in rats.

Benzalkonium chloride (BZK) is a cationic surfactant used widely as a disinfectant, preservative and sanitizer in hospitals, at home and many public places. The toxicity of BZK is not well established although several human fatalities have been reported over the years. In this study, distribution and disposition of BZK following oral administration (PO) and intravascular (jugular vein (JV), femoral artery (FA), femoral vein (FV) and jugular artery (JA)) administration in rats were investigated along with pathological examinations. Toxic doses of 250 and 15 mg/kg of BZK were used for PO and intravascular administration, respectively. The fatal effects of BZK appeared soon in JV-, FV- or JA-rats, but took hours in PO or FA-rats. No rat receiving BZK via FA survived longer than 1 day. The PO-rats that aspirated BZK into their lungs had some systemic symptoms and higher blood and tissue concentrations of BZK. The blood BZK levels and kinetics were similar among the different routes of intravascular administration, but the lung and kidney levels were higher in JV-rats. Pathological examinations confirmed severe congestion and edema in the lungs and kidneys. These results suggest that (1) the toxic effects of BZK varied depending on the route of administration, (2) the degree of toxicity correlated with peak blood and tissue concentrations in orally dosed rats, (3) different toxicological progressions and manifestations were observed in FA- and JV-dosed rats even though these groups had similar blood concentration profiles, and (4) lung and kidney are reservoirs for BZK and considered to be the target organs of BZK.

Skin analysis following dermal exposure to kerosene in rats: the effects of postmortem exposure and fire.

To evaluate the usefulness of skin analysis for the forensic examination of cases involving postmortem dermal exposure to kerosene and/or fire, an experimental study using rats was performed. Rats received dermal exposure to kerosene before or after death, and the effect of fire was determined by burning an area of exposed skin after death. Kerosene concentrations in skin and blood were determined by gas chromatography-mass spectrometry and microscopic observation was performed for skin samples. No differences were observed in skin kerosene levels between antemortem and postmortem exposure. Kerosene concentrations in mildly burned skin where the stratum corneum (SC) was retained were approximately 84% compared to those in non-burned exposed skin, whereas concentrations in severely burned skin where the SC was almost completely burned off were 28% of non-burned skin. Even in non-exposed control skin 14% of the original kerosene concentrations could be detected, which was considered to be caused by contamination during the experimental protocol combined with kerosene's property of a high affinity for the SC. These results suggest that (1) skin analysis is useful in estimating the type of petroleum product involved in crimes or accidents even for postmortem exposure, (2) whether the SC is retained or not primarily determined the kerosene levels in burned skin, and (3) attention must be paid to evaluate the results obtained from skin samples in the light of the circumstances surrounding the case.

Dermal absorption of kerosene components in rats and the influence of its amount and area of exposure.

The influences of amount and area of dermal exposure to kerosene upon the levels of kerosene components in biological samples were examined in vivo and in vitro. Thirty-two rats were randomly divided into four groups and exposed to kerosene through the abdominal skin for 2h. The amounts (soaked in cotton) and area of kerosene exposed were 1 ml/4 cm(2) in Group I, 4 ml/4 cm(2) in Group II, 4 ml/16 cm(2) in Group III and 16 ml/64 cm(2) in Group IV. Before, then 5, 10, 20, 30, 45, 60, 90 and 120 min after exposure, 0.5 ml of blood was collected. Solid tissue samples, including the exposed skin area, were harvested at 120 min. Kerosene components were analyzed by gas chromatography/mass spectrometry. Trimethylbenzens (TMBs) that are easily absorbed kerosene components, appeared at 5-20 min. The time course changes in TMB levels in blood were significantly different between Groups I and II or Groups I and III, and almost identical between Groups II and III. Similar trends were observed in tissue samples at 120 min. High concentrations of aliphatic hydrocarbons (AHCs) were detected in the exposed skin and the AHC levels were dependent on the amount of kerosene exposed per unit area. These results suggest that (1) dermal absorption of kerosene occurs soon after dermal exposure started, (2) absorption of TMBs is influenced by the total amount of kerosene rather than area of exposure, and (3) AHCs remaining in the skin at significant levels are influenced by the amount of kerosene per unit area exposed.

Simple method of methylation for gas chromatographic analysis of S-benzyl-N-acetylcysteine, a metabolite of toluene, in human urine.

A simplified method of methylation for the determination of urinary S-benzyl-N-acetylcysteine (benzylmercapturic acid, SBAC), a metabolite of toluene, by gas chromatography (GC) was developed. Acidified urine samples (pH 2) were extracted once with ethyl acetate and then derivatized to methyl ester (ME) using HCl-methanol. The optimum conditions for derivatization for SBAC and the internal standard (S-phenethyl-N-acetylcysteine) were reaction at 60 degrees C for 20 min. The SBAC-ME was measured by capillary GC. The calibration curve showed good linearity over the range of 0.2 to 5.0 mg/L (r = 0.986). This method was compared with a previously developed diazomethane methylation method for testing urine from subjects who had sniffed toluene. The values obtained by the two different methods were in good accordance. These results suggest that this technique for the methylation of SBAC by means of HCI-methanol is simpler and time-saving, thus making it feasible to determine SBAC and other mercapturic acids in urinary samples obtained from subjects who have been exposed to organic solvents.

Sensitive determination of benzalkonium chloride in blood and tissues using high-performance liquid chromatography with solid-phase extraction.

A sensitive and simple high-performance liquid chromatography assay for benzalkonium chloride (BZK) in biological samples was developed. The biological samples, spiked with domiphen used as an internal standard, were purified by solid-phase extraction. The major homologues of BZK in pharmaceutical products (C(12) and C(14)) were eluted at 24 and 36 min using a YMC-Pack CN column (4.6 x 250 mm, 5 microm) with a mobile phase, mixture of acetonitrile and sodium acetate buffer (48:52), and monitored at 254 nm. The most dominant component C(12) was selected as an indicator to quantify BZK, and adjustment was then done based on the proportion of C(12) in the BZK product. Good linearity was obtained in the range of 0.1-3 microg, and the limit of detection was 20 ng as a loaded amount on column. The recoveries of BZK in serum and tissues ranged from 54 to 90%. In a practical case, 0.16 microg/ml of BZK was quantified in serum collected several hours after accidental ingestion. The method is simple, sensitive and reliable for determining BZK levels in practical biological samples.