Highly sensitive analysis of methamphetamine and amphetamine in human whole blood using headspace solid-phase microextraction and gas chromatography-mass spectrometry.

A simple and highly sensitive method for analysis of derivatized methamphetamine (MA) and amphetamine (AM) in whole blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry electron impact ionization selected ion monitoring (GC-MS-EI-SIM). A whole blood sample, deuterated-MA (d(5)-MA), as an internal standard (IS), tri-n-propylamine and pentafluorobenzyl bromide were placed in a vial. The vial was heated and stirred at 90 degrees C for 30min. Then the extraction fiber of the SPME was exposed at 90 degrees C for 30min in the headspace of the vial while being stirred. The derivatives adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC-MS. The calibration curves showed linearity in the range of 0.5-1000ng/g for both MA and AM. The time for analysis was about 80min per sample. In addition, this proposed method was applied to two autopsy cases where MA ingestion was suspected. In one case, MA and AM concentrations in the mixed left and right heart blood were 165 and 36.9ng/g, respectively. In the other case, MA and AM concentrations were 1.79 and 0.119 microg/g in the left heart blood, and 1.27 and 0.074 microg/g in the right heart blood, respectively.

Simple and simultaneous analysis of fenfluramine, amphetamine and methamphetamine in whole blood by gas chromatography-mass spectrometry after headspace-solid phase microextraction and derivatization.

A simple and sensitive method for the simultaneous analysis of fenfluramine, amphetamine and methamphetamine in whole blood was developed using a headspace-solid phase microextraction (SPME) and derivatization. A 0.5 g whole blood sample, 5 microl d(5)-methamphetamine (50 micrig/ml) as an internal standard, and 0.5 ml sodium hydroxide (1 M) were placed into a 12 ml vial, and sealed rapidly with a silicone septum and an aluminum cap. Immediately after the vial was heated to 70 degrees C in an aluminium block heater, the needle of the SPME device was inserted through the septum of the vial, and the extraction fiber was exposed in the headspace for 15 min. First, heptafluorobutyric anhydride was injected into the injection port of the GC-MS, and the compounds extracted by the fiber were then desorbed and derivatized simultaneously by exposing the fiber in the injection port. The calibration curves, using an internal standard method, demonstrated good linearity throughout the concentration range from 0.01 to 1.0 microg/g. The detection limits of this method were 5.0 ng/g for fenfluramine and methamphetamine, and 10 ng/g for amphetamine. No interferences were found, and the time for analysis was about 30 min for one sample. This method was applied to a suicide case in which the victim ingested fenfluramine. Fenfluramine was detected in the blood sample collected from the victim at the concentration of 7.7 microg/g.

Sensitive determination of pentazocine in human tissues by high-performance liquid chromatography.

The authors developed a simple, reliable and sensitive method for the determination of pentazocine in human solid tissues using high-performance liquid chromatography, combined with a three-step liquid-liquid extraction procedure. Levallorphan tartrate served as the internal standard. The extract was evaporated to dryness and dissolved in the mobile phase of acetonitrile/10 mM phosphate buffer (pH 4.0). The eluent was pumped at a flow rate of 0.4 ml/min through a Spherisorb Ph (2.1 mm I.D. x 150 mm) column. A fluorescence detector with excitation at 247 nm and emission at 320 nm was used. The lower limit of detection was about 0.5 ng/g. The calibration curve was linear over the concentration ranges from 1 to 500 ng/g in each tissue examined and could be determined up to at least 10.0 microg/g by means of reduction of injected volumes. Using this method, the concentrations of pentazocine could be determined in the tissues of an autopsied individual for toxicological evaluation.

Traumatic avulsion fracture of the occipital condyles and clivus: a case report.

A 25-year-old male was found dead on a river bank. Wounds and injuries were found on the corpse, and a bloodstain was found on the face from blood discharged from the oral and nasal cavities. Since the man was considered the victim of a traffic accident, a forensic autopsy was carried out. At the time of autopsy, severe bruises and wounds were found on the occiput, the nape and the dorsal side of the lower extremities. There was a fracture line in the bilateral occipital condyles through the lower part of the clivus and the head was unstable in relation to the neck. The medulla oblongata was severed near the fracture site and the basilar artery was also torn at the branching end of the posterior cerebral arteries. The fracture of the occipital condyles and clivus was considered to be avulsed due to hyperflexion of the neck joint by a blunt impact onto the occiput. Anterior traction of the skull following the fracture was supposed to have accompanied the injuries of the medulla oblongata and the basilar artery. In this autopsy case, the authors observed an unusual type of fracture in the posterior cranial fossa.

Direct colorimetric method for determination of organophosphates in human urine.

A simple and sensitive method for determination of organophosphorus pesticides in human urine was developed by detecting the color complexes which resulted from reactions of organophosphorus pesticides and 4-(4-nitrobenzyl)pyridine (NBP) in urine. Based on studies of reaction conditions, e.g. reaction temperature and time, and reagent concentration, a colorimetric method was established. A 0.1-ml volume of NBP (45% in acetone) was added to a 1.0-ml volume of a urine sample, and the mixture was heated at 100 degrees C for 20 min. After cooling, 0.1 ml of tetraethylenepentamine was added. The organophosphorus pesticides showed a characteristic purplish blue color and the coloring complexes which were produced were stable for several hours. Furthermore, these complexes could be determined spectrophotometrically. The detection limits were 0.10-10 microg/ml in urine. The required time for analysis was approximately 30 min for one sample. Comparing the result of the proposed method with those of the GC-MS method, the results were similar for the 12 poisoning cases studied. Thus, the proposed method is useful for detection of these pesticides in critical care practices.

Liquid chromatography-fast atom bombardment mass spectrometry for detection and determination of pentazocine in human tissues.

A reliable and sensitive method was developed for the detection and determination of pentazocine in human solid tissues using liquid chromatography-dynamic fast atom bombardment (FAB) mass spectrometry, combined with a three-step liquid-liquid extraction procedure. Levallorphan tartrate served as an internal standard. The extract was evaporated to dryness and dissolved in the mobile phase, acetonitrile-10 mM ammonium acetate solution (20:80, pH 4.0) containing 0.5% glycerol as FAB matrix. The eluent was pumped at a flow rate of 25 microl/min and split before introduction to FAB mass spectrometer. Quantitative analysis was carried out by means of monitoring quasi-molecular ions with m/z 286 for pentazocine and m/z 284 for levallorphan. The lower limit of detection of pentazocine in each tissue tested was 1 ng/g with scan mode and 0.1 ng/g with SIM mode. Using this method, the concentrations of pentazocine were determined in the tissues of an autopsied individual to perform toxicological evaluation.

Simple and sensitive analysis of nereistoxin and its metabolites in human serum using headspace solid-phase microextraction and gas chromatography-mass spectrometry.

A simple method for the analysis of nereistoxin and its metabolites in human serum using headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) is developed. A vial containing a serum sample, 5M sodium hydroxide, and benzylacetone (internal standard) is heated to 70 degrees C, and an SPME fiber is exposed for 30 min in the headspace of the vial. The compounds extracted by the fiber are desorbed by exposing the fiber in the injection port of the GC-MS. The calibration curves show linearity in the range of 0.05-5.0 micrograms/mL for nereistoxin and N-methyl-N-(2-methylthio-1-methylthiomethyl)ethylamine, 0.01-5.0 micrograms/mL for S,S'-dimethyl dihydronereistoxin, and 0.5-10 micrograms/mL for 2-methylthio-1-methylthiomethylethylamine in serum. No interferences are found, and the analysis time is 50 min for one sample. In addition, this proposed method is applied to a patient who attempted suicide by ingesting Padan 4R, a herbicide. Padan 4R contains 4% cartap hydrochloride, which is an analogue of nereistoxin. Nereistoxin and its metabolites are detected in the serum samples collected from the patient during hospitalization. The concentration ranges of nereistoxin in the serum are 0.09-2.69 micrograms/mL.

Determination of acrolein in human urine by headspace gas chromatography and mass spectrometry.

A rapid and sensitive headspace gas chromatographic and mass spectrometric (GC-MS) method was developed for the determination of acrolein in human urine. A 0.5-ml urine sample in a glass vial containing propionaldehyde as an internal standard was heated at 80 degrees C for 5 min. A 0.1-ml volume of headspace vapor was injected into a GC-MS instrument. Acrolein and propionaldehyde were coeluted at 3.1 min using a DB-1 capillary column, and well separated by selective ion monitoring (SIM) mode using ions m/z 56.05 and m/z 58.05. The interassay and intraassay coefficient of variation were 0.99% and 3.3%. The calibration curve demonstrated a good linearity throughout concentrations ranging from 1 to 1000 nM. However, due to a wide variation of acrolein evaporation rates from human urine, a calibration curve must be established for each urine specimen using a standard addition method and detection limit varied from 1 to 5 nM. The total analysis time for two samples from one urine specimen required about 15 min. Therefore, this method is convenient for the urgent monitoring of urinary acrolein in patients to whom alkylating agents are administered.

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography-mass spectrometry.

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC-MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C18 disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol-water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform-isopropanol (3:1, v/v) from the cartridge. The eluate (1 microl) was injected into the GC-MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 microg ml(-1) for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.

[Difficulty of getting correct dental findings for a doctor of medicine: deciduous molars found in a 16-year-old female].

The first victim of four serial murders occurring in Hiroshima in 1996 was an unidentified young female when she was found. In the lower jaw, the left and right deciduous second molars were identified, and the left and right permanent second premolars were revealed to be congenitally defective by X-ray examination. Comparison of the dental findings and study model after death with those before death identified the victim as a 16-year old female.

Simple analysis of tetracyclic antidepressants in blood using headspace-solid-phase microextraction and GC-MS.

A simple and sensitive method for analysis of three tetracyclic antidepressants, maprotiline, mianserin, and setiptiline, in human whole blood was developed using headspace-solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). A vial containing a blood sample, sodium hydroxide, and imipramine as an internal standard was heated at 120 degrees C. The extraction fiber of the SPME was exposed for 45 min in the headspace of the vial. The compounds absorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC-MS. The calibration curves, using an internal standard method, demonstrated good linearity throughout the concentration range from 0.005 to 5.0 microg/g for mianserin and setiptiline and from 0.025 to 25 microg/g for maprotiline. No interferences were found, and the time for analysis was 60 min for one sample. In addition, this proposed method was applied to a medicolegal case in which the cause of death was suspected to be acute setiptiline poisoning. Setiptiline was detected in the left and right heart blood samples of the victim at concentrations of 1.77 and 0.78 microg/g, respectively.

Simple analysis of local anaesthetics in human blood using headspace solid-phase microextraction and gas chromatography-mass spectrometry-electron impact ionization selected ion monitoring.

A simple method for analysis of five local anaesthetics in blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry-electron impact ionization selected ion monitoring (GC-MS-EI-SIM). Deuterated lidocaine (d10-lidocaine) was synthesized and used as a desirable internal standard (I.S.). A vial containing a blood sample, 5 M sodium hydroxide and d10-lidocaine (I.S.) was heated at 120 degrees C. The extraction fiber of the SPME system was exposed for 45 min in the headspace of the vial. The compounds adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC-MS system. The calibration curves showed linearity in the range of 0.1-20 microg/g for lidocaine and mepivacaine, 0.5-20 microg/g for bupivacaine and 1-20 microg/g for prilocaine in blood. No interfering substances were found, and the time for analysis was 65 min for one sample. In addition, this proposed method was applied to a medico-legal case where the cause of death was suspected to be acute local anaesthetics poisoning. Mepivacaine was detected in the left and right heart blood samples of the victim at concentrations of 18.6 and 15.8 microg/g, respectively.

On the influence of postmortem alcohol diffusion from the stomach contents to the heart blood.

Alcohol concentrations in the mixed left and right heart blood, urine and stomach contents of 186 cadavers were analyzed by gas chromatography in order to find the influence of postmortem diffusion of alcohol from the stomach contents to the heart blood. In 39 cases where blood alcohol concentrations (BACs) were less than 0.10 mg/g, alcohol in the stomach contents was suggested to be due to postmortem production, and the postmortem diffusion of alcohol from the stomach contents to the heart blood was less than 10%. In 147 where BACs were 0.10 mg/g and more, ratios of BAC to urine alcohol concentration (UAC) were 1.0 and more in 47 cases (32%), and less than 1.0 in 100 cases (68%). In 17 of these 147 cases, alcohol concentrations in stomach contents (SACs) were more than ten times as high as BACs. Where the highest ratio of SAC/BAC was 60.1, the BAC of 0.14 mg/g was suspected to be due to drinking. In the case where the highest SAC was 50.8 mg/g, the BAC of 5.18 mg/g, the highest in this study, seemed to be little affected by the diffusion. These results suggest that it is important to compare BAC, UAC and SAC to assess the influence of postmortem diffusion of alcohol from the stomach contents to the heart blood.

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography-mass spectrometry.

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography-mass spectrometry (GC-MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C18 disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol-water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform-isopropanol (3:1, v/v) from the cartridge. The eluate (1 microl) was injected into a GC-MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 microg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.

Pyrrole butyric acid derivatives as inhibitors of steroid 5 alpha-reductase.

A series of pyrrole butyric acid derivatives was synthesized and evaluated for inhibitory activity on human and rat steroid 5 alpha-reductase in vitro and ex vivo. 3-Benzoyl-4-alkylpyrrole-1-butyric acids and 1-methyl-2-alkyl-3-benzoylpyrrole-5-butyric acid derivatives were effective inhibitors. Structure activity relationships were evaluated among the 37 compounds synthesized. Compound 37 (HQL-1069) shows potent inhibitory activities against both rat and human 5 alpha-reductase.

Rapid quantitative analysis of oleandrin in human blood by high-performance liquid chromatography.

A simple and rapid method for quantitation of oleandrin in human blood has been developed using an Extrelut column and high-performance liquid chromatography (HPLC). Blood samples spiked with oleandrin were mixed with distilled water and applied to an Extrelut column for elution with ethyl acetate before HPLC. The HPLC separation of the compound from endogeneous impurities was generally satisfactory with the use of a reversed-phase column. Oleandrin showed excellent linearity in the range of 0.05-10 micrograms/g and the limit of detection was 0.02 microgram/g for human blood. The recovery of the compound, which had been spiked to human blood, was more than 90%. The present method is simple and more rapid than those so far reported, and seems useful for analysis of oleandrin in the cases of oleander poisoning.

Rapid analysis of malathion in blood using head space-solid phase microextraction and selected ion monitoring.

A simple and rapid method for analysis of malathion in blood was developed using head space-solid phase microextraction (HS-SPME) and gas chromatography mass spectrometry/ electron impact ionization-selected ion monitoring (GC-MS/EI-SIM). A vial containing a blood sample, ammonium sulphate, sulphuric acid and fenitrothion as an internal standard, was heated at 90 degrees C for 15 min. The extraction fiber of the SPME was exposed for 5 min in the head space of the vial. The compounds absorbed on the fiber were detached by exposing the fibre in the injection port of GC-MS. A straight calibration curve was obtained between malathion concentrations of 2.5 to 50.0 micrograms g-1 in blood. No interfering substances were found, and the time for analysis was 40 min for one sample.

Random amplified polymorphic DNA analysis and variation of essential oil components of Atractylodes plants.

Total DNAs were prepared from the leaves of Atractylodes lancea DE CANDOLLE, A. chinensis KOIDZUMI, A. lancea var. simplicifolia KITAMURA, A. japonica KOIDZUMI ex KITAMURA and A. ovata DE CANDOLLE. The DNAs were subjected to random amplified polymorphic DNA (RAPD) analysis. Some primers showed the definitive polymorphic DNA patterns in A. lancea, A. japonica and A. ovata. The RAPD of A. lancea var. simplicifolia and one of A. chinensis gave similar patterns to those of A. lancea, but one of the other A. chinensis gave a similar pattern to A. japonica. Furthermore, quantitative analysis of atractylon, hinesol, beta-eudesmol and atractylodin in the rhizomes was done using gas chromatography. Though atractylon was detected not only in A. japonica and A. ovata but also in some of A. lancea, their RAPD profiles revealed the presence of intraspecific variation with A. lancea.

Clonal propagation of Cnidium officinale by shoot tip culture.

Multiple shoots were induced from the apical domes of shoot tips of Cnidium officinale Makino (Apiaceae) by culturing them on Murashige and Skoog (MS) 1 static media solidified with 0.2% gelrite and supplemented with 6-benzylaminopurine (BAP) 10 (-6)M. An average of 5.3 shoots per segment were obtained within 6 weeks and this ability did not decline even after two years of subculture. Subsequent transfer of these regenerated shoots on MS media supplemented with alpha-naphthaleneacetic acid (NAA) 10 (-7)M and BAP 10 (-7)M resulted in root formation. Rooted plantlets were able to grow in soil after a short period of acclimatization. Cytological observation in root tip cells of cultivated, as well as in vitro propagated plantlets revealed that in both cases the cells had 2n = 22 chromosomes indicating the homogeneity of the clonally propagated plants.