Stereoselective analyses of selegiline metabolites: possible urinary markers for selegiline therapy.

The stereoselective analysis of selegiline metabolites in human urine and plasma by gas chromatography using the chiral column with the non-chiral reagent was investigated for the differentiation of selegiline therapy from the methamphetamine (MA) abuse. This method gave clear separations of MA and amphetamine (AM) isomers without any artifactual optical-opposite peaks due to the reagent. After the administration of selegiline tablets, desmethylselegiline (DMS), MA and AM were observed as (-)-isomers in the urine and plasma. Within the first 48 h after dosing, approximately 40% of selegiline administered was excreted in urine as these three metabolites. The parent drug, selegiline, was not detected in any urine or plasma samples. On the other hand, MA and AM were observed only as (+)-isomers in the urine of MA abusers. For the distinction of selegiline users from street MA abusers in urinalysis, (-)-DMS, a specific metabolite of selegiline, was not a suitable marker. (-)-DMS rapidly disappeared from urine and was excreted only 1% of the given dose. By the moment analysis with the trapezoidal integration, the mean residence times of (-)-DMS in plasma and urine were 2.7 and 3.8 h, respectively, which were 5-20 times shorter than those of (-)-MA or (-)-AM. The values of AM/MA in the urine increased from 0.24 to 0.67 (r = 0.857) along with time after the selegiline administration. This ratio was not a sufficient marker to differentiate selegiline users from MA abusers, although the values of AM/MA in 74% of MA abusers were less than 0.24. The present GC technique improved the chiral analyses of MA and AM. This chiral analysis is the most useful technique to avoid the misinterpretation in the discrimination between clinical selegiline therapy and illicit MA use.

Chiral and nonchiral determination of ketoprofen in pharmaceuticals by capillary zone electrophoresis.

The new method for the enantiomeric resolution of various 2-arylpropionic acids by capillary zone electrophoresis (CZE) using heptakis-tri-O-methyl-beta-cyclodextrin as chiral selector was applied to the determination of ketoprofen in different commercially-available pharmaceutical preparations. The analyte was determined under chiral and nonchiral conditions (viz. in the presence and absence of 50 mM heptakis-tri-O-methyl-beta-cyclodextrin in the background electrolyte), with significantly similar results and relative standard deviations from 1.2 to 6.5% in both cases. The limits of detection and determination for the inactive enantiomer, R-(-)-ketoprofen, were calculated to be 7.0 x 10(-7) and 1.6 x 10(-6) M, respectively. The proposed method was successfully used to determine enantiomeric purity in the drugs studied, with results comparable to those provided by the chiral HPLC method.

Separation of profen enantiomers by capillary electrophoresis using cyclodextrins as chiral selectors.

A method for resolving the enantiomers of various 2-arylpropionic acids (viz. ketoprofen, ibuprofen and fenoprofen) by capillary zone electrophoresis (CZE) using a background electrolyte (BGE) containing a cyclodextrin as chiral selector is proposed. The effects of the type of cyclodextrin used and its concentration on resolution were studied and heptakis-2,3,6-tri- O-methyl-beta-cyclodextrin was found to be the sole effective choice for the quantitative enantiomeric resolution of all the compounds tested. The influence of pH, BGE concentration, capillary temperature and addition of methanol to the BGE on resolution and other separation-related parameters was also studied. The three compounds studied can be enantiomerically resolved with a high efficiency in a short time (less than 20 min) with no capillary treatment. This makes the proposed method suitable for assessing the enantiomeric purity of commercially available pharmaceuticals.

A fatal disaster case based on exposure to hydrogen sulfide--an estimation of the hydrogen sulfide concentration at the scene.

Four adult men fell into an artificial lake which was being used to raise flatfish, after a water pipe had been connected to a tube allowing seawater to flow into the lake. Forensic autopsies were carried out on three of the four men, who died soon after the incident. From autopsy findings, the cause of death was diagnosed to be suffocation after aspirating seawater in the three victims. To clarify why the men fell into the lake, a chemical analysis for hydrogen sulfide was carried out using the extractive alkylation technique combined with gas chromatography/mass spectrometry. The sulfide was detected as its derivative, bis(pentafluorobenzyl)sulfide, in body tissues taken from all the victims, and the concentration of hydrogen sulfide gas at the scene was estimated as having been nearly fatal.

Rapid and sensitive quantitation of cyanide in blood and its application to fire victims.

We developed a head-space method for the determination of blood cyanide by gas chromatography with electron-capture detection. In this technique, a reaction precolumn packed with chloramine-T was used for the conversion of hydrogen cyanide into cyanogen chloride. Since the reaction precolumn eliminated the necessity of trapping hydrogen cyanide from biological samples, blood cyanide could be analyzed quickly by acidification only. Using this method, blood cyanide levels of fire victims were determined at autopsy. The serum values of cyanide ranged from 0.11 micrograms/ml to 18.12 micrograms/ml. However, a significantly higher cyanide content was detected in the left ventricular blood than in the right. This indicates that death was caused by the fire and suggests that the collecting point of the blood sample is an important factor in the determination of inhaled cyanide. There was a positive correlation between blood cyanide and carboxyhemoglobin contents.

"First pass phenomenon" of inhaled gas in the fire victims.

We investigated the differences in the levels of carboxyhemoglobin (COHb), cyanide (HCN) and petroleum fuels (gasoline and kerosene) between left and right ventricular bloods from fire victims. COHb was slightly, and HCN and petroleum fuels were markedly higher levels in the left than those in the right. These effects were so called 'first pass phenomena' due to the circulation, diffusion and metabolization before the deaths of fire victims.

[Determination of blood cyanide using head-space gas chromatography with electron capture detection].

Two head-space gas chromatographic methods for the determination of hydrogen cyanide (HCN) in blood were demonstrated using electron capture detector (ECD). One was the double head-space gas sampling technique and the other used the reaction pre-column packed with Chloramine-T for the conversion of HCN into cyanogen chloride (CNC1). In the former method, HCN in blood was firstly released by acidification, and then the HCN gas was replaced into the another vial in which Chloramine-T solution had been placed. The head-space gas in the second vial was analyzed by gas chromatograph (GC). In the latter, HCN gas released was directly injected into the GC pre-connected the reaction column. The conversion of HCN into CNC1 was easily achieved by both methods. The use of dichloromethane as an internal standard and PTFE-faced septum for the sealing of vial greatly improved the precision and reliability of these analytical methods. The calibration curves by these methods gave good linearity and the lower detection limit taken from these plots was ca. 0.05 micrograms/ml. In particular, the reaction pre-column method, by which blood cyanide can be analyzed rapidly and accurately with only simple alteration of apparatus, is superior in the routine analysis of blood cyanide.

Improved gas chromatography with electron-capture detection using a reaction pre-column for the determination of blood cyanide: a higher content in the left ventricle of fire victims.

We developed a head-space method for the determination of blood cyanide by gas chromatography with electron-capture detection. In this technique, a reaction pre-column, packed with chloramine-T, was used for the conversion of hydrogen cyanide into cyanogen chloride. Since the reaction pre-column eliminated the necessity for trapping hydrogen cyanide from the biological samples, blood cyanide was quickly analysed by acidification only. The reaction pre-column was durable for at least several months. The calibration curve gave good linearity when dichloromethane was used as the internal standard, and the lower detection limit taken from this plot was ca. 0.05 micrograms/ml. The relative standard deviation of spiked blood samples was in the range 0.6-3.9%. We determined blood cyanide levels at autopsy in victims who had died from fire using this method. A significantly higher cyanide content was detected in the left ventricular blood than in the right. There was a positive correlation between blood cyanide and carboxylhaemoglobin contents. This simple and sensitive technique could be very useful for the determination of cyanide in various samples.

Increased plasma free fatty acid and triglyceride levels after single administration of toluene in rabbits.

Changes of plasma lipids (triglyceride, TG: total cholesterol, Cho; and phospholipids, PL), free fatty acid (FFA), and blood glucose (BG) were studied in male rabbits after toluene administration (0.5 g/kg per os). Hypertriglyceridemia was observed at and after 2 h. Plasma FFA and BG were elevated temporarily during the early stage and lowered gradually thereafter. Initially, plasma Cho and PL were virtually unchanged, but the Cho level increased slowly after 6 h. The hypertriglyceridemia observed may have some adverse effects on heart function.

Gas chromatographic determination for forensic purposes of petroleum fuel inhaled just before fatal burning.

The determination of petroleum fuel in the blood of burned bodies was carried out by three different gas chromatographic procedures. Seven components of gasoline (isopentane, n-pentane, 2-methylpentane, benzene, 2-methylhexane, 3-methylhexane and toluene) and five of kerosene (xylene, C9H20, mesitylene, pseudocumene and C11H24) were chosen as indicators with a coefficient of variation of 5-24%. The methods were applied to four autopsy cases with a relatively low carboxyhaemoglobin (HbCO) content. When gasoline exposure had occurred, the blood concentrations determined were almost identical whatever the components selected. Great variations in the components determined were found after kerosene exposure, and hydrocarbons greater than or equal to C14 were hardly inhaled by the victims. A higher content of fuel in the left than in the right ventricular blood observed in the autopsy cases suggests fuel inhalation just before death. The same phenomenon was also observed in the content of blood HbCO. Determinations of petroleum fuel and HbCO in both the right and left ventricular blood would be useful for the forensic diagnosis on burned bodies with a low HbCO content.

Derivative spectrophotometric studies on cytotoxic effects of carbon monoxide.

Characteristics of cytochrome oxidase prepared from hearts of Sprague-Dawley male rats were studied with the use of the fourth derivative spectrophotometry in respect to the cytotoxic effects of carbon monoxide (CO). CO-exposed rats tended to show lower specific activities of cytochrome oxidase than control and recovered rats. Moreover, there was a significant difference in the fourth derivative spectral features of the enzyme: CO-exposed groups indicated peaks at 412 nm in the spectra while controls at 408 nm. This spectral difference seemed to reflect specific effect of CO on cytochrome oxidase, though such trace remained for not more than a day after death.

Detection of delta 9-THC in saliva by capillary GC/ECD after marihuana smoking.

A method is described for the determination of delta 9-tetrahydrocannabinol (delta 9-THC) in the saliva by the use of a combination of moving-precolumn injector and glass capillary gas chromatograph with electron capture detector (GC/ECD). There were no interfering peaks due to impurities around the peak of pentafluoropropyl derivative of delta 9-THC (delta 9-THC-PFP). This GC/ECD method was linear over the range of 5-200 ng/ml of delta 9-THC-PFP. The lower detection limit was approximately 1 ng/ml. delta 9-THC content in the saliva after experimental marihuana smoking was measured by this method. It was demonstrated that for at least 4 h after smoking the level of delta 9-THC was sufficient for detection.

Salsolinol in the urine of nonalcoholic individuals after long-term moderate drinking.

Urine samples were collected before breakfast from 94 normal volunteers (41 males and 53 females) aged 25-70 years. Salsolinol (SA) was analyzed by gas chromatography-mass spectrometry (GC/MS), and dopamine, norepinephrine and epinephrine were determined using high performance liquid chromatography (HPLC). SA levels were significantly higher in the urine of male moderate drinkers (MDs) than in male seldom or non drinkers (SNDs). In females, however, a significant difference of urinary SA levels was not observed between MDs and SNDs. There was a sex difference of urinary SA levels among SND subjects, i.e., females showed a higher SA than males. Urinary catecholamines were not significantly altered by long-term moderate alcohol drinking in either sex. There was no correlation between urinary levels of dopamine and SA. These results indicate that urinary SA can be increased by long-term drinking even in normal, not alcoholic subjects.

Quantitation of cocaine, benzoylecgonine and ecgonine methyl ester by GC-CI-SIM after Extrelut extraction.

A method of gas chromatography-chemical ionization selected ion monitoring (GC-CI-SIM) is described for the determination of cocaine, benzoylecgonine and ecgonine methyl ester in biological materials using an Extrelut extraction technique. Recoveries of cocaine, benzoylecgonine and ecgonine methyl ester by this technique were 95, 81 and 97%, respectively. The method uses cocaine-d5, benzoylecgonine-d5 and lidocaine as internal standards, and isobutane as reagent gas for chemical ionization. Sensitivity of the method proved to be 1 ng/ml for cocaine and benzoylecgonine, and 10 ng/ml for ecgonine methyl ester when used in a 10-ml urine sample. With animal experiments, ecgonine methyl ester as well as benzoylecgonine was confirmed as a major metabolite of cocaine.

Chromophoric labeling of cannabinoids with 4-dimethylaminoazobenzene-4'-sulfonyl chloride.

A simple and sensitive assay for the cannabinoids is presented using a dabsylation procedure. Dabsyl derivatives of delta 9-tetrahydrocannabinol (delta 9-THC) and cannabinol (CBN) were prepared by reacting with 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride) in acetone in the presence of sodium carbonate-sodium bicarbonate buffer (pH 10). Crystalline dabsylcannabinoids gave intense absorption in the visible region. With these derivatives, analysis by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) were tested. These techniques gave good separation and nanogram detection of dabsyl-THC and -CBN by using n-hexane-ethyl acetate-diethylamine (20:5:1) for TLC and MeOH--H2O (95:5) at 450 nm for HPLC.