A fatal case of suspected anaphylaxis with cefoperazone and sulbactam: LC-MS analysis.

Cefoperazone and sulbactam are prescribed in combination and used in the treatment of moderate to severe bacterial infections. Serious anaphylaxis is a rare side effect. This report describes a fatal case of suspected anaphylaxis after intravenous administration of a combination of the two drugs. Heart blood was analyzed for cefoperazone by protein precipitation with acetonitrile and by liquid-liquid precipitation for sulbactam after protein precipitation with aqueous acetonitrile, followed by tandem mass spectrometry in the product ion scan mode for identification and by liquid chromatography mass spectrometry in the selected ion monitoring mode for quantitation. Calibration curves for cefoperazone and sulbactam were linear over the range 0.07 to 1.93 and 0.046 to 0.914 microg/ml respectively. The decedent's blood concentrations of cefoperazone and sulbactam were 0.368 and 0.143 microg/ml respectively. As these concentrations were below concentrations reported after single dosing studies and below those considered to be minimally inhibitory, death was presumed to have been caused by hypersensitivity and not an overdose. In conclusion, this procedure is useful for detecting and quantitating cefoperazone and sulbactam in postmortem blood and may be useful in the evaluation of anaphylaxis.

Uptake of 3,4-methylenedioxymethamphetamine and its related compounds by a proton-coupled transport system in Caco-2 cells.

3,4-methylenedioxymethamphetamine (MDMA) is an illegal amphetamine-type stimulant (ATS) that is abused orally in the form of tablets for recreational purposes. The aim of this work is to investigate the absorption mechanism of MDMA and other related compounds that often occur together in ATS tablets, and to determine whether such tablet components interact with each other in intestinal absorption. The characteristics of MDMA uptake by the human intestinal epithelial Caco-2 cell line were investigated. The Michaelis constant and the maximal uptake velocity at pH 6.0 were 1.11 mM and 13.79 nmol/min/mg protein, respectively, and the transport was electroneutral. The initial uptake rate was regulated by both intra- and extracellular pH. MDMA permeation from the apical to the basolateral side was inferior to that in the reverse direction, and a decrease in apical pH enhanced MDMA permeation from the basolateral to the apical side. These facts indicate that this transport system may be an antiporter of H+. However, under physiological conditions, the proton gradient cannot drive the MDMA uptake because it is inwardly directed. Large concentration differences of MDMA itself drive this antiporter. Various compounds with similar amine moieties inhibited the uptake, but substrates of organic cation transporters (OCT1-3) and an H+-coupled efflux antiporter, MATE, were not recognized.

Comparison and classification of methamphetamine seized in Japan and Thailand using gas chromatography with liquid-liquid extraction and solid-phase microextraction.

Methamphetamine (MA) is one of the most frequently abused drugs worldwide. The aim of this study is to improve the analytical method for profiling MA impurity in order to compare and classify MA crystals seized in different countries and to investigate the relationships between seizures. To compare MA samples seized in Japan and Thailand, the following analytical method was adopted. A 50mg sample of MA.HCl was dissolved in 1ml of buffer solution (four parts 0.1M phosphate buffer, pH 7.0, and one part 10% Na(2)CO(3)), impurities were extracted with 0.5ml of ethyl acetate containing four internal standards (n-decane, n-pentadecane, n-eicosane and n-octacosane) and analyzed by gas chromatography with a flame ionization detector on a DB-5 capillary column (0.32mm i.d.x30m, film thickness 1.0mum). Fourteen characteristic peaks on chromatograms were selected for the comparison and classification of samples, and the data were evaluated by the Euclidean distance of the relative peak areas after logarithmic transformation. Sixty-nine samples seized in Japan and 42 seized in Thailand were analyzed. The samples were classified into four groups roughly by cluster analysis. In addition, when it was difficult to compare samples that had fewer impurities on chromatograms obtained from liquid-liquid extraction (LLE), solid-phase microextraction (SPME) was effective. Because many characteristic peaks were detected using SPME, SPME made it easy to compare samples of high purity. The combination of LLE and SPME was useful for impurity profiling of MA samples seized in different countries.

Interactions between 3,4-methylenedioxymethamphetamine, methamphetamine, ketamine, and caffeine in human intestinal Caco-2 cells and in oral administration to rats.

Amphetamine-type stimulants (ATSs) are often abused orally in the form of tablets for recreational purposes. The ATS tablets contain one or more active ingredients such as 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine (MA), ketamine (KA), and caffeine (CF). The aim of this work is to determine whether such components in tablets interact with each other in intestinal absorption. The interactions between MDMA, MA, KA, and CF in the uptake and permeation by human intestinal epithelial Caco-2 cell line were investigated in monolayer cultures. MDMA, MA, and KA mutually inhibited the uptakes by Caco-2 cells. The inhibition of MA uptake by KA was the greatest of all combinations (72.6% inhibition). Similarly, MDMA, MA, and KA mutually inhibited the permeation from the apical to the basolateral side through Caco-2 cells. Although CF did not affect the uptakes of MDMA, MA, and KA, CF enhanced the permeation of MDMA in comparison to MDMA alone. In addition, the interaction of MA with KA and that of MDMA with CF in intestinal absorption were investigated by oral administration to rats. The area under the plasma concentration-time curve of MA significantly decreased by co-administration with KA in comparison to MA alone, while that of MDMA significantly increased by co-administration with CF in comparison to MDMA alone. The results in rats were similar to those in Caco-2 cells. These findings suggest that the intestinal absorption of similar compounds with amine moieties such as MDMA, MA, and KA are mediated by a common transport system, and that CF affects the absorption of MDMA in a different way from the transport system. In human, intakes of ATS tablets mixed with such components might result in similar interactions in intestinal absorption to those in Caco-2 cells and rats.

Determination of muscimol and ibotenic acid in Amanita mushrooms by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry.

A reliable analytical method was developed for the quantification and identification of muscimol (MUS) and ibotenic acid (IBO), the toxic constituents of Amanita muscaria and Amanita pantherina. MUS and IBO were extracted from mushrooms by aqueous methanol and derivatized with dansyl chloride (DNS-Cl). After extraction with ethyl acetate and evaporation of the solvent, the residue was ethylated with 1.25 M hydrogen chloride in ethanol. The resulting derivatives were quantified by high-performance liquid chromatography with UV detection and identified by liquid chromatography electrospray ionization tandem mass spectrometry. Calibration curves were linear in the range of 25-2500 ppm for MUS and 40-2500 ppm for IBO, respectively. This method was successfully applied to identify and quantify MUS and IBO in Amanita mushrooms naturally grown and circulated in the drug market.

Contribution of thermal desorption and liquid-liquid extraction for identification and profiling of impurities in methamphetamine by gas chromatography-mass spectrometry.

Impurity profiling of methamphetamine (MA) using thermal desorption (TD) and gas chromatography-mass spectrometry (GC-MS) was examined. Using TD/GC-MS, impurities were extracted and separated under various conditions. Optimal chromatograms were obtained when a 20 mg MA sample was extracted at 120 degrees C for 3 min using a TD instrument, followed by separation of the extracts using a non-polar capillary column coated with (5%phenyl)-methylpolysiloxane. MA samples from nine different batches were analyzed under optimized conditions. Compounds related to the structure of MA, such as benzaldehyde, benzyl alcohol, amphetamine, cis- and trans-1,2-dimethyl-3-phenylaziridine, dimethylamphetamine, and N-acetylephedrine, were detected in the chromatograms without any laborious extraction procedure. Compounds such as ethanol, diethyl ether, and acetic acid, which are considered reagents and solvents for MA synthesis, were also detected in some of the chromatograms. The numbers and intensities of the peaks detected were different among the samples. Impurity profiling of MA using TD was compared with that using liquid-liquid extraction (LLE). Better reproducibility of peak areas was obtained using LLE, whereas higher intensities and numbers of peaks were detected using TD. Solvents were extracted more effectively using TD. The nine batches of MA were classified using both extraction procedures. The nine batches were divided roughly into two groups using data from LLE. Subsequently, the groups were classified in detail using data from TD. TD can be used to provide supplemental information for LLE, and the combination of these extraction methods can be helpful for impurity profiling of MA.

Forensic application of chiral separation of amphetamine-type stimulants to impurity analysis of seized methamphetamine by capillary electrophoresis.

The applicability of capillary electrophoresis (CE) with a UV detector using highly sulfated gamma-cyclodextrin as a chiral selector was examined for analysis of impurities in seized methamphetamine. Samples of methamphetamine-hydrochloride dissolved in water at a high concentration (20 mg/mL) were analyzed. Electrokinetic injection has an advantage over hydrodynamic injection for improving the detection of trace impurities. Small peaks of the precursor impurities, such as (1R,2S)-(-)-ephedrine and (1S,2S)-(+)-pseudoephedrine, were detected and quantified without extraction. The seized drugs could be classified into three groups based on the contents of the two impurities.

Metabolism and the urinary excretion profile of the recently scheduled designer drug N-Benzylpiperazine (BZP) in the rat.

The metabolism of N-benzylpiperazine (BZP), a recently scheduled designer drug, in the rat has been studied by analyzing its urinary metabolites. p-Hydroxy-BZP (p-OH-BZP) was unequivocally identified as the main metabolite along with a minor metabolite m-hydroxy-BZP (m-OH-BZP), using gas chromatography-mass spectrometry and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS). The time-course excretion profiles of BZP, p-OH-BZP, and m-OH-BZP in the rats were investigated after a single intraperitoneal dosing of 5 mg/kg BZP, by using an optimized analytical procedure that combines solid-phase extraction and LC-ESI MS determination. The cumulative amounts excreted within the first 48 h were approximately 25% for p-OH-BZP and 2% for m-OH-BZP, whereas 6.7% dose of the parent drug BZP was excreted unchanged within 36 h post-dosing. The concentration ratio of p-OH-BZP to m-OH-BZP was 11.6 in the first 4 h, but it increased to 22.7 in 48 h with the elapsed time post-dosing. Most of p-OH-BZP was excreted in urine within approximately 36 h post-dosing, with approximately 50% appearing as the glucuronide conjugate. The present results suggest that p-OH-BZP is the most relevant metabolite to be detected for the proof of BZP intake in the forensic and clinical analysis of human urine.

Analysis of hallucinogenic constituents in Amanita mushrooms circulated in Japan.

The constituents of seven mushrooms sold as Amanita muscaria or Amanita pantherina (five A. muscaria and two A. pantherina) and four "extracts purported to contain A. muscaria" products that are currently circulated in Japan were determined. All mushroom samples were identified as A. muscaria or A. pantherina by macroscopic and microscopic observation. The dissociative constituents, ibotenic acid (IBO) and muscimol (MUS), were extracted with 70% methanol twice and determined by gas chromatography/mass spectrometry. The IBO (as the hydrate)/MUS contents were in the range of <10-2845ppm/46-1052ppm in the cap of A. muscaria and 188-269ppm/1554-1880ppm in the cap of A. pantherina. In the caps, these compounds had a tendency to be more concentrated in the flesh than in the cuticle. On the other hand, the IBO/MUS contents in the stem were far lower than in the caps. In the "extracts purported to contain A. muscaria" products, IBO/MUS were detected below the lower limit of calibration curve (<10ppm/<25ppm) or not detected. However, these samples contained other psychoactive compounds, such as psychoactive tryptamines (5-methoxy-N,N-diisopropyltryptamine and 5-methoxy-N,N-dimethyltryptamine), reversible monoamine oxidase inhibitors (harmine and harmaline) and tropane alkaloids (atropine and scopolamine), which were not quantified. This is the first report of the chemical analysis of Amanita mushrooms that are circulated in the drug market.

Identification of impurities and the statistical classification of methamphetamine using headspace solid phase microextraction and gas chromatography-mass spectrometry.

The profiling of impurities in methamphetamine (MA) using headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) is described. The extraction of the impurities with an SPME fiber was examined under varying conditions. Optimal chromatograms were obtained when a 50 mg MA sample at 85 degrees C for 30 min was extracted using a fiber coated with divinylbenzene/carboxen/polydimethylsiloxane. MA samples from nine different origins were analyzed under optimized extraction conditions. Compounds related to MA such as benzaldehyde, benzyl alcohol, amphetamine, benzyl methyl ketone, cis- and trans-1,2-dimethyl-3-phenylaziridine, dimethylamphetamine, N-acetylamphetamine, N-acetylmethamphetamine and N-formylmethamphetamine were detected in the chromatograms. Trace amounts of ethanol, diethyl ether and acetic acid were also detected in some of the chromatograms. The numbers and intensities of the peaks detected were different, depending on the sample. After the areas of the eight principal peaks were converted to their square root and logarithm, similarities among the samples were evaluated by Euclidian distance, cosine distance and correlation coefficient. The results showed that a combination of logarithmic conversion and cosine distance was the most suitable for discriminating and classifying the samples. HS-SPME/GC-MS is a simple and effective method for the extraction and identification of impurities. The present method, in combination with an appropriate statistical analysis, would be useful for developing a profile of impurities in MA.

A method for screening for various sedative-hypnotics in serum by liquid chromatography/single quadrupole mass spectrometry.

A screening method for the detection of sedative-hypnotics in serum is described. The target drugs, which include practically all the sedative-hypnotics distributed in Japan, consisted of 5 barbiturates, 30 benzodiazepine-related drugs and 11 other sedative-hypnotics (i.e., apronalide, bromisovalum, chloral hydrate, triclofos, chlorpromazine, promethazine, diphenhydramine, hydroxyzine, zopiclone, zolpidem and tandospirone). Thirty-nine analytes, selected in terms of the pharmacokinetics of the target drugs, in human serum were screened using a combination of mixed-mode solid-phase extraction and liquid chromatography/electrospray-ionization single-quadrupole mass spectrometry. The detection limits (non-basic analytes, 1-50 ng/ml; basic analytes, 0.1-5 ng/ml) were sufficient to permit the screening of a single therapeutic administration of a target drug.

Urinary excretion profiles of two major triazolam metabolites, alpha-hydroxytriazolam and 4-hydroxytriazolam.

The objective of this study was to examine urinary excretion profiles of two major triazolam metabolites, alpha-hydroxytriazolam (alpha-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ) in humans. Urine samples were collected from three healthy male volunteers who had been previously administered single 0.25- and 0.5-mg doses of triazolam 24 h and 48 h, respectively, before sample collection. After enzymatic hydrolysis and extraction, each sample was analyzed by liquid chromatography-mass spectrometry. alpha-OHTRZ was rapidly excreted, with the maximum concentrations appearing in the first or second sample collected after ingestion, with the majority of the drug being excreted within 12 h. Meanwhile, 4-OHTRZ was excreted more slowly than alpha-OHTRZ. The alpha-OHTRZ/4-OHTRZ ratios were initially greater than 19.7, then decreased rapidly, reaching a nearly constant value for times in excess of 12 h.

Morphological and chemical analysis of magic mushrooms in Japan.

Morphological and toxicological analyses were performed on hallucinogenic mushrooms that are currently circulated in Japan. Scanning electron microscope (SEM) indicated a three-dimensional microstructures in the mushrooms. The complementary use of SEM with an optical microscope was effective for observing characteristic tissues, such as basidiomycetes, spores, cystidia and basidia. Hallucinogenic alkaloids were extracted with methanol and determined by high performance liquid chromatography (HPLC) with a UV detector set at 220 nm. The psilocin/psilocybin contents in Psilocybe cubensis were in the range of 0.14-0.42%/0.37-1.30% in the whole mushroom (0.17-0.78%/0.44-1.35% in the cap and 0.09-0.30%/0.05-1.27% in the stem), respectively. The hallucinogenic alkaloids in Copelandia were 0.43-0.76%/0.08-0.22% in the whole mushroom (0.64-0.74%/0.02-0.22% in the cap and 0.31-0.78%/0.01-0.39% in the stem). It thus appears that P. cubensis is psilocybin-rich, whereas Copelandia is psilocin-rich.

Methamphetamine impurity profiling using a 0.32 mm i.d. nonpolar capillary column.

Classification of seized methamphetamine by impurity profiling can provide very useful information in criminal investigations of drug traffic routes, sources of supply and relationships between seizures. The aim of this study is to improve and develop an analytical method for detecting impurities such as starting materials and by-products in illegally prepared methamphetamine.HCl samples. A 50mg sample of methamphetamine.HCl was dissolved in 1 ml of buffer solution (four parts 0.1M phosphate buffer pH 7.0 and one part 10% Na2CO3). Impurities were extracted with 0.5 ml of ethyl acetate containing four internal standards (ISs) (n-decane, n-pentadecane, n-nonadecane and n-hexacosane) and analyzed by gas chromatography (GC) using a flame ionization detector (FID) on a DB-5 capillary column (0.32 mmi.d. x 30 m, film thickness 1.0 microm). The use of a middle-bore column offered better separation of the impurity peaks. The correction of the retention times of impurity peaks with four ISs made peak identification very accurate for subsequent data processing. Twenty-four characteristic peaks were selected for comparison and similarity and/or dissimilarity between samples, and the data were evaluated by the Euclidean distance of the relative peak areas after logarithmic transformation. The results indicate that the present method would be useful for methamphetamine impurity profiling.

Chiral analysis of amphetamine-type stimulants using reversed-polarity capillary electrophoresis/positive ion electrospray ionization tandem mass spectrometry.

Reversed-polarity (RP) capillary electrophoresis/positive ion electrospray ionization mass spectrometry (CE-ESI+ MS) and tandem mass spectrometry (MS/MS) were utilized for simultaneous chiral separation of nine amphetamine-type stimulants (ATS) (dl-norephedrine, dl-norpseudoephedrine, dl-ephedrine, dl-pseudoephedrine, dl-amphetamine, dl-methamphetamine, dl-methylenedioxyamphetamine, dl-methylenedioxymethamphetamine, and dl-methylenedioxyethylamphetamine). Using highly sulfated gamma-cyclodextrin (SU(XIII)-gamma-CD) as a chiral selector, the nine ATS were completely separated within 50 min. The migrated ATS-CD complex was dissociated at the ESI interface, and only ATS molecules went into the MS detector so that all 18 individual enantiomers were identified by their mass spectra. The detection limit of MS/MS was 10 times more sensitive than those for single MS. Seized d-methamphetamine hydrochloride samples dissolved at high concentration (20 mg/mL) were analyzed. Impurities originating in the precursor such as l-ephedrine and d-pseudoephedrine were detected and identified by tandem mass spectra.

The use of a highly sulfated cyclodextrin for the simultaneous chiral separation of amphetamine-type stimulants by capillary electrophoresis.

We investigated the simultaneous chiral separation of nine amphetamine type stimulants (dl-norephedrine, dl-norpseudoephedrine, dl-ephedrine, dl-pseudoephedrine, dl-amphetamine, dl-methamphetamine, dl-methylenedioxyamphetamine (MDA), dl-methylenedioxymethamphetamine (MDMA), and dl-methylenedioxyethylamphetamine (MDEA)) by capillary electrophoresis using highly sulfated gamma-cyclodextrin (SU(XIII)-gamma-CD) as a chiral selector. Three different approaches using SU(XIII)-gamma-CD with 50 mM phosphate background electrolyte were designed; (I) high CD concentration (10 mM SU(XIII)-gamma-CD) at neutral pH (pH 7.0) in the normal polarity mode, (II) low CD concentration (1.0 mM) at low pH (pH 2.6) in the normal polarity mode and (III) high CD concentration at low pH (pH 2.6) in the reversed-polarity mode. In mode (II), the effects of adding three neutral CDs (beta-CD, dimethyl-beta-CD and hydroxypropyl-beta-CD) were also investigated. The best separation was obtained after optimizing mode (III) as follows: run buffer of 10 mM SU(XIII)-gamma-CD with 50 mM phosphate background electrolyte at pH 2.6, applied voltage of -12 kV and capillary temperature of 15 degrees C.

In vivo metabolism of 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in the rat: identification of urinary metabolites.

The in vivo metabolism of 4-bromo-2,5-dimethoxyphenethylamine (2C-B), a ring-substituted psychoactive phenethylamine, in the rat was studied. Male Wistar rats were administered 10 mg/kg of 2C-B hydrochloride orally, and 24 h urine fractions were collected. After enzymatic hydrolysis of the urine samples, the metabolites were extracted by liquid-liquid extraction and analyzed by gas chromatography-mass spectrometry. 2-(4-Bromo-2,5-dimethoxyphenyl)-ethanol, 4-bromo-2,5-dimethoxyphenylacetic acid, 2-(2-hydroxy-4-bromo-5-methoxyphenyl)-ethylamine, 2-(2-methoxy-4-bromo-5-hydroxyphenyl)-ethylamine, 1-acetoamino-2-(2-hydroxy-4-bromo-5-methoxyphenyl)-ethane, and 1-acetoamino-2-(2-methoxy-4-bromo-5-hydroxyphenyl)-ethane were identified as 2C-B metabolites. These findings suggest that at least two metabolic pathways for 2C-B are operative in rats. The first pathway leads to the corresponding aldehyde metabolite by deamination, which is subsequently reduced or oxidized, to give the corresponding alcohol and carboxylic acid metabolites. The second pathway leads to the corresponding 2-O-desmethyl or 5-O-desmethyl metabolites in which the amino group is subsequently acetylated.