Medical Findings and Toxicological Analysis in Infant Death by Balloon Gas Asphyxia: A Case Report.

In recent years, the increasing number of asphyxiation cases due to helium inhalation is remarkable. All described cases in the literature where diagnosed as suicide. In this article, however, we describe a triple infant homicide in which helium, as balloon gas, was administered to three young children after sedation causing asphyxiation and death through the medical findings and toxicological analysis. During autopsy, in addition to standard toxicological samples, gas samples from lungs as well as lung tissue itself were directly collected into headspace vials. Besides routine toxicological analysis, which revealed toxic levels of doxylamine, qualitative analysis on gas and lung samples was performed using headspace gas chromatography-mass spectrometry. As carrier gas, the commonly used helium was replaced by nitrogen. In gas samples from lungs of all three children, no helium was found. Nevertheless, lung tissue samples were found positive on helium. Therefore, sedation followed by asphyxia due to helium inhalation can strongly be assumed as the cause of death of all three children.

Inactivation of anthracyclines by serum heme proteins.

We have previously shown that the anticancer agent doxorubicin undergoes oxidation and inactivation when exposed to myeloperoxidase-containing human leukemia HL-60 cells, or to isolated myeloperoxidase, in the presence of hydrogen peroxide and nitrite. In the current study we report that commercial fetal bovine serum (FBS) alone oxidizes doxorubicin in the presence of hydrogen peroxide and that nitrite accelerates this oxidation. The efficacy of inactivation was dependent on the concentration of serum present; no reaction was observed when hydrogen peroxide or serum was omitted. Peroxidase activity assays, based on oxidation of 3,3',5,5'-tetramethylbenzidine, confirmed the presence of a peroxidase in the sera from several suppliers. The peroxidative activity was contained in the >10000 MW fraction. We also found that hemoglobin, a heme protein likely to be present in commercial FBS, is capable of oxidizing doxorubicin in the presence of hydrogen peroxide and that nitrite further stimulates the reaction. In contrast to intact doxorubicin, the serum + hydrogen peroxide + nitrite treated drug appeared to be nontoxic for PC3 human prostate cancer cells. Together, this study shows that (pseudo)peroxidases present in sera catalyze oxidation of doxorubicin by hydrogen peroxide and that this diminishes the tumoricidal activity of the anthracycline, at least in in vitro settings. Finally, this study also points out that addition of H2O2 to media containing FBS will stimulate peroxidase-type of reactions, which may affect cytotoxic properties of studied compounds.

Suicide through doxylamine poisoning.

Doxylamine is an antihistamine of the ethanolamine class. It is used primarily as a sleep-inducing agent. Only a few reports can be found in the literature about lethal intoxications with doxylamine, but many with combined intoxications. Doxylamine is, aside from diphenhydramine, the only chemically defined active ingredient in some sleeping medications which is available without a prescription in the Federal Republic of Germany. Two cases of doxylamine poisoning are presented, in which high doxylamine concentrations were found in the blood and organs.

Metabolism of doxylamine succinate in Fischer 344 rats. Part III: Conjugated urinary and fecal metabolites.

Elimination and metabolic profiles of the glucuronide products of doxylamine and its N-demethylated metabolites were determined after the oral administration of (14C)-doxylamine succinate (13.3 and 133 mg/kg doses) to male and female Fischer 344 rats. The cumulative urinary and fecal eliminations of these conjugated doxylamine metabolites at the 13.3 mg/kg dose were 44.4 +/- 4.2% and 47.3 +/- 8.1% of the total recovered dose for male and female rats, respectively. The cumulative urinary and fecal eliminations of conjugated doxylamine metabolites at the 133 mg/kg dose were 55.2 +/- 2.6% and 47.9 +/- 2.5% of the total recovered dose for male and female rats, respectively. The conjugated doxylamine metabolites that were isolated, quantitated, and identified are doxylamine O-glucuronide, N-desmethyl-doxylamine O-glucuronide, and N,N-didesmethyldoxylamine O-glucuronide.

Characterization of doxylamine and pyrilamine metabolites via thermospray/mass spectrometry and tandem mass spectrometry.

Analysis of doxylamine N-oxide and pyrilamine N-oxide as synthetic standards and biologically derived metabolites by thermospray mass spectrometry (TSP/MS) provided [M + H]+ ions for each metabolite. TSP/tandem mass spectrometry (TSP/MS/MS) of the [M + H]+ ions provided fragment ions characteristic of these metabolites. In addition, TSP mass spectrometry and TSP/MS/MS analysis of ring-hydroxylated N-desmethyldoxylamine, N-desmethylpyrilamine and O-dealkylated pyrilamine is also reported. A fragmentation pathway for analysis by MS/MS of pyrilamine and its metabolites is also proposed. The results demonstrate the utility of TSP/MS for biologically derived metabolites of pyrilamine and doxylamine.

Identification in in vivo acetylation pathway for N-dealkylated metabolites of doxylamine in humans.

1. Metabolites of doxylamine in human urine were separated by g.l.c. and h.p.l.c. and tentatively identified by their mass spectrometric behaviour. 2. N-Desmethyldoxylamine, N,N-didesmethyldoxylamine and their N-acetyl conjugates were identified. This is believed to be the first report of acetylation in vivo of primary and secondary aliphatic amines in man.

Identification of two glucuronide metabolites of doxylamine via thermospray/mass spectrometry and thermospray/mass spectrometry/mass spectrometry.

Analysis of a high-pressure liquid chromatography fraction containing two urinary glucuronide metabolites of doxylamine by thermospray mass spectrometry (TSP/MS) provided [MH]+ ions for each metabolite. TSP/MS/MS of the [MH]+ ions provided a fragment ion characteristic of these metabolites. The results demonstrate the utility of TSP/MS analysis for biologically derived glucuronide metabolites.

Metabolism of doxylamine succinate in Fischer 344 rats. Part II: Nonconjugated urinary and fecal metabolites.

Elimination and metabolic profiles of doxylamine and its nonconjugated metabolites were determined after the oral administration of [14C]-doxylamine succinate (13.3 mg/kg and 133 mg/kg doses) to male and female Fischer 344 rats. Total urine and fecal recovery of the administered dose was greater than 90% regardless of sex or dose. The cumulative urinary and fecal elimination of these nonconjugated doxylamine metabolites at the 13.3 mg dose was 44.4 +/- 4.4% and 36.0 +/- 5.8% of the total recovered dose for male and female rats, respectively. The cumulative urinary and fecal elimination of the doxylamine nonconjugated metabolites at the 133 mg/kg dose was 38.7 +/- 2.7% and 41.4 +/- 1.0% of the total recovered dose for male and female rats, respectively. In order to determine the contribution of mammalian and bacterial enzymes in the overall metabolism and excretion patterns for doxylamine, two in vitro techniques were investigated. Incubation of [14C]-doxylamine succinate with human and rat intestinal microflora indicated that anaerobic bacteria were not capable of effecting the degradation of [14C]-doxylamine succinate. However, the incubation of [14C]-doxylamine succinate with isolated rat hepatocytes generated several metabolites similar to those observed in vivo. The nonconjugated doxylamine metabolites isolated and identified include: doxylamine N-oxide, desmethyldoxylamine, didesmethyldoxylamine and ring-hydroxylated products of doxylamine and desmethyldoxylamine. The studies demonstrate the role of hepatic metabolism in the elimination of doxylamine succinate in the rat.

Desorption chemical ionization and fast atom bombardment mass spectrometric studies of the glucuronide metabolites of doxylamine.

Three glucuronide metabolites of doxylamine succinate were collected in a single fraction using high-performance liquid chromatography (HPLC) from the urine of dosed male Fischer 344 rats. The metabolites were then separated using an additional HPLC step into fractions containing predominantly a single glucuronide metabolite. Analysis of the metabolites by methane and ammonia desorption chemical ionization, with and without derivatization, revealed fragment ions suggestive of a hydroxylated doxylamine moiety. Identification of the metabolites as glucuronides of doxylamine, desmethyldoxylamine and didesmethyldoxylamine was accomplished, based on determination of the molecular weight and exact mass of each metabolite using fast atom bombardment (FAB) ionization. This assignment was confirmed by the fragmentation observed in FAB mass spectrometric and tandem mass spectrometric experiments. Para-substitution of the glucuronide on the phenyl moiety was observed by 500-MHz nuclear magnetic resonance (NMR) spectrometry. A fraction containing all three glucuronide metabolites, after a single stage of HPLC separation, was also analysed by FAB mass spectrometry, and the proton- and potassium-containing quasimolecular ions for all three metabolites were observed.

Doxylamine metabolism in rat and monkey.

Metabolites of doxylamine obtained with rat-liver homogenate in vitro and from urine of Wistar rats and squirrel monkeys in vivo were examined. The metabolites were separated by g.l.c., h.p.l.c. and t.l.c., and tentatively identified through interpretation of their mass-spectrometric behaviour. N-Desmethyldoxylamine was identified in vitro while both N-desmethyl and N,N-didesmethyldoxylamine were detected in rat and monkey urine. The N-acetyl conjugates of N-desmethyl and N,N-didesmethyldoxylamine were tentatively identified both in rat urine and in vitro. Only the N-acetyl conjugate of N,N-didesmethyldoxylamine was detected in monkey urine. In addition, nine other metabolites were tentatively identified in rat urine: N,N-dimethyl-2-[1-(?-hydroxyphenyl)-1-(2-pyridyl)ethoxy]ethanamine; N-methyl-2-[1-(?-hydroxyphenyl)-1-(2-pyridyl)ethoxy]ethanamine; 1-phenyl-1-(2-pyridyl)ethanol; 1-(?-hydroxyphenyl)-1-(2-pyridyl)ethanol; 1-phenyl-1-(2-pyridyl)ethane; 1-(?-hydroxyphenyl)-1-(2-pyridyl)ethane; 2-phenyl-2-(2-pyridyl)ethanol; N,N-dimethyl-2-[1-phenyl-1-(2-pyridyl)-2-hydroxyethoxy]ethanamine; doxylamine pyridine N-oxide. The excretion of doxylamine aliphatic N-oxide in rat urine was confirmed by comparison with the authentic synthetic compound.

Metabolism of 14C-labeled doxylamine succinate (Bendectin) in the rhesus monkey (Macaca mulatta).

The time-course of the metabolic fate of [14C]doxylamine was determined after the p.o. administration of 13 mg/kg doxylamine succinate as Bendectin plus [14C]doxylamine succinate to the rhesus monkey. Urine and plasma samples were analyzed by reversed-phase high performance liquid chromatography (HPLC), chemical derivatization, and mass spectrometry. The cumulative 48-hr urinary metabolic profile contained 81% of the administered radiolabeled dose and consisted of at least six radiolabeled peaks. They were peak 1: unknown polar metabolites (8% of dose); peak 2: 2-[1-phenyl-1-(2-pyridinyl)ethoxy] acetic acid, 1-[1-phenyl-1(2-pyridinyl)ethoxy] methanol, and another minor metabolite(s) (31%); peak 3: doxylamine-N-oxide (1%); peak 4a: N,N-didesmethyldoxylamine (17%); peak 4b: doxylamine (4%); and peak 5: N-desmethyldoxylamine (20%). The plasma metabolic profile was the same as the urinary profile except for the absence of doxylamine-N-oxide. The maximum plasma concentrations and elapsed time to attain these concentrations were as follows. Peak 1: 540 ng/mL, 4 hr; peak 2: 1700 ng/mL, 1 hr; peak 4a: 430 ng/mL, 4 hr; peak 4b: 930 ng/mL, 2 hr; and peak 5: 790 ng/mL, 2 hr. These data suggest that in the monkey, doxylamine metabolism follows at least four pathways: a minor pathway to the N-oxide; a minor pathway to unknown polar metabolites; a major pathway to mono- and didesmethyldoxylamine via successive N-demethylation; and a major pathway to side-chain cleavage products (peak 2) via direct side-chain oxidation and/or deamination.

Metabolism of doxylamine succinate in Fischer 344 rats. Part I: Distribution and excretion.

Experiments were conducted with male and female rats (12 per group) dosed by gavage with 2 or 20 mg (based on the free amine) doxylamine succinate containing about 10 microCi 14C-doxylamine succinate to determine distribution and excretion of the activity as a function of dose and sex with time. Urine and feces were collected at intervals up to 72 hr. Most of the dose (approximately equal to 70%) was eliminated in the first 24 hr after dosing and 95 to 100% of the dose was recovered during the 72-hr course of the experiments with both sexes and dose levels. Less than 1% of the total dose remained in the rats at the end of the test period. The urinary route of elimination was more predominant than the fecal route in both sexes given the 20-mg dose. The fecal route predominates in low-dose males whereas there is no significant difference between urinary and fecal routes of elimination in low-dose females. Preliminary characterization of urinary metabolite form using extraction techniques shows 99% of the metabolites to be in the polar conjugated form.

The pharmacokinetics of doxylamine: use of automated gas chromatography with nitrogen-phosphorus detection.

Sixteen healthy male volunteers received a single oral dose of 25 mg doxylamine succinate. Doxylamine concentrations in plasma were measured by a newly developed gas chromatographic methodology, utilizing direct alkaline extraction into hexane:isoamyl alcohol followed by concentration and autoinjection. Doxylamine kinetics were determined from multiple plasma doxylamine concentrations measured during the 24 hours postdose. Mean kinetic variables were: peak plasma level, 99 ng/mL; time of peak, 2.4 hours postdose; elimination half-life, 10.1 hours; and apparent oral clearance, 217 mL/min. Analogous analytic methodology can be used to study the pharmacokinetics of other drugs of this class.

Mass spectral characterization of doxylamine and its rhesus monkey urinary metabolites.

This study describes the use of mass spectrometry (MS), high-performance liquid chromatography (HPLC) and chemical derivatization techniques for the identification of doxylamine and five rhesus monkey urinary metabolites. The analyses were performed using chemical ionization mass spectrometry with either methane or ammonia as the reagent gas. The confirmation of the structures of two of these urinary metabolites was aided by the synthesis of doxylamine N-oxide and desmethyldoxylamine and by the use of methylation and acetylation derivatization techniques. Doxylamine N-oxide, desmethyldoxylamine, didesmethyldoxylamine, and two metabolites which resulted from the cleavage of the aliphatic tertiary nitrogen side chain to the subsequent 2-[1-phenyl-1-(2-pyridinyl)ethoxy]acetic acid or 2-[1-phenyl-1-(2-pyridinyl)ethoxy]methanol compounds were isolated and identified from rhesus monkey urine. Additional data concerning the mass spectral analysis of derivatization or reaction products from the three chloroformate reactions with doxylamine, and the synthesis and separation techniques which afforded mass spectral identification of the urinary metabolites are also presented.

[Biotransformation of doxylamine: isolation, identification and synthesis of some metabolites (author's transl)]. / Zum Stoffwechsel des Doxylamin: Isolierung, Identifizierung und Syntheseeiniger Metabolite.

After administration of therapeutic doses of doxylamine, the unchanged drug(I) and five degradation products were detected in human urine; their chemical structures are discussed and - to some extent - confirmed by synthesis. The results show that biotransformation of doxylamine in man takes place by the following routes: successive dealkylations at the nitrogen atom, giving N-demethyl-doxylamine(II) and N.N-didemethyl-doxylamine(II); cleavage at the benzhydrylether-function, resulting in the formation of 1-phenyl-1-(2-pyridyl)-ethanol(V), 1-phenyl-1-(2-pyridyl)-ethane(VI) and 1-phenyl-1-(2-pyridyl)-ethene(VII). VI and VII may be artefacts. Identification of an additional degradation product(=IV) was not possible, because the isolated quantities were too small. The analytical properties (hydrolysis!) of the pure substance and free base are thoroughly discussed, as well as the role of Chemical Ionization Mass Spectrometry with various reagent gases for the examination of biological extracts.