Sodium-Dependent Growth of Azotobacter chroococcum

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.     

Sodium-Dependent Growth of Azotobacter chroococcum

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.     

Assay of 2-Hydroxyputrescine in Various Regions of Rat Brain by Gas Chromatography-Mass Spectrometry

2-Hydroxyputrescine in seven regions of single rat brains was measured with a sensitive, specific assay by gas chromatography-mass spectrometry. The regions were the cerebral cortex, cerebellum, medulla oblongata, hypothalamus, striatum, hippocampus, and midbrain. The level of 2-hydroxyputrescine was very high in the cerebral cortex and cerebellum, high in the medulla oblongata, hypothalamus, and hippocampus, and low in the striatum and midbrain. The level of 2-hydroxyputrescine in the cerebellum was significantly higher than in the striatum and midbrain.     

Detection of fluoxetine in brain, blood, liver and hair of rats using gas chromatography-mass spectrometry

This study reports the measurements of fluoxetine in discrete brain regions, blood, liver and hair of male rats injected with 10 mg/kg fluoxetine HCl for 15 consecutive days. Concentrations of the antidepressant were obtained by gas chromatography-mass spectrometry (GC-MS) methodology. In brain, fluoxetine levels were unevenly distributed, with the raphé nucleus containing the highest amounts relative to the hypothalamus or striatum. Fluoxetine was also measured in blood and liver roughly paralleling those ratios described in previous rodent studies. Of potential interest, fluoxetine was found to accumulate in rat hair after chronic treatment. Detection of fluoxetine in hair by GC-MS could be used as a marker for probative analyses.     

Metabolism of leukotriene B4 in isolated rat hepatocytes. Identification of a novel 18-carboxy-19,20-dinor leukotriene B4 metabolite

Isolated rat heptocytes were found to metabolize leukotriene B4 (LTB4) to a number of products which could be separated by reverse phase high performance liquid chromatography (HPLC). After incubation of LTB4 with hepatocytes for 15 min, the known omega-oxidized metabolites, 20-hydroxy- and 20-carboxy-LTB4, were identified by HPLC retention time and gas chromatography-mass spectrometry. An early fraction corresponding to 15% of the initial LTB4 was structurally characterized as a novel metabolite, 18-carboxy-19,20-dinor-LTB4, by ultraviolet spectroscopy and gas chromatography-mass spectrometry of the derivatized and derivatized, reduced metabolite. The short HPLC retention time of this metabolite was consistent with its reduced lipophilicity. An additional minor metabolite was tentatively identified as 3-hydroxy-LTB4. These two novel metabolites provide evidence for beta-oxidation as an important route of hepatic biotransformation of LTB4 and 20-hydroxy-LTB4.     

The cholecalciferol sulphate system in mammals

7-Dehydrocholesterol sulphate has been identified in human and rat skin. The compound was isolated by anion exchange chromatography and following hydrolysis it was characterized by high-performance liquid chromatography and gas chromatography-mass spectrometry. Experiments with rats showed that 7-dehydrocholesterol sulphate can serve as a precursor of cholecalciferol sulphate and 25-hydroxy-cholecalciferol 3-sulphate, the latter compound being present in significant amounts in human blood. The sulphated sterols identified represent a previously unknown secosteroid system in mammals.     

Studies on the metabolism and toxicological detection of the Eschscholtzia californica alkaloids californine and protopine in urine using gas chromatography-mass spectrometry

Eschscholtzia californica preparations are in use as phytopharmaceuticals and as herbal drugs. Studies are described on the metabolism and the toxicological analysis of the Eschscholtzia californica alkaloids californine and protopine in rat urine using gas chromatography-mass spectrometry. The identified metabolites indicated that californine is extensively metabolized by N-demethylation and/or single or double demethylenation with consecutive catechol-O-methylation of one of the hydroxy groups. Protopine, however, only undergoes extensive demethylenation of the 2,3-methylenedioxy group followed by catechol-O-methylation. All phenolic hydroxy metabolites were found to be partly conjugated. The authors' systematic toxicological analysis procedure using full-scan gas chromatography-mass spectrometry after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of the main metabolites of californine and protopine in rat urine after a dose which should correspond to that of drug users. Therefore, use of Eschscholtzia californica preparations should also be detectable in human urine by the authors' systematic toxicological analysis procedure.     

Quantitative determination of carbamazepine and carbamazepine-10,11-epoxide in rat brain areas by multiple ion detection mass fragmentography.

A specific and sensitive method is described for the simultaneous determination of carbamazepineand carbamazepine-10,11-epoxide in biological specimens by combined gas chromatography-mass spectrometry. Cytenamide is used as the internal standard for quantitation. Kinetics of the distribution of the drug and its metabolite in plasma and in different areas of rat brain are reported. Determinations are possible at the nanogram level.     

A comparative study in cultured rat liver cells of the metabolism of different analogues and metabolites of a natural hepatocarcinogen: safrol]

Metabolism by sexed rat liver cell lines of safrole, a natural hepatocancerogen and of metabolites and analogs were studied and compared by gas chromatography-mass spectrometry. The epoxide-diol route was evidenced for safrole, isosafrole and eugenol as well as the induction of drug metabolizing enzymes by tested compounds.     

Gas chromatography-mass spectrometry analysis of tert.-butyldimethylsilyl derivatives of 2-acetylaminofluorene and metabolites in isolated rat hepatocytes

A new technique for the conversion of 2-acetylaminofuorene and several ring-hydroxylated metabolites to mono- and di-tert.-butyldimethylsilyl derivatives was developed to permit their analysis by gas chromatography-mass spectrometry in order to quantify the metabolism of 2-acetylaminofluorene incubated in freshly isolated rat hepatocytes. This new gas chromatography-mass spectrometry method allowed the separation, identification and quantitation of seven known metabolites comprising five arylhydroxylated compounds, 2-aminofuorene and N-hydroxy-2-acetylaminofuorene.Abbreviations 2-AAF 2-acetylaminofluorene - 2-AF 2-aminofluorene - DMF dimethylformamide - El electron impact ionization - FBS fetal bovine serum - GC-MS gas chromatography-mass spectrometry - MtBSTFA N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide - MU methylene unit - N-OH-2-AAF N-hydroxy-2-acetylaminofluorene - 4,4-OH-BP 4,4-hydroxybiphenyl - tBDMS tert.-butyldimethylsilyl     

Identification of a pyrovalerone metabolite in the rat by gas chromatography-mass spectrometry and determination of pyrovalerone by gas chromatography-nitrogen-phosphorus detection

Pyrovalerone and its hydrolated metabolite have been identified by gas chromatography-mass spectrometry in rat urine and plasma. A sensitive gas chromatographic method for the quantitative analysis of pyrovalerone in rat urine and plasma is described. The method also permits the quantitative monitoring of the urinary excretion of the drug and its metabolite. Pyrovalerone and its hydroxylated metabolite are detected up to 18 h after a single oral administration to the rat at a dose of 20 mg/kg.     

Alterations in the rat renal glycosaminoglycans in streptozotocin-induced diabetes

Incubation of rat lung cytosol with arachidonic acid produced 12-hydroxy-5,8,10,14-eicosatetraenoic acid as a major product, which was identified by gas chromatography-mass spectrometry. By ammonium sulfate fractionation and DEAE-cellulose chromatography the arachidonate 12-lipoxygenase was purified about 30-fold from the rat lung cytosol. The partially purified enzyme was mostly free of the glutathione peroxidase activity and transformed arachidonic acid to its 12-hydroperoxide. 5,8,11,14,17-Eicosapentaenoic acid was also an active substrate, and the oxygenation at C-12 was confirmed by mass spectrometry. A significant amount of 12-lipoxygenase activity was also found in the microsomes and other particulate fractions.     

Isolation and quantitation of isopentenyladenosine in an anise cell culture by single-ion monitoring,radioimmunoassay and bioassay

Using three different techniques, isopentenyladenosine was identified and quantitated in an anise cell line (Pimpinella anisum L.), growing on a medium without cytokinin. A method to quantitate cytokinins was developed which utilizes extraction of cytokinins in the presence of a deuterated reference compound and subsequent quantitation of the cytokinins by single-ion monitoring on a gas chromatography-mass spectrometry system. The results were compared with those obtained by radioimmunoassay as well as a bioassay. Quantitations by gas chromatography-mass spectrometry and radioimmunoassay correlated well, whereas the estimates by the bioassay gave considerably lower values.     

Release of N-Acetylaspartylglutamate from Slices of Rat Cerebellum, Striatum, and Spinal Cord, and the Effect of Climbing Fiber Deprivation

Abstract: The release of endogenous N -acetylaspartylglutamate (NAAG) from slices of rat cerebellum, striatum, and spinal cord upon depolarization with 50 m M K⁺ was investigated. NAAG in superfusates was prepurified using an ion exchanger, esterified, and then quantified by gas chromatography-mass spectrometry. Deuterated NAAG was used as internal standard. A depolarization-induced release of NAAG was found in all three regions. The release was Ca²⁺ dependent to over 85% in cerebellum and striatum, but only to approximately 70% in spinal cord. In addition, the effect of lesions of the olivocerebellar pathway on the K⁺-induced release of NAAG was studied: Treatment of the animals with 3-acetylpyridine reduced the release of NAAG from cerebellar hemispheres significantly, by about 40% compared with controls. These results suggest that part of the NAAG released from cerebellar slices on depolarization is related to climbing fibers. Implications of these findings concerning possible physiological roles of NAAG in the three CNS regions are discussed.     

Metabolic activation by adrenal tissue in rats of a liver carcinogen: safrole]

In vivo and in vitro metabolisms by the rat adrenal tissue of safrol, a natural hepatocarcinogen, were studied by gas chromatography-mass spectrometry and compared to the hepatic metabolism. In the adrenals the epoxide-diol pathway was found besides demethylase and hydroxylase activities, suggesting that the adrenal cortex may participate to the metabolic activation of a procarcinogen.     

Specificity and the reliability of quantitation in the analysis of neurotransmitters

The unique advantages of selected ion monitoring, when using gas chromatography-mass spectrometry (GC-MS) as an analytical method for neurotransmitters are presented, using two examples: (1) paired quantitations of GABA from 11 brain regions, using different ion ratios of native and deuterated GABA: (a) m/z 190/194 and (b) m/z 204/210. Of 44 data pairs of GABA concentration calculated using these ratios, only 2 differed by > 10%, 23 differed by <5%, 15 differed by <2.0% and 3 were identical. In no case was the difference statistically significant. (2) The analysis of dopamine in the developing nervous system of rat. Based on its GC-MS characteristics, a dopamine-like compound was detected in the central nervous system (CNS) of rat, in high concentration, during embryonic and neonatal life. It co-eluted with dopamine, and generated two (m/z 387,415) of the three (m/z 387,415,428) ions normally monitored from authentic dopamine. The analysis of this compound by GC alone, with an electron capture detector, using the preparative procedure described, would have lead to the false, positive conclusion of high concentrations of dopamine in the CNS of rat during fetal and neonatal life.     

Norcocaine: a pharmacologically active metabolite of cocaine found in brain

N-Demethylation of cocaine results in the production of norcocaine, which has been identified by gas chromatography-mass spectrometry in the brains of monkeys given repeated doses of cocaine. This metabolite is about as active as cocaine in inhibiting uptake of ³H-norepinephrine by synaptosomes prepared from rat brain. Other cocaine derivatives have been found to be relatively inactive in inhibiting uptake of this amine.     

IDENTIFICATION AND QUANTIFICATION OF 3-METHOXY-4-HYDROXYPHENYLETHANOL (MOPET) IN HUMAN CEREBROSPINAL FLUID AND RAT BRAIN BY MEANS OF GAS CHROMATOGRAPHY-MASS SPECTROMETRY

Abstract— The identification of 3-methoxy-4-hydroxyphenylethanol (MOPET) in human cerebrospinal fluid and in rat brain is described. Use was made of the high sensitivity and selectivity provided by gas chromatography-mass spectrometry. Concentrations of MOPET in human cerebrospinal fluid, rat brain and rat urine together with those of some other catecholamine metabolites are given. The effect of intraperitoneal administration of deuterium-labelled MOPET and haloperidol on rat brain and urine MOPET levels was studied. The quantitative importance of MOPET as an end product of central and peripheral dopamine metabolism in man and rat is discussed.     

Amino acid sequencing by gas chromatography-mass spectrometry using trifluoro-dideuteroalkylated peptide derivatives: C. The primary structure of the carboxypeptidase inhibitor from potatoes

The carboxypeptidase inhibitor from potatoes has been used to demonstrate the utility of gas chromatography-mass spectrometry for the determination of the primary structure of such large polypeptides. Two mixtures of oligopeptide fragments, obtained by limited acid hydrolysis and enzymatic digestion of this polypeptide, were transformed into the corresponding mixtures of O-trimethyl-silylated trifluoro-dideuteroethyl polyamino alcohols which were then analyzed by gas chromatography-mass spectrometry. The resulting mass spectral and retention index data allowed the identification of 61 oligopeptide fragments which were assembled by the computer by positioning all 39 amino acid residues in a unique sequence (with the exception of the assignment of the primary amide groups of Asn and Gln).     

Dihydrozeatin riboside,a minor cytokinin from the leaves of Phaseolus vulgaris L.

Dihydrozeatin riboside has been identified in the leaves of decapitated bean plants by Sephadex LH20 chromatography and combined gas chromatography-mass spectrometry. The relationship between the cytokinins isolated and identified from this system and those previously reported in Phaseolus is discussed.Abbreviations DHZ dihydrozeatin - DHZOG dihydrozeatin-O--D-glucoside - DHZR dihydrozeatin riboside - GCMS combined gas chromatography-mass spectrometry - GLC gas-liquid chromatography - TIC total ion current - TMS trimethylsilyl - Z zeatin - ZR zeatin riboside