Gas Chromatography-mass Fragmentography Research Articles

Cellular Distribution of the GABAA Receptor‐Modulating 3α‐Hydroxy, 5α‐Reduced Pregnane Steroids in the Adult Rat Brain

The 3alpha-hydroxy,5alpha-reduced pregnane steroids, allopregnanolone and allotetrahydrodeoxycorticosterone, are the most potent endogenous positive modulators of GABA(A) receptor-mediated inhibition. This study presents the first immunohistochemical examination of the cellular distribution of 3alpha-hydroxy,5alpha-reduced pregnane steroids across the brain. We found a widespread distribution in the adult rat, with dense immunolabelling in the olfactory bulb, striatum and cerebral cortex, and lower density labelling in the brainstem reticular formation. In general terms, this distribution accords with the regional concentrations of 3alpha-hydroxy,5alpha-reduced steroids determined, in other laboratories, by brain region sampling and either gas chromatography-mass fragmentography or radioimmunoassay. However, immunohistochemistry allowed for a more detailed examination of regional distribution and cellular specificity. All immunoreactivity was confined to the cell bodies and thick dendrites of neurones; no identifiable glia were labelled. In most brain areas, the location and morphology of labelled cells identified them as excitatory neurones. In addition, cell populations known to be projecting GABAergic neurones (e.g. cerebellar Purkinje cells) were immunoreactive, whereas local inhibitory neurones generally were not. The cellular distribution of 3alpha-hydroxy,5alpha-reduced steroids suggests that sensory, motor, limbic and homeostatic systems can be influenced by neurosteroids at multiple stages of processing.

Poor evidence for depolarization block but uncoupling of nigral from striatal dopamine metabolism after chronic haloperidol treatment in the rat

Chronic haloperidol treatment induces depolarization block in midbrain dopamine neuronal systems. We studied the effect of this treatment on nigrostriatal dopamine catabolism using microwave fixation in situ of the brain to prevent post-mortem changes. Male Sprague-Dawley rats were given haloperidol (0.4 mg/kg/day, i.p.) or vehicle for 21 days. On day 22, some rats in each group received a haloperidol challenge (0.4 mg/kg, i.p.), and the remaining rats were given the vehicle. Dopamine metabolite levels 60 min after the challenge were assayed by combined gas chromatography-mass fragmentography. Haloperidol pretreatment significantly modified haloperidol challenge effect on regional dopamine metabolite contents. The challenge elevated all striatal metabolites studied similarly in the chronic vehicle- or chronic haloperidol-pretreated rats. In contrast, it did not significantly affect nigral dopamine metabolites except it elevated 3,4-dihydroxyphenylacetic acid in the haloperidol-pretreated rats. A linear correlation between the nigral and striatal contents of 3-methoxytyramine (R = 0.72, p = 0.03), and a trend for correlation (R = 0.65, p = 0.06) between the respective 3,4-dihydroxyphenylacetic acid contents were found after the haloperidol challenge in the vehicle-pretreated rats only. These results suggest that chronic haloperidol treatment uncouples somatodendritic dopamine turnover and release from those in the axon terminals of nigrostriatal dopamine neurons.

Enantiomeric analysis of D- and L-pipecolic acid in plasma using a chiral capillary gas chromatography column and mass fragmentography.

Enantiomeric analysis of D- and L-pipecolic acid in plasma using a chiral capillary gas chromatography column and mass fragmentography.

Busulphan level and early mortality in thalassaemia patients after BMT.

The aim of the study was to correlate busulphan (BU) levels of thalassaemia patients with outcome of allogeneic transplant. BU levels were measured by gas chromatography mass fragmentography. All patients received a standardised dose of BU 16 mg/kg, and cyclophosphamide 150 or 200 mg/kg. For area-under-the-curve analysis (AUC), blood samples were obtained at 0, 1, 2, 3, 4 and 6 h after the first and fifth dose for all patients, and additional levels were measured after ninth and/or 13th dose in most patients. Outcome parameters examined included veno-occlusive disease of liver (VOD), idiopathic interstitial pneumonitis, chimerism, and day 90 survival. Twenty consecutive thalassaemia patients who underwent haematopoietic stem cell transplantation were studied. The median age at transplant was 11.2 years (range 3-21 years). Mean BU AUC levels were correlated with age at transplant (r = 0.58, P = 0.007). Nine patients developed VOD and six had mixed chimerism, but these did not correlate with mean BU AUC level. Four patients died before day 50 from VOD and interstitial pneumonitis. Patients with BU AUC levels greater than the median (908 micromol x min/l) had significantly lower probability of survival at day 90 (60%), whereas patients with BU AUC level less than the median all survived beyond day 90. No patient had graft rejection. In conclusion, a high BU AUC level was associated with a higher treatment-related mortality in thalassaemia patients after transplant.

Metabolism of coenzyme Q10: biliary and urinary excretion study in guinea pigs.

Biliary and urinary metabolites were examined after intravenous administration of 14C-coenzyme Q10 (14C-CoQ) to guinea pigs. Cumulative recovery of administered radioactivity for up to 8 hours by bile drainage was 4.8%. The greater part of radioactivity was detected in conjugate form. After hydrolyzing with beta-glucuronidase, aglycone fragments were subjected to methylation and reductive acetylation. The main metabolite was demonstrated to be Q acid-1 1,4-hydroquinone diacetate methyl ester (M-1) on HPLC. Then, the main metabolite was assumed to be glucuronide of 2,3-dimethoxy-5-methyl-6-(3'-methyl-5'-carboxy-2'-pentenyl)-1, 4-benzohydroquinone [Q acid-I hydroquinone]. The cumulative urinary recovery of the administered radioactivity over 48 hours was 8.3%. The labeled samples were treated similarly to bile. The urinary metabolites of CoQ10 consisted of unconjugated and conjugated forms. Lyophilized urine was treated as a bile sample and analyzed. The two major metabolites were assigned to be M-1 and Q acid-II 1,4-hydroquinone diacetate methyl ester (M-2). Then, the two metabolites were assumed to be composed of Q acid-I and 2,3-dimethoxy-5-methyl-6-(3'-carboxypropyl)-1,4-benzoquinone (Q acid-II) in free and corresponding hydroquinone conjugate forms. To investigate the effect of exogenous labeled CoQ10 on unlabeled CoQ10 (endogenous) metabolites in urine, simultaneous quantitative determination was performed using deuterium labeled CoQ10 (CoQ10-d5). Urine collected over a 72-hour period after intravenous administration of CoQ10-d5 was processed similarly to that described above and two derivatized metabolites (M-1 and M-2) were quantified by gas chromatography-mass fragmentography with the multi-ion detection method. The analytical results showed that the addition of exogenous labeled CoQ10 did not influence the metabolism (or breakdown) of unlabeled (endogenous) CoQ10.

Routine analysis of plasma busulfan by gas chromatography–mass fragmentography

Busulfan (BU) is a widely used alkylating agent for antineoplastic therapy and marrow ablation in preparation for bone marrow transplantation (BMT). High-dose BU often leads to successful preparation and low relapse but is associated with veno-occlusive disease of liver. We established a protocol to determine postdosage plasma BU concentrations by gas chromatography-mass fragmentography in an attempt to relate clinical outcome to plasma BU concentrations. We used nonisotopic pusulfan as the internal standard. After extraction into ethyl acetate, BU and pusulfan were iodinated into 1, 4-diiodobutane and 1,5-diiodopentane, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out on an Hewlett-Packard (HP) 5890II gas chromatograph with a 30-m 100% methyl silicon narrow bore, fused-silica capillary column interfaced with an HP 5970A mass spectrometer. Helium was the carrier gas. The sample molecules were identified by total ion monitoring and quantified by selective ion monitoring of m/z 183 and 197. The calibration curve was linear to 4 mg/L. The limit of quantification was 0.04 mg/L, and the analytical recovery was approximately 97%. The within-day and between-day imprecision (CV) was <6% and 9%, respectively. In a preliminary study of 12 children, the BU areas under the BU-time curve were 616-949 micromol. min/L after the first dose and 793-1143 micromol. min/L after the fifth dose. We conclude that the GC-MS procedure is suitable for routine analysis of plasma BU.

23] Measurement of pantothenic acid and hopantenic acid by gas chromatography-mass spectroscopy

This chapter investigates the properties of hopantenic acid (HOPA)—a natural homolog of pantothenic acid (PaA) containing γ -aminobutyric acid (GABA) in place of β -alanine. Its calcium salt and pharmacologic properties have been used to improve blood circulation and metabolism in the brain. Because hopantenic acid is used pharmacologically it is important to be able to determine HOPA and PaA separately for pharmacokinetic studies. Gas chromatography–mass fragmentography (GC–MF) has been used to assay HOPA after administration of calcium hopantenate. Other methods for the assay of HOPA have also been developed, such as the GC–MF determination of pantoyllactone, which is a product of HOPA hydrolysis, the colorimetric determination of GABA with sodium 1,2-naphthoquinone 4-sulfate, and a high-performance liquid chromatographic (HPLC) method with 9-anthryldiazomethane as a fluorescent derivative. A bioassay method using Lactobacillus arabinosus for the determination of trimethylsilylated pantoyllactone by GC–MF and for the assay of PaA is developed. The simultaneous rapid microanalysis for PaA and HOPA in biological samples and natural products by GC–MF with a wide-bore column and multiple ion detection (MID) is described in the chapter. 5-[(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)amino]pentanoic acid calcium salt is used as an internal standard (IS) to measure PaA and HOPA simultaneously in plasma samples from humans, monkeys, dogs, pigs, rabbits, mice, rats, chickens, and soft-shelled turtles; in brain samples of chicken and soft-shelled turtle; and also in natural products.

Dopamine release in the rat cerebellum and hippocampus: a tissue 3-methoxytyramine study

Multiple lines of evidence indicate dopamine is a neurotransmitter or neuromodulator in the cerebellum and hippocampus. In this study, we explored the utility of 3-methoxytyramine as an index of dopamine release in these regions. We found that: (1) cerebellar and hippocampal 3-methoxytyramine levels can be measured by combined gas chromatography-mass fragmentography with negative chemical ionization; (2) basal 3-methoxytyramine accumulation rates following monoamine oxidase inhibition, but not the steady-state tissue levels, are several times lower in these regions than in the frontal cortex; (3) accumulation of 3-methoxytyramine in the hippocampus and cerebellum can be enhanced following electroconvulsive shock, but not acute haloperidol (0.4 mg/kg) treatment. We conclude that 3-methoxytyramine accumulation may be a useful index of dopamine release in the cerebellum and hippocampus, but dopamine release is regulated differently in these regions than in the frontal cortex and striatum.

Plasma and leukemic cell pharmacokinetics of high-dose N4-behenoyl-1-beta-D-arabinofuranosylcytosine in acute leukemia patients.

The pharmacokinetics of N4-behenoyl-1-beta-D-arabinofuranosylcytosine (BHAC), a lipophilic antitumor analog of 1-beta-D-arabinofuranosylcytosine (ara-C), was investigated, by assay of plasma and leukemic cells of ten acute leukemic patients receiving 60-minute intravenous (IV) infusion of 700 mg/m2 BHAC, for BHAC and 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) by high-performance liquid chromatography, ara-C by radioimmunoassay, and 1-beta-D-arabinofuranosyluracil (ara-U) by gas chromatography-mass fragmentography. The plasma concentration of BHAC reached a maximum (173.4 +/- 75.3 micrograms/mL) at the end of the infusion and then declined in a biphasic pattern with an initial-phase half-life (t1/2 alpha) of 1.00 +/- .36 hours and a second-phase half-life (t1/2 beta) of 4.28 +/- 2.35 hours. That of ara-C similarly reached a maximum (102.2 +/- 39.9 mg/mL) at the end of the infusion and then declined with t1/2 alpha of 1.37 +/- 1.11 hours and t1/2 beta of 11.2 +/- 4.31 hours. Intracellular ara-CTP concentration increased in a linear-accumulation manner for the first 4 hours after the infusion, reached a maximum of .081 +/- .112 micrograms/10(7) cells at approximately 7 hours, and then declined very slowly in accordance with a one-compartment model with t1/2 of 13.56 +/- 9.62 hours.

Gas chromatographic-mass fragmentographic determination of serum 1α,25 dihydroxyvitamin D3

We extracted 1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] from 10 mL serum using Sep-Pak C18 and Sep-Pak Silica mini-columns and normal-phase high performance liquid chromatography (HPLC) separation for analysis by gas chromatography-mass fragmentography (GC-MS). A GC-MS method was optimised using manual tuning for ion mass calibration and selective ion monitoring (SIM) for quantitation. Serum 1 alpha,25(OH)2D3 was identified by superimposition of the m/z 452 and 501 ion peaks and by overlapping the m/z 452 ion peak with that of its authentic standard. It was quantitated from the relative peak areas of its m/z 452 ion and the m/z 363 ion of vitamin D2, the internal standard. Twenty picograms of 1 alpha,25(OH)2D3 gave a peak with a signal-to-noise ratio of 26:1. Between-batch coefficient of variation (CV) for 1 alpha,25(OH)2D3 standard was < 13%. However, serum analysis was less precise, within-batch CV being 20%. The analytical recovery was about 70% and detection limit 0.5 pg/mL. When compared with a commercial radioreceptor assay we still found our method to be sensitive, specific, and adequate for confirmative and semiquantitative analysis of serum 1 alpha,25(OH)2D3.

Mutagenicity of nitrodibenzofurans in Salmonella typhimurium strains

Heavy-duty diesel-exhaust particles were collected, extracted and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA100, TA98, TA98NR and TA98/1,8-DNP6 in the presence and absence of a 9000 × g post-mitochondrial supernatant from Aroclor-induced rat liver (S9 mix). The neutral and acidic fractions showed high mutagenicity with TA98 in the absence of S9 mix, the acidic fraction having the highest specific activity. In the absence of S9 mix, the mutagenicity of crude, neutral and acidic fractions was greater in TA98 than in TA98NR and TA98/1,8-DNP6.Chemically-synthesized nitroacetoxypyrenes and nitrohydroxypyrenes were fractionated into the neutral and acidic fractions, respectively. These nitroarenes were purified by high-performance liquid chromatography and their mutagenicity was measured with the 4 strains. With TA98 in the absence of S9 mix, 1-nitro-3-acetoxypyrene, 1-nitro-6/8-acetoxypyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6/8-hydroxypyrene induced 16 700, 336, 992, 94 His+ revertants per plate per nmole, respectively. In the absence of S9 mix, the level of mutagenicity of these nitroarenes was highest in TA98, lowest in TA98/1,8-DNP6 and intermediate in TA98NR.The neutral and acidic fractions of diesel-exhaust particles were analyzed by gas chromatography-mass spectrometry and gas chromatography-mass fragmentography. The neutral fraction was found to contain nitroacetoxypyrenes, 1-nitropyrene, 1,6-dinitropyrene, while nitrohydroxypyrenes were detected in the acidic fraction. The amounts of 1-nitro-3-acetoxypyrene, 1-nitropyrene, 1,6-dinitropyrene and 1-nitro-3-hydroxypyrene were 6.3, 62, 0.81, and 70 ng per mg of crude extract, and accounted for 12, 3.6, 8.0, and 9.0%, respectively, of mutagenicity of the crude extract in TA98 in the absence of S9 mix.

Quantification of dimethindene in plasma by gas chromatography—mass fragmentography using ammonia chemical ionization

A gas chromatographic—mass fragmentographic method using ammonia chemical ionization for the determination of dimethindene in human plasma is described. The drug was isolated from plasma by liquid—liquid extraction with hexane—2-methylbutanol. Plasma components were separated on a capillary column coated with chemically bonded methyl silicone. For detection of dimethindene, its quasi-molecular ion (M + H +) was mass fragmentographically monitored after chemical ionization with ammonia as reagent gas. Dimethindene was quantified using methaqualone as the internal standard: the quantification limit in plasma was 0.2 ng/ml, the within-run precision was 8.0% and the inter-run precision 5.6%. The plasma concentration—time profile was established after a single dose of 4 mg of dimethindene with an average maximum concentration of 5.5 ng/ml, detectable up to 48 h post application.

Analytical studies on the chiral separation and simultaneous determination of pantothenic acid and hopantenic acid enantiomers in rat plasma by gas chromatography—mass fragmentography: a reply

Analytical studies on the chiral separation and simultaneous determination of pantothenic acid and hopantenic acid enantiomers in rat plasma by gas chromatography—mass fragmentography: a reply

Analytical studies on the chiral separation and simultaneous determination of pantothenic acid and hopantenic acid enantiomers in rat plasma by gas chromatography—mass fragmentography: Reply to Küsters et al.

Analytical studies on the chiral separation and simultaneous determination of pantothenic acid and hopantenic acid enantiomers in rat plasma by gas chromatography—mass fragmentography: Reply to Küsters et al.

Analytical studies on the chiral separation and simultaneous determination of pantothenic acid and hopantenic acid enantiomers in rat plasma by gas chromatography—mass fragmentography

The chiral separation and simultaneous determination of D- and L-pantothenic acids and D- and L-hopantenic acids in rat plasma using gas chromatography-mass fragmentography are described. The method is based on deproteinization by ion-exchange resin, extraction with ethyl acetate under acidic conditions, and derivatization to form several interesting compounds. After methyl esterification for carboxylic acid of D- and L-pantothenic acids, D- and L-hopantenic acids could be derivatized to trifluoroacetate, cyclic sulphite, and cyclic n-butylboronate for hydroxy groups. D- and L-forms of these derivatives were completely separated by mass fragmentographic technique with quasi-molecular ions. Calcium salt of D-5-[(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)amino]pentanoic acid was used as an internal standard for the determination of DL-pantothenic acids and DL-hopantenic acids. The detection limits of DL-pantothenic acids and DL-hopantenic acids in this method were 5 and 12 ng, respectively. This method could be applied to the study of plasma levels of D-, L-pantothenic acids and D-, L-hopantenic acids in rat.

Simultaneous determination of pantothenic acid and hopantenic acid in biological samples and natural products by gas chromatography-mass fragmentography

A method for the simultaneous determination of pantothenic acid and hopantenic acid in plasma samples was developed using gas chromatography-mass spectrometry with multiple ion detection. Plasma samples were directly purified without deproteinization on an ion-exchange resin, and the eluate was extracted with ethyl acetate under acidic conditions. The organic layer was evaporated to dryness under a stream of nitrogen, and the residue was dissolved in an internal standard solution. Pantothenic and hopantenic acids were converted into their trimethylsilyl derivatives by treating with bis(trimethylsilyl)trifluoroacetamide. Aliquots of this solution were injected into the gas chromatograph-mass spectrometer, which was equipped with a wide-bore fused-silica column (DB-17) and analysed by the multiple ion detection method. The detection limits for pantothenic acid and hopantenic acid in plasma were 1 ng/ml each at a signal-to-noise ratio of 5. This method was applied to a study of the assay of pantothenic acid and hopantenic acid in biological samples and natural products.

Constant infusion of [ 13C 6]glucose: Simultaneous measurement of turnover of GABA and glutamate in defined regions of the brain of individual animals

A new method has been developed to measure simultaneously the turnover rates of glutamate and GABA in individual areas of the brain of the rat. Rats received a constant infusion of [ 13C 6]glucose, such that the flux of this stable isotope label through the pools of glucose, glutamate and GABA in the central nervous system (CNS) could be monitored by gas chromatography-mass fragmentography. The ratios of glucose/GABA and glucose/glutamate labelling were then used to calculate the fractional rate constants for GABA and glutamate, respectively. Using this approach, baclofen (20 mg/kg) decreased the turnover rates of both glutamate and GABA in the cerebellum, prefrontal cortex, striatum and hippocampus of the rat. In contrast, only the turnover of GABA was decreased in the septum and superior colliculus. Muscimol decreased the turnover rates of both amino acids in all regions of the brain examined. These data, therefore, provide in vivo support for the results of previous in vitro studies which indicated that cortical glutamatergic nerve endings and/or cell bodies possess inhibitory GABA B receptors. The present data further suggest that not all glutamatergic projections possess these receptors.

Determination of a new hypolipidemic agent, 2-(trans-4-isobutylcyclohexyl)-2-oxoethyl benzenesulphonate, and its metabolite by high-performance liquid chromatography and gas chromatography—mass fragmentography in human biological materials

Determination of a new hypolipidemic agent, 2-(trans-4-isobutylcyclohexyl)-2-oxoethyl benzenesulphonate, and its metabolite by high-performance liquid chromatography and gas chromatography—mass fragmentography in human biological materials

The effects of scopolamine and traumatic brain injury on central cholinergic neurons.

This study examined the effects of scopolamine and fluid percussion traumatic brain injury (TBI) on the activity of cholinergic neurons in specific areas of the rat brain 12 min, 4 h, and 24 h after injury. Acetylcholine (ACh) turnover, used as an index of cholinergic neuronal activity, was determined using gas chromatography-mass fragmentography. Scopolamine pretreatment prevented significant increases in dorsal pontine ACh turnover at 12 min and 4 h after TBI, suggesting that the drug's protective actions against the neurologic deficits following TBI may involve blockade of cholinergic neuronal activation as well as postsynaptic muscarinic blockade. The responses of thalamic, hippocampal, and amygdaloid cholinergic neurons to TBI did not differ substantially in scopolamine-pretreated rats from those studied previously in untreated fluid-percussion-injured rats. However, cholinergic neurons in the cingulate-frontal cortex of rats receiving TBI did respond in a different manner to scopolamine than those of rats receiving sham injury, suggesting a disruption of regulation of cortical cholinergic neurons following this model of TBI.

Detection and quantitation of urinary 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid, a metabolite of tetrahydrocannabinol, by capillary gas chromatography and electron impact mass fragmentography.

A procedure for detection and quantitation of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid, a major metabolite of delta-9-tetrahydrocannabinol in urine, has been described. Since the metabolite is present in both conjugated and unconjugated forms, hydrolysis of urine was carried out to increase the sensitivity of detection. The acidic metabolite was isolated by strongly basic anion exchange resin, and subsequently derivatized to methyl 1-dehydroxy-1-methoxy-11-nor-delta-9-tetrahydrocannabinol-9-carbox ylate (1a) by methyliodide in the presence of tetramethylammonium hydroxide. The derivatized product was separated in a capillary column gas chromatograph, and finally detected by a mass spectrometer under electron impact mode. Confirmation of the product was carried out by monitoring three ions that represent the major portions of the molecule and comparing their relative abundances to that of a standard. Quantitation was based on 5'-2H3-11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid as internal standard. Excellent linearity was obtained over the range of 2 to 1000 ng/mL. The overall yield of extraction using the anion exchange resin was 50 to 60%. This extraction process is rapid and suitable for a large number of sample analyses. The methylated product (1a) is stable for at least 72 hr at room temperature.