Gas Chromatography-electron Impact Mass Spectrometry Research Articles

Simultaneous analysis of diphenylmethoxyacetic acid, a metabolite of diphenhydramine, and its deuterium-labeled stable isotope analog in ovine plasma and urine.

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydramine in monkeys, dogs, and humans. The metabolic fate of diphenhydramine (DPHM) in sheep is not yet well understood; however, preliminary studies have demonstrated the presence of DPMA in the plasma and urine of sheep following an intravenous bolus of DPHM. Our current studies employ the simultaneous intravenous co-administration of DPHM and the stable isotope analog of DPHM to investigate the pharmacokinetics of DPHM in sheep. In these studies, in order to investigate the pharmacokinetics of the DPMA metabolite, measurement of both unlabeled and stable-isotope labeled DPMA is required. Thus, a stable isotope analog of DPMA ([2H10]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chromatography-electron-impact mass spectrometry (GC-EI-MS) analysis of DPMA and [2H10]DPMA used a single step liquid-liquid extraction procedure using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide. A 1.0-microliter aliquot of the prepared sample was injected into the GC-MS system and quantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenylacetic acid, m/z 183 for DPMA, and m/z 177 for [2H10]DPMA. The ion chromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The intra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10% at all other concentrations). This method was used to measure the concentration of DPMA and [2H10]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [2H10]DPHM were administered by an intravenous bolus dose via the femoral vein. DPMA appeared to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [2H10]DPMA in biological samples obtained from sheep (e.g. plasma and urine).

Effects of hypolipidemic drugs on hepatic degradation of apolipoprotein B and production of apo B-containing lipoproteins

Applicability of the gas chromatography-electron impact-mass spectrometry and data system (GC-EI-MS) for fatty acid analysis of formalin-fixed human tissues was evaluated by comparison of formalin-fixed and unfixed brain tissues, and the results of GC-EI-MS analyses of the formalin-fixed brain and adrenal cortex from cases with adrenoleukodystrophy (ALD) and the cerebro-hepato-renal syndrome of Zellweger (ZWS) were presented. When formalin-fixed and unfixed materials were compared, a reduction of polyunsaturated fatty acids was observed after formalin fixation, while saturated and monounsaturated fatty acids remained unchanged. The chromatographic patterns of brain tissues fixed in 10% formalin for 3 days or 17 months were basically the same. In ALD, increased C26:0C22:0 ratios in the cerebral white matter (2.05 in ALD versus 0.74 in control) and in the adrenal gland (15.2 in ALD versus 0.1 in control), and the presence of unusual very long chain fatty acids (VLCFA), longer than C29 : 0, were demonstrated. In the ZWS brain, all of the VLCFAs were severely depleted. Thus, formalin-fixed tissue may be a reliable subject to fatty acid analysis.

Cuticular hydrocarbons ofAedes hendersoni cockerell andA. triseriatus (SAY)

Field-caught adult male and femaleAedes hendersoni are difficult to distinguish from the sibling speciesA. triseriatus. We found that mosquitoes from the same sex of the sibling species can not be readily separated either by unique cuticular hydrocarbon components or by differences in percent composition of those components. Multivariate analysis of the cuticular hydrocarbon data does not provide good separation. Cuticular hydrocarbons were identified using gas chromatography electron-impact mass spectrometry and gas chromatography chemical-ionization mass spectrometry. Flame-ionization capillary gas chromatography was used for quantitative analysis of individual mosquitoes. Sixty-four hydrocarbons with chain lengths from C16 to greater than C46 were common to both species. Identified hydrocarbon components weren-alkanes, monomethylalkanes, dimethylalkanes, trimethylalkanes, and alkenes.

Different mechanisms of regioselection of fatty acid hydroxylation by laurate (omega-1)-hydroxylating P450s, P450 2C2 and P450 2E1.

P450 2C2 as well as P450 2E1 [Fukuda, T. et al. (1993) J. Biochem. 113, 7-12] catalyzed the hydroxylation of medium chain fatty acids, although the regioselectivity of substrates of the former contrasted with that of the latter. Whereas P450 2E1 hydroxylated C9-C18 fatty acids at the omega-1 position and to a much lesser extent at the omega and omega-2 positions, P450 2C2 hydroxylated C9-C13 fatty acids at different positions dependent on the chain length of fatty acids. Among the fatty acids used as the substrate, undecanoate was hydroxylated at the omega-1 position almost exclusively by P450 2C2. The proportion of omega-hydroxylated products produced by P450 2C2 was markedly increased with decreasing chain length of fatty acids, while the hydroxylation positions were enlarged to the omega-3 position with tridecanoate. When the conserved Thr at the putative distal helix was replaced with Ser, the substrate regioselectivity of the two P450s was affected in different manners. The mutation of P450 2C2 did not change the hydroxylation positions of C9-C12 fatty acids, but caused a significant decrease in the proportion of the omega-1 hydroxy analog in the total products. In sharp contrast to P450 2C2, the mutated P450 2E1 gave additional products to those with the wild-type P450, and the number of different products increased with increasing chain length of the fatty acids. Thus, the products of palmitate hydroxylation were identified as omega-1, omega-2, omega-3, omega-4, omega-5, omega-6, and omega-7 monohydroxy isomers using gas chromatography-electron impact mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of clonidine in human plasma by gas chromatography—electron-impact mass spectrometry

A new and sensitive gas chromatographic—mass spectrometric (GC—MS) assay for routine analysis of clonidine in human plasma is described. Quantification is carried out with simple electron-impact ionization mass spectrometry and solid-phase cartridges for preliminary extraction from plasma. The pentafluorobenzyl derivative of clonidine yields an intense ion fragment at m/z 354, and the lower limit of detection is 0.025 ng/ml for a 1-ml plasma sample. The practical applicability of this method is demonstrated by determining plasma concentrations of clonidine in a clinical study of a new transdermal delivery system for clonidine.

Catalytic properties of rabbit kidney fatty acid ω-hydroxylase cytochrome P-450ka2 (CYP4A7)

We have examined in detail the substrate specificity of a rabbit kidney fatty acid ω-hydroxylase, designated cytochrome P-450ka2 (CYP4A7). The hydroxylation products were identified as ω- and (ω - 1)-hydroxy fatty acids mainly using gas chromatography-electron impact mass spectrometry. [1]Straight-chain saturated fatty acids ranging from 10 to 19 carbons were effectively hydroxylated at the ω- and (ω - 1)-position. The ratios of ω- to (ω - 1)-hydroxylation activity decreased with increasing the carbon chain length of fatty acids. [2]Both isomyristate and anteisomyristate, and isopalmitate were hydroxylated several fold more rapidly than myristate and palmitate, respectively, with iso-branched chain fatty acids being hydroxylated at the ω-position solely. [3]Both palmitoleate and palmitoelaidate, and both oleate and elaidate were hydroxylated much more rapidly than palmitate and stearate, respectively. [4]Linoleate, γ-linolenate, and arachidonate were also excellent substrates for this enzyme. [5]Prostaglandin (PG) A 1 and PGA 2 were efficiently hydroxylated at the ω-position solely, with PGE 1 and PGE 2 being much less active. [6]Arachidonic acid not only showed a K m value significantly lower than those for lauric acid, γ-linolenic acid and PGA 1, but also it is a potent competitor for lauric acid and PGA 1, showing a very high affinity for the enzyme. It is possible that arachidonic acid is the physiological substrate for kidney P-450ka2.

Geographic variation of cuticular hydrocarbons among fourteen populations of Aedes albopictus (Diptera: Culicidae).

Cuticular hydrocarbons were used to differentiate among fourteen Aedes albopictus (Skuse) populations from Asia, Brazil, and the United States. Forty cuticular hydrocarbon peaks from each North American population were previously identified using gas chromatography electron impact-mass spectrometry. The same cuticular hydrocarbon peaks were identified in the Asian and Brazilian populations. Samples from three populations were analyzed using capillary gas chromatography chemical ionization mass spectrometry. Total carbon number and branch points were found for four peaks which occurred at equivalent chain lengths > 38.65. Quantitative analyses were performed by flame ionization gas chromatography. All populations contained the same cuticular hydrocarbons but showed differences in percentage composition. Nine of the 14 populations were separable by discriminant analysis of hydrocarbon components. Overlap of the 95% CIs occurred between the two Brazilian populations and among three North American populations. Individual specimens from three North American populations were used as a test group in the discriminant analysis. Correct classification of these individuals by population ranged from 80 to 100%.

Quantitation of Hydroperoxy-Eicosatetraenoic Acids and Hydroxy-Eicosatetraenoic Acids as Indicators of Lipid Peroxidation Using Gas Chromatography-Mass Spectrometry

Peroxidation of cellular membrane lipids has been implicated in a wide variety of acute and chronic pathologies. We have developed a method for quantifying lipid peroxidation products using gas chromatography-mass spectrometry (GC-MS) in order to help elucidate the role of lipid peroxidation in such disorders. The method involves analysis of the methyl ester, trimethylsilyl ether derivatives of various hydroxyeicosatetraenoic acids (HETEs). 16-Hydroxy-9, 12,14-heneicosatrienoic acid was synthesized for use as an internal standard. The assay involves the following steps: The internal standard is added to the sample and cellular lipids are extracted and transesterified. Next, any hydroperoxides are reduced with triphenylphosphine and the samples are subjected to two steps of solid phase extraction. The samples are then hydrogenated and the trimethylsilyl ether derivative of the hydroxyls formed. The derivatized HETEs are analyzed by electron impact GC-MS. 12-HETE, 11-HETE, 9-HETE, and 8-HETE are assayed by monitoring ions at m/ z 301, 287, 259, and 271, respectively. Standard curves were constructed for each HETE and were linear over the range 1 to 250 ng; correlation coefficients were typically greater than 0.99. The assay has been applied to the study of autoxidation of lipids in both in vitro and in vivo systems.

Determination of four metabolites of the plasticizer di(2-ethylhexyl)phthalate in human urine samples.

A method for biological monitoring of exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) is described. In this method the four main metabolites of DEHP [i.e., mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate, mono(2-ethyl-5-oxohexyl)phthalate, and mono(2-ethyl-5-hydroxyhexyl)-phthalate] are determined in urine samples. The procedure includes enzymatic hydrolysis, ether extraction, and derivatization with triethyloxonium tetrafluoroborate. Analysis is performed by gas chromatography-electron impact mass spectrometry. The detection limit for all four metabolites is less than 25 micrograms/l urine. The coefficient of variation based on duplicate determinations of urine samples of workers occupationally exposed to DEHP was 16% for MEHP (mean concentration 0.157 mg/l) and 6%-9% for the other three metabolites (mean concentrations 0.130-0.175 mg/l). The method described here was used to study DEHP metabolism in man. Most persons excrete mono(2-ethyl-5-oxohexyl)-phthalate and mono(2-ethyl-5-hydroxyhexyl)phthalate as a (glucuronide) conjugate. Mono(5-carboxy-2-ethylpentyl)phthalate is mainly excreted in free form, while for MEHP a large interindividual variation in conjugation status was observed. Of the four metabolites quantified, 52% are products of a (omega-1)-hydroxylation reaction of MEHP [i.e., mono(2-ethyl-5-oxohexyl)phthalate and mono(2-ethyl-5-hydroxylation reaction of MEHP [i.e., mono(5-carboxy-2-ethylpentyl)phthalate], and 26% is not oxidized further (i.e., MEHP). A good correlation is obtained when the amount of MEHP omega-hydroxylation products is compared with the amount of MEHP (omega-1)-hydroxylation products in urine samples. When the internal dose of DEHP has to be established we recommend that the levels of all four metabolites of DEHP be studied in urine samples.

Determination on the microscale of plasmatic lactic acid as its t-butyldimethylsilyl derivative by stable-isotope dilution using capillary gas chromatography/mass spectrometry.

A new sensitive and precise method for the determination of lactic acid in plasmatic microsamples (50 microL) has been developed. Lactic acid was directly extracted from plasma by ethyl acetate in acidic conditions, and analysed as its di-t-butyldimethylsilyl derivative by capillary gas chromatography/electron-impact mass spectrometry (GC/MS). The internal standard was a previously synthesized deuterated compound, 3-[2H]-(2R)-lactic acid. The method gives good reproductibility and precision, the overall standard deviation being better than 3%. The GC/MS assay was in good agreement with the enzymatic determination.

Determination of dioxopiperazine metabolites of quinapril in biological fluids by gas chromatography—mass spectrometry

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane—dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography—electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a signal-to-noise ratio of > 3 and > 5, respectively. The proposed method is applicable to pharmacokinetic studies.

Pyrrolizidine alkaloid composition of three Chinese medicinal herbs, Eupatorium cannabinum, E. japonicum and Crotalaria assamica.

The pyrrolizidine alkaloid composition of three Chinese herbs, "pei lan", "cheng gan cao" and "zi xiao rong," identified respectively as Eupatorium cannabinum, Eupatorium japonicum (Compositae) and Crotalaria assamica (Leguminosae), were studied by fast atom bombardment mass spectrometry and gas chromatography-electron impact mass spectrometry. Viridiflorine, cynaustraline, amabiline, supinine, echinatine, rinderine and isomers of these alkaloids were found in the Eupatorium species. Monocrotaline was the only pyrrolizidine alkaloid detected in the Crotalaria species.

Cuticular Hydrocarbon Geographic Variation Among Seven North American Populations of Aedes albopictus (Diptera: Culicidae)

Cuticular hydrocarbons were used to differentiate among the following North American populations of Aedes albopictus: Chicago, Ill.; Milford, Del.; Jacksonville, Fla.; San Antonio, Tex.; New Orleans, La.; Houston, Tex.; and Lexington, Ky. Forty cuticular hydrocarbon peaks were identified from each population using gas chromatography electron impact-mass spectrometry: 16 n-alkanes, 21 monomethylalkanes, and 3 dimethylalkanes. Fourteen peaks occurred at equivalent chain lengths greater than 38.65 and consisted of complex mixtures of dimethyl- and trimethylalkanes. Quantitative analyses were performed on pooled samples of five females from each population using flame ionization gas chromatography. All populations contained the same cuticular hydrocarbons, but showed differences in percent composition. Five of seven populations were separable by discriminant analysis of hydrocarbon components.

Liquid chromatography-thermospray tandem mass spectrometry for identification of a heptabarbital metabolite and sample work-up artefacts

An unknown heptabarbital metabolite, observed in the liquid chromatogram of rat plasma and urine samples after administration of heptabarbital, was identified by liquid chromatography-thermospray tandem mass spectrometry. By applying the parent scan mode for screening and the daughter scan mode for structure elucidation, the metabolite was determined to be 5-ethyl-5-(1′-3′- or 6′-cycloheptadienyl)barbituric acid. It was demonstrated that artefact formation occurred when hydrochloric acid was used for conjugate hydrolysis in the sample clean-up. Identification of the artefacts was achieved by the same method. Confirmation of the structures of the metabolite and the artefacts was obtained by gas chromatography-electron impact mass spectrometry.

High-temperature GC/MS characterization of porphyrins and high molecular weight saturated hydrocarbons

Determination of the exact structure and relative concentrations of metalloporphyrins and high molecular weight cyclic and acyclic hydrocarbon is of importance since these are useful «biomarkers» in petroleum exploration. Description of the recent efforts in high-temperature gas chromatography /electron impact mass spectrometry (GC/EIMS) and gas chromatography/field ionisation mass spectrometry (GC/FIMS) analysis of these relatively high molecular weight components in petroleum and petroleum sediments

Gas chromatographic—mass spectrometric characterisation of some novel hydroxyeicosatetraenoic acids formed on incubation of arachidonic acid with microsomes from induced rat livers

The biotransformation of arachidonic acid by rat liver microsomes from both control animals and animals pretreated with known inducers of cytochrome P-450 isoenzymes has been studied using a combination of reversed- and normal-phase high-performance liquid chromatography and combined gas chromatography—electron-impact mass spectrometry. The metabolite profiles observed were found to be dependent upon the inducing agent. Five metabolites were identified, namely 16-, 17-, 18-, 19- and 20-hydroxylated arachidonic acids. Of these the 16- and 17-isomers have not been reported as products of arachidonic acid metabolism by any biological system and the 18-isomer has not been reported as a product of liver metabolism.

Cytokinins in the Phloem Sap of White Lupin (Lupinus albus L.)

Cytokinin-like activity in samples of xylem and phloem sap collected from field-grown plants of white lupin (Lupinus albus L.) over a period of 9 to 24 weeks after sowing was measured using the soybean hypocotyl callus bioassay following paper chromatographic separation. The phloem sap was collected from shallow incisions made at the base of the stem, the base of the inflorescence (e.g. stem top), the petioles, and the base and tip of the fruit. Xylem sap was collected as root exudate from the stump of plants severed a few centimeters above ground level. Concentration of cytokinin-like substances was highest in phloem sap collected from the base of the inflorescence and showed an increase over the entire sampling period (from week 10 [61 nanogram zeatin equivalents] to week 24 [407 nanogram zeatin equivalents]). Concentrations in the xylem sap and in the other phloem saps were generally lower. Relatively high concentrations of cytokinin-like substances in petiole phloem sap (70 to 130 nanogram zeatin equivalents per milliliter) coincided in time with high concentrations in sap from the base of the inflorescence (see above). Concentrations in sap (phloem or xylem) from the base of the stem were very much lower. This finding is consistent with movement of cytokinins from leaves into the developing inflorescence and fruit, rather than direct input to the fruit from xylem sap. However, an earlier movement of cytokinins from roots into leaves via the xylem cannot be ruled out. Sap collected at an 18-week harvest was additionally separated by sequential C(18) reversed-phase high performance liquid chromatography --> NH(2) normal phase high performance liquid chromatography, bioassayed, and then analyzed by electron impact gas chromatography-mass spectrometry. Identification of zeatin riboside and dihydrozeatin as two of the major cytokinins in combined sap samples was accomplished by gas chromatography-mass spectrometry-selected ion monitoring.

Surface hydrocarbon components of two species ofNasutitermes from Trinidad

Colonies ofNasutitermes costalis (Holmgren) andN. ephratae (Holmgren) were collected from five locations in Trinidad. Cuticular hydrocarbons were characterized by gas chromatography-electron impact mass spectrometry and quantified by capillary gas chromatography. Sixteen major components were identified; all but one component (12, 16-dimethyltriacontane) were common to both species. The methyl-branched hydrocarbons were predominant inN. costalis, while the majority of the hydrocarbon components inN. ephratae weren-alkanes. One hydrocarbon (11,15-dimethylheptacosane) was found in abundance in samples ofN. ephratae from Trinidad but was not previously reported from collections of this species in Panama. In addition to the morphology of the soldiers and alates and the architecture of the arboreal nests,N. costalis andN. ephratae from Trinidad can easily be separated by chromatograms of the hydrocarbons.N. costalis has an enormous 13,17-dimethylhentriacontane peak (mean = 42.4% of total hydrocarbon). InN. ephratae this peak is much smaller and the 12,16-dimethyltriacontane peak is completely missing.N. costalis from Trinidad andN. corniger from Panama appear to have cuticular hydrocarbon profiles that are more similar to one another than are those ofN. ephratae from Trinidad and Panama.

7-Substituted pterins. A new class of mammalian pteridines.

Three novel pteridines have been isolated from the urine of patients with a new variant of 6-(L-erythro-1',2'-dihydroxypropyl)-5,6,7,8-tetrahydropterin (tetrahydrobiopterin) deficiency, showing hyperphenylalaninemia. From the results of high performance liquid chromatography, oxidative degradation, and gas chromatography-electron impact mass spectrometry, their structures were identified as 7-(D-erythro-1',2',3'-trihydroxypropyl)-pterin (7-neopterin), 7-(L-erythro-1',2'-dihydroxypropyl)-pterin (7-biopterin), and 6-oxo-7-(L-erythro-1',2'-dihydroxypropyl)-pterin (6-oxo-7-biopterin). The ratio of biopterin to 7-biopterin in the patients' urines was 1:1, and after oral loading with tetrahydrobiopterin, 7-biopterin excretion rose parallel to biopterin. This finding suggests that 7-substituted pterins may be formed endogenously by a yet unknown isomerization reaction. The cause of hyperphenylalaninemia is still unclear. The activities of the enzymes involved in tetrahydrobiopterin biosynthesis and regeneration were found to be normal in the patients, and no effect of 7-biopterin on these enzymes was observed in vitro. However, compared with the normal cofactor, tetrahydrobiopterin, the Km values of tetrahydro-7-biopterin for phenylalanine hydroxylase and dihydropteridine reductase are 20 and 5 times higher, respectively.

Purification, Primary Structure Characterization, and Cellular Distribution of Two Forms of Cellular Retinol-binding Protein, Type II from Adult Rat Small Intestine

Cellular retinol-binding protein type II (CRBP(II)) is a major protein in the small intestine, accounting for more than 1% of the soluble protein recovered from rat jejunal mucosa. Two forms of the protein, called CRBP(II)A and CRBP(II)B, were purified from rat small intestine using a three-column procedure. The two forms were present in equal abundance. The primary structures of CRBP(II)A and CRBP(II)B were determined using a combination of techniques including amino acid composition and sequence analyses, and fast atom bombardment and gas chromatography-electron impact mass spectrometry. The primary structures of both proteins were found to be identical, but they differed in their NH2-terminal processing. CRBP(II)B was acetylated at its NH2 terminus, while CRBP(II)A was not. The results also confirmed the amino acid sequence of CRBP(II)A that was deduced from the cDNA sequence by Li et al. (Li, E., Demmer, L. A., Sweetser, D. A., Ong, D. E., and Gordon, J. I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5770-5783). Antibodies capable of distinguishing between the two forms of CRBP(II) were used for immunohistochemical studies which indicated that the organ and cellular distributions of the two forms were identical. The 50% acetylation observed here in vivo fits the pattern predicted by recent in vitro studies which described the effect of NH2-terminal sequence on cotranslational NH2-terminal processing of cytosolic proteins (Boissel, J. P., Kasper, T. J., and Bunn, H. F. (1988) J. Biol. Chem. 263, 8443-8449). Our results provide a basis for investigating the possibility of different roles of CRBP(II)A and CRBP(II)B within cells, as well as the importance of acetylation of the amino terminus for these biological functions.