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Biomedical subjects

P Vouros

Publications and source records attributed to P Vouros.

At least 19 recordsLinked to original sources
PubMed1992

Monitoring cholesterol autoxidation processes using multideuteriated cholesterol.

Deuterium-labeled cholesterol is used to monitor for artifactually produced cholesterol oxidation products during analysis. [2H9]Cholesterol, labeled on the side chain, is added to the sample immediately upon isolation, and the ratios of labeled to unlabeled oxides and of labeled to unlabeled cholesterol are monitored by capillary gas chromatography-mass spectrometry. The analytical methodology involves an initial solvent extraction followed by silica gel LC and reversed-phase HPLC to isolate and concentrate the oxide fraction. The feasibility of the technique for analysis of cholesterol oxides in foods and biological samples at the part per million level with an accuracy of better than +/- 5% is demonstrated.

Animals↗
PubMed1992

Collisionally induced dissociation in the study of A-ring hydroxylated vitamin D type compounds.

Collisionally induced dissociation (CID) is often used to determine the structure of ions based on comparison with the CID spectra of known ions. The latter are generated from judiciously selected compounds taking into account basic principles of ion chemistry. We report here on the use of this approach toward determination of the site of A-ring hydroxylation of vitamin D. Although not intrinsically an aromatic compound, vitamin D gives rise in its mass spectrum to an aromatic methylstyryl cation at m/z 118. A-ring hydroxylated metabolites of vitamin D would thus incorporate the extra OH group on the ion at m/z 118, shifting it to m/z 134. The position of substitution of the extra OH group on a metabolite could then be ascertained by comparing the CID spectrum of its m/z 134 fragment to those of the four possible (hydroxymethyl)styryl cations generated from synthesized authentic compounds. Because of their propensity to polymerize, these cations were generated in situ via the McLafferty rearrangement of the corresponding (hydroxyphenyl)ethanols. For optimum differentiation of isomeric ions, preparation of permethylated derivatives of vitamin D was necessary. The validity of the hypothesis was verified using 1,25-dihydroxy-vitamin D3 as a test compound. This method provides a viable approach for the characterization of A-ring hydroxylated metabolites of vitamin D as well as for related aromatic compounds.

Binding Sites↗
PubMed1991

Hydrogen-deuterium exchange in fused-silica capillary columns.

Ketones deuteriated alpha to the carbonyl have been observed to undergo significant isotope exchange on fused-silica capillary columns during gas chromatography-mass spectrometry. The residence time of the compounds on the column was found to influence the extent of isotope exchange. The degree of exchange was examined using a variety of columns and the isotope exchange was found to occur even with brand new columns. Conversion of the keto compounds into the methyloxime derivatives resulted in retention of the "correct" isotope content during gas chromatography-mass spectrometry.

Deuterium↗
PubMed1990

Gas chromatography-mass spectrometry of conjugated dienes by derivatization with 4-methyl-1,2,4-triazoline-3,5-dione.

The dienophile 4-methyl-1,2,4-triazoline-3,5-dione forms stable adducts with conjugated dienes by generating Diels-Alder cycloaddition products. The reaction is rapid, highly selective for conjugated dienes and the derivatives are suitable for analysis by gas chromatography. Their mass spectra are marked by their simplicity and by the presence of abundant fragment ions diagnostic of the diene position in the parent compound.

Alkenes↗
PubMed1990

Detection and structural characterization of amino polyaromatic hydrocarbon-deoxynucleoside adducts using fast atom bombardment and tandem mass spectrometry.

Fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS) are shown to be useful methods for the detection and structural characterization of nanogram amounts of amino polyaromatic hydrocarbon-nucleoside DNA adducts. The positive ion spectra of four aromatic amine guanosine adducts were studied in detail. The FAB spectra of these adducts exhibit an [MH]+ ion and a more abundant aglycon fragment ion, [AH2]+, which results from the loss of the deoxyribose sugar. The sensitivity of the adducts to FAB was enhanced by preparing trimethylsilyl (TMS) ether derivatives. High-quality full-scan spectra could be obtained on less than 70 ng of the derivatized adducts without signal averaging. With a B/E-linked scan of the [MH]+ ion for the TMS2 species, these same adducts could be detected by examination of their metastable ion spectra at levels as low as 4-5 ng (S/N greater than 10). Collision-induced dissociation (CID) of the [MH]+ ion yields the aglycon fragment and an ion, S1, which results from cleavage through the sugar. The CID spectrum of the aglycon [AH2]+ ion is much more useful, providing structural information relating to the base, the polyaromatic hydrocarbon, and, possibly, the site of covalent attachment. Differentiation of isomeric aminophenanthrene-guanine adducts was demonstrated on the basis of the CID spectra of their respective [AH2]+ ions. The use of TMS derivatives also improves the sensitivity of these methods.

2-Acetylaminofluorene↗
PubMed1990

Selection of electrophoric derivatives of 1-aminopyrene and 2-aminofluorene for determination by gas chromatography with electron-capture negative-ion mass spectrometry.

Several electrophoric derivatives of 1-aminopyrene and 2-aminofluorene were prepared. Reagents such as heptafluorobutyryl chloride, pentafluorobenzoyl chloride, pentafluorobenzyl bromide and pentafluorobenzaldehyde, alone and in certain combinations, were employed. The ease of formation, yield, stability and fragmentation by gas chromatography with electron-capture negative-ion (ECNI) mass spectrometry of the derivatives were compared. This allowed the most promising ones to be selected for future work on the sensitive detection of aminopolyaromatics by this detection technique. Pentafluorobenzylidene (first choice) and N-pentafluorobenzyl-N-heptafluorobutyryl (second choice) derivatives emerged as the best ones. The origins of losses of HF and 2HF from some of the derivatives were elucidated in the ECNI mass spectra by studies of deuterium-labeled analogues.

Chemistry Techniques, Analytical↗
PubMed1990

Mass spectrometric assay for determination of pancuronium and vecuronium in biological fluids utilizing the moving belt introduction system.

An assay for the determination of the neuromuscular blocking agents pancuronium bromide and vecuronium bromide in plasma or urine has been developed. This method is based on syringe application of sample extracts to the moving belt surface and single metastable transition monitoring of the elimination of acetic acid from the ion of m/z 543 during chemical ionization. The analysis shows good linearity over three orders of magnitude and is capable of analyzing for the compounds below the 5 ng ml-1 level. The assay has been used in preliminary pharmacokinetic studies of the biological fluids of surgical patients. The utility of the method for simultaneous determination of the deacetylated metabolites of these two drugs has also been investigated.

Chromatography, Liquid↗
PubMed1990

Reduction processes in fast atom bombardment mass spectrometry: interdependence of analyte and matrix redox potentials.

The reduction of selected redox indicator dyes during fast atom bombardment mass spectrometry was investigated in several different matrices. Reduction is indicated by the incorporation of one or more hydrogens into the cation of the dye. The extent of reduction of the analyte can be related to the E 1/2 value of the reduction potential of the dye. Selection of matrix also influences the reduction, the reducing power decreasing in the order glycerol greater than sulfolane greater than thioglycerol greater than hydroxyethyl disulfide greater than nitrobenzyl alcohol. Evidence is given suggesting that reduction is initiated by exposure of the analyte-matrix mixture to atom bombardment. It is shown that reduction can be controlled by the addition of metal ions with a very low reduction potential to the matrix to act as electron scavengers.

Azo Compounds↗
PubMed1989

Ester and related derivatives of ring N-pentafluorobenzylated 5-hydroxymethyluracil. Hydrolytic stability, mass spectral properties, and trace detection by gas chromatography-electron-capture detection, gas chromatography-electron-capture negative ion mass spectrometry, and moving-belt liquid chromatography-electron-capture negative ion mass spectrometry.

One consequence of radiation damage to DNA is the conversion of thymine to 5-hydroxymethyluracil (HMU). In order to sensitively detect this DNA adduct by gas chromatography (GC) or high-performance liquid chromatography (HPLC) with electron-capture detection techniques, it is necessary to derivatize it. This study was designed to select an optimum ester derivative of the aliphatic hydroxyl group on HMU. N1, N3-Bis(pentafluorobenzyl)-HMU was formed as a parent derivative, and from this a series of esters. Also O-pentafluorobenzyl and O-tetrafluorobenzyl ether derivatives were prepared. Of the esters the pivalyl derivative was the best choice because it formed easily, was relatively stable to aqueous hydrolysis (t 1/2 = 9.8 days at pH 11.5, 24 degrees C) and gave a response at fmol levels by GC and LC comparable to that of the ethers. Unanticipated was a good response as well for the parent derivative, a free hydroxyl compound, by GC and LC at this level. The work also demonstrates a high performance by LC-electron-capture negative ion mass spectrometry with a belt interface for the trace detection of derivatives of this type.

Chemical Phenomena↗
PubMed1989

Trace detection of modified DNA bases via moving-belt liquid chromatography-mass spectrometry using electrophoric derivatization and negative chemical ionization.

Electrophoric derivatives of 5-methylcytosine and 5-hydroxymethyluracil nucleobases are determined using high-performance liquid chromatography-mass spectrometry coupled via a moving-belt interface. Standards as well as samples derived from DNA are analysed. As little as 9.9 pg (signal-to-noise ratio 5) and 180 fg (signal-to-noise ratio 10) of the respective nucleobases are detected in the electron-capture negative chemical ionization mode, and linear responses are observed over a moderate dynamic range. In a comparison study, liquid chromatography-electron-capture negative chemical ionization mass spectrometry is found to have a sensitivity comparable to gas chromatography-electron-capture negative chemical ionization mass spectrometry for 5-hydroxymethyluracil. A detection limit of 60 fg (signal-to-noise ratio 5) by gas chromatography-mass spectrometry is only three-fold better than the amount detected by liquid chromatography-mass spectrometry using the same mass spectrometer.

5-Methylcytosine↗
PubMed1989

Preparation and mass spectral characterization of pentafluorobenzyl derivatives of alkyl and hydroxyalkyl-nucleobase DNA adducts.

Pentafluorobenzyl (PFBz) derivatives of the following nucleobases were prepared: cytosine, 5-methylcytosine, O2-methylcytosine, O2-ethylthymine, O4-ethylthymine, 5-hydroxymethyluracil, N6-methyladenine, O6-methylguanine, O6-hydroxyethylguanine and O6-hydroxyethylpurine. 13C nuclear magnetic resonance was diagnostic for O- versus N-attachment of the PFBz moiety: the resonance of the methylene carbon appeared in the range 29.15-42.13 ppm for NCH2C6F5, and 58.45-69.01 for OCH2C6F5. Considerable structural information was provided by mass spectrometry with ionization by electron impact. All of the derivatives were detected with high sensitivity and specificity by gas chromatography with detection by electron capture negative ion mass spectrometry, reflecting not only their chemical and physical stability, but also their strong tendency to form a structurally diagnostic anion, [M - PFBz]-, in high yield under these ionization conditions. PFBz derivatives are therefore attractive forms of alkyl-substituted nucleobases for analysis by mass spectrometry.

DNA↗
PubMed1988

Pentafluorobenzylation of alkyl and related DNA base adducts facilitates their determination by electrophore detection.

The DNA adduct O4-ethylthymine can be alkylated under mild conditions with pentafluorobenzyl bromide. The product has good gas chromatographic characteristics and also forms a structurally characteristic anion in high yield when subjected to electron capture mass spectrometry. Related adducts for other DNA bases behave similarly. These properties stimulated us to develop a general analytical method based on the derivatization reaction. Important for this method is an oxidation-elimination reaction that mildly releases a base from a nucleoside. Thus, a general analytical method based on pentafluorobenzylation is now available for determining many alkyl and related DNA adducts.

Alkylation↗
PubMed1987

Replacement of aromatic fluorine by a methoxy group during reaction with methyl iodide in N,N-dimethylformamide solvent.

The DNA base uracil was derivatized with pentafluorobenzoyl chloride, followed by methylation with methyl iodide in the presence of N,N-dimethylformamide (DMF). In addition to a 3-pentafluorobenzoyl-1-methyl derivative of uracil, GC/MS analysis of the reaction mixture revealed the formation of an unusual product, whose molecular weight was 12 U higher than that of the prior derivative. This unexpected product has been identified as the 3-(para-methoxytetrafluorobenzoyl)-1-methyl derivative of uracil. Isotopic labeling and related experiments have revealed that the DMF solvent contributes the oxygen atom of the methoxy group that replaces the para fluorine atom. This work allowed a single derivative to be obtained for the methylation reaction by changing the solvent to acetonitrile.

Acylation↗
PubMed1986

Aporphines 65: chemical, microbial synthesis and characterization by gas chromatography/mass spectrometry of (R)-(-)-10-hydroxy 11-methoxy-N-n-propylnoraporphine, a potential metabolite of N-n-propylnorapomorphine.

The title compound has been synthesized by a multistep sequence from (R)(-)-morphine, and compared with the product obtained by microbial O-demethylation of (R)- and (S)-10,11-dimethoxy-N-n-propylnoraporphine (N-n-propylnorapomorphine dimethyl ether). The comparison was based on an analysis of the trifluoroacetyl derivatives of the microbial products and the synthesized compounds using gas chromatography/mass spectrometry in the electron impact mode. Examination by gas chromatography/electron impact/mass spectrometry of the trifluoroacetyl derivatives of 11-hydroxy-10-methoxyaporphine (apocodeine, 3) and 10-methoxy-11-hydroxyaporphine (isoapocodeine, 5) has revealed their excellent chromatographic resolution, and the preferential loss of the methyl group as in 3 [M-15]+ or the trifluoroacetyl group from the 10-position of the aporphine ring as in 5 [M-97]+. Characteristic fragmentations of these isomeric aporphines were used to confirm their structures and were compared with the spectra of authentic synthetic samples.

Apomorphine↗
PubMed1985

Isolation and structural identification of 1,25-dihydroxyvitamin D3 produced by cultured alveolar macrophages in sarcoidosis.

Hypercalcemia and hypercalciuria in sarcoidosis are thought to result from the endogenous overproduction of an active vitamin D metabolite. We employed primary cultures of pulmonary alveolar macrophages from two patients with biopsy-proven pulmonary sarcoidosis and a recent or current clinical abnormality in calcium metabolism to synthesize in vitro a 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-like metabolite from 25-hydroxyvitamin D3 (25OHD3). The macrophage metabolite cochromatographed with [3H]1,25-(OH)2D3 on normal phase and reverse phase high performance liquid chromatography and was bound with high affinity by the chick intestinal receptor for 1,25-(OH)2D3. On UV spectroscopy, the metabolite possessed the carbon-5,7,10 (19) cis-triene chromophore characteristic of a vitamin D sterol. Electron impact mass spectrometry of trimethylsilyl ether derivatives of the metabolite revealed a mass fragmentation pattern similar to that of the trimethylsilyl ether derivative of authentic 1,25-(OH)2D3. The incubation of cultured macrophages from two patients with idiopathic pulmonary fibrosis and two with scleroderma with [3H]25OHD3 did not result in production of a metabolite with the chromatographic identity of 1,25-(OH)2D3. These data indicate that the metabolite of 25OHD3 synthesized by sarcoid macrophages in vitro is 1,25-(OH)2D3 and that the macrophage is a synthetic source of the sterol metabolite in sarcoidosis.

Adult↗
PubMed1979

Influence of the 6-trimethylsilyl group on the fragmentation of the trimethylsilyl derivatives of some 6-hydroxy- and 3,6-dihydroxy-steroids and related compounds.

The 25 eV mass spectra of the trimethylsilyl derivatives of a number of 6-hydroxy and 3,6-dihydroxy steroids together with deuterium and 18O-labeled analogs have been examined to determine the influence of the 6-OTMS group on fragmentation patterns. Ions in the cholestane series at m/z 321 and 403 were the most characteristic ions derived from the 6-OTMS function; their relative abundances, although low in the spectra of 6-OTMS steroids themselves, were considerably elevated when a 3-OTMS or 3-oxo group was present. Similar ions were present in the spectra of androstane and pregnane derivatives. No correlation was found between the abundance of these ions and the stereochemistry at C3, C5, or C6. Fragmentation mechanisms and gas chromatographic data are discussed.

Cholestanols↗
PubMed1979

Aporphines. 25-trimethylislyl derivatives of N-methyl and N-propyl aporphines: gas chromatographic and mass spectrometric properties.

A series of synthetic, semisynthetic and naturally occurring aporphine alkaloids were converted to the O-trimethylsilyl derivatives, and analyzed by gas chromatography mass spectrometry. A discussion of structurally informative fragment ions along with gas chromatographic data is presented. Their applicability to the analysis of biological metabolites of apomorphine, a CNS active structural analog of dopamine, is discussed.

Apomorphine↗