Gas Chromatography-electron Capture Mass Spectrometry Research Articles

Validation of the LC-MS/MS method for the quantification of mevalonic acid in human plasma and determination of the matrix effect

A simple, specific, and sufficiently sensitive liquid chromatography-tandem mass spectrometry (negative-ion electrospray ionization) methodology to determine mevalonic acid (MVA) in human plasma is described, and its application to the analysis of rat plasma MVA levels after rosuvastatin administration is demonstrated. The method was validated over the linearity range of 0.5-50.0 ng/ml (r(2) > 0.99) using deuterated MVA as an internal standard. The lower limit of quantification was 0.5 ng/ml. The assay procedure involved the isolation of MVA from plasma samples using solid-phase extraction. Chromatographic separation was achieved on a HyPurity Advance column with a mobile phase consisting of ammonium formate buffer (10 mM, pH 8.0) and acetonitrile (70:30, v/v). Excellent precision and accuracy were observed. MVA and deuterated mevalonolactone were stable in water and plasma under different storage and processing conditions. The recovery observed was low, which was attributable to a significant matrix effect. A significant decrease (30-40%; P < 0.05) was observed in rat plasma MVA levels after rosuvastatin administration.

Measurement of N7-(2‘-Hydroxyethyl)guanine in Human DNA by Gas Chromatography Electron Capture Mass Spectrometry

An improved method is presented, based on gas chromatography-electron capture mass spectrometry (GC-EC-MS), for measuring N7-(2'-hydroxyethyl)guanine (N7-HEG) in DNA from an in vivo sample. The method was used to detect this adduct in amounts of human DNA ranging from 0.07 to 11.5 microg isolated from granulocytes. In this method, the DNA is spiked with a stable isotope internal standard (N7-HEG-d4) and heated in water to release the adduct in a nucleobase form. After the adduct is extracted into 1-butanol, it is purified by reverse phase HPLC and derivatized with HONO, pentafluorobenzyl bromide, and pivalic anhydride. Further purification by silica solid phase extraction and reverse phase HPLC is done prior to injection into a GC-EC-MS. Relatively clean GC-EC-MS chromatograms result, contributing to the high sensitivity that is observed. In the samples tested, from 1.6 to 240 N7-HEG adducts in 10(7) nucleotides were observed, a 150-fold range.

Synthesis of releasable electrophore tags for applications in mass spectrometry.

Releasable electrophore mass tags (electrophore tags) are compounds for use as labels in ligand assays such as hybridization assays and immunoassays. In such assays, the electrophore-tagged reagent (e.g., DNA probe or antibody) is quantified at the conclusion of the assay by cleaving a bond in the attached tag so that the electrophore part can be brought into the gas phase (usually thermally) for detection by electron capture mass spectrometry (EC-MS) or a related technique. Interest in these tags is promoted mainly by their potential to provide highly sensitive and multiplexed assays. The high multiplexing arises from the opportunity to measure many such tags simultaneously in the mass spectrometer, where each tag has an electrophore part with a unique mass. In this study five precursors of electrophore mass tags are presented. Each precursor can lead to a large library of electrophore tags in a practical way, since each precursor can be converted to many different electrophore tags by reaction with commonly available phenols that provide a variation in mass. The phenol-reactive part of the tag is either a polyfluorobiphenyl or a benzyl chloride moiety. Representative library compounds are prepared and detected in an inert ester form by gas chromatography electron capture mass spectrometry (GC-EC-MS). Further, one tag is conjugated to DNA, and the resulting product is detected by laser-induced electron capture time-of-flight mass spectrometry on a silver surface. A calculation by the semiempirical method AM1 for an ion formed by one of the electrophores suggests that ring rotation promotes dissociative electron capture. The features of practical synthesis, simple composition, physicochemical stability, high multiplicity, high sensitivity, and potential for high throughput detection make releasable electrophore mass tags attractive for highly multiplexed assays. This includes their use in SNP assays or dideoxy DNA sequencing for detection of mutations in individuals, where the combination of high accuracy and speed is essential.

Brominated flame retardants in archived serum samples from Norway: a study on temporal trends and the role of age.

The temporal trends and influence of age and gender on levels of selected brominated flame retardants (BFRs) in human serum have been assessed by analyzing archived samples from Norway. Serum from 40 to 50 year old men collected at six time periods during 1977 to 1999 and from eight groups of differing age and gender sampled in 1998 were pooled into six and eight samples, respectively. The BFRs were isolated using solid-phase extraction (SPE) and the serum lipids decomposed bytreatmentwith concentrated sulfuric acid directly on the polystyrene-divinylbenzene SPE column, prior to elution of the BFRs. Following diazomethane derivatization, the samples were analyzed by gas chromatography-electron capture mass spectrometry. Eight BFRs were quantified in the serum samples: 2,4,4'-tribromodiphenyl ether (BDE-28), 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), 2,2',4,4',6-pentabromodiphenyl ether (BDE-100), 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153), 2,2',4,4',5,6'-hexabromodiphenyl ether (BDE-154), 2,4,6-tribromophenol (TriBP), and tetrabromobisphenol A (TBBP-A). The serum concentrations of all the BFRs, increased during the entire period with the exception of TriBP, and the sum of the six polybrominated diphenyl ethers increased from 0.44 ng/g lipids in 1977 to 3.3 ng/g lipids in 1999. The BFR concentrations in the serum from the different age groups were relatively similar, except for the age group 0-4 years, which had 1.6-3.5 times higher serum concentrations. Women older than 25 years had lower serum concentrations of BFRs compared to the corresponding group of men. No trend related to age or gender, nor time during the period 1977 to 1999 was observed for TriBP. The present study indicates an ongoing increase in human exposure to BFRs, and the current body burden appears to be independent of age, except for infants (0-4 years old), who seem to experience elevated exposure.

Brominated flame retardants in plasma samples from three different occupational groups in Norway.

Brominated flame retardants (BFRs) are widely used in plastics, textile coatings, electrical appliances and printed circuit boards to prohibit the development of fires. In order to investigate how exposure to BFRs is related to specific occupations, samples were obtained from Norwegian individuals working at an electronics dismantling facility, in the production of printed circuit boards, or as laboratory personnel. Nine BFRs were quantified in the plasma samples: 2,4,4'-tribromodiphenyl ether (BDE-28), 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), 2,2',4,4',6-pentabromodiphenyl ether (BDE-100), 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153), 2,2',4,4',5,6'-hexabromodiphenyl ether (BDE-154), 2,2',3,4,4',5',6-heptabromodiphenyl ether (BDE-183), 2,4,6-tribromophenol (TriBP) and tetrabromobisphenol A (TBBP-A). The BFRs were extracted from plasma using solid-phase extraction (SPE). The plasma lipids were decomposed by treatment with concentrated sulfuric acid directly on the SPE column, prior to the elution of the BFRs. Following diazomethane derivatization, the samples were analysed by gas chromatography-electron capture mass spectrometry. The subjects working at the electronics dismantling plant had significantly higher plasma levels of TBBP-A and BDE-153 compared to the other groups, and the heptabrominated congener BDE-183 was only detected in plasma from this group. TriBP was generally the most abundant BFR present, and the plasma concentrations were in the range 0.17-81 ng g-1 lipids. BDE-47 was the dominant BDE congener in all the individual samples and the levels were in the range 0.43-14.6 ng g-1 lipids. The total amounts of the seven BDEs were 8.8, 3.9 and 3.0 ng g-1 lipids for the group of electronics dismantlers, circuit board producers and laboratory personnel, respectively. Generally, large variations in the individual concentration levels were found within the groups, especially in the group of electronics dismantlers, where the relative standard deviations for BDE concentrations were in the range 23-164%. The levels of BFRs were not correlated to age or the level of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153). The present work indicates that the population in Norway is exposed to several BFRs, probably with food as a major source. The elevated level of higher brominated BDEs and TBBP-A in the plasma from the workers at the dismantling plant suggests an additional occupational exposure for these individuals. Thus, human exposure to BFRs seems to originate from a combination of different sources; however, further studies investigating plasma samples from a larger number of individuals are necessary for a more complete assessment of human exposure pathways to these environmental contaminants.

Determination of phenolic flame-retardants in human plasma using solid-phase extraction and gas chromatography–electron-capture mass spectrometry

A method for determination of phenolic flame-retardants in human plasma utilizing solid-phase extraction (SPE) and gas chromatography with electron-capture mass spectrometric detection (GC–ECMS), has been developed. The plasma lipids were decomposed by application of concentrated sulphuric acid directly on the polystyrene–divinylbenzene SPE column. The method has been validated for 2,4,6-tribromophenol (TriBP), pentabromophenol (PeBP), tetrachlorobisphenol-A (TCBP-A) and tetrabromobisphenol-A (TBBP-A) in the concentration range 1.2–25, 0.4–40, 4–200 and 4–200 pg g −1 plasma, respectively. The average absolute recovery of the analytes ranged from 51 to 85%. Tetrabromo- o-cresol and chlorotribromobisphenol-A were found suitable as internal standards, and the average recovery of the analytes relative to the internal standards was in the range 93–107%. The repeatability of the method was in the range 4–30% relative standard deviation. The estimated detection limits of TriBP, PeBP, TCBP-A and TBBP-A were 0.3, 0.4, 3.0 and 0.8 pg g −1 plasma, respectively. The method has been used for analysis of plasma samples from potentially occupationally exposed human individuals.

Analysis of [formula omitted]α-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H 3 receptor agonist, N α-methylhistamine ( N α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10 7–10 8 CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.

Mass spectrometric analysis of pentafluorobenzyl oxime derivatives of reactive biological aldehydes

We recently demonstrated that a battery of reactive aldehydes can be generated by human neutrophils through the action of myeloperoxidase on α-amino acids. To determine the aldehydes, we formed the pentafluorobenzyloxime (PFBO) derivatives by reacting them with pentafluorobenzylhydroxylamine (PFBHA) and submitting the derivatives to gas chromatography electron-capture mass spectrometry (GC/EC/MS). Two geometric isomers are formed for each of the aldehydes, and they are separable by gas chromatography (GC) and exhibit distinguishable electron-capture (EC) mass spectra. Major fragment ions include [M − HF] · −, which probably has a six-membered ring formed via HF loss from the molecular radical anion. Subsequent decomposition of this intermediate yields other characteristic ions (e.g. those formed by elimination of NO and the anion of m/ z 178). We proposed fragmentation pathways and mechanisms that are consistent with the data derived from collisionally-activated dissociations (CAD) coupled with tandem mass spectrometry, exact-mass measurements, studies of deuterium-labeled analogs, and calculations by PM3 semiemperical and ab initio methods. The generality of the structurally informative fragmentation pattern together with GC separation is the basis of a powerful means for the identification of reactive aldehydes in biological processes and the pathogenesis of disease.

21] Measuring DNA adducts by gas chromatography-electron capture-mass spectrometry: Trace organic analysis

This chapter discusses the practical experience in method development for the determination of trace amounts of DNA adducts, by gas chromatography-electron capture-mass spectrometry (GC-EC-MS). It has detected femtomole amounts of such analytes, by optimizing sample preparation (involving extraction, chemical reaction, and purification steps, starting with a biological sample) and low-attomole amounts of pure, derivatized standards, by GC-EC-MS. Although such methodology is already useful, the concepts and techniques described extend the sample preparation to the attomole level. In this chapter, the work on chemical transformation is emphasized as a part of sample preparation. This is a means to broaden the range of compounds that can be detected by GC-EC-MS. Also, the experience, in operating a GC-EC-MS to achieve attomole detection limits routinely (for standards), is discussed in the chapter. New ionization techniques for MS, such as electrospray and matrix-assisted laser desorption, are increasing the ability of MS to analyze “nonvolatile” substances present even in aqueous samples. Less new but of continuing importance, as a desorption/ionization technique, in this respect is fast atom bombardment, In contrast, this chapter discusses the procedures, in which significant chemical treatment of the sample precedes the “old technique” of GC, to deliver the analyte into the MS. The desorption approaches are attractive, because they can minimize sample preparation. They are also unique in their ability to achieve the direct detection of medium to high molecular weight biopolymers by MS.

General method for determining ethylene oxide and related N 7-guanine DNA adducts by gas chromatography-electron capture mass spectrometry

A 112-μg sample of DNA was spiked with 103 pg of N 7-(2′-hydroxyethyl)guanine and 100 pg of N 7-(2′-hydroxyethyl-d4)guanine, the internal standard. The sample was subjected to the following sequence of steps: heating at 100°C, precipitation of the DNA with HCl, reaction with nitrous acid to form the corresponding xanthines, reaction twice with pentafluorobenzyl bromide (first to derivatize NH, then OH), solid-phase extraction on silica and detection by gas chromatography-electron capture mass spectrometry. The absolute, overall yield of final product for both the analyte and internal standard was 9.7%. Conveniently, the three chemical reactions are conducted sequentially in the same vial and, aside from a washing step, are separated only by evaporations. Corresponding N 7-guanine methyl, phenyl and styrene oxide adducts were detected at about the 50-ng level by the procedure, to indicate the generality of the method.

Rapid identification of 4-hydroxy-2-alkylquinolines produced by Pseudomonas aeruginosa using gas chromatography-electron-capture mass spectrometry.

The 4-hydroxy-2-alkylquinolines and their N-oxides are secondary metabolites produced by Pseudomonas aeruginosa which inhibit the growth of a number of Gram-positive organisms including Staphylococcus aureus. To facilitate the identification of these compounds in biological fluids, we have developed a rapid profiling system based on gas chromatography-electron-capture mass spectrometry of the O-bistrifluoromethylbenzoyl derivatives. Using the technique, over twenty hydroxyalkylquinolines have been identified from a culture obtained from a strain of P. aeruginosa obtained from a patient with severe bronchiectasis.

Direct correlation between in vivo cholesterol synthesis and hepatic secretion of apolipoprotein B-100 in normolipidaemic subjects

The regulation of apolipoprotein B-100 (apo B) metabolism in man is not fully understood. In vitro studies suggest a key role for the hepatic availability of cholesterol substrate. We therefore examined whether there was a direct association between plasma mevalonic acid (MVA) concentration (an index of in vivo cholesterol synthesis) and hepatic secretion of very-lowdensity lipoprotein (VLDL) apo B in eight normolipidemic, healthy adult subjects. Hepatic secretion of VLDL apo B was estimated by endogenous labeling of apo B with an 8-hour primed, constant infusion of 1-13C-leucine. Isotopic enrichment of VLDL apo B was measured by gas chromatography-mass spectrometry (GCMS), from which the fractional secretion rate (FSR) was derived by a modified monoexponential function. Plasma concentration of MVA was measured by gas chromatographyelectron-capture mass spectrometry in blood samples taken at 9 AM. The absolute secretion rate (ASR) of VLDL apo B (mean -+ SD) was 9.7 -+ 2.6 mg/kg/d, and MVA concentration was 5.0 +_ 2.5 ng/mL. There was a highly significant positive correlation between ASR of VLDL apoB and plasma MVA (r = .88, P = .004), which persisted after adjusting for apo E phenotype. The findings suggest that in vivo cholesterol synthesis is a determinant of hepatic secretion of apo B in normolipidemic subjects, Copyright © 1995 by W,B. Saunders Company POLIPOPROTEIN B-100 (apo B) is the major structural protein of the proatherogenic plasma lipoproteins. I Apo B is synthesized almost exclusively in the liver, and its hepatic secretion is an important determinant of both plasma lipid levels and risk of atherosclerosis. 1-3 The respective roles of cholesterol ester and triglyceride in regulating apo B secretion have recently been reviewed. 4 Results of experimental studies suggest that the availability of intracellular cholesterol plays a key role in regulating apo B secretion. 5-8 However, the opposite conclusion was reached in more recent studies with Hep G2 cells, which showed that triglyceride synthesis was the major determinant. 9 The availability of free fatty acids may also regulate apo B secretion by controlling the rate of both cholesterol and triglyceride synthesis. 4,6 A perfused-rat liver preparation suggested that cholesterol ester competes with triglyceride for the lipid core of very-low-density lipoprotein (VLDL) particles and that cholesterol feeding can stimulate VLDL secretion3 ° Furthermore, in a patient with cholesterol ester storage disease, pharmacologic inhibition of cholesterol synthesis decreased VLDL apo B secretion, u However, there is as yet no evidence that under physiologic conditions rates of cholesterol synthesis and of hepatic secretion of apo B are closely coupled in man. The principal apo B-containing lipoprotein secreted by the liver is VLDL. Hepatic secretion of VLDL apo B may

Quantitation of plasma mevalonic acid using gas chromatography-electron capture mass spectrometry.

Circulating concentrations of mevalonic acid (MVA) change in parallel with, and may be used as a marker of cholesterol biosynthesis. Plasma MVA levels have been quantified using a sensitive and specific capillary gas chromatography-electron capture mass spectrometric assay. The detection limit for MVA in plasma is 100 pg/ml; the intra-assay variation is 5.11%; the inter-assay variation is 7.7%. Using this assay, the mean plasma MVA in 15 normolipidemic subjects was 2.37 +/- 1.2 ng/ml (range 0.41-5.31 ng/ml). Administration of 40 mg of simvastatin (an HMG-CoA reductase inhibitor) significantly accenutated the diurnal decrease in plasma MVA levels. This assay may be useful in investigating cholesterol synthesis rates in different dyslipidemias and individual responses of HMG-CoA reductase-inhibiting drugs.

Gas chromatography-electron capture mass spectrometry of steroids of human origin

Gas chromatography-electron capture mass spectrometry of steroids of human origin

Bradykinin-stimulated prostaglandin synthesis in conscious rabbits.

1 Bradykinin was infused intravenously into conscious rabbits to determine its effect on the concentration of prostaglandins in plasma. 6-Oxo-prostaglandin (PG) F1 alpha, the stable hydrolysis product of prostacyclin, and 13,14-dihydro-15-oxo-PGF2 alpha, a metabolite derived from PGE2 and PGF2 alpha, were measured by gas chromatography-electron capture mass spectrometry. 2 Incremental infusions of bradykinin (0.4-3.2 micrograms kg-1 min-1) increased plasma concentrations of both 6-oxo-PGF1 alpha and 13,14-dihydro-15-oxo-PGF2 alpha. 3 Aspirin (10 mg kg-1, i.v.) inhibited bradykinin-stimulated 6-oxo-PGF1 alpha and 13,14-dihydro-15-oxo-PGF2 alpha synthesis at 30 min and 6 h. At 24 h, the mean bradykinin-stimulated 13,14-dihydro-15-oxo-PGF2 alpha concentration was 66% of its original value, whilst 6-oxo-PGF1 alpha remained substantially inhibited. 4 The different rates of recovery of bradykinin-stimulated production of the two prostaglandins after inhibition by aspirin suggests that intravenous bradykinin stimulates prostacyclin and PGE2/PGF2 alpha production in distinct cell populations which synthesize cyclo-oxygenase at different rates.