Gas Chromatography-negative Chemical Ionization Mass Research Articles

Evidence for the Formation of Dinor Isoprostanes E1from α-Linolenic Acid in Plants

The free radical oxidation of arachidonic acid is known to generate complex metabolites, termed isoprostanes, that share structural features of prostaglandins and exert potent receptor-mediated biological activities. In the present study, we show that alpha-linolenic acid can undergo a similar oxidation process, resulting in a series of isomeric dinor isoprostanes E1. E-ring dinor isoprostane formation from linolenate was found to be catalyzed by soybean lipoxygenase. The main enzymatic products were 13- and 9-hydroperoxylinolenate but in addition, two dinor isoprostane E1 regioisomers were formed with a yield of 0.31%. Identification and quantification of two dinor isoprostane E1 regioisomers in plant cell cultures was achieved by a negative chemical ionization gas chromatography-mass spectrometry method using [18O]dinor isoprostanes E1 as internal standards. Endogenous levels of these compounds were determined in four taxonomically distant plant species and found to be in the range of 4.5 to 60.9 ng/g of dry weight. Thus analogous pathways in animals and plants exist, each leading to a family of prostaglandin-like compounds derived from polyunsaturated fatty acids. It remains to be shown whether the dinor isoprostanes exert biological activities in plants as has been demonstrated for their C20 congeners in mammals.

Tracing Gluconeogenesis with Deuterated Water: Measurement of Low Deuterium Enrichments on Carbons 6 and 2 of Glucose

The contribution of gluconeogenesis to glucose productionin vivocan be measured by enriching body water with 0.5%2H2O and measuring the glucose labeling ratio C6/C2 (Landauet al., J. Clin. Invest.95, 172–178, 1995). We present further refinements of the measurements of the2H enrichments on C6 and C2 of glucose. The transfer of2H from C6 of glucose to hexamethylenetetramine (HMT) and extraction in preparation for gas chromatography–mass spectrometry can be done in a single test tube, without distillation of the intermediate formaldehyde. In addition, extraction of small amounts of HMT is greatly improved by making a HMT–iodine adduct. For C2, glucose is reduced to sorbitol, and2H on C2 is transferred enzymatically to [U-13C3]pyruvate, forming [U-13C3,2-2H]lactate. The latter is assayed by negative chemical ionization gas chromatography–mass spectrometry of the pentafluorobenzyl derivative. The natural enrichment of the [U-13C3]lactyl ion is only 0.4%, allowing measurements of2H enrichment down to 0.1%. These techniques were used in dogs infused with2H2O and in isolated rat livers perfused with buffer containing 1 to 5%2H2O. Our data reveal a difference in the rate of labeling of C6 and C2 of glucosein vivo.Lastly, in cows infused with [6,6-2H2]glucose, we show that the turnover of glucose can be economically measured by assaying low tracer enrichment (down to 0.1%) via hexamethylenetetramine.

Determination of S-1 (combined drug of tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate) and 5-fluorouracil in human plasma and urine using high-performance liquid chromatography and gas chromatography-negative ion chemical ionization mass spectrometry.

A high-performance liquid chromatography (HPLC) and gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI-MS) method was developed for the analysis of the combined antitumor drug S-1 (tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate) and active metabolite 5-fluorouracil in human plasma and urine. Tegafur was fractionated from biological fluids by extraction with dichloromethane and analyzed by HPLC. 5-Fluorouracil and 5-chloro-2,4-dihydroxypyridine were extracted with ethyl acetate from the residual layer after extraction of tegafur, and converted to pentafluorobenzyl (PFB) derivatives. Potassium oxonate was cleaned up with an anion-exchange column (Bond Elut NH2). The extracted potassium oxonate was degraded to 5-azauracil and converted to PFB derivatives. The PFB derivatives were analyzed by GC-NICI-MS. A stable isotope was employed as the internal standard in the GC-NICI-MS analysis. The limits of quantitation of tegafur, 5-fluorouracil, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in plasma were 10, 1, 2 and 1 ng/ml, respectively. The reproducibility of the analytical method according to the statistical coefficients is approximately 10%. The accuracy of the method is good; that is, the relative error is < 10%. The methods were applied to pharmacokinetic studies of S-1 in patients.

Characterization of a novel diclofenac metabolite in human urine by capillary gas chromatography-negative chemical ionization mass spectrometry.

A sensitive analytical method was developed to characterize diclofenac metabolites in small amounts of body fluids. Desalted and lyophilized urine samples were extracted with supercritical carbon dioxide directly or after acidic hydrolysis. The extracts were derivatized with N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide. The derivatives were separated by capillary gas chromatography and identified by negative chemical ionization mass spectrometry. Full mass spectra were obtained at a level of 1.10(-9) g/ml. With direct extraction, the metabolites could be analysed in one step as open-chained acids and as (cyclic) oxindoles. By acidic hydrolysis the conjugates were transformed to the oxindoles. With both methods, a new main metabolite, [2-[2,6-dichloro-4-hydroxy-3-methoxyphenyl)amino]phenyl]acetic acid, was identified The mechanism of its formation is discussed.

Serum total testosterone: immunoassay compared with negative chemical ionization gas chromatography-mass spectrometry

We have developed an electron capture negative chemical ionization gas chromatography-mass spectrometry (GC-MS) procedure to quantify serum testosterone in the clinically relevant range 0.69-69.3 nmol/L and used this procedure to assess Ciba Corning Diagnostics ACS:180 testosterone immunoassay. The GC-MS method involves liquid-liquid extraction of serum samples and synthesis of a pentafluorobenzyloxime/silyl ether derivative of testosterone with excellent chromatographic and electron capturing properties. The ACS testosterone assay is the first fully automated nonradioactive testosterone immunoassay approved by the US Food and Drug Administration. Patients' specimens (101, 57 males, 44 females) were analyzed by both techniques. A plot of the GC-MS (x) vs ACS (y) testosterone concentrations for men was linear (y = 1.07x + 0.19 nmol/L), showing excellent correlation (r2 = 0.98) between the two assays. Agreement of the two assays for female specimens was poor (y = 0.72x + 1.2 nmol/L), with a poor correlation (r2 = 0.31).

Determination of blood lead by electron-capture negative chemical ionization gas chromatography-mass spectrometry

An electron-capture negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for determination of lead (Pb) in blood samples is described. Extraction of Pb from the sample does not involve hot digestion but is based on treatment at ambient temperature. The blood sample is supplemented with a known amount of internal standard (204Pb) for isotope dilution and is treated with concentrated nitric acid. After adjusting the pH to 7, the Pb is extracted into toluene as the pyrrolidine-dithiocarbamate chelate. Samples are then derivatized with 4- fluorophenylmagnesium bromide to form Pb(FC6H4)4. The use of NCI offers enhanced sensitivity (by 75-fold better than previously used electron ionization), gives good precision and accuracy, and has no observable memory effect. The isotope dilution GCoff methodology typically agreed within 2-3% of expected values for the College of American Pathologists blood Pb specimens and the National Institute of Standards and Technology Standard Reference Material 955a.

Quantitative analysis of fluorimated ethylchloroformate derivatives of non-protein amino acids using positive and negative chemical ionization gas chromatography-mass spectometry

The GC-MS characterization of the ethylchloroformate derivatives of amino acids in an aqueous medium has been applied to non-protein amino acids. Derivatization of non-protein amino acids using ethylchloroformate, trifluoroethanol, and pyridine produced strong [M + 1] + and [M - 1] − ions in positive and negative chemical ionization (CI) modes, respectively. Twenty-one out of the twenty-three non-protein amino acids studied produced detectable ion chromatograms in both ionization modes when methane was used as the CI reagent gas. Mass spectra of these non-protein amino acid derivatives showed characteristic [M - 19] +, [M + 1] +, [M + 29] +, and [M + 41] + peaks in the positive chemical ionization mode, and [M - 1] −, and [M + 35] − peaks in the negative chemical ionization mode. The detection limits and the linear dynamic range of trifluorethanol ethylchloroformate derivatives of non-protein amino acids were studied using positive chemical ionization. The detection limits are mostly in the femtomole range.

Determination of Ro 19-6327 (Lazabemide) in human plasma and urine by gas chromatography-negative chemical ionization mass spectrometry.

A sensitive and specific analytical method was developed for determination of Ro 19-6327 (Lazabemide) in human plasma and urine samples to provide pharmacokinetic data from clinical trials. The new method employs a simple liquid-liquid extraction to isolate the drug from biological samples. The extract is reacted to form the trifluoroacetyl derivative of Ro 19-6327 and then analyzed by gas chromatography-negative chemical ionization mass spectrometry (GC-NCIMS). The lower limit of quantitation of the assay is 0.05 ng/ml for plasma and 5.0 ng/ml for urine, based on 1-ml aliquots. No interferences from anticoagulants, collection devices, or endogenous constituents of plasma and urine were observed. Recovery (64.3%), inter-assay precision (< 8% R.S.D.), and accuracy (> 85%) of the method were considered acceptable. The assay proved reliable enough to be automated for unattended sample analysis of approximately 50 samples daily. In an additional series of tests, Ro 19-6327 was shown to be stable under conditions that might be encountered during the analysis of samples from clinical trials.

Fractional synthesis rates of retinol-binding protein, transthyretin, and a new peptide measured by stable isotope techniques in neonatal pigs

Our objective was to develop a stable isotopic method to measure the synthesis rates of retinol-binding protein (RBP) and transthyretin (TTR). Both proteins were isolated from human and pig plasma by sequential immunoprecipitation and purified by SDS-polyacrylamide gel electrophoresis under denaturing conditions. Both human and pig anti-RBP precipitates contained a peptide (TTR2) that had a molecular mass that was similar but not identical to that of TTR subunit. The N-terminal amino acid sequence of porcine TTR2 was highly but not completely homologous with porcine TTR. Human TTR2 showed no homology with TTR but was completely homologous with an internal sequence of human fibrinogen alpha chain. To measure the fractional rates of synthesis (FRS) of these peptides, six infant pigs were infused with [2H3]leucine at a constant rate for 6 h, and the amount of [2H3]leucine incorporated into the proteins was measured by negative chemical ionization gas chromatography-mass spectrometry. The plateau isotope ratio of plasma very low density lipoprotein apoB-100-bound leucine was used to estimate the isotopic enrichment of hepatic protein synthetic precursor pool. The mean FRS (% h +/- S.E.) of TTR (1.97 +/- 0.13) and RBP (3.89 +/- 0.07) were significantly different. The FRS of TTR2 was low (0.31 +/- 0.19) relative to that of RBP and TTR. Thus, three different peptides with different turnover rates seem to be involved in the transport of retinol.

Is arterial pressure a determinant of renal prostaglandin release?

The effect of changes in renal perfusion pressure (RPP) on renal prostaglandin (PG) release was investigated in conscious dogs (n = 10). PGE2, PGF2 alpha, and 6-keto-PGF1 alpha levels in renal venous and aortic plasma were measured in response to controlled reductions of RPP by an inflatable cuff implanted around the renal artery. PG plasma concentrations were determined by gas chromatography-negative ion chemical ionization mass spectrometry. At control RPP, PGE2 and PGF2 alpha concentrations in renal venous plasma were severalfold higher than in aortic plasma (PGE2, 54.2 +/- 13.4 vs. 9.3 +/- 2.7 pg/ml, P < 0.01; PGF2 alpha, 40.3 +/- 10.4 vs. 10.4 +/- 3.4 pg/ml, P < 0.01), whereas only a small secretion rate was found for 6-keto-PGF1 alpha (renal vein, 24.3 +/- 2.6 pg/ml; aorta, 17.4 +/- 3.4 pg/ml; P < 0.01). Concentrations of all three PGs in renal venous or aortic plasma did not change in response to reductions of RPP within the normal range of renal blood flow (RBF) autoregulation. The mean difference between PG release at a RPP of 70 mmHg and control was for PGE2 -7.9 pg/ml with a 95% confidence interval (CI) from -20.7 to + 5.0 pg/ml; for PGF2 alpha it was -13.7 pg/ml (95% CI -29.4 to +2.0 pg/ml); and for 6-keto-PGF1 alpha it was -0.9 pg/ml (95% CI -6.0 to +4.1 pg/ml). Reductions of RPP below the lower limit of RBF autoregulation (< 66 mmHg) had no effect on 6-keto-PGF1 alpha secretion rate but decreased secretion rates of PGE2 and PGF2 alpha (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

VLDL Apolipoprotein B-100, a Potential Indicator of the Isotopic Labeling of the Hepatic Protein Synthetic Precursor Pool in Humans: Studies with Multiple Stable Isotopically Labeled Amino Acids ,

Four adult men received a 48-h constant intravenous infusion of [2H4]lysine, [2H3]leucine, L-[ring-13C6]phenylalanine, and L-[1,2,3,-13C3]alanine. Subjects ingested hourly meals for two 12-h periods, separated to two 12-h fasting periods. The isotopic enrichments of free amino acids in venous plasma and in VLDL apolipoprotein B-100 (apoB)-bound amino acids, plasma alpha-keto isocaproic acid (alpha-KIC) and plasma pyruvic acid (PYR) were measured by negative chemical ionization gas chromatography-mass spectrometry. By 7 h of infusion, all four amino acids achieved an equilibrium isotopic enrichment (EIE) in plasma and in apoB. In the fed state, the EIE of the amino acids in apoB was lower than that in plasma free amino acids. The ratio EIE-apoB:EIE-plasma differed significantly among amino acids in the fed state (alanine 0.30; lysine 0.64; leucine 0.70; phenylalanine 0.81). In the postabsorptive state, the EIE-apoB:EIE-plasma ratio rose significantly compared with the fed state (alanine 0.38; lysine 0.73; leucine 0.94; phenylalanine 1.05). Plasma PYR and apoB-alanine were in isotopic equilibrium irrespective of nutritional state. The EIE-apoB-leucine:EIE-plasma-alpha-KIC ratio rose from 0.75 in the fed state to near 1 in the postabsorptive state. We conclude that the contribution of systemic amino acids to apoB-100 synthesis is sensitive to nutritional state, and that systemic essential amino acids seem to be preferentially incorporated into apoB.

Evaluation of gas chromatography with electrolytic conductivity detection and electron capture detection and use of negative chemical ionization GC-MS for the analysis of PCBs in effluents

Gas chromatography with electrolytic conductivity detection and electron capture detection in combination with gas chromatography-mass spectrometry, operated in the electron capture negative chemical ionization mode, were evaluated as techniques for the analysis of polychlorinated biphenyls in wastewater from an industrial facility. The specificity of the electrolytic conductivity detector reduced sample turnaround time because extracts could be analyzed without fractionation or cleanup Using a 2 L sample, this methodology had a quantification limit, based on Aroclor 1260, of 0.1 μg/L and a detection limit of approximately 0.03 μg/L The electron capture detector was subject to interferences from nonhalogenated compounds and required additional sample cleanup Electron capture negative chemical ionization gas chromatography-mass spectrometry was highly specific and provided full mass spectra of polychlorinated biphenyl congeners at the same quantification limit. Effluents from the facility had polychlorinated biphenyl concentrations of 0.1 to 1 μg/L.

Preparation of 4,4′-diaminodiphenylmethane-(2H4) for use as internal standard in the quantification of 4,4′-diaminodiphenylmethane

A simple method has been developed for the synthesis of 4,4′-diaminodiphenylmethane-(2H4) for analytical purposes in gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICIMS). It consists in a proton-deuterium exchange reaction of the diamine using deutero-hydrochloric acid in deuterated methanol. The reaction was conducted at 60°C. Deuteration of the diamine in the ortho positions of the aromatic rings was observed after eight days in these conditions, using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.

EVALUATION OF GAS CHROMATOGRAPHY WITH ELECTROLYTIC CONDUCTIVITY DETECTION AND ELECTRON CAPTURE DETECTION AND USE OF NEGATIVE CHEMICAL IONIZATION GC-MS FOR THE ANALYSIS OF PCBs IN EFFLUENTS

Gas chromatography with electrolytic conductivity detection and electron capture detection in combination with gas chromatography-mass spectrometry, operated in the electron capture negative chemical ionization mode, were evaluated as techniques for the analysis of polychlorinated biphenyls in wastewater from an industrial facility. The specificity of the electrolytic conductivity detector reduced sample turnaround time because extracts could be analyzed without fractionation or cleanup Using a 2 L sample, this methodology had a quantification limit, based on Aroclor 1260, of 0.1 μg/L and a detection limit of approximately 0.03 μg/L The electron capture detector was subject to interferences from nonhalogenated compounds and required additional sample cleanup Electron capture negative chemical ionization gas chromatography-mass spectrometry was highly specific and provided full mass spectra of polychlorinated biphenyl congeners at the same quantification limit. Effluents from the facility had polychlorinated biphenyl concentrations of 0.1 to 1 μg/L.

Gas chromatography/negative chemical ionization mass spectrometry of intact glucuronides

Gas chromatography-negative chemical ionization mass spectrometry (GC–NCIMS) has been applied to the analysis of O- and S-glucuronides. The glucuronides were derivatized with pentafluorobenzyl (PFB) bromide and Tri-Sil, and analyzed by GC–NCIMS. All glucuronides gave a large [M—PFB]− ion indicative of the molecular weight of the compound. This methodology has been successfully applied to the identification of a glucuronide of unknown structure.

Pharmacokinetics of Rioprostil in Human Plasma as Assayed by Combined Capillary Gas Chromatography-Negative Ion Chemical Ionization Mass Spectrometry

To investigate the pharmacokinetics of rioprostil in man an assay is developed to analyse levels of rioprostil in plasma. After extraction and purification by solid-phase cartridges rioprostil is measured by negative ion chemical ionization mass spectrometry using a deuterated internal standard. The method is validated by a recovery experiment. Levels of rioprostil in the plasma of four volunteers following a single oral dose of 600 micrograms rioprostil are found as always below 100 pg/ml. The data presented here suggest that rioprostil is transformed rapidly in man to its more polar metabolites.

Harman in alcoholic beverages: pharmacological and toxicological implications.

Harman, a beta-carboline compound, was identified and quantitated in beer and wine samples using a combination of high performance liquid chromatography with fluorescence detection and gas chromatography-negative chemical ionization mass spectrometry. The concentration of harman in beer (7.3-140.0 ng/ml) was greater than in wine (0.8-10.5 ng/ml) and was not related to alcohol content. The pharmacological and toxicological implications of harman in alcoholic beverages are discussed.

Analysis of tryptamine at the femtomole level in tissue using negative ion chemical ionization gas chromatography-mass spectrometry

An ultra sensitive method for the detection of tryptamine, an endogenous amine in mammalian neuronal systems, at the femtomole level has been developed using negative chemical ionization gas chromatography-mass spectrometry (NCI-GC—MS). The amine is converted into a perfluorinated spirocyclic derivative, e.g. 1-pentafluoro-2-methylenepyrrolidine-3-spiro-3′-(3 H-indole) which is detected using selected-ion monitoring of the (M — 2HF) ions of the endogenous and deuterated internal standard compounds. Two mass spectrometers were compared; they gave minimum detectable quantities from tissue samples of 40 pg (VG-7070F) and 0.9 pg (VG-70S) respectively. These detection levels are approximately 5–200 times lower than have been obtained by previous MS methods.

Depletion of brainstem epinephrine stores by α-methyldopa: Possible relation to attenuated sympathetic outflow

The antihypertensive effect of α-methyldopa (MD) is believed to be critically dependent on its ability to deplete endogenous catecholamines or cause the synthesis of false neurotransmitters. We used liquid chromatography with electrochemical detection (LCEC) and negative chemical ionization gas chromatography-mass spectrometry (GC-MS) for quantitation of catecholamines and MD metabolites in rat. MD intraperitoneally (100 mg/kg q12 hr × 12 days), significantly increased α-methylnorepinephrine (MNE) in brain (1.02 ± 0.33 μg/g), heart (1.67 ± 0.57 μg/g) and adrenal glands (114.93 ± 50.47 μg/g) Endogenous norepinephrine (NE), epinephrine (E) and dopamine (DA) were reduced. ME levels were 2.19 ± 0.44 μg/g (n=6) in the adrenal gland but only 99 ± 26 pg/g (n=3) in the brainstem. MD-induced endogenous brainstem NE depletion was more than compensated by MNE production, but brainstem E depletion was not compensated for by a stoichiometric production of brainstem ME.

Organochlorine pesticides and polychlorinated biphenyls in the atmosphere of Southern Sweden

High volume air samples were taken in Stockholm and at a rural site approximately 100 km south of the city using a glass fiber filter-polyurethane foam (PUF) collector. The samples were analyzed for polychlorinated biphenyls (PCB), DDT compounds, hexachlorocyclohexanes (HCH), hexachlorobenzene (HCB), chlordanes and polychlorocamphenes (PCC) by capillary gas chromatography with electron capture detection (GC-ECD), and for chlordanes and PCC by capillary gas chromatography-negative chemical ionization mass spectrometry (GC-MS). Geometric mean concentrations (pg m −3) were: PCB (Aroclors 1242 + 1254) = 165, p,p'-DDT + p,p'-DDE = 7.2, HCH = 489, HCB = 64, chlordane = 8.4 and PCC = 25. Examination of air trajectories suggested that some of the PCC in the air over Sweden may be derived from sources in eastern Europe.