Determination of N-acetylneuraminic acid by gas chromatography-mass spectrometry with a stable isotope as internal standard

N-Acetylneuraminic acid was determined by gas chromatography-mass spectrometry using selected ion-monitoring technique with N-[²H₃]acetylneuraminic acid as an internal standard. M-COOTMS fragments at $\text{m}\text{z}$ 624 of trimethylsilyl derivatives of N-acetylneuraminic acid and at $\text{m}\text{z}$ 627 of that of the internal standard were used as monitoring ions. The standard curve obtained was linear in the range of over 10³, and the lower limit for quantitation was estimated to be a few hundred picograms. This method was used to measure total N-acetylneuraminic acid in the plasma of healthy humans and patients with lung cancer. The total N-acetylneuraminic acid level in the plasma was two to three times higher in the patients than in controls. A few hundred nanoliters of plasma was sufficient for the analysis. The mass fragmentogram of plasma gave a good signal/noise ratio, and measurements were very specific, accurate, and reproducible.     

Indole-3-acetic acid determination in cultured crown-gall and genetic tumour tissues by gas chromatography-mass spectrometry

Indole-3-acetic acid (IAA) was identified and quantified in three cultured tumour lines, which included crown-gall tissue of Datura (25.4 ng/g fresh wt.) and two genetic tumour lines of tobacco (4.6 and 8.0 ng/g fresh wt.). The analysis was carried out using a stable isotope dilution assay in combination with gas chromatography-mass spectrometry. This is the first unambiguous determination of endogenous IAA in genetic tumour tissues.     

Detection of cholesteryl ester hydroperoxide isomers using gas chromatography-mass spectrometry combined with thin-layer chromatography blotting

Oxidative modification of low-density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. During the oxidation of LDL, cholesteryl esters, the major lipid components in LDL, are oxidized to cholesteryl ester hydroperoxides (CEOOH). The isomers of CEOOH may reflect the reactive species that initiate the peroxidation reaction. In the current study, a novel analytical method for the determination of CEOOH isomers, especially cholesteryl linoleate hydroperoxide isomers, was developed using the combination of two chromatographic techniques: (i) thin-layer chromatography blotting with diphenyl-1-pyrenylphosphine (DPPP) fluorescent detection (DPPP-TLC blotting) and (ii) gas chromatography-electron ionization-mass spectrometry (GC-EI-MS). CEOOH was applied to DPPP-TLC blotting, the obtained DPPP-derived fluorescent spots containing cholesteryl ester hydroxides were extracted and derivatized (hydrogenation, transmethylation, and trimethylsilylation), and the formed methyl ester/trimethylsilylether derivatives of hydroxyoctadecenoic acid were then analyzed by GC-EI-MS. The CEOOH isomers were determined by selected ion monitoring of isomer-specific fragment ions originated from the alpha-cleavage of the trimethylsilyloxyl group. Using these two chromatographic techniques, we were able to detect isomeric CEOOH in the oxidized human LDL. Our results indicated that GC-EI-MS analysis combined with DPPP-TLC blot is a specific method for analyzing cholesteryl ester hydroperoxide isomers in biological samples such as oxidized LDL.     

Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes

Metabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography-mass spectrometry (GC-MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization-based GC-MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U-(13)C]glucose. The reproducibility of this method was similar to that found for other GC-MS-based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC-MS method has been applied to compare the metabolism of wild type and well-characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism.     

Analysis of modified bases in DNA by stable isotope dilution gas chromatography-mass spectrometry: 5-Methylcytosine

A sensitive assay for 5-methylcytosine in DNA has been developed based on stable isotope dilution gas chromatography-mass spectrometry with selected ion monitoring. 5-([²H₃]-Methyl)cytosine and [methyl-²H₃]thymine have been synthesized as internal standards for analysis of DNA following acid digestion, conversion of pyrimidines to volatile t-butyldimethylsilyl derivatives, and separation in 3 min by gas chromatography. Submicrogram amounts of DNA have been analyzed for 5-methylcytosine content in the range 0.02–1.5 mol%. The estimated limit of quantitative measurement is 0.3 pmol of methylated base in a DNA hydrolysate. The method is compared with other techniques for quantitative measurement of methylated bases in DNA, and 5-methylcytosine levels and precision of analysis for calf thymus, pBR322, and ΦX-174 DNAs are reported and compared with literature values. The method can readily be adapted to the accurate high-sensitivity analysis of other methylated bases in DNA.     

A sensitive method for 4-hydroxybutyric acid in urine using gas chromatography-mass spectrometry

4-Hydroxybutyric acid (4HB) was analyzed by gas chromatography-mass spectrometry. Under acidified conditions, 4HB is difficult to detect due to lactonization. Using a urine sample containing 0.01 mg creatinine, we performed trimethylsilyl derivatization without extraction, only adding dimethylsuccinic acid as an internal standard and 10 microl of 0.1 N NaOH methanol solution with adequate evaporation. Urine 4HB levels in a patient with 4-hydroxybutyric aciduria was determined to be 1258 mmol/mol Cr (control, 0.28-2.81 mmol/mol Cr) in this method. Direct derivatization of samples without extraction showed good reproducibility and linearity. Only a small sample of urine was required. Alkalinization by NaOH prevented not only lactonization of 4HB, but also loss of the compounds during evaporation.     

Determination of abscisic acid in Eucalyptus haemastoma leaves using gas chromatography/mass spectrometry and deuterated internal standards

Abscisic acid and 2-trans-abscisic acid each with three deuterium atoms in the C-3 methyl group, have been synthesized chemically and used as internal standards in selected ion monitoring experiments to establish the endogeneous concentrations of these compounds and their conjugates in turgid and wilted Eucalyptus haemastoma leaves. The analytical procedure used GC/CIMS(methane) to detect the methyl esters of abscisic acid, 2-trans-abscisic acid and their deuterated internal standards. A three-fold increase in the concentration of abscisic acid occurred on wilting and the amounts of 2-trans-abscisic acid and conjugates of both compounds were determined.     

Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography

A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH₃ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.     

Identification and quantitative analysis of beta-sitosterol oxides in vegetable oils by capillary gas chromatography-mass spectrometry

As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of beta-sitosterol oxides using capillary gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify beta-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time.     

Facts about the artifacts in the measurement of oxidative DNA base damage by gas chromatography-mass spectrometry

Recently, several papers reported an artifactual formation of a number of modified bases from intact DNA bases during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC/MS). These reports dealt with 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra) and 5-formyluracil that represent only a small percentage of the 20 or so modified DNA bases that can be analyzed by GC/MS. Removal of intact DNA bases by prepurification of calf thymus DNA hydrolysates using HPLC was shown to prevent artifactual formation of these modified bases during derivatization. It needs to be emphasized that the procedures for hydrolysis of DNA and derivatization of DNA hydrolysates used in these papers substantially differed from the established procedures previously described. Furthermore, a large number of relevant papers reporting the levels of these modified bases in DNA of various sources have been ignored. Interestingly, the levels of modified bases reported in the literature were not as high as those reported prior to prepurification. Most values for the level of 5-OH-Cyt were even lower than the level measured after prepurification. Levels of 8-OH-Ade were quite close to, or even the same as, or smaller than the level reported after prepurification. The same holds true for 5-OHMeUra and 8-OH-Gua. All these facts raise the question of the validity of the claims about the measurement of these modified DNA bases by GC/MS.

A recent paper reported a complete destruction of 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) by formic acid under the conditions of DNA hydrolysis prior to GC/MS. The complete destruction of FapyGua and FapyAde by formic acid is in disagreement with the data on these compounds in the literature. These two compounds were measured by GC/MS following formic acid hydrolysis for many years in our laboratory and by other researchers with no difficulties. These facts clearly raise the question of the validity of the claims made about the previous measurements of these compounds by GC/MS.     

Formation of 2-hydroxy-4-methylpentanoic acid from l-leucine by Clostridium butyricum

Abstract Five Clostridium butyricum strains of different origin were grown in trypticase-yeast extract-hemin medium with or without d-glucose (TGYH or TYH medium, respectively) and in a synthetic basal medium with d-glucose (BMG medium). 2-Hydroxy-4-methylpentanoic acid was detected by gas chromatography-mass spectrometry (GC-MS) for the five strains whether grown in TGYH or TYH medium (270 or 170 μM, respectively). In BMG medium supplemented with l-leucine (10 mM), the concentration of this metabolite was strongly increased (2.8 mM versus 10 μM in the control). After culture in TGYH or TYH medium supplemented with l-( methyl -²H₃)leucine, 2-hydroxy-4-([²H₃]methyl)pentanoic acid was detected by GC-MS. This observation demonstrates that C. butyricum is able to convert l-leucine into the corresponding 2-hydroxy acid and opens a new aspect in the study of C. butyricum metabolism.     

Detection of nitrous oxide in the neuronal nitric oxide synthase reaction by gas chromatography-mass spectrometry

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.     

Comparison of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods to quantify alpha-tocopherol and alpha-tocopherolquinone levels in human plasma.

Two mass spectrometric methods were established for the quantitative analyses of alpha-tocopherol (TH) and its oxidation product alpha-tocopherolquinone (TQ) in human plasma. Both methods make use of isotopically labeled internal standards of different levels of deuteration (d3-TH and d6-TQ). Plasma (100 microl) was saponified in the presence of a mixture of antioxidants, and then TH and TQ were extracted with hexane. With the GC-MS method, the analytes were first converted into O-trimethylsilyl derivatives before analysis in the selective ion monitoring mode. The derivatization procedure led to the quantitative conversion of TQ into the O-trimethylsilyl derivative of tocopherolhydroquinone, giving rise to a more stable molecule with less fragmentation than for TQ. The increased stability of the molecule resulted in an enhanced contribution of the base peak to the total observed ions and therefore an increased sensitivity of the base peak for quantification. With the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, TH and TQ were detected by multiple reaction monitoring after positive electrospray ionization. The GC-MS and LC-MS/MS methods showed nearly the same accuracy (>95%) and the same within-day precisions, with less than 5 and 10% for TH and TQ, respectively. The between-day precision and the limit of quantification for TQ in plasma were better by LC-MS/MS (4%; 3 nM) than by GC-MS (21%; 10 nM). Analysis and method validation were carried out with plasma samples obtained from a male volunteer pre- and postexercise. Both techniques showed that the ratio of TQ/TH was elevated by 35% immediately after exercise and had returned to basal levels when measured 24 h later.     

Identification by gas-liquid chromatography-mass spectrometry of 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid,a new sialic acid from horse submandibular gland

The novel sialic acid 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid has been identified as a constituent of horse submandibular gland glycoproteins in addition to the already know equine sialic acids, N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, 4,9-di-O-acetyl-N-acetylneuraminic acid, N-glycolylneuraminic acid, 4-O-acetyl-N-glycolylneuraminic acidand 9-O-acetyl-N-glycolylneuraminic acid. The structure has been established by combined gas-liquid chromatography-mass spectrometry.     

Docosahexaenoic acid (22:6, n-3) is metabolized to lipoxygenase reaction products in the retina

Docosahexaenoic acid (22:6, n-3), a major component of retinal phospholipids, is a substrate for active lipoxygenation in intact canine retinas incubated in vitro with [U-14C]docosahexaenoic acid. The major lipoxygenase reaction product was identified by high performance liquid chromatography and gas chromatography-mass spectrometry as 11-hydroxy-4,7,9-(trans)13,16,19 docosahexaenoic acid. Other mono- and di-hydroxy derivatives also were detected. The synthesis of these compounds was inhibited by the antioxidant and lipoxygenase inhibitor, nordihydroguaiaretic acid, but was not inhibited by indomethacin or esculetin.     

Determination of N-7-[2H3]methyl guanine in rat urine by gas chromatography-mass spectrometry following administration of trideuteromethylating agents or precursors

A gas chromatographic-mass spectrometric method has been developed for the determination of N-7-[2H3]methyl guanine in urine in the presence of large natural levels of N-7-methyl guanine. Urine is fractionated on heptanesulfonic acid-treated C-18 Sep-pak cartridges, followed by derivatization to give a volatile N-heptafluorobutyryl-O6-2,3,4,5, 6-pentafluorobenzyl derivative which is separated on an SE52 fused silica capillary column. Using N-7-ethyl guanine as an internal standard, the total amount of N-7-methyl guanine is determined by gas chromatography-flame ionization detection. The percentage of N-7-[2H3]methyl guanine is then measured by gas chromatography-mass spectrometry, enabling the amount of deuterated base to be determined. Preliminary experiments with [2H3]methyl methanesulfonate in rats showed measurable excretion of N-7-[2H3]methyl guanine. 4-(Di[2H3]methylamino)antipyrine alone gave no detectable amount of alkylated base, but coadministration of nitrite resulted in excretion of deuterated N-7-methyl guanine.     

Rapid and sensitive detection of urinary 4-hydroxybutyric acid and its related compounds by gas chromatography-mass spectrometry in a patient with succinic semialdehyde dehydrogenase deficiency

We describe the rapid and sensitive detection of 4-hydroxybutyric acid, which is a marker compound for succinic semialdehyde dehydrogenase (SSADH) deficiency. Urinary 4-hydroxybutyric acid and 3,4-dihydroxybutyric acid were targeted, quantified by gas chromatography-mass spectrometry after simplified urease digestion in which lactone formation from gamma-hydroxy acids is minimized. The recovery of 4-hydroxybutyric acid using this method was over 93%. 2,2-Dimethylsuccinic acid was used as an internal standard. The detection limit of this method was 1 nmol ml(-1) for both 4-hydroxybutyric acid and 3,4-dihydroxybutyric acid. The urinary concentrations of 4-hydroxybutyric acid and of 3,4-dihydroxybutyric acid from the patient with an SSADH deficiency were 880-3628 mmol mol(-1) creatinine (control; 3.3+/-3.3 mmol mol(-1) creatinine) and 810-1366 mmol mol(-1) creatinine (control; 67.4+/-56.2 mmol mol(-1) creatinine), respectively. The simplified urease digestion of urine is very useful for quantifying 4-hydroxybutyric acid and its related compounds in patients with 4-hydroxybutyric aciduria.     

Temperature effects on endogenous indole-3-acetic acid levels in leaves and stamens of the normal and male sterile 'stamenless-2' mutant of tomato (Lycopersicon esculentum Mill.)

The indole-3-acetic acid (IAA) concentration in leaves and stamens of the normal and a temperature-sensitive male sterile ‘stamenless-2′ (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.), grown under three temperature conditions, was measured by gas chromatography — mass spectrometry — selected ion monitoring (GC-MS-SIM) and by enzyme-linked immunosorbant assay (ELISA). At low (LTR, 18°C day/15°C night), intermediate (ITR, 23°C day/18°C night), and high temperatures (HTR, 28°C day/23°C night), the mutant leaves had approximately 10 to 20 times higher IAA concentrations, respectively, than the normal leaves under these temperature regimes. Similarly, the stamens of mutant flowers had approximately five and eight times higher IAA concentration at ITR and HTR, respectively, than the normal flowers. In the low temperature reverted mutant stamens, however, the level of IAA was similar to that in normal stamens. Also, with an increase in temperature, there was an increase in the level of IAA in the leaves and stamens of mutant plants. However, different temperatures had no appreciable effect on the IAA content of leaves and stamens of normal plants. It is suggested that the high IAA content in leaves and stamens of the stamenless-2 mutant is one of the factors associated with male sterility and carpellization of stamens in this mutant.     

Determination of the cyanide metabolite 2-aminothiazoline-4-carboxylic acid in urine and plasma by gas chromatography-mass spectrometry

The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) is a promising biomarker for cyanide exposure because of its stability and the limitations of direct determination of cyanide and more abundant cyanide metabolites. A simple, sensitive, and specific method based on derivatization and subsequent gas chromatography-mass spectrometry (GC-MS) analysis was developed for the identification and quantification of ATCA in synthetic urine and swine plasma. The urine and plasma samples were spiked with an internal standard (ATCA-d(2)), diluted, and acidified. The resulting solution was subjected to solid phase extraction on a mixed-mode cation exchange column. After elution and evaporation of the solvent, a silylating agent was used to derivatize the ATCA. Quantification of the derivatized ATCA was accomplished on a gas chromatograph with a mass selective detector. The current method produced a coefficient of variation of less than 6% (intra- and interassay) for two sets of quality control (QC) standards and a detection limit of 25 ng/ml. The applicability of the method was evaluated by determination of elevated levels of ATCA in human urine of smokers in relation to non-smokers for both males and females.