The identification of 5,6-dihydrouridine in normal human urine by combined gas chromatography/mass spectrometry

The identification of 5,6-dihydrouridine in normal human urine is reported. Partial purification and isolation of the compound by boronate gel affinity chromatography and reversed-phase high-performance liquid chromatography preceded its characterization as a trimethylsilyl derivative by combined gas chromatography/mass spectrometry. Structure proof is based upon a comparison of mass spectral and chromatographic features of the urinary component to that of an authentic reference sample. Additional data derived from high resolution mass measurements and deuterium isotope-labeling experiments provide confirmation of fragment ion structure. The poor detectability inherent in the HPLC/uv analysis of nucleosides is also discussed.     

Characterization of mixtures of dipeptides by gas chromatography/mass spectrometry

The gas chromatographic and mass spectrometric behavior of over 120 different dipeptides has been investigated. The dipeptides were analyzed as their N,O-perfluoropropionyl methyl ester derivatives by combined gas chromatography/mass spectrometry. Mass spectra of the dipeptides were obtained using both electron impact and chemical ionization. Gas chromatographic retention times were obtained for each of the dipeptides studied and utilized for the prediction of the retention times for most of the 400 common dipeptides. These techniques enable the unambiguous identification of dipeptides in mixtures.     

Simultaneous measurement of urinary polyols using gas chromatography/mass spectrometry

In the present study, we simultaneously measured several polyols, such as adonitol, arabitol, dulcitol, glucose, myo-inositol, mannitol, sorbitol, and xylitol, in urine by gas chromatography/mass spectrometry-positive chemical ionization. We also examined possible relationship between the levels of these metabolites and age in normal individuals. In order to proceed to its quantification by GC/MS, 200 microL of a urine sample were diluted with 3 ml of distilled water, lyophilized, acetylated, and then analyzed them. Using this method, we were able to quantify as little as 0.5-1.0 ng/microL, and we made the calibration curves to be linear from 0.25 to 250 ng/microL (r(2)>0.991). Analytical recoveries were over 89.4%, and the inter-day and intra-day variability for accuracy and reproducibility was less than 20%. In the normal urine sample, the levels of polyols were gender-differentiated and age-related. This simple GC/MS method is sensitive and allows the measurement of wide ranges of polyols using small amounts of urine. We conclude that the quantitation of urinary polyols using GC/MS appears to be a clinically useful method for assessing polyol-pathway activity.     

The analysis of trenbolone and the human urinary metabolites of trenbolone acetate by gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry.

The electron impact mass spectrometric properties of trimethylsilyl ether and fluoroacyl ester derivatives of trenbolone, combined or not combined with a methoxime group, are presented. Some derivatization problems were observed and were due to the formation of enol derivatives at the 3C-position in several tautomeric forms, which in their turn were not stable and lost two or four hydrogens under the conditions studied. The enolization could be minimized by carefully selecting the reaction conditions or could be prevented by the introduction of a methoxime group at the 3C-position. The limits of detection and identification of the methoxime heptafluorobutyryl ester and the methoxime trimethylsilyl ether derivative of trenbolone were determined using a mass selective detector in the electron impact mode and a triple-stage quadrupole in the methane positive chemical ionization mode. Selected reaction monitoring in tandem mass spectrometry did not improve the limit of detection, but because of the gain in selectivity did improve the limit of identification. The glucuronides of trenbolone and epitrenbolone could be identified in three urine specimens out of 200 samples in routine doping control.     

Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry

Derivatization of 4-hydroxyproline (Hyp) in collagen using trifluoroacetylation and methanol esterification produces two derivatives when analyzed by gas chromatography/mass spectrometry (GC/MS). The diacyl derivative N,O-bis(trifluoroacetyl)-4-hydroxy-L-proline methyl ester (N,O-TFA-Hyp) formed in this manner has a shorter retention time and different fragmentation pattern by GC/MS as compared to the slower eluting monoacetylated species N-trifluoroacetyl-4-hydroxy-L-proline methyl ester (N-TFA-Hyp). By selected ion monitoring of the appropriate ions of either N,O-TFA-Hyp (m/z 164, 278) or N-TFA-Hyp (m/z 164, 182) efficient quantitation of Hyp in collagen is possible within the broad range of 5-1000 ng with a lower limit of detection of 0.5 ng per injection. Measurement of 18O2 incorporation into collagen is possible by selected ion monitoring of the m/z 182 ion formed only from the monoacetylated derivative, N-TFA-Hyp, produced by methanol solvolysis of the N,O-TFA-Hyp derivative, as proposed herein.     

Stable isotope dilution gas chromatography/mass spectrometry of prostaglandins and leukotrienes

The group of arachidonic acid metabolites comprising the prostaglandins, thromboxanes, and leukotrienes (eicosanoids) are extremely potent, biologically active compounds. Their properties include proaggregatory anti-aggregatory activity for platelets, chemotactic activity for neutrophils, vasoactive activity, and contractile activity to smooth muscle. In order to determine the role of these substances in pathophysiological conditions, it is essential to have highly sensitive methods available for their analysis. It is generally accepted that combined gas chromatography/mass spectrometry is the most specific technique available for the quantitative analysis of eicosanoids. However, methods based on electron impact ionization and positive ion chemical ionization are relatively insensitive, and many investigators have preferred the use of less specific but more sensitive methods based on radioimmunoassay. We have explored the use of negative ion chemical ionization mass spectrometry to improve sensitivity coupled with capillary column chromatography to maximize specificity. Conversion of the terminal carboxyl group (present in all eicosanoids) to the pentafluorobenzyl ester derivative confers excellent electron capturing properties to the molecule. The derivative undergoes highly efficient thermal electron capture in the gas phase, and any fragmentation that occurs subsequently is directed almost entirely away from the analyte molecule. The stabilized carboxylate anion that results carries at least 30% of the total ion current. Using selected ion monitoring techniques it is possible to detect eicosanoids in the range 1–8 pg on column. This methodology has been applied to the development of stable isotope dilution assays for plasma 6-oxo-prostaglandin (PG) F_1α (1) and for the simultaneous analysis of six biologically important PGs in biological fluids (2). In addition, stable isotope dilution techniques have been developed for the analysis of serum thromboxane B₂ and serum leukotriene B₄ (3). The application of this technology to understanding the role of arachidonic acid metabolism in humans will be discussed.     

Quantitative analysis of albuterol in human plasma by combined gas chromatography chemical ionization mass spectrometry

A highly sensitive and specific quantitative assay for the determination of albuterol in human plasma, based on selected ion monitoring gas chromatography chemical ionization mass spectrometry, has been developed. The [MH]+ ions from the tri-TMS derivatives of albuterol (m/z 456) and the internal standard (2H3)albuterol (m/z 459), were assayed simultaneously by selected ion monitoring. The lower limit of quantitation is 0.25 ng ml-1 and the average assay precision (CV) for albuterol concentrations ranging from 0.25 ng ml-1 to 25 ng ml-1 is approximately 4%. This method is currently being employed for the routine quantitation of albuterol in plasma following the administration of doses therapeutically effective to man.     

Affinity chromatography as a method for sample preparation in gas chromatography/mass spectrometry.

Analytical chemistry aims at developing analytical methods and techniques for unequivocal identification and accurate quantitation of natural and synthetic compounds in a given matrix. Analytical methods based on the mass spectrometry (MS) technology, e.g., GC/MS and LC/MS and their variants, GC/tandem MS and LC/tandem MS, are best suited both for qualitative and quantitative analyses. GC/MS methods not only serve as reference methods, e.g., in clinical chemistry, but they are now widely and routinely used for quantitative determination of numerous analytes. However, despite inherent accuracy, analytical methods based on GC/MS commonly consist of several analytical steps, including extraction and derivatization of the analyte. In general, unequivocal identification and accurate quantification of an analyte in very low concentrations in complex matrices require further chromatographic techniques, such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for sample purification. In recent years, affinity chromatography (e.g., boronate and immunoaffinity chromatography) has been developed to a superior technique for sample preparation of numerous classes of compounds in GC/MS. In this article, the application and importance of affinity chromatography as a method for sample preparation in modern quantitative GC/MS method is described and discussed, using as examples various natural and synthetic compounds, such as arachidonic acid derivates, nitrosylated and nitrated proteins, steroids, drugs, and toxins.     

Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin

High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH(4). Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure.     

Identification of 4-methoxybenzoyl-N-glycine in urine by gas chromatography/mass spectrometry.

An unusual metabolite was detected in the urine of two children with neurological dysfunctions of unclear aetiology by using gas chromatography/mass spectrometry (GC/MS). On the basis of the analysis of its fragmentation pathways, synthesis of tentative compound and its GC/MS analysis it was stated that the unknown metabolite is 4-methoxybenzoyl-N-glycine.     

The decomposition of benzodiazepines during analysis by capillary gas chromatography/mass spectrometry

A capillary gas chromatography column directly interfaced to a mass spectrometer was used for the analysis of sixteen benzodiazepines. The thermal stability of the drugs was found to be related to their chemical structure. Nine of the benzodiazepines were thermally unstable indicating that care should be taken in the interpretation of gas chromatographic data from this class of drugs. The unstable benzodiazepines were: ketazolam which decomposes to diazepam; N-4 oxides (chlordiazepoxide and demoxepam) which lose an oxygen radical; aromatic 7-nitro compounds (nitrazepam and clonazepam) which are partially reduced to the corresponding amine; alpha-hydroxy ketones (lorazepam and oxazepam) which decompose with the loss of water and N-methyl-alpha-hydroxy ketones (lormetazepam and temazepam) which partially decompose with the loss of a hydrogen molecule to produce the corresponding alpha, beta-diketones. Few problems were encountered in distinguishing the drugs by their mass spectra, the exceptions being ketazolam which decomposes to diazepam and demoxepam which decomposes to desmethyldiazepam. In general, good spectra were obtained from 20-50 ng of drug injected. However, for those compounds where the decompositions were not quantitative (nitrazepam, clonazepam, lormetazepam, temazepam) detection limits were poor.     

Quantification of isoflavones and lignans in urine using gas chromatography/mass spectrometry

Phytoestrogens (isoflavones and lignans) are of increasing interest due to their potential to prevent certain types of complex diseases. However, epidemiological evidence is needed on the levels of phytoestrogens and their metabolites in foods and biological fluids in relation to risk of these diseases. We report an assay for phytoestrogens which is sensitive, accurate, and uses low volumes of sample. Suitable for epidemiological studies, the assay consists of a simple sample preparation procedure and has been developed for the analysis of five isoflavones (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone), which requires only 200 microl of urine and utilizes one solid-phase extraction stage for sample preparation prior to derivatization for GC/MS analysis. Limits of detection were in the region 1.2 ng/ml (enterodiol) to 5.3ng/ml (enterolactone) and the method performed well in the UK Government's Food Standards Agency-sponsored quality assurance scheme for phytoestrogens. For the first time, average levels of all the above phytoestrogens were measured in samples of urine collected from a free living population sample of women. Results show a large range in both the amount and the type of phytoestrogens excreted.     

用气相色谱-质谱联用比较牛耳草代谢物的提取方法

通过比较不同的提取方法对牛耳草新鲜和脱水叶片中代谢物的提取效率,旨在建立一种可以有效鉴定并分析牛耳草脱水过程中关键小分子代谢物的种类和含量变化的方法,为研究植物耐脱水分子机制提供技术方法。本研究以气相色谱-质谱联用(GC-MS)为分析方法,对复苏植物牛耳草代谢物提取方法进行比较。从提取总色谱峰数目、提取效率、代谢物保留时间和提取效率稳定性等方面比较甲醇溶液(A法)和甲醇-氯仿-水溶液(B法)两种提取方法的提取效果。对牛耳草新鲜样品提取结果表明,B法提取的总色谱峰数目多于A法;对9种共有代谢物的提取效率比较结果表明,B法的提取效率高于A法;对10种色谱峰的保留时间和提取效率的方法学考察结果表明,两者保留时间RSD(相对标准偏差)值均小于1%,A法提取效率的RSD值≤10%的比例为50%,B法的为100%。A法对干样的提取色谱峰数目远少于鲜样,而B法对干样的提取色谱峰数目和鲜样没有显著差异,保留时间RSD值均小于1%,提取效率的RSD值与鲜样没有差异,稳定性良好。     

Quantification of plasma phenylethylamine by combined gas chromatography/electron capture negative ion mass spectrometry of the N-acetyl-N-pentafluorobenzoyl derivative

An ultrasensitive method capable of detection and quantification of beta-phenylethylamine in 1 ml of human plasma has been developed using gas chromatography/electron capture negative ion mass spectrometry. Phenylethylamine and tetra-deutero phenylethylamine internal standard in plasma were acetylated, extracted into organic solvent and then further acylated with pentafluorobenzoyl chloride. The N-acetyl-N-pentafluorobenzoyl-phenylethylamines were detected by high-resolution single ion monitoring of the molecular ions. Normal plasma levels were found to be 41.5 +/- 10.7 pg ml-1, in accordance with results of a previous high-performance liquid chromatographic method.     

Comprehensive analysis of metabolites in Corynebacterium glutamicum by gas chromatography/mass spectrometry

An analytical method based on gas chromatography/mass spectrometry was developed for metabolome investigation of Corynebacterium glutamicum. For the first time a fast method for metabolic screening that can be automated is described for this organism. More than 1000 compounds could be detected per experiment, ca. 330 of those showed a peak area significantly above background. Out of these 164 compounds were identified so far, representing derivatives of 121 different metabolites, which were quantified in one sample. In spite of the different chemical nature of metabolites and high matrix content, a measurement reproducibility in the range of 6% error was achieved. The application of this method for the analysis of the adaptation of C. glutamicum to different growth conditions is demonstrated.     

Chiral analysis by online coupling of reversed-phase liquid chromatography to gas chromatography and mass spectrometry

Enantiomeric composition of selected chiral compounds present in complex mixtures is determined by using the online coupling of reversed-phase liquid chromatography (LC) to gas chromatography (GC) and mass spectrometry. Integration of sample preparation into GC analysis, in a completely automated way, is achieved by means of the effective clean-up resulting from both the LC fractionation step and the eluent elimination provided by the through oven transfer adsorption desorption system used for LC-GC interfacing. The possibilities of the technique are illustrated through some examples concerning the stereodifferentiation in essential oils of major and minor chiral compounds via LC-GC transfer of different volume fractions, ranging from 0.5 to 1.9 ml, which show the significance of the window size for the determination of enantiomeric profiles.     

A high-throughput method for microbial metabolome analysis using gas chromatography/mass spectrometry

An analytical high-throughput method based on gas chromatography/mass spectrometry (GC/MS) was developed for fast metabolome investigation. By parallelization and partial automation the time needed for the preanalytical steps could be reduced. In addition a strong decrease of the relative standard deviation of metabolite concentrations from independent samples on the same microtiter plate from 25 to 13% was achieved. Between different plates the relative standard deviation is comparable to the one observed in standard experiments with shaking flasks. Using a fast GC the time need for the full GC/MS-based metabolome analysis could be decreased from 60 to 18 min per run, allowing the measurement of 72 single samples per day and GC/MS machine. In samples of the model organism Corynebacterium glutamicum more than 1000 peaks in the total ion current could be detected in a single fast GC/MS run of which 650 were strong enough to be quantified. Approximately 150 compounds of these were identified using our metabolite MS-library. Correlation analysis of the concentration vectors of independent wild-type samples raised under the same conditions show very high correlations of 0.99+/-0.01 (logs). In conclusion this method allows screenings of large mutant libraries for genetically induced metabolic perturbations.     

Estimation of deanol and choline by gas chromatography mass spectrometry

Analytical methods are described which permit the measurement of both deanol and choline in the same sample by gas chromatography mass spectrometry when either compound may be present in large excess (100:1). Deuterium labelling is employed for internal standards, to distinguish endogenous from tracer variants and to distinguish deanol in the sample from deanol formed by derivatization of choline. The limit of detection of both compounds is about 50 pmol.     

Quantitation of positional isomers of deuterium-labeled glucose by gas chromatography/mass spectrometry.

A method for determining the site and extent of deuterium (D) labeling of glucose by GC/MS and mass fragmentography was developed. Under chemical and electron impact ionization, ion clusters m/z 328, 242, 217, 212, and 187 of glucose aldonitrile pentaacetate and m/z 331 and 169 of pentaacetate derivative were produced. From the mass spectra of 13C- and D-labeled reference compounds, glucose carbon and hydrogen (C-H) positions included in these fragments were deduced to be m/z 328 = C1-C6, 2,3,4,5,6,6-H6; m/z 331 = C1-C6, 1,2,3,4,5,6,6-H7; m/z 169 = C1-C6, 1,3,4,5,6,6-H6; m/z 187 = C3-C6, 3,4,5,6,6-H5; m/z 212 = C1-C5, 2,3,4,5-H4; m/z 217 = C4-C6, 4,5,6,6-H4; and m/z 242 = C1-C4, 2,3,4-H3. After correction for isotope discrimination and deuterium-hydrogen exchange, the D enrichment of these fragments can be quantitated using selective ion monitoring, and the D enrichment of all C-H positions can be obtained by the difference in enrichment of the corresponding ion pairs. The validity of this approach was tested by examining D enrichment of known mixtures of 1-d1-, 2-d1-, 3-d1-, and 5,6,6-d3-glucose with unlabeled glucose and D enrichment of perdeuterated glucose using these fragments. This method was used to determine deuterium incorporation in C1 through C6 of blood glucose in fasted (24 h) rats infused with deuterated water. The distribution of deuterium was similar to that found by Postle and Bloxham (1980, Biochem. J. 192, 65-73). Approximately one deuterium atom was incorporated into C5 and only 75% deuterium atom was incorporated into C2. The enrichment of C2 and C6 of glucose relative to that of water indicated that 74 +/- 9% of plasma glucose was newly formed 4 h after the onset of deuterium infusion, and gluconeogenesis accounted for about 76 +/- 7% of the glucose 6-phosphate flux.