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.     

Gas chromatography and mass spectrometry of disaccharides from glycoproteins.

Partial acid hydrolysis and methanolysis released disaccharides and disaccharide methylglycosides from the glycoproteins, ovomucoid and porcine gastric mucin in amounts of 0.5--7 microgram disaccharide per mg of glycoprotein. These disaccharides were fractionated by gas chromatography as the trimethylsilyl (Me3Si) derivatives. The composition of recovered disaccharides has been determined by hydrolysis and rechromatography of the Me3Si monosaccharides. The intersaccharide linkages of the disaccharides have been determined by electron impact mass spectrometry. This simple and rapid method can give structural information on small glycoprotein samples.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.     

Electrospray mass spectrometry of human hair wax esters

Wax esters extracted from human hair have been examined by capillary GC-MS and by nano electrospray ionization (ESI) mass spectrometry using a tandem quadrupole mass spectrometer. Initially, the wax esters were examined by capillary GC-MS using conventional means, thus revealing an incomplete chromatographic resolution of the complex array of >200 wax esters ranging from 28 to 40 carbons in length, including saturated/straight-chained, unsaturated/straight-chained, saturated/branched, and unsaturated/branched molecular species. ESI of wax esters produced ammonium adduct ions [M+NH4]+, and collisional activation of these ions formed abundant [RCO2H2]+ product ions. Wax esters containing a double bond in the fatty acyl or fatty alcohol portion of the molecule revealed identical behavior, suggesting little influence of the double bond on the ionization process or subsequent decomposition. The wax ester mixture was analyzed by ESI and tandem mass spectrometry using multiple reaction monitoring and neutral loss scanning. The neutral loss experiment [loss of NH3 and CH2=CH-(CH2)nCH3] was particularly effective at rapidly surveying the complex biological mixture, identifying>160 different wax esters that range from 24 to 42 total carbons.     

Detection and identification of gibberellins in extracts of Begonia leaves by bioassay, radioimmunoassay and gas chromatography – mass spectrometry

Gibberellins A₁ (GA₁), GA₄, GA₉, GA₁₉, and GA₂₀ were identified in extracts of leaves of Begonia x cheimantha Everett cv. Nova (Christmas or Lorraine Begonia). GA-like substances were purified by reverse phase and normal phase high performance liquid chromatography (HPLC) and detected by Tan-ginbozu dwarf rice bioassay and binding to antibodies raised against GA₁, GA₄ and GA₉. The final identifications were made by gas chromatography—mass spectrometry (GC-MS).     

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.     

气相色谱-质谱定量检测水产品腐败菌群体感应DKPs信号分子

【目的】为了分析水产品腐败菌群体感应的新型信号分子二酮哌嗪(DKPs)化合物,建立一种简便、灵敏的气相色谱-质谱(GC-MS)定量检测方法。【方法】通过优化气相色谱和质谱条件、培养基和提取溶剂建立定量检测方法,确定Cyclo-(L-Pro-L-Gly)、Cyclo-(L-Pro-L-Leu)、Cyclo-(L-Leu-L-Leu)和Cyclo-(L-Pro-L-Phe)4种DKPs标准品的特征离子,并检测水产品腐败菌荧光假单胞菌(Pseudomonas fluorescens)和波罗的海希瓦氏菌(Shewanella baltica)中的DKPs。【结果】4种DKPs化合物在1-200 mg/L范围内线性良好,检测限为0.06、0.10、0.06和0.04 mg/L,定量限为0.16、0.18、0.14和0.12 mg/L,回收率为51.8%-88.5%,标准偏差为1.4%-8.3%。以LB培养基为细菌培养基,氯仿作为萃取溶剂,检测的DKPs含量较高。两种水产品腐败菌都检测到DKPs活性,主要种类为Cyclo-(L-Pro-L-Leu)和Cyclo-(L-Pro-L-Phe)。随着两种细菌的生长,培养上清中DKPs含量显著增加,在12 h达到最高。【结论】建立了检测细菌DKPs的GC-MS定量方法,具有较高精密度、准确度,能够准确定量分析4种DKPs的含量。为探究水产品特定腐败菌DKPs的调控机制奠定基础。     

Detection and identification of gibberellins in Sitka spruce (Picea sitchensis) of different ages and coning ability by bioassay, radioimmunoassay and gas chromatography – mass spectrometry

Gibberellins Al (GA₁), GA₃, GA₄, GA₉, and after enzymatic hydrolysis of GA-conjugate-like fractions, GA₉ and GA₁₅, were identified in shoots of Sitka spruce [ Picea sitchensis (Bong.) Carr.] of different ages by combined gas chromatography-mass spectrometry (GC-MS). The purification and separation of the GAs involved the use of reverse phase and normal phase high performance liquid chromatography (HPLC). The Tan-ginbozu dwarf rice bioassay and binding to antibodies raised against GA₁, GA₄ and GA₉ were used for detection of GA-like substances. The qualitative differences between the three ages of plant material were the presence of GA₃ and GA₁ in the 48-year-old material and the absence of detectable amounts of GA₄ in the same material. This indicates a difference in GA metabolism which may reflect the difference in ability to form reproductive buds.     

Thermospray liquid chromatography/mass spectrometry for the analysis of l-carnitine and its short-chain acyl derivatives

A method utilizing thermospray high-performance liquid chromatography/mass spectrometry for the separation and direct analysis of carnitine, acetylcarnitine, and propionylcarnitine is described. On-column analysis of mixtures of the acylcarnitines with their corresponding stable, isotope-labeled analogs at nanomolar concentrations has indicated that isotope dilution assays can be applied towards the analysis of carnitine and short-chain acylcarnitines present in biological samples.     

Ion-pairing reversed-phased chromatography/mass spectrometry of heparin

Heparin and heparin-derived components are widely applied anticoagulant drugs used for amongst other applications medical treatment of deep vein thrombosis and pulmonary embolism. Depolymerisation of native heparin results in complex mixtures of sulfated linear oligosaccharides that are usually not well characterised. In order to further characterise such mixtures, two on-line ion-pairing reverse-phased chromatography electrospray ionisation (ESI) mass spectrometry methods have been developed. One of the systems allows the determination of more than 200 components in a medium molecular weight heparin preparation, whereas the other system can be used to separate isomeric heparin oligosaccharides after previous separation according to size. This latter system allows semi-preparative isolation of isomeric heparin oligosaccharides. The experimental setup includes on-line cation exchange in order to prevent the ion-pairing reagent from entering the mass spectrometer.     

Comparison of human axillary odour profiles obtained by gas chromatography/mass spectrometry and skin microbial profiles obtained by denaturing gradient gel electrophoresis using multivariate pattern recognition

Several studies have shown that microbial action is responsible for many compounds responsible for human odour. In this paper, we compare the pattern of microbial profiles and that of chemical profiles of human axillary odour by using multivariate pattern matching techniques. Approximately 200 subjects from Carinthia, Austria, participated in the study. The microbial profiles were represented by denaturing gradient gel electrophoresis (DGGE) analysis and the axillary odour profiles were determined in the sweat samples collected by a stir-bar sampling device and analysed by gas chromatography/mass spectrometry (GC/MS). Both qualitative and quantitative distance metrics were used to construct dissimilarity matrices between samples which were then used to represent the patterns of these two types of profiles. The distance matrices were then compared by using the Mantel test and the Procrustean test. The results show that on the overall dataset there is no strong correlation between microbial and chemical profiles. When the data are split into family groups, correlations vary according to family with a range of estimated p values from 0.00 to 0.90 that the null hypothesis (no correlation) holds. When 32 subjects who followed four basic rules of behaviour were selected, the estimated p-values are 0.00 using qualitative and <0.01 using quantitative distance metrics, suggesting excellent evidence that there is a connection between the microbial and chemical signature.     

Rapid and comprehensive evaluation of microalgal fatty acids via untargeted gas chromatography and time‐of‐flight mass spectrometry

Due to their high triacylglyceride content, microalgae are intensively investigated for bio‐economy and food applications. However, lipid analysis is a laborious task incorporating extraction, transesterification and typically gas chromatographic measurement. Co‐elution induces a significant risk of fatty acid misidentification and thus, additional purification steps like thin layer chromatography are needed. Contrary to database matching approaches, solely targeted analysis is facilitated as compound identification is driven by matching retention times or indices with standard substances. In this context, a rapid workflow for the analysis of algal fatty acids is presented. In‐situ transesterification was used to simplify sample preparation and conditions were optimized towards fast processing. If results are needed at the very day of sampling, direct processing without a preceding drying step is feasible to obtain a rough estimate about the occurrence of the major compounds. Coupling gas chromatography and time‐of‐flight mass spectrometry enables untargeted analysis. Unknown compounds may be identified by structural reconstruction of their respective fragmentation patterns and by database matching to routinely avoid mismatches by co‐elution of disturbing agents. The developed workflow was successfully applied to derive the exact stereochemistry of all fatty acids from Chlorella vulgaris and a systematic shift depending on physiological state of the cells was confirmed.     

Global analysis of metabolites in rat and human urine based on gas chromatography/time-of-flight mass spectrometry

Sediment in urine may contain low-molecular-weight compounds that should be included in the analysis. To date, no systematic investigation has addressed this issue. We investigated three primary factors that influence the extraction efficiency of metabolites during preparation of urine samples for metabolomic research: centrifugation, pH, and extraction solvents. Obtained with the use of gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) technique and principal component analysis (PCA), our results indicate that (1) conventional centrifugation causes an apparent loss of some metabolites, indicating that urine samples for metabolomic research should not be centrifuged before procedures are undertaken to recover the metabolites; (2) pH adjustment has a large impact on the recovery of metabolites and is therefore not encouraged; (3) with design of experiment analysis, methanol and water yield the optimal extraction efficiency. Differences between rat and human urine were observed and are discussed. Ninety-nine metabolites identified in rat and human urine are presented. An efficient protocol is proposed for the pretreatment of urine samples.     

A gas chromatography/mass spectrometry assay to measure estradiol, catecholestradiols, and methoxyestradiols in plasma

We have developed a gas chromatography/mass spectrometry (GC/MS) assay to measure 17beta-estradiol (E) and its biologically active metabolites 2-hydroxyestradiol (2OHE) and 4-hydroxyestradiol (4OHE), and 2-methoxyestradiol (2MEOE) and 4-methoxyestradiol (4MEOE) in rat plasma. All analytes are well separated and show a linear relationship between concentration (0.25-5 pg/microl) and signal, and coefficients of variation (CVs) are low. Intra-assay CV for the lowest quality control samples (QCs) (0.375 pg/microl) were on average for 17beta-estradiol 20.5%, for 2-hydroxyestradiol 15.6%, for 4-hydroxyestradiol 16.5%, for 2-methoxyestradiol 16.5%, and for 4-methoxyestradiol 11.5%. The inter-assay CVs for the lowest QCs were for 17beta-estradiol 12.1%, for 2-hydroxyestradiol 7.1%, for 4-hydroxyestradiol 15.5%, for 2-methoxyestradiol 16.7%, and for 4-methoxyestradiol 9.7%. The highest sensitivity for this assay was observed for hydroxyestradiols followed by the methoxyestradiols and 17beta-estradiol. In summary, we describe a convenient, sensitive, and specific assay to measure 17beta-estradiol and its biologically active metabolites.     

Gas chromatography mass spectrometry based metabolic profiling reveals biomarkers involved in rice-gall midge interactions

The Asian rice gall midge (Orseolia oryzae Wood-Mason) is a serious pest of rice that causes huge loss in yield. While feeding inside the susceptible host, maggots secrete substances that facilitate th...     

A proteomic view of Bifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gut where they exert several health-promoting effects. The present paper reports the use of a strong cation exchange-reversed-phase-tandem mass spectrometry strategy to catalogue the most abundantly expressed proteins of a probiotic Bifidobacterium infantis strain. A global view of the B. infantis proteome was obtained. The bimodal representation of the proteins identified by mass spectrometry provides the first theoretical two-dimensional map of protein distribution for this organism. Among the 136 proteins identified by multidimensional protein identification technology (MudPIT) analysis, 118 showed the highest similarity with the translated sequences of B. longum genome, two proteins were similar to other Bifidobacterium species and the remaining 16 were similar to different genera. Specific biological activities have been assigned to 115 identified proteins, whereas 21 have been referred to the group of hypothetical proteins. The MudPIT approach allowed us to identify high mass and basic isoelectric point proteins that are generally challenging to visualize using the traditional two-dimensional electrophoresis technique. Redundancy in peptide and protein identification using the double chromatography technique was also evaluated.     

Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry

High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats.

In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market.     

Characterization of a noncovalent lipocalin complex by liquid chromatography/electrospray ionization mass spectrometry.

Nanoscale liquid chromatography coupled to electrospray ionization mass spectrometry was used to identify the nature of the ligand that binds noncovalently to siderocalin (lipocalin 2). The folded state siderocalin-ligand complex was separated from free, unfolded siderocalin using reversed phase chromatography, and the molecular weight of the siderocalin ligand was then determined from the deconvoluted molecular weights of the complex and of the free protein. The ligand was identified as dihydroxybenzoyl-serine, a breakdown product of enterobactin, an iron-chelating compound ("siderophore") synthesized in bacteria. These results demonstrate that, in some cases, electrostatic noncovalent protein complexes can survive the denaturing conditions of reversed phase liquid chromatography and the gas phase transfer occurring during electrospray ionization.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Identification of carbonyl sulfide and sulfur dioxide in porcine coronary artery by gas chromatography/mass spectrometry, possible relevance to EDHF

Incubation of porcine coronary artery rings and cardiac muscle tissue in Krebs buffer followed by GC/MS analysis of the headspace gas revealed two gases, carbonyl sulfide (COS) and sulfur dioxide (SO(2)). The gases were identified by characteristic ions obtained by electron ionization, and by comparison of the retention time on a chromatographic column (GS GasPro) with standards of these gases. Stimulation of the arterial rings with acetylcholine and calcium ionophore A23187 increased the levels of SO(2) and COS in the vascular tissue. We also provide evidence that SO(2) could originate from disproportionation of a very unstable gas, sulfur monoxide (S=O). We suggest potential origins of these gases and discuss their relevance to endothelium-derived hyperpolarizing factor.