Analysis of galanthamine-type alkaloids by capillary gas chromatography-mass spectrometry in plants

Galanthamine, an acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease, and galanthamine-type alkaloids are synthesised in different plants of the family Amaryllidaceae. A capillary gas chromatographic-mass spectroscopic (CGC-MS) method for the separation of 7 galanthamine type alkaloids, including galanthamine and epigalanthamine, is described in the present paper. A simple method for the routine quantification of galanthamine in plants was developed using pre-packed columns with diatomaceous earth (Isolute HM-N), allowing simultaneous preparation of a large number of samples. Galanthamine showed excellent linearity in the range from 50 to 1000 microg/mL and the limit of quantification was 5 microg/mL in total ion current mode and 1.6 ng/mL in selected ion monitoring mode. The recovery of galanthamine was more than 90%. Interday reproducibility (RSD) of the extraction was 2.74%. A method to find and to microextract Amaryllidaceae alkaloids in low-mass plant samples is also described.     

A phytochemical study of the quinolizidine alkaloids from Genista tenera by gas chromatography-mass spectrometry

Gas chromatography coupled with mass spectrometry has been used to analyse the alkaloids present in the aerial parts of Genista tenera. Anagyrine, cytisine, N-formylcytisine, N-methylcytisine and lupanine were the major compounds, the last two alkaloids being known for their hypoglycaemic activity. Dehydrocytisine, 5,6-dehydrolupanine, rhombifoline, aphylline and thermopsine were the minor alkaloids. The characterisation of the constituents was based on comparison of their Kovats retention indexes and electron impact-mass spectrometric data recorded on-line with those of reference compounds and literature data.     

Identification and quantification of isoquinoline alkaloids in the genus Sarcocapnos by GC-MS

Six cularine alkaloids, cularicine, O-methylcularicine, celtisine, cularidine, cularine and celtine, three isocularine alkaloids, sarcophylline, sarcocapnine and sarcocapnidine, and five non-cularine alkaloids, glaucine, protopine, ribasine, dihydrosanguinarine and chelidonine, were identified and quantified by GC-MS in nine taxa of the genus Sarcocapnos (Fumariaceae). The chemotaxonomic significance of the results is discussed.     

Metabolomic analysis of biofluids from rats treated with Aconitum alkaloids using nuclear magnetic resonance and gas chromatography/time-of-flight mass spectrometry

The Aconitum alkaloids aconitine, mesaconitine, and hypaconitine are the main toxic components in a commonly used traditional Chinese herbal medicine Fu Zi. To provide guidelines for the safe use of this medicine, metabolic changes in Wistar rats caused by these compounds were investigated by means of integrated analysis of two metabonomic approaches: ¹H nuclear magnetic resonance (NMR) and gas chromatography/time-of-flight mass spectrometry (GC/TOF–MS). Rats were given a single dose of aconitine, mesaconitine, hypaconitine, or vehicle. The largest metabolic changes were observed 6 h after treatment. Every group receiving a dose had higher urine concentrations of glucose, acetate, dimethylglycine, succinate, and alanine and had lower concentrations of creatinine, citrate, 2-oxoglutarate, N-acetylated metabolites, and trimethylamine-N-oxide (TMAO) than did the control group. These results may reflect the perturbation of renal tubular function within the first 24 h after treatment. The results also revealed a larger perturbation of metabolic profiles in the aconitine group than in the mesaconitine and hypaconitine groups, illustrating how these alkaloids exhibit different toxicities. An analysis of plasma samples collected 7 days postdose showed that there were higher levels of lactate, alanine, and lipids along with lower levels of glucose, β-hydroxybutyrate, and creatine in the plasma of the aconitine and mesaconitine groups than there were in the control and hypaconitine groups. The GC/TOF–MS data from the plasma samples showed that the number of metabolites, with significant changes or with a tendency to change, in the aconitine and mesaconitine groups were dissimilar, suggesting a possible difference in the acute toxicity mechanisms of these alkaloids.     

Detection of bacterial contamination in cultures of eucaryotic cells by gas chromatography-mass spectrometry

The use of gas chromatography-mass spectrometry for early detection of bacterial contaminations in cultures of baker's yeast, Penicillium chrysogenum, and an animal cell line was evaluated; muramic acid and characteristic cellular fatty acids were used as analytes. By analyzing branched-chain and cyclopropane-substituted fatty acids as methyl esters, Staphylococcus epidermidis, Bacillus subtilis, Lactobacillus reuteri, Enterobacter cloacae, and Pseudomonas fluorescens were detected in a 500-fold excess (w/w) of baker's yeast; the amounts injected corresponded to 300 ng (dry mass) of the bacteria. Contamination with Bacillus was detected in cultures of Penicillium chrysogenum and animal cells by analyzing muramic acid, both as its alditol acetate derivative, using electron impact ionization, and its trifluoroacetyl methyl glycoside derivative, using negative ion-chemical ionization. The trifluoroacetylated derivative was detected in injected amounts corresponding to 1 x 10(3) bacterial cells in the contaminated animal cell line, whereas amounts corresponding to 1 x 10(5) bacteria were required for detection of the alditol acetate derivative; the amounts in the original samples were 5 x 10(5) and 5 x 10(6), respectively. However, the alditol acetate method exhibited lower chemical interferences than the trifluoroacetyl methyl glycoside procedure. The results show the potential of using gas chromatographic-mass spectrometric analysis of cellular constituents for the detection of bacterial contaminations in eucaryotic cultures as an alternative to conventional microbiological methods. (c) 1993 John Wiley & Sons, Inc.     

Methyl chanofruticosinate alkaloids from Kopsia arborea

Six alkaloids belonging to the methyl chanofruticosinate group, viz., prunifolines A-F, in addition to six other known methyl chanofruticosinate alkaloids, were isolated from the leaf extract of Kopsia arborea. The structures were determined using NMR and MS analysis and comparison with known related compounds.     

Effects of co-existed proteins on measurement of pesticide residues in blood by gas chromatography-mass spectrometry

Accurate measurement of pesticides in biological fluids such as blood is important for quantifying environmental exposures. Beyond sample enrichment and separation, the method presented here is focused on studies of interactions between pesticides and co-existed proteins. It was experimentally demonstrated that entrapped or adsorbed pesticide residues within the folded native structures of proteins were poorly recovered using direct solvent extraction solely. We described here an effective approach termed Enzymatic Digestion-Organic Solvent Extraction (eDOSE) that utilizes the enzymatic approach to disrupt the folded structures of proteins and release entrapped or adsorbed pesticide residues. In this approach, samples were first reduced, alkylated, tryptically digested and then diluted 10 times before the subsequent extraction using an n-hexane solution. Resultant pesticide residues were determined by capillary gas chromatography coupled with a mass spectrometer. Mean recoveries of the 5 organophosphorus pesticides pre-spiked in fish blood including diazinon, parathion-methyl, malathion, parathion-ethyl and ethion were 85%, 95%, 84%, 103%, and 43% respectively using eDOSE strategy but only 24%, 45%, 40%, 27%, and 29% respectively using direct solvent extraction approach. The eDOSE approach was effective for demonstrating the critical role of folded native structure of serum albumin in adsorption of exogenous chemicals. It provides an alterative means for denaturation of proteins when the target analytes are not stable in acidic solution or entrapped within the protein aggregates caused by organic solvents such as acetone that have been applied for protein denaturation. The eDOSE approach should be able to combine with other advanced techniques of enrichment and separation for more efficient and accurate measurement of target compounds present in the context of complex biological systems. This approach can provide wide applications to the analysis of a variety of small molecules including environmental pesticide residues and metabolites as well as other toxins present in cells, tissues and biofluids.     

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.     

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.     

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。     

Quantification of histamine in blood plasma and cell culture supernatants: a validated one-step gas chromatography-mass spectrometry method

A novel one-step ethylchloroformate (ECF) derivatization of histamine in biological liquid matrices that allows the sensitive quantification by gas chromatography and mass spectroscopic detection (GC-MS) from small volumes of blood plasma or cell culture supernatants within 15 min is described. After addition of ECF/chloroform directly to the crude sample, histamine has been found to be quantitatively derivatized within seconds. Following centrifugation, the organic phase is transferred to a fresh vial, dried by addition of anhydrous sodium sulfate, and subjected to GC-MS analysis. The reliability of the results is verified by use of two different ion pairs for detection. The method is validated according to DIN 38402. Linearity is given from 0.0054 to 13 microg/ml and the limit of detection is 2 ng/ml (10 pg absolute, at a signal to noise ratio of 3:1). The limit of quantification, as calculated at a confidence level of 95%, is 15.6 ng/ml. Practical application is exemplified by the determination of the histamine content in blood plasma of birch pollen-sensitized mice and in the culture supernatant of rat basophil leukemia cells after Ca(2+) ionophore-mediated degranulation.     

Identification and quantification of methyl jasmonate in leaf volatiles of Arabidopsis thaliana using solid-phase microextraction in combination with gas chromatography and mass spectrometry

Static headspace sampling with solid-phase microextraction has been used in combination with GC-FID and GC-MS for the specific enrichment, identification and quantification of volatile methyl jasmonate secreted by wounded leaves of Arabidopsis thaliana. The microsample method of analysis was found to be precise, accurate, sensitive and rapid. The detection limit of the procedure is 1.5 ppb (approximately 1.3 ng) per injection, which is of adequate sensitivity to detect the natural baseline levels of methyl jasmonate (approximately 10-100 ng/g) present in plant tissues. The method can be applied to most plants, requires a minimum of sample material, and shows the additional advantage that it is suitable for automation and could thus be used for high-throughput screening.     

Analysis of ergot alkaloids — a review

Methods for detection and determination of ergot alkaloids in grains, grasses, feeds and grain foods are reviewed. They incorporate simple detection procedures - colorimetry, thin layer chromatography and enzyme-linked immunosorbent assay - or instrumental procedures such as liquid chromatography with fluorescence, mass spectrometric (MS) or MS/MS detection, capillary zone electrophoresis, and direct MS/MS.     

High-temperature gas chromatography and gas chromatography-mass spectrometry of glycoprotein and glycosphingolipid oligosaccharides

High-temperature gas chromatography and gas chromatography-inass spectrometry for the analyses of oligosaccharides derived from glycoproteins or glycosphingolipids has been developed. Pcrmethylatcd oligosaccharides with up to about 12 sugar residues and masses up to 2500 Daltons can be analyzed. This approach is discussed and exemplified.     

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.     

Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction-ultracentrifugation-liquid chromatography-mass spectrometry

The membrane protein components of photosystem I (PSI) and II (PSII) from different species were prefractionated by liquid extraction and sucrose gradient ultracentrifugation and subsequently analyzed by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) using poly-(styrene-divinylbenzene)-based monolithic capillary columns. The analytical method was shown to be very flexible and enabled the identification of antenna proteins as well as most of the proteins of the reaction center from PSI and PSII in various plant species with few RP-HPLC-ESI-MS analyses necessitating only minor adaptations in the gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid. The membrane proteins, ranging in molecular mass (Mr) from 4196 (I protein) to more than 80,000 (PSI A/B) as well as isoforms were identified on the basis of their intact Mr and comparison with Mr deduced from known DNA or protein sequences. High quality mass spectra enabled the identification and quantitation of the nonphosphorylated and phosphorylated reaction center subunits D1, D2, and CP43 of PSII, containing five to seven membrane-spanning alpha-helices. Because of its high flexibility and suitability for proteins having a very wide range of Mr and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins.     

Analysis of dialkyl phosphate metabolites in hair using gas chromatography-mass spectrometry: A biomarker of chronic exposure to organophosphate pesticides

The aim of our study was to develop and validate an analytical approach for the quantitative determination of three dialkyl phosphate (DAP) metabolites, dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP) and diethyl phosphate (DEP), of organophosphate pesticides (OPs) in hair samples. The proposed methodology comprises a decontamination step, solid–liquid extraction, followed by liquid–liquid extraction, pentafluorobenzyl bromide derivatization, clean-up on Florisil/PSA column and analysis by gas chromatography–mass spectrometry (GC-MS). Extraction recovery, obtained from 50?mg hair samples spiked at two concentration levels, ranged from 56.1 to 107.9% and the within-day precision ranged from 13.5 to 17.5%. Limits of detection (LODs) ranged from 0.02 to 0.10?ng mg^?1. The results obtained from the analysis of hair samples of 30 agricultural workers show the suitability of the proposed method for monitoring people occupationally exposed to OPs. The most frequently detected compound was DEP followed by DMP. This is the first report on the detection of dialkyl phosphates in human hair which reflects the ability of hair testing to assess chronic exposure to OPs.     

一种基于LC-MS的抗生素敏感性快速确定方法

目的探索利用液相色谱-质谱（LC—MS）进行细菌快速药敏试验的方法。方法选择肠杆菌科和非发酵菌,以头孢他啶作为目标抗生素,测定不同时间段培养肉汤内头孢他啶的残存量作为判断药敏结果的依据。结果不同敏感性的细菌培养基中残留的头孢他啶量差异有统计学意义（P〈0．05）。结论LC—MS技术可以用于细菌的快速药敏试验。     

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography–mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.     

Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2-(13)C]cytosine and [2-(13)C]5-methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 microg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.