Determination of Aconitum alkaloids in blood and urine samples: II. Capillary liquid chromatographic–frit fast atom bombardment mass spectrometric analysis

Determination of fourteen alkaloids, toxic Aconitum alkaloids, aconitine, mesaconitine, jesaconitine, hypaconitine and deoxyaconitine, and their hydrolysis products, benzoylaconines and aconines, have been established using capillary liquid chromatography (LC) fast atom bombardment mass spectrometry (FAB-MS) with a frit interface. Protonated molecular ions were observed as base peaks in the FAB-MS for these fourteen alkaloids. All the alkaloids were simultaneously quantified with linear gradient LC elution by solvent mixture of acetonitrile and 0.3% trifluoroacetic acid using selected ion monitoring of the protonated molecular ions. The calibration curves of these alkaloids were linear in injection amounts ranging from 5 to 500 pg, and their detection limits were 1 pg per injection (S/N=3). Solid-phase extraction using Sep-Pak Plus PS-1 was also investigated to clean-up and concentrate alkaloids in blood and urine samples, and showed satisfactory recoveries. This capillary LC–frit-FAB-MS method enables determination of low levels of Aconitum alkaloids in blood and urine samples, coupled with solid-phase extraction.     

Determination of Aconitum alkaloids in blood and urine samples. I. High-performance liquid chromatographic separation, solid-phase extraction and mass spectrometric confirmation

Determination of four toxic Aconitum alkaloids, aconitine, mesaconitine, hypaconitine and jesaconitine, in blood and urine samples has been established using high-performance liquid chromatography (HPLC) combined with ultraviolet absorbance detection, solid-phase extraction and mass spectrometry (MS). These alkaloids were hydrolyzed rapidly in alkaline solution (half lives (t_1/2)<one day), were stable in solutions of acetonitrile, tetrahydrofuran and diluted hydrochloric acid (t_1/2>five months) and were unstable in solutions of methanol and ethanol (t_1/2<one month). These alkaloids were separated on an octadecylsilica column with isocratic elution using a solvent mixture of tetrahydrofuran and 0.2% trifluoroacetic acid (14:86, v/v), which was found to be the optimal solvent of the elution systems examined. Calibration curves with UV detection were linear on injection of amounts ranging from 2.5 to 500 ng, and the limit of detection was 1 ng (S/N = 3). These four alkaloids in aqueous solution were recovered almost totally by solid-phase extraction using the styrene polymer resin, Sep-Pak Plus PS-1, and were eluted using a mixture of acetonitrile and hydrochloric acid. These Aconitum alkaloids were confirmed by HPLC coupled with fast atom bombardment MS, giving their protonated molecular ions as base peaks. These alkaloids were detected by HPLC with UV detection from blood samples spiked with more than 50 ng ml⁻¹ of alkaloids, but were not detectable from urine samples spiked with 5 μg ml⁻¹ of alkaloids because of severe sample interference.     

Neutral Fragment Filtering for Rapid Identification of New Diester-Diterpenoid Alkaloids in Roots of Aconitum carmichaeli by Ultra-High-Pressure Liquid Chromatography Coupled with Linear Ion Trap-Orbitrap Mass Spectrometry

A rapid and effective method was developed for separation and identification of diester-diterpenoid alkaloids (DDA) in the roots of Aconitum carmichaeli by ultra-high-pressure liquid chromatography coupled with high resolution LTQ-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MSⁿ). According to accurate mass measurement and the characteristic neutral loss filtering strategy, a total of 42 diester-diterpenoid alkaloids (DDA) were rapidly detected and characterized or tentatively identified. Meanwhile, the proposed fragmentation pathways and the major diagnostic fragment ions of aconitine, mesaconitine and hypaconitine were investigated to trace DDA derivatives in crude plant extracts. 23 potential new compounds were successfully screened and characterized in Aconitum carmichaeli, including 16 short chain fatty acyls DDA, 4 N-dealkyl DDA and several isomers of aconitine, mesaconitine and hypaconitine.     

Quantitative determination of Aconitum alkaloids in blood and urine samples by high-performance liquid chromatography

An HPLC method has been developed for the simultaneous determination of the toxic Aconitum alkaloids, aconitine, mesaconitine and hypaconitine in blood and urine samples. The samples were initially subjected to solid phase extraction using Oasis MCX cartridges, and the alkaloids were separated on an XTerra RP18 column, gradient-eluted with acetonitrile: ammonium hydrogen carbonate buffer. Calibration curves were linear in the range 2.75-550 ng for aconitine and hypaconitine, and 3-600 ng for mesaconitine: the limit of detection was 0.1 ng (signal-to-noise ratio of 3) for each alkaloid. The described analysis proved to be sensitive, rapid and economical, and will be applied in the identification and determination of these alkaloids in forensic and therapeutic drug monitoring.     

1H NMR and GC/MS metabolomics of earthworm responses to sub-lethal DDT and endosulfan exposure

The metabolic response of the earthworm Eisenia fetida to two pesticides, dichlorodiphenyltrichloroethane (DDT) and endosulfan, was characterized in contact tests using proton nuclear magnetic resonance (¹H NMR) and principal component analysis (PCA). PCA loading plots suggested that maltose, leucine and alanine were important metabolites contributing to the differences in dosed and control earthworms for both compounds at doses of 0.5, 1.0 and 2.0 μg/cm². Gas chromatography/mass spectrometry (GC/MS) was used to quantify the metabolites identified in E. fetida and determine if the changes in maltose, leucine and alanine following exposure to DDT and endosulfan (at 0.5 and 1.0 μg/cm²) were reproducible and greater than the natural variability. Quantification by GC/MS suggested that maltose was not a reliable biomarker since it both increased and decreased in earthworms exposed to DDT and increased by just 3% with exposure to endosulfan. Leucine was not stable with the GC/MS derivitization method used in this study and could not be confirmed as a reliable biomarker. However, alanine consistently increased for both DDT and endosulfan exposed E. fetida. Alanine showed considerable variability in control earthworms (±41.6%), yet the variability in alanine to glycine ratios was just ±10.5%. Increases in the alanine to glycine ratio were statistically significant at the P = 0.05 level for the 1.0 μg/cm² DDT dose and both the 0.5 and 1.0 μg/cm² endosulfan doses, suggesting that deviations from the normal homeostatic ratio of 1.5 for alanine to glycine is a potential biomarker of DDT and endosulfan exposure warranting further study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Environmental Metabolomics Special Issue of Metabolomics.     

Report on the preparation of deuterium-labelled aconitine and mesaconitine and their application to the analysis of these alkaloids from body fluids as internal standard

Improved analysis of aconitine and mesaconitine, highly toxic compounds from Aconitum species, in body fluids by gas chromatography–selected ion monitoring with their deuterium-labelled analogues as internal standards (I.S.s) is described. Deuterium-labelled analogues of aconitine and mesaconitine were synthesized by the substitution of the N-alkyl group for a deuterium-labelled one. The mass spectra of the derivatives of the deuterium labels closely resembled that of the nonlabelled compounds except for an obvious mass shift produced by substitution of the deuterium atoms at N-alkyl groups. Using these deuterium-labelled compounds as I.S.s, the standard curves for aconitine and mesaconitine were linear (r²=0.999 each) in the concentration range of 50 pg to 50 ng, respectively. The detection limit of the alkaloids was 10 pg each per injection. The recovery, accuracy and precision of the analysis were evaluated with three different concentration of spiked human blood and urine (n=5 each). The recovery rates ranged from 97.6% to 101.3% and the standard deviations of the interseries ranged from 2.1% to 3.9%. These I.S.s give us a more precise analysis and may be useful in examining the behavior of these alkaloids in the human body.     

Phytochemical differentiation of Galanthus nivalis and Galanthus elwesii (Amaryllidaceae): A case study

The alkaloid patterns of two occasionally sympatric Galanthus nivalis and Galanthus elwesii populations were studied by GC/MS. Thirty-seven alkaloids were detected, 25 for G. nivalis and 17 for G. elwesii. Only five alkaloids were found to occur in both species. The populations of Galanthus differed in their alkaloid biosynthetic pathways. Thus, the alkaloid pattern of G. nivalis was dominated by compounds coming from a para–para′ oxidative coupling of O-methylnorbelladine. The predominant alkaloids in the roots of this species were found to belong to the lycorine and tazettine structural types; bulbs were dominated by tazettine, leaves by lycorine and flowers by haemanthamine type alkaloids. In contrast, the alkaloid pattern of G. elwesii was dominated mainly by compounds coming from an ortho–para′ oxidative coupling. The predominant alkaloids in G. elwesii roots, bulbs and leaves were those of homolycorine type, whereas the flowers accumulated mainly tyramine type compounds. The chemotaxonomical value of the alkaloids found in the studied species is discussed.     

Studies on the stability of diester-diterpenoid alkaloids from the genus Aconitum L. by high performance liquid chromatography combined with electrospray ionisation tandem mass spectrometry (HPLC/ESI/MSn)

The stability of diester-diterpenoid alkaloids (DDA) from plants of the genus Aconitum L. has been studied in different solvents and pH buffers. The HPLC/ESIMS method for analysing the concentration of DDA was established and DDA's decomposition products were elucidated by HPLC/ESI-MS/MS(n). In different solvents, e.g. dichloromethane, ether, methanol and distilled water, the decomposition pathways of DDA are quite different and their difference in stabilities depends on the difference of their structures, in which substituents at the N atom and substituents at C-3 are different. The pyrolytic products of DDA, such as deacetoxy aconitine-type alkaloids, have been observed in the above solvents, whereas 8-methoxy-14-benzoyl aconitine-type alkaloids have been obtained only in methanol. Furthermore, the experimental results demonstrate that the stability of DDA depends on pH values of the buffer. Aconine as hydrolysate has been only found in pH 10.0 buffer, and the other hydrolysates and the pyrolyzates of DDA, such as benzoylaconine and deacetoxy aconitine, have been observed in all pH aqueous solutions. The decomposition pathways of DDA in buffers are related to the substituent on the C-3 position. The decomposition pathway of aconitine is similar to that of mesaconitine, but different from that of hypaconitine.     

Development and validation of a rapid capillary zone electrophoresis method for the determination of aconite alkaloids in aconite roots

Introduction – Aconites, with aconite alkaloids as the major therapeutic and toxic components, are used for the treatment of analgesic, antirheumatic and neurological symptoms. Quantification of the aconite alkaloids is important for the quality control of aconite‐containing drugs. Objective – To establish a validated capillary zone electrophoresis (CZE) method for the simultaneous determination of six major alkaloids, namely aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine, in crude and processed aconite roots. Methodology – The CZE method was optimised and validated using a stability‐indicating method. The optimised running buffer was a mixture of 200 mm Tris, 150 mm perchloric acid and 40% 1,4‐dioxane (pH 7.8) with the capillary thermostated at 25°C. Results – Using the optimised method, six aconite alkaloids were well separated. The established method showed good precision, accuracy and recovery. Contents of these alkaloids in crude and processed aconites were determined and it was observed that the levels of individual alkaloids varied between samples. Conclusion – The developed CZE method was reliable for the quality control of aconites contained in herbal medicines. The method could also be used as an approach for toxicological studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Biodegradation of Methyl red by Galactomyces geotrichum MTCC 1360

Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane, azo and reactive high exhaust textile dyes. At shaking condition this strain showed 100% decolorization of a toxic azo dye Methyl red (100 m gl⁻¹) within 1 h in deionized water at 30 °C. The degradation of Methyl red was possible through a broad pH (3–12) and temperature (5–50 °C) range. Glucose and mycelium concentration had increased the decolorization rate, but the addition of 1 gl⁻¹ molasses in deionized water made decolorization possible in only 10 min. Induction in the NADH–dichloro phenol indophenol (NADH–DCIP) reductase, Malachite green reductase, laccase and lignin peroxidase (Lip) activities were observed in the cells obtained after complete decolorization, showing that there is direct involvement in the degradation of Methyl red. The absence of N-N′-dimethyl-p-phenylenediamine (DMPD) in 5 °C, 2-aminobenzoic acid (ABA) in 50 °C and both the compounds in 30 °C sample have shown the differences in the metabolic fate of Methyl red at different temperatures. The untreated dye at 300 mg l⁻¹ concentration showed 88% germination inhibition in Sorghum bicolor, whereas it was 72% in Triticum aestivum. There was no germination inhibition for both the plants by Methyl red metabolites at 300 mg l⁻¹ concentration.

The scientific relevance of the paper

The azo dye Methyl red (100 mg l⁻¹) was decolorized by G. geotrichum MTCC 1360 within 1 h at shaking condition in deionized water. This organism could decolorize Methyl red at wide pH and temperature ranges. Decolorization time was reduced to 10 min by the addition of molasses to deionized water. There was induction in laccase and Lip, NADH–DCIP reductase and Malachite green reductase activities. The metabolic fate of Methyl red changes with temperature which can be evidenced by the formation of 2-ABA at 5 °C, N-N′-DMPD at 50 °C and both the compounds were absent at 30 °C. Phytotoxicity showed that metabolites of dye had induced shoot and root length of both the tested plants.     

Quantification of Aconitum alkaloids in aconite roots by a modified RP-HPLC method

The three Aconitum alkaloids, aconitine (1), mesaconitine (2) and hypaconitine (3), are pharmacologically active but also highly toxic. A standardised method is needed for assessing the levels of these alkaloids in aconite roots in order to ensure the safe use of these plant materials as medicinal herbs. By optimising extraction, separation and measurement conditions, a reliable, reproducible and accurate method for the quantitative determination of all three Aconitum alkaloids in unprocessed and processed aconite roots has been developed. This method should be appropriate for use in the quality control of Aconitum products. The three Aconitum alkaloids were separated by a modified HPLC method employing a C18 column gradient eluted with acetonitrile and ammonium bicarbonate buffer. Quantification of Aconitum alkaloids, detected at 240 nm, in different batches of samples showed that the content of 1, 2 and 3 varied significantly. In general, the alkaloid content of unprocessed roots was higher than that of processed roots. These variations were considered to be the result of differences in species, processing methods and places of origin of the samples.     

The antitumoral, trypanocidal and antileishmanial activities of extract and alkaloids isolated from Duguetia furfuracea

The alkaloid extract and five alkaloids isolated from subterranean stem bark of Duguetia furfuracea (Annonaceae) were investigated for the following activities: antitumoral, trypanocidal and leishmanicidal. Dicentrinone showed weak cytotoxicity, but it had the strongest leishmanicidal activity (IC₅₀ 0.01 μM). Duguetine and duguetine β-N-oxide caused considerable antitumoral activity in every cell lines evaluated, although duguetine was more active against trypomastigote forms (IC₅₀ 9.32 μM) than other alkaloids tested.     

Genetic transformation of <Emphasis Type="Italic">Ruta graveolens</Emphasis> L. by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: hairy root cultures a promising approach for production of coumarins and furanocoumarins

Ruta graveolens L. is a source of pharmacologically active compounds such as coumarins, furanocoumarins and furoquinolone alkaloids. Hypocotyls, callus and shoots of R. graveolens were inoculated with bacteria from two Agrobacterium rhizogenes strains. Hairy root cultures were established after inoculation of hypocotyls with wild A. rhizogenes strain LBA 9402. The transgenic nature of the regenerated tissue was confirmed by PCR amplification. Coumarins, furanocoumarins and alkaloids present in the hairy root tissue were identified by GC and GC-MS and compared with those present in in vitro shoot cultures. The level of pinnarin and rutacultin, bergapten, isopimpinelin and xanthotoxin was approximately twofold higher in hairy root than in shoot cultures. Two additional coumarins: osthole and osthenol, never been found in R. graveolens, were identified in hairy root tissue. Besides coumarins, alkaloids were identified: dictamnine, skimmianine, kokusaginine, rybalinine and an isomer of rybalinine. The levels of nearly all coumarins and alkaloids in hairy roots cultured in the darkness were higher than those accumulated under a photoperiod mode.     

Effects of Microdesmis keayana roots on sexual behavior of male rats

In the present study, the aphrodisiac properties of Microdesmis keayana J. Léonard root extract and major isolated alkaloids were evaluated by observing the sexual behavior of male rats.Aqueous extract (150 mg/kg body weight) and pure alkaloids (3 mg/kg body weight) were administered orally by gavage to male rats. Latent times of observation, intromission and ejaculation, mounting behavior, number of intromissions and mating performances were evaluated and compared to those obtained with untreated rats in the presence of receptive and non-receptive females. The results have shown that aqueous extract and alkaloids of M. keayana stimulate sexual parameters in rats’ sexual behavior.A short-term toxicity study undertaken to establish the therapeutic index of aqueous extract, showed that a high dose of the extract (2 g/kg body weight) caused no mortality or changes in rats’ behavior.     

Application of the high-performance liquid chromatographic method for separation, purification and characterisation of p-bromophenylacetylurea and its metabolites

This study presents a HPLC method for the separation and purification of p-bromophenylacetylurea (BPAU) and its metabolites. The method effectively separated and purified BPAU and its metabolites. Three metabolites of BPAU, M1, M2 and M3 were characterised by mass spectroscopy and nuclear magnetic resonance. They are named as N′-hydroxy-p-bromophenylacetylurea, 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione and N′-methyl-p-bromophenylacetylurea, respectively. The major metabolic pathways of BPAU were proposed. The establishment of the HPLC method and characterisation of BPAU metabolites make it possible for further pharmacokinetic studies to explore the mechanism of BPAU-induced delayed neuropathy.     

Synergistic hepatoprotective effect of Schisandrae lignans with Astragalus polysaccharides on chronic liver injury in rats

The aim of this study was to investigate the synergistic hepatoprotective effect of lignans from Fructus Schisandrae chinensis (LFS) with Astragalus polysaccharides (APS) on chronic liver injury in male Sprague-Dawley rats. Subcutaneous injection of 10% CCl₄ twice a week for 3 months resulted in significantly (p<0.001) elevated serum alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP) activities compared to controls. In the liver, significantly elevated levels (p<0.001) of malondialdehyde (MDA), lowered levels of reduced glutathione (GSH) (p<0.05) and catalase (CAT) (p<0.001), superoxide dismutase (SOD) (p<0.01)were observed following CCl₄ administration. ‘LFS+ASP’ treatment of rats at doses of ‘LFS (45 mg/kg)+APS (150 mg/kg)’ and ‘LFS (135 mg/kg)+APS (450 mg/kg)’ displayed hepatoprotective and antioxidative effects than the administration of either LFS or APS, as evident by lower (p<0.005 or 0.001) levels of serum ALT, AST, ALP and hepatic MDA (p<0.001) concentration, as well as higher SOD (p<0.05 or 0.005), CAT activities(p<0.01 or 0.005), GSH concentration (p<0.05 or 0.005) compared to the toxin treated group. Histopathological examinations revealed severe fatty degeneration in the toxin group, and mild damage in groups treated with ‘LFS+APS’ were observed. The coefficients drug interaction (CDI) between each individual drug and their combination (at the same dose of their single treatment) of these foregoing parameters were all less than 1, indicating that LFS and APS display hepatoprotective and antioxidant properties and act in a synergistic manner in CCl₄ induced liver injury in rats.     

基于LC-MS/MS分析马缨杜鹃花代谢物的变化

为分析马缨杜鹃(Rhododendron delavayi)花开花至凋谢过程中的代谢产物差异及其通路,该文采用LC-MS/MS技术对其花苞期、开裂期、传粉期、盛开期、衰老期和凋谢期的化学成分进行非靶向代谢组学分析。结果表明:(1)共鉴定到973种代谢物,主要包含黄酮类、有机酸、酚酸类、氨基酸及其衍生物、脂类、生物碱等。(2)主成分分析(PCA)表明样本间代谢物存在差异,结合正交偏最小二乘判别分析(OPLS-DA)、t检验的P值和单变量分析的差异倍数(fold-change)筛选差异代谢物(VIP>1,P<0.05,Fc>2或Fc<0.5),涉及591种,在马缨杜鹃花期进入衰老期和凋谢期后差异代谢物数量和表达量显著上升,其中花苞期至开裂期差异代谢物的表达主要呈现下调,而进入衰老期和凋谢期后差异代谢物的表达主要呈现上调。(3)KEGG注释到68条代谢通路,其中差异代谢物极显著富集(P < 0.01)通路3条,包括苯丙素类生物合成、植物激素的生物合成和类黄酮生物合成。(4)结合苯丙素类、黄酮类等有效成分生物合成通路共筛选到10种代谢物包括苯丙氨酸(L-phenylalanine)、反式肉桂酸(trans-cinnamic acid)、查耳酮(chalcone)、柚皮素(naringenin)、对香豆酰基莽草酸(p-coumaroyl shikimic acid)、阿魏酸(ferulic acid)、松柏醇(coniferyl alcohol)、芥子酸(sinapic acid)、紫丁香苷(syringin)、槲皮素(quercetin)。此外,有效成分的差异代谢物表明苯丙素类生物合成代谢活动随马缨杜鹃花的发育逐渐增强,而黄酮类化合物生物合成逐渐减弱,这些关键差异代谢物可能对马缨杜鹃花的发育有重要的调控作用。该研究为马缨杜鹃花开花至凋谢进程中的有效成分代谢途径活性物质的研究提供了代谢组学基础,为进一步研究马缨杜鹃花花期调控的分子机理提供参考。     

Gas chromatographic method using nitrogen–phosphorus detection for the measurement of tramadol and its O-desmethyl metabolite in plasma and brain tissue of mice and rats

A method that allows the measurement of plasma and brain levels of the centrally-acting analgesic tramadol and its major metabolite (O-desmethyl tramadol) in mice and rats was developed using gas chromatography equipped with nitrogen–phosphorus detection (GC–NPD). Plasma samples were extracted with methyl tert.-butyl ether (MTBE) and were injected directly into the GC system. Brain tissue homogenates were precipitated with methanol, the resulting supernatant was dried then acidified with hydrochloric acid. The aqueous solution was washed with MTBE twice, alkalinized, and extracted with MTBE. The MTBE layer was dried, reconstituted and injected into the GC system. The GC assay used a DB-1 capillary column with an oven temperature ramp (135 to 179°C at 4°C/min). Dextromethorphan was used as the internal standard. The calibration curves for tramadol and O-desmethyl tramadol in plasma and brain tissue were linear in the range of 10 to 10 000 ng/ml (plasma) and ng/g (brain). Assay accuracy and precision of back calculated standards were within ±15%.     

Simultaneous quantitation of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine in human plasma by liquid chromatography-tandem mass spectrometry and pharmacokinetics evaluation of "SHEN-FU" injectable powder

A rapid, specific and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for simultaneous quantitation of six Aconitum alkaloids, i.e. aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) in human plasma collected from 18 healthy volunteers after intravenous drop infusion of "SHEN-FU" injectable powder in three different dosages. Lappaconitine was selected as the internal standard (IS). LC/MS/MS system coupled with an electrospray ionization (ESI) source was performed in multiple-reaction monitoring (MRM) mode. The transitions of the Aconitum alkaloids executed as following: m/z 646.3-->586.0 for AC; m/z 632.4-->573.1 for MA; m/z 616.2-->556.1 for HA; m/z 604.2-->104.8 for BAC; m/z 590.1-->104.8 for BMA; m/z 574.1-->104.8 for BHA; m/z 585.2-->161.8 for IS. Sample preparation was performed with solid-phase extraction (SPE) on a 1 mL HLB cartridge prior to analysis. The separation was applied on a Waters C(18) column (1.7 microm, 2.1 mm x 100 mm) and a gradient elution of methanol and 0.1% formic acid-water was used as mobile phase. The retention time was less than 4.5 min. The concentrations ranged from 0.1 to 1000 ng/mL for all six Aconitum alkaloids and showed a good linearity with the correlation coefficient (r(2)) >0.995. The validated method was employed to simultaneous quantitation and successfully used for the first time for the pharmacokinetic evaluation of the six Aconitum alkaloids after intravenous drop administration of "SHEN-FU" injectable powder in phase I clinical trial.     

The yeast metabolome addressed by electrospray ionization mass spectrometry: Initiation of a mass spectral library and its applications for metabolic footprinting by direct infusion mass spectrometry

Mass spectrometry (MS) has been a major driver for metabolomics, and gas chromatography (GC)-MS has been one of the primary techniques used for microbial metabolomics. The use of liquid chromatography (LC)-MS has however been limited, but electrospray ionization (ESI) is very well suited for ionization of microbial metabolites without any previous derivatization needed. To address the capabilities of ESI-MS in detecting the metabolome of Saccharomyces cerevisiae, the in silico metabolome of this organism was used as a template to present a theoretical metabolome. This showed that in combination with the specificity of MS up to 84% of the metabolites can be identified in a high mass accuracy ESI-spectrum. A total of 66 metabolites were systematically analyzed by positive and negative ESI-MS/MS with the aim of initiating a spectral library for ESI of microbial metabolites. This systematic analysis gave insight into the ionization and fragmentation characteristics of the different metabolites. With this insight, a small study of metabolic footprinting with ESI-MS demonstrated that biological information can be extracted from footprinting spectra. Statistical analysis of the footprinting data revealed discriminating ions, which could be assigned using the in silico metabolome. By this approach metabolic footprinting can advance from a classification method that is used to derive biological information based on guilt-by-association, to a tool for extraction of metabolic differences, which can guide new targeted biological experiments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.