Determination of bupivacaine and metabolites in rat urine using capillary electrophoresis with mass spectrometric detection

A method using capillary electrophoresis-mass spectrometry (CE-MS) was developed for the structural elucidation of bupivacaine and metabolites in rat urine. Prior to CE-MS analysis, solid-phase extraction (SPE) was used for sample cleanup and preconcentration purposes. Exact mass and tandem mass spectrometric (MS/MS) experiments were performed to obtain structural information about the unknown metabolites. Two instruments with different mass analyzers were used for mass spectrometric detection. A quadrupole time-of-flight (Q-TOF) and a magnetic sector hybrid instrument were coupled to CE and used for the analysis of urine extracts. Hydroxybupivacaine as well as five other isomerically different metabolites were detected including methoxylated bupivacaine.     

Assessment of metastable atom bombardment (MAB) ionization mass spectrometry for the fast determination of heterocyclic aromatic amines in cooked meat

An investigation of metastable atom bombardment (MAB) ionization mass spectrometry for the fast characterization of mutagenic/carcinogenic heterocyclic aromatic amines (HAAs) formed during heating processes of meats is presented. The aim of our study was to use the selective ionization of MAB to develop a detection method for HAAs in non-purified meat extracts, thus avoiding purification and concentration steps and reducing analysis time. Sample introduction into the MAB ion source was achieved by pyrolysis, allowing the direct and fast insertion of complex food extracts into the mass spectrometer. Analysis conditions were optimized on standard HAAs by using different ionization gases for the MAB process. Metastable nitrogen was selected as the best MAB gas for the analysis of HAAs. Ionization selectivity is shown by the detection of heterocyclic amines in non-purified chicken meat extracts spiked with HAAs. A quantitative approach is also presented by using pyrograms as chromatograms for quantification purposes. HAAs determination using Py-MAB-ToF was finally performed on cooked chicken breast extracts and compared to an LC-APCI-MS/MS method. Although Py-MAB-ToF sensitivity remains to be improved in the present state of development of our prototype device, only 2 h from the cooking were required to obtain quantitative results in good agreement with HAAs concentrations measured by LC-MS/MS in 36 h. Figure Experimental set-up for pyrolysis-MAB-ToF mass spectrometry experiments.     

High-performance thin-layer chromatography/mass spectrometry for rapid analysis of neutral glycosphingolipids

Direct coupling of high-performance thin-layer chromatography (HPTLC) to matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MS) was shown to be a reliable and reproducible method to obtain structural information and fundamental properties of glycosphingolipids (GSLs). We report a protocol for the preparation of neutral GSL extracts from mouse tissues and demonstrate the applicability of HPTLC/MS to these preparations. The protocol consists of lipid extraction and ion exchange chromatography followed by a mild alkaline treatment and a reversed-phase cartridge extraction. Possible structures for each GSL are proposed based on HPTLC/MS analyses. This fast and simple method can be used to screen neutral GSL extracts obtained from tissues and cells without isolation and purification into individual GSLs.     

Rapid analysis of fungal cultures and dried figs for secondary metabolites by LC/TOF-MS

A liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS) method has been developed for profiling fungal metabolites. The performance of the procedure in terms of mass accuracy, selectivity (specificity) and repeatability was established by spiking aflatoxins, ochratoxins, trichothecenes and other metabolites into blank growth media. After extracting, and carrying out LC/TOF-MS analysis, the standards were correctly identified by searching a specially constructed database of 465 secondary metabolites. To demonstrate the viability of this approach 11 toxigenic and four non-toxigenic fungi from reference collections were grown on various media, for 7-14 days. The method was also applied to two toxigenic fungi, A. flavus (200-138) and A. parasiticus (2999-465) grown on gamma radiation sterilised dried figs, for 7-14 days. The fungal hyphae plus a portion of growth media or portions of dried figs were solvent extracted and analysed by LC/TOF-MS using a rapid resolution microbore LC column. Data processing based on cluster analysis, showed that electrospray ionization (ESI)-TOF-MS could be used to unequivocally identify metabolites in crude extracts. Using the elemental metabolite database, it was demonstrated that from culture collection isolates, anticipated metabolites. The speed and simplicity of the method has meant that levels of these metabolites could be monitored daily in sterilised figs. Over a 14-day period, levels of aflatoxins and kojic acid maximised at 5-6 days, whilst levels of 5-methoxysterigmatocystin remained relatively constant. In addition to the known metabolites expected to be produced by these fungi, roquefortine A, fumagillin, fumigaclavine B, malformins (peptides), aspergillic acid, nigragillin, terrein, terrestric acid and penicillic acid were also identified.     

Detection, characterization, and quantification of resveratrol glycosides in transgenic arabidopsis over-expressing a sorghum stilbene synthase gene by liquid chromatography/tandem mass spectrometry

Transgenic Arabidopsis plants were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) to investigate the glycosylation patterns of resveratrol derived from expression of a sorghum stilbene synthase gene. In negative ionization mode, the different resveratrol derivatives fragmented to yield the diagnostic deprotonated resveratrol ion at m/z 227.2. The use of precursor ion scanning led to the identification of precursor ions for different resveratrol glycosides through rapid differentiation from other phytochemical constituents. Structural information was generated simultaneously from the low-collision-energy product ion spectra using hybrid linear ion-trap mass spectrometry. Three additional resveratrol-related metabolites - a resveratrol diglucoside (M1) and trans- and cis-resveratrol acetylhexosides (M2 and M3) - were detected in the crude plant extracts. The identities of M1, M2, and M3 were confirmed by accurate mass analysis on a quadrupole time-of-flight mass spectrometer as well as beta-glucosidase digestion or UV-induced isomerization. Quantitative analyses by LC/MS in multiple reaction monitoring mode revealed that resveratrol diglucoside and cis-resveratrol acetyhexoside accumulated up to 2.79 and 10.38 microg/g, respectively, while trans-resveratrol acetylhexoside was barely detectable. This study demonstrated the power of the hybrid linear ion-trap technology for simultaneous profiling and structural characterization of stilbene-related metabolites, which would be useful to understand how resveratrol is modified in sorghum and other plants.     

The identification of three human metabolites of a peptide-doxorubicin conjugate using HPLC-MS-MS in positive and negative ionization modes

A peptide-doxorubicin conjugate (I) is a drug candidate that has been evaluated for the treatment of prostate cancer. During the high-performance liquid chromatographic (HPLC)-fluorescence analysis of clinical samples for compound I and its two known metabolites [doxorubicin (II) and leucine-doxorubicin (III)], additional metabolites are observed in postdose human plasma extracts. Using neutral loss, precursor, and product ion tandem mass spectrometric (MS-MS) experiments, two of these metabolites are identified as doxorubicinol (IV) and leucine-doxorubicinol (V), the active 13-hydroxy metabolites of doxorubicin and leucine-doxorubicin, respectively. A third metabolite, 7-deoxydoxorubicinol aglycone (VI), is detected using single-ion monitoring at m/z 399 in the negative ionization mode. The product ion mass spectrum of this metabolite contains a major fragment at m/z 351, resulting from the loss of water and formaldehyde from the pseudomolecular ion. An HPLC-MS-MS method for simultaneous analysis of II, III, IV, V, and VI is developed utilizing gradient HPLC with a combination of positive/negative ionization MS in the multiple reaction monitoring mode and monitoring the appropriate MS-MS transitions. Using this methodology, rat, dog, and human plasma metabolite profiles are compared and found to be qualitatively similar. Simultaneous fluorescence and MS detection experiments confirm that the peaks observed in the HPLC-fluorescence chromatograms of plasma extracts correspond to each of the five metabolites (II-VI).     

Rapid screening for cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. using liquid chromatography/tandem mass spectrometry

A simple and rapid qualitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for screening cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. at sub-ppm levels. An electrospray ionization orbitrap mass spectrometer, which provides accurate full scan MS and MS/MS data, was used in this study. After simple extraction with ethanol and purification by AB-8 resin, the extracts were subjected to LC/MS/MS analysis. A high mass tolerance (10 ppm) was used in the initial screening to filter the full scan MS data. The cyclo-dopa and diketopiperazine alkaloid standards gave limits of detection (LODs) at or below 5 ng/mL. The results also indicated that the method had an acceptable precision for day-to-day use in the identification of compounds. The alkaloids could be identified based on their MS/MS data, elemental compositions, and retention behavior. This system was used to assay trace amounts of cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L., leading to the identification of 5/2 confirmed/unconfirmed cyclo-dopa and 7/6 confirmed/unconfirmed diketopiperazine alkaloids, respectively. The screening method considerably reduces the time and cost involved in the identification of cyclo-dopa and diketopiperazine alkaloids in Portulaca oleracea L., as well as being a simple and convenient approach to the identification of other structural families of natural products.     

Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry

Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI‐MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,‐trimethoxyphenyl)phosphonium acetic acid (TMPP‐AA) to improve liquid chromatography (LC)/ESI‐MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl‐sn‐glycerol, dihydrotachysterol, octadecanol, and alpha‐tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI‐MS instrumentation and demonstrates the potential for targeted metabolomics analysis. Published in 2009 by John Wiley & Sons, Ltd.     

Identification of conjugated metabolites of pyrazolone drugs in human urine by pyrolysis-mass spectrometry

It is shown that a combination of pyrolysis electron ionization and soft ionization mass spectrometry represents a direct approach for the elucidation of the complex biotransformation pattern of two pyrazolone analgesics, antipyrine and dipyrone. In a first screening procedure crude extracts from biological material can readily be utilized. Urinary conjugates of structural hydroxy isomers formed from antipyrine can be differentiated by specific thermolytic reactions of the pyrazolone moiety. As a rule, sulpho conjugates are found to exhibit a lower stability against thermal stress than the corresponding gluco conjugates. Using this integrated mass spectrometric approach, investigation of the phase II metabolism of dipyrone succeeded in identifying five unknown metabolites.     

Phytochemical Profile,Antioxidant Activity,and Cytotoxicity Assessment of Tagetes erecta L. Flowers

Tagetes erecta L. is a popular ornamental plant of the Asteraceae family, which is widely cultivated not only for its decorative use, but also for the extraction of lutein. Besides carotenoid representatives, which have been extensively studied, other important classes of secondary metabolites present in the plant, such as polyphenols, could exhibit important biological activities. The phytochemical analysis of a methanolic extract obtained from T. erecta inflorescences was achieved using liquid chromatography–mass spectrometry (LC-MS) techniques. The extract was further subjected to a multistep purification process, which allowed the separation of different fractions. The total extract and its fractions contain several polyphenolic compounds, such as hydroxybenzoic and hydroxycinnamic acid derivatives, flavonols (especially quercetagetin glycosides), and several aglycons (e.g., quercetin, patuletin). One of the fractions, containing mostly quercetagitrin, was subjected to two different antioxidant assays (metal chelating activity and lipoxygenase inhibition) and to in vitro cytotoxicity assessment. Generally, the biological assays showed promising results for the investigated fraction compared to the initial extract. Given the encouraging outcome of the in vitro assays, further purification and structural analysis of compounds from T. erecta extracts, as well as further in vivo investigations are justified.     

Sub-parts-per-billion determination of nitro-substituted polynuclear aromatic hydrocarbons in airborne particulate matter and soil by electron capture-Tandem mass spectrometry

A procedure for the determination of nitro-substituted polynuclear aromatic hydrocarbons (nitro-PAH) on crude air-particulate and soil extracts is introduced. Elimination of purification and fractionation procedures was made possible by the use of both a selective ionization method, such as electron-capture chemical ionization, and a specific fragmentation process, in an experiment of tandem mass spectrometry (gas chromatography-electron capture tandem mass spectrometry). Different mass spectrometric procedures were compared. The best performance was observed when the nitro-PAH molecular ions [M]⁻ were mass-selected by the first analyzer under multiple reaction monitoring conditions and then fragmented to NO⁻₂ (m/z 46). Detection limits were on the order of hundreds of femtograms, as determined in extracts of real environmental samples. This corresponds approximately to 5-15 pg of nitro-PAH per cubic meter of air sampled. Calibration curves were linear over 3 orders of magnitude. Applications to contamination from motor vehicle combustion and the iron industry are briefly discussed.     

Identification of new phase II metabolites of xanthohumol in rat in vivo biotransformation of hop extracts using high-performance liquid chromatography electrospray ionization tandem mass spectrometry

Polyphenolic compounds occurring in hop extracts and their phases I and II metabolites formed during in vivo rat biotransformation have been analyzed using HPLC/MS/MS with electrospray ionization (ESI). Two main groups of polyphenolics are present in the hops, i.e., xanthohumol related compounds and so called α- and β-bitter acids (humulones and lupulones). In our study, hybrid quadrupole-time-of-flight (QqTOF) analyzer is used for the identification of both natural phenolics and their metabolites due to the possibility of accurate mass measurements in full scan and tandem mass spectra supported by MSⁿ data obtained with the ion trap analyzer. Both ESI polarity modes are used for the determination of molecular weights based on [M+H]⁺ and [M−H]⁻ ions in the full scan spectra and the structural information in subsequent tandem mass spectra. The emphasis is given on the elemental composition determination of individual metabolites based on accurate masses typically better than 5 ppm even with the external calibration. Advanced software tools are used for the metabolite identification using the comparison of the blank chromatogram with the real incubation sample together with the software prediction and detection of possible metabolites. Chromatograms of rat incubations are also compared with chromatograms of pure rat feed, rat feed enriched with hop extracts and the placebo experiment. More than ten compounds originating from the hops are identified in rat feces, two of them belong to phase I metabolites and five compounds are phase II metabolites.     

Liquid chromatography–mass spectrometry for analysis of a novel β2‐adrenoceptor agonist trantinterol and its metabolites in beagle dog urine

A liquid chromatography–tandem mass spectrometry method was developed for the identification of metabolites of trantinterol, a novel β₂‐adrenoceptor agonist, in beagle dog urine. The separation of metabolites was performed on a reversed‐phase C₈ column using 0.1% formic acid in water and methanol (70 : 30, v/v) as the mobile phase. The structural information and elemental information of metabolites were acquired by an electrospray ionization tandem mass spectrometer and a quadrupole time‐of‐flight mass spectrometer, respectively. A total of 13 metabolites were detected and characterized on the basis of their tandem MS/MS fragmentation patterns. The accurate masses of nine metabolites were determined and two metabolites were further confirmed by comparing with reference standards. The metabolic pathways of trantinterol in beagle dog are proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

基于液相色谱-质谱技术的肾脏代谢组学分析方法研究

建立了一种基于高效液相色谱-超高分辨质谱技术的肾脏代谢组学分析方法。肾脏组织于液氮中研磨成粉后,采用甲基叔丁基醚/甲醇/水溶剂体系进行提取,分别得到强极性部分(下层)和弱极性部分(上层)提取物,依次采用HILIC亲水色谱柱和反相C18色谱柱进行梯度洗脱分离后,进行高分辨质谱分析。采用电喷雾离子源(ESI),正、负离子模式检测,扫描方式为全扫描,扫描范围为100~1000 Da,质量分辨率为120000。结果表明,该方法灵敏度高,专属性强,稳定性良好,可同时获取肾脏组织中的强极性和弱极性代谢物信息,可为肾脏疾病和药物肾毒性生物标志物的发现提供一种新的方法。     

Comprehensive multiresidue method for the simultaneous determination of 74 pesticides and metabolites in traditional Chinese herbal medicines by accelerated solvent extraction with high-performance liquid chromatography/tandem mass spectrometry

In this paper, a multiresidue method for the simultaneous target analysis of 74 pesticides and metabolites in traditional Chinese herbal medicines (TCHMs) was developed using accelerated solvent extraction (ASE) coupled with HPLC/MS/MS. Pesticide residues were extracted from the different samples using ASE, then purified by gel permeation chromatography and graphitized carbon black/primary, secondary amine SPE. Gradient elution was used in conjunction with positive mode electrospray ionization MS/MS to detect 74 pesticides and metabolites from Cortex Cinnamomi, Flos Carthami, Folium Ginkgo, Herba Pogostemonis, Radix Ginseng, and Semen Ginkgo using a single chromatographic run. The analytical performance was demonstrated by the analysis of extracts spiked at three concentration levels ranging from 0.005 to 0.125 mg/kg for each pesticide and metabolite. In general, recoveries ranging from 70 to 110%, with RSDs better than 15%, were obtained. The recovery and repeatability data were in good accordance with European Union guidelines for pesticide residue analysis. The LOD for most of the targeted pesticides and metabolites tested was below 0.01 mg/kg.     

Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

Structural investigation and elucidation of new communesins from a marine‐derived Penicillium expansum Link by liquid chromatography/electrospray ionization mass spectrometry

Penicillium expansum is a ubiquitous species for which there are only few reports for chemical investigation in marine environments. Among the numerous secondary metabolites produced by this species, communesins represent a new class of cytotoxic and insecticidal indole alkaloids. In this study, we investigated a marine P. expansum strain exhibiting neuroactivity on a Diptera larvae bioassay. Bio‐guided purification led to the isolation and the identification of communesin B as the main active compound by HRMS and ¹H and ¹³C NMR. Liquid chromatography analyses with detection by electrospray ionization coupled with tandem mass spectrometry (LC/ESI‐MS/MS) and high‐resolution tandem mass spectrometry (LC/HRMS/MS) allowed the identification and characterization of four other known communesins (A, D, E and F) in the crude extract. A fragmentation model for dimethyl epoxide communesins was proposed after detailed interpretation of their MS/MS spectra. Further analyses of the extract using the modelled fragmentations led to the detection of seven new communesins found as minor compounds. Chemical structural elucidation of these new derivatives is discussed based on their fragmentation characteristics. Copyright © 2009 John Wiley & Sons, Ltd.     

Use of the bromine isotope ratio in HPLC-ICP-MS and HPLC-ESI-MS analysis of a new drug in development

A combination of inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization mass spectrometry (ESI-MS) was deployed for the metabolite profiling and metabolite identification of a new antituberculosis compound (R207910, also known as TMC207) that is currently in drug development. R207910 contains one bromine atom, allowing the detection by ICP-MS. Fluctuations in the Br sensitivity caused by the HPLC gradient were counteracted by the use of species-unspecific isotope dilution. In order to evaluate the method developed, the results obtained were compared with those acquired via radioactivity detection. HPLC-ESI-MS was used for the structural identification of R207910 and its metabolites. The ⁷⁹Br/⁸¹Br isotope ratio is also valuable in the search for metabolites in the complex background of endogenous compounds obtained using HPLC-ESI-MS analyses. Data-dependent scanning using isotope recognition with an ion trap mass spectrometer or processing of Q-Tof data provides HPLC-ICP-MS-like “bromatograms”. The combination of accurate mass measurements and the fragmentation behavior in the MS² spectra obtained using the Q-Tof Ultima mass spectrometer or MSⁿ spectra acquired using the LTQ-Orbitrap allowed structural characterization of the main metabolites of R207910 in methanolic dog and rat faeces extracts taken 0–24 h post-dose. Figure Analyses of a rat faeces extract taken 0–24 h post-dose: a HPLC-ICP-MS using isotope dilution, b corresponding Br mass flow chromatogram, c radio-HPLC, d Q-Tof ESI-MS TIC, e Q-Tof ESI-MS bromatogram after Br stripping, f LTQ-Orbitrap ESI-MS² TIC obtained with isotopic-data-dependent scanning     

Homolog separation, a necessity for the proper identification of fungal metabolites

Monitoring of fungal extracts for the production of novel metabolites, using a modular analytical system combining HPLC with UV-MS-ELS detection, identified culture LL-W1278 as a fungus producing new biopolymers. Only a non-routine HPLC analysis of a culture extract revealed that the standard water-acetonitrile elution method did not separate all members of the metabolite complex. Fine-tuning the eluting solvents established that it was essential to include acid with the water-methanol system to separate the new materials. The routinely used water-acetonitrile system, with or without acid, was incapable of separating all homologues. With the modified method the new homologues W1278-Ax, Bx, and Cx were separated. LC/MS analysis indicated that these compounds had molecular weights of 706, 900, and 1094, respectively, 44 mass units lower than their three major homologues, W1278-A, B, and C, identified previously. UV and NMR data as well as mass fragmentation patterns established unambiguously that the new compounds lacked a carboxyl group at the terminal resorcinol unit of the biopolymer, consisting of several catenated hydroxymellein residues. A time study concerning the stability of these fungal metabolites showed a slow, but complete degradation of the primary metabolites over several months when kept as a DMSO solution.     

液相色谱-飞行时间质谱同时测定粮食中13种真菌毒素

建立了液相色谱-飞行时间质谱(LC-TOF MS)联用技术同时检测小麦和玉米中镰刀菌、曲霉菌和青霉菌产生的13种真菌毒素的分析方法。样品经乙腈-水-乙酸(84∶15∶1,体积比)混合溶剂提取,My-cosep 226多功能净化柱和强阴离子交换柱净化后,采用LC-TOF MS检测。在电喷雾正离子模式下,以保留时间和化合物精确分子离子质量对真菌毒素进行识别,以10 ppm为提取离子窗口进行定量。结果表明,13种真菌毒素在一定的线性范围内线性关系良好,相关系数均大于0.99,质量精确度均小于5 ppm,回收率为70%～113%,相对标准偏差为0.2%～14.5%。该方法可用于粮食中多种真菌毒素的同时测定。