应用超高效液相色谱-四极杆-飞行时间质谱法建立谷物中18种真菌毒素非靶向筛查数据库及确证方法

建立了基于超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF/MS)的18种真菌毒素非靶向筛查方法。真菌毒素标准物质用HSS T3色谱柱进行色谱分离后在UPLC-Q-TOF/MS MS^E模式下分别用正、负离子模式采集,获取MS和MS/MS的信息,记录对应保留时间、加合物离子、碎片离子精确质量数等信息,设置保留时间偏移为0.3 min,加合物离子和碎片离子的精确质量匹配容差为5×10^-6,在UNIFI中建立18种真菌毒素的数据库。在稻谷、小麦基质中,以筛查检出限(SDL)作为主要参数对筛查方法进行了验证。18种真菌毒素分为有最大限量和无最大限量两种类型,结果有最大限量的真菌毒素均能在其限量水平被准确筛查,无最大限量的真菌毒素其SDL的范围为2～800μg/kg。基质效应考察表明,稻谷中有14种真菌毒素有中等基质效应,小麦中有11种真菌毒素有中等基质效应。样品经乙腈提取后用QuEChERS萃取盐包和HLB净化柱净化,用建立的方法对25批稻谷、小麦进行筛查,结果2批稻谷中检出4种真菌毒素,2批小麦中检出2种真菌毒素。该方法能准确筛查SDL水...     

Thermospray high performance liquid chromatographic/mass spectrometric analysis of some fusarium mycotoxins

Thermospray high performance liquid chromatography/mass spectrometry (TSP HPLC/MS) was used to analyze five Fusarium mycotoxins in porcine plasma and urine. Four cytotoxic trichothecene mycotoxins, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), T2 tetraol, and the fungal estrogen zearalenone (F-2 toxin) were analyzed. The thermospray mass spectrum contained molecular weight information with few, if any, fragment signals. Detection limits ranging from 1 to 10 ng of mycotoxin injected onto the HPLC column were obtained using selected ion monitoring (SIM) HPLC/MS. Neither the plasma nor the urine matrix interfered with TSP HPLC/MS analysis of these mycotoxins and no sample derivatization was necessary for the analysis. The TSP HPLC/MS technique appears to be ideal for very sensitive analysis of mycotoxins in biological samples.     

超高效液相色谱串联质谱法测定花生、粮油中18种真菌毒素

采用同位素稀释法并结合凝胶色谱净化技术,建立了花生、粮油中18种常见真菌毒素污染的超高效液相色谱串联质谱分析方法.样品中添加同位素内标U-[13C17]-黄曲霉毒素 B1和U-[13C15]-脱氧雪腐镰刀菌烯醇,经乙腈-水溶液(84:16,体积比)均质提取,凝胶渗透色谱净化,Waters ACQUITY UPLCTM ...     

The use of tandem mass spectrometry for the differentiation of bile acid isomers and for the identification of bile acids in biological extracts

Tandem mass spectrometric (MS/MS) techniques hae been widely used for the differentiation of isomeric compounds, since their spectra may show differences sufficient to distinguish between them. There are several different ways by which the MS/MS data can be obtained depending on the energies of the ions and the collisions. In this paper MS/MS spectra have been obtained for a group of isomeric bile acids using: 1, low-energy ions and low-energy collisions in a triple quadrupole mass spectrometer by liquid chromatography/MS/MS; 2, high-energy ions and low-energy collisions in a hybrid mass spectrometer by fast-atom bombardment MS/MS. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) has also been used to identify the bile acids present in biological matrices such as bile extracts.     

Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology

A standardised LC-UV-MS micro-scale method for screening of fungal metabolites and mycotoxins in culture extracts is presented. The paper includes data for detection and dereplication of > 400 fungal metabolites to facilitate detection and identification when standards are not available. The data also shows the types of components that can be analysed by positive electrospray (ESI+) mass spectrometry (MS) along with common fragments and adducts of these, as well as giving suggestions on whether UV or ESI+-MS methods should be used. Examples of dereplication of penitrems and macro-cyclic ichothecenes, and detection of several novel compounds are shown. This was done by UV spectroscopy combined with accurate mass determination of adduct and fragment ions obtained by high-resolution orthogonal time-of-flight MS.     

Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography-atmospheric pressure ionisation mass spectrometry

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that are growing on agricultural commodities. Their frequent presence in food and their severe toxic, carcinogenic and estrogenic properties have been recognised as potential threat to human health. A reliable risk assessment of mycotoxin contamination for humans and animals relies basically on their unambiguous identification and accurate quantification in food and feedstuff. While most screening methods for mycotoxins are based on immunoassays, unambiguous analyte confirmation can be easily achieved with mass spectrometric methods, like gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS). Due to the introduction of atmospheric pressure ionisation (API) techniques in the late 80s, LC/MS has become a routine technique also in food analysis, overcoming the traditional drawbacks of GC/MS regarding volatility and thermal stability. During the last few years, this technical and instrumental progress had also an increasing impact on the expanding field of mycotoxin analysis. The aim of the present review is to give an overview on the application of LC-(API)MS in the analysis of frequently occurring and highly toxic mycotoxins, such as trichothecenes, ochratoxins, zearalenone, fumonisins, aflatoxins, enniatins, moniliformin and several other mycotoxins. This includes also the investigation of some of their metabolites and degradation products. Suitable sample pre-treatment procedures, their applicability for high sample through-put and their influence on matrix effects will be discussed. The review covers literature published until July 2006.     

Detection of oxidative species for 4-phenoxyphenol derivatives during the electrospray ionization process

Analyses by flow injection as well as liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were performed with four 4-phenoxyphenol derivatives. When ambient temperature nitrogen gas was used to facilitate solvent evaporation, [M + H]+, [M + NH4]+, and [2M + NH4]+ ions were observed as the major ions. As the nitrogen gas temperature increased from ambient to 250 and 450 degrees C, [M]+*, [M - 1]+ and [M + 15]+ ions were the predominant ions. Heat-induced oxidation was found to be the primary source for the formation of oxidative species. Aqueous solvents were found to be essential for the formation of the [M + 15]+ ions. The [M]+* and [M + 15]+ ions were further characterized by tandem mass spectrometry. Based on the MS/MS data, it was proposed that the [M + 15]+ ions were the in-source generated 1,2-quinone ions.     

Development and in-house validation of a robust and sensitive solid-phase extraction liquid chromatography/tandem mass spectrometry method for the quantitative determination of aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods

A sensitive and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods. Samples were extracted with a mixture of acetonitrile/water (84:16, v/v) and cleaned up through a polymeric solid-phase extraction column. Detection and quantification of the nine mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS), using fully (13)C-isotope-labelled mycotoxins as internal standards. The method was validated in-house for five different cereal processed products, namely barley, oat and durum wheat flours, rye- and wheat-based crisp bread. Recoveries and repeatability of the whole analytical procedure were evaluated at contamination levels encompassing the EU maximum permitted levels for each tested mycotoxin. Recoveries ranged from 89 to 108% for deoxynivalenol, from 73 to 114% for aflatoxins, from 85 to 114% for T-2 and HT-2 toxins, from 64 to 97% for zearalenone, from 74 to 102% for ochratoxin A. Relative standard deviations were less than 16% for all tested mycotoxins and matrices. Limits of detection (signal-to-noise ratio 3:1) ranged from 0.1 to 59.2 μg/kg. The trueness of the results obtained by the proposed method was demonstrated by analysis of reference materials for aflatoxins, deoxynivalenol, zearalenone. The use of inexpensive clean-up cartridges and the increasing availability of less expensive LC/MS/MS instrumentation strengthen the potential of the proposed method for its effective application for reliable routine analysis to assess compliance of tested cereal products with current regulation.     

The unanticipated loss of SO2 from sulfonamides in collision-induced dissociation

A potent and selective sulfonamide beta3 agonist with an excellent pharmacokinetic profile has recently been synthesized. During the analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of metabolites of the sulfonamide N-[4-[2-(2-hydroxy-2-pyridin-3-ylethylamino)ethyl]phenyl]-4-[4-(4-trifluoromethylphenyl)thiazol-2-yl]benzulfonamide (compound A), we observed loss of 64 Da for a few of the metabolites in the negative ion mode. Accurate mass measurements performed with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and quadrupole time-of-flight (Q-TOF) mass spectrometry suggested that the loss of 64 Da corresponded to the loss of SO(2). The same phenomenon was observed for a group of structurally related and commercially available compounds that also contain a sulfonamide moiety. MS/MS analysis of the fragment ions that had lost SO(2) in the ion source suggested that these ions were covalently bound rather than ion-molecule complexes. The neutral loss involving the cleavage of two bonds was unanticipated and suggested a complex rearrangement process. A mechanism for the loss of SO(2) has been proposed.     

Simultaneous determination of multi-component mycotoxin contaminants in foods and feeds by ultra-performance liquid chromatography tandem mass spectrometry

The present study developed an improved analytical method for the simultaneous quantification of 17 kinds of Aspergillus, Fusarium and Penicillium mycotoxin contaminants in foods and feeds by ultra-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UPLC-MS/MS) under the multiple reaction monitoring (MRM) mode, and especially focused on the optimization of extraction, clean-up, UPLC separation and MS/MS parameters of analytes. Homogenized samples were sequentially extracted by 84% (v/v) of acetonitrile aqueous solution with the selected internal standard (zearalanone) spiking, SPE clean-up with Mycosep 226 Aflazon+ Multifunctional cartridges, filtration, concentration and secondary filtration. Using double sample injection method, the analytes were separated by UPLC BEH C18 column (100 mm x 2.1 mm I.D., 1.7 microm), and eluted with ammonium acetate/methanol and aqueous ammonia/methanol for the ESI+ and ESI- analysis, respectively. The 10 positive ions and 7 negative ions of mycotoxins were separated by gradient elution with the retention time of 6.5 and 4 min, respectively. The LOQ of selected analytes ranged from 0.01 to 0.70 microg kg(-1), which was lower than the criteria of EU, USA and other countries on the determination of the minimum limiting level of various mycotoxins in foods including baby foods and feed stuffs. Meanwhile, high correlation coefficients (r2>0.99) of 17 mycotoxins were obtained within their respective linear ranges (0.05-20 microg kg(-1) for 10 positive ions and 0.5-50 microg kg(-1) for 7 negative ions) and reasonable recoveries (70.6-119.0%) of them were also demonstrated in different spiked levels. This quantitative method has many advantages including simple pretreatment, rapid determination and high sensitivity, which could be applied to the determination and quantification of multi-component mycotoxin contaminants in complex matrixes. Meanwhile, the method successfully fulfilled the minimum limiting level requests from various countries.     

Determination of polychlorodibenzo-p-dioxins and polychlorodibenzofurans by gas chromatography/tandem mass spectrometry and gas chromatography/triple mass spectrometry in a quadrupole ion trap

The determination of tetra- to octachlorodibenzo-p-dioxins and tetra- to octachlorodibenzofurans (PCCD/Fs) by high-resolution gas chromatography/tandem mass spectrometry (HRGC/MS/MS) and high-resolution gas chromatography/triple mass spectrometry (HRGC/MS(3)) in a quadrupole ion trap, equipped with an external ion source, is presented. MS/MS involves a typical four-step process, namely ionization, parent ion isolation, collision-induced dissociation (CID) and mass analysis of the daughter ions. For the MS(3) experiment, the MS/MS scan function is used with the addition of selected daughter ion isolation, their CID and the mass analysis of second-generation product ions called 'grand-daughter ions.' For both methods, the energies necessary for the CID of the 17 PCDD/Fs were determined and optimized using multiple scan functions with different CID amplitudes. The CID efficiency, defined as the signal ratio of fragment ions detected from the major dissociation channels to molecular ions isolated, was 1.15-2.40 V for parent ion dissociation (MS/MS) and 1.05-1.50 V for daughter ion dissociation (MS(3)) and for all the chloro congeners. The same sensitivity (1 pg microl(-1)) can be reached with both the MS/MS and MS(3) methods and linear responses were obtained between 1 and 100 pg microl(-1) injected.     

Generation of average mass values and end group information of polymers by means of a combination of matrix-assisted laser desorption/ionization-mass spectrometry and liquid secondary ion-tandem mass spectrometry

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and liquid secondary ion-tandem mass spectrometry (LSI-MS/MS) have been applied to the analysis of synthetic polymers to generate values for the average mass and the mass of the end groups. The average mass values were calculated for polymethylmethacrylate and polystyrene standards from the MALDI-MS data. Abundant fragment ions of the polymers, generated by means of LSI-MS/MS, were consistent with the known structures of the end groups of the polymers. Furthermore, losses from the side chains of the polymers were also observed in the LSI-MS/MS spectra.     

表面解吸常压化学电离质谱快速分析六味地黄丸

采用新型表面解吸常压化学电离(Surface Desorption Atmospheric Pressure Chemical Ionization, SDAPCI)质谱法, 在敞开环境下, 对潮湿的空气进行电晕放电产生试剂离子, 进而在六味地黄丸表面发生解吸电离过程, 在无需复杂预处理的前提下对六味地黄丸中的待测物进行离子化, 从而获得了六味地黄丸在正负离子模式下的化学指纹图谱, 并利用主成分分析法对质谱指纹数据进行处理, 可对6个厂家生产的多个批次产品进行较好的区分. 结果表明, SDAPCI-MS技术能够快速测定六味地黄丸的剂型和生产厂家信息, 并能够对目标组分做多级串联质谱鉴定, 发现痕量目标组分. 研究方法可望应用于中成药药品生产质量控制和成品检测等领域.     

Use of doubly protonated molecules in the analysis of cathedulins in crude extracts of khat (Catha edulis) by liquid chromatography/serial mass spectrometry

Analysis of crude methanolic extracts of fresh khat (Catha edulis) by liquid chromatography/mass spectrometry (LC/MS) revealed the presence of 62 cathedulin alkaloids (compared with 15 published structures). Many cathedulins generated doubly protonated molecules following electrospray ionisation and the ratio of doubly to singly protonated species could be manipulated by adjusting the electrospray capillary position and source conditions. By selecting the doubly protonated species for serial mass spectrometric analysis (MS/MS), it was possible to use an ion trap mass spectrometer to observe singly charged product ions at lower m/z values than ion trap MS/MS analysis of [M+H](+) would have allowed. These spectra were particularly valuable in elucidating the acylation patterns of cathedulins where MS/MS analysis of [M+H](+) resulted in loss of a large neutral species to yield a small singly charged fragment below the lower limit for ion trapping. Acylation patterns for most of the 62 cathedulins are proposed from mass spectrometric analysis, and the data obtained for a major unreported cathedulin of mass 1001 Da suggest that it belongs to a new group of cathedulins having a cathate dilactone bridge but not an evoninate bridge.     

On-Line HPLC-UV-mass spectrometry and tandem mass spectrometry for the rapid delineation and characterization of differences in complex mixtures: a case study using toxic oil variants

An integrated differential approach to the characterization of complex mixtures is presented which includes the targeting of liquid chromatography (LC) peaks for identification using characteristic UV adsorption of the LC peak, subsequent molecular weight and formula determination using accurate mass LC mass spectrometry (MS), and structure characterization using accurate mass LC-tandem mass spectrometry. The use of differential UV adsorption aids in narrowing the scope of the study to only specific peaks of interest. Accurate mass measurement of the molecular ion species provides molecular weight information as well as atomic composition information. The tandem MS (MS/MS) spectra provide fragmentation information which allows for structural characterization of each component. Accurate mass assignment of each of the fragment ions in the MS/MS spectrum provides atomic composition for each of the fragment ions and thus further aids in the structural characterization. These experiments are facilitated through the use of on-line LC-MS and LC-MS/MS with in-line UV detection. A synthetic toxic oil (STO) related to Toxic Oil Syndrome is studied with a focus on possible contaminants resulting from the interaction of aniline, used as a denaturant, with the normal components of the oil. A differential analysis between the STO and a control oil is performed. LC peaks were targeted using UV absorbance to indicate the possible presence of the aniline moiety. Further differential analysis was performed through the determination of the MS signals associated with each component separated on the LC. Finally, the MS/MS data was also used to determine if the fragmentation of the targeted components indicated the presence of aniline. The MS/MS and accurate mass data were used to assign the structures for the targeted components.     

Dual parallel electrospray ionization and atmospheric pressure chemical ionization mass spectrometry (MS), MS/MS and MS/MS/MS for the analysis of triacylglycerols and triacylglycerol oxidation products

Two mass spectrometers, in parallel, were employed simultaneously for analysis of triacylglycerols in canola oil, for analysis of triolein oxidation products, and for analysis of triacylglycerol positional isomers separated using reversed-phase high-performance liquid chromatography. A triple quadrupole mass spectrometer was interfaced via an atmospheric pressure chemical ionization (APCI) interface to two reversed-phase liquid chromatographic columns in series. An ion trap mass spectrometer was coupled to the same two columns using an electrospray ionization (ESI) interface, with ammonium formate added as electrolyte. Electrospray ionization mass spectrometry (ESI-MS) under these conditions produced abundant ammonium adduct ions from triacylglycerols, which were then fragmented to produce MS/MS spectra and then fragmented further to produce MS/MS/MS spectra. ESI-MS/MS of the ammoniated adduct ions gave product ion mass spectra which were similar to mass spectra obtained by APCI-MS. ESI-MS/MS produced diacylglycerol fragment ions, and additional fragmentation (MS/MS/MS) produced [RCO](+) (acylium) ions, [RCOO+58](+) ions, and other related ions which allowed assignment of individual acyl chain identities. APCI-MS of triacylglycerol oxidation products produced spectra like those reported previously using APCI-MS. APCI-MS/MS produced ions related to individual fatty acid chains. ESI-MS of triacylglycerol oxidation products produced abundant ammonium adduct ions, even for those molecules which previously produced little or no intact molecular ions under APCI-MS conditions. Fragmentation (MS/MS) of the [M+NH(4)](+) ions produced results similar to those obtained by APCI-MS. Further fragmentation (MS/MS/MS) of the diacylglycerol fragments of oxidation products provided information on the oxidized individual fatty acyl chains. ESI-MS and APCI-MS were found to be complementary techniques, which together contributed to a better understanding of the identities of the products formed by oxidation of triacylglycerols.     

Electrospray ionization mass spectrometry of AZT H-phosphonates conjugated with steroids

AZT H-phosphonates conjugated with steroids were synthesized and determined by positive and negative ion electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). The fragmentation pathways were investigated in detail. There are very different characteristic fragment ions in the positive and negative ion MS/MS spectra. The azide group of compounds 6a and 6b underwent either elimination of HN(3) or rearrangement to an amine in both positive and negative ion mass spectrometry.     

Evaluation of automated single mass spectrometry to tandem mass spectrometry function switching for comprehensive drug profiling analysis using a quadrupole time-of-flight mass spectrometer

A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.     

Simultaneous determination of aflatoxins, ochratoxin A and Fusarium toxins in maize by liquid chromatography/tandem mass spectrometry after multitoxin immunoaffinity cleanup

A liquid chromatography/tandem mass spectrometry method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, fumonisins (B(1), B(2)), deoxynivalenol, zearalenone, T-2 and HT-2 toxins in maize. A double extraction approach, using a phosphate-buffered solution followed by methanol, was applied to achieve effective co-extraction of the 11 mycotoxins under investigation having quite different polarities and chemical structures. A new multitoxin immunoaffinity column containing antibodies for all these mycotoxins was used to clean up the extract. Detection and quantification of the 11 mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS) using, as chromatographic mobile phase, a linear gradient of methanol/water containing 0.5% acetic acid and 1 mM ammonium acetate. Method performances were quite satisfactory for all tested mycotoxins at contamination levels close to or below the relevant EU maximum permitted or recommended levels. Limits of detection in maize ranged from 0.3 to 4.2 microg/kg. Recoveries higher than 79% were obtained for all tested mycotoxins with relative standard deviations less than 13%.     

Identification of isobaric product ions in electrospray ionization mass spectra of fentanyl using multistage mass spectrometry and deuterium labeling

Isobaric product ions cannot be differentiated by exact mass determinations, although in some cases deuterium labeling can provide useful structural information for identifying isobaric ions. Proposed fragmentation pathways of fentanyl were investigated by electrospray ionization ion trap mass spectrometry coupled with deuterium labeling experiments and spectra of regiospecific deuterium labeled analogs. The major product ion of fentanyl under tandem mass spectrometry (MS/MS) conditions (m/z 188) was accounted for by a neutral loss of N‐phenylpropanamide. 1‐(2‐Phenylethyl)‐1,2,3,6‐tetrahydropyridine (1) was proposed as the structure of the product ion. However, further fragmentation (MS³) of the fentanyl m/z 188 ion gave product ions that were different from the product ion in the MS/MS fragmentation of synthesized 1, suggesting that the m/z 188 product ion from fentanyl includes an isobaric structure different from the structure of 1. MS/MS fragmentation of fentanyl in deuterium oxide moved one of the isobars to 1 Da higher mass, and left the other isobar unchanged in mass. Multistage mass spectral data from deuterium‐labeled proposed isobaric structures provided support for two fragmentation pathways. The results illustrate the utility of multistage mass spectrometry and deuterium labeling in structural assignment of isobaric product ions. Copyright © 2010 John Wiley & Sons, Ltd.