实时直接分析质谱新技术及其应用

由于不需要复杂的样品前处理过程,并具有实时、原位分析等特点,常压敞开式离子化技术成为近年来质谱研究的热点之一.实时直接分析(direct analysis in real time,DART)是近年来出现的一种常压敞开式离子化新技术,在食品安全、环境监测、爆炸物检测以及生物医药等诸多研究领域均有广泛的应用.本文简单介绍了常压敞开式离子化方法的发展,DART的基本原理和研究现状.进而介绍了我国质谱研究人员在基于DART的质谱仪器改进、联用技术以及生物药物等检测分析方面取得的新进展和新成果.     

The coupling of direct analysis in real time ionization to Fourier transform ion cyclotron resonance mass spectrometry for ultrahigh‐resolution mass analysis

Direct Analysis in Real Time (DART) is an ambient ionization technique for mass spectrometry that provides rapid and sensitive analyses with little or no sample preparation. DART has been reported primarily for mass analyzers of low to moderate resolving power such as quadrupole ion traps and time‐of‐flight (TOF) mass spectrometers. In the current work, a custom‐built DART source has been successfully coupled to two different Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometers for the first time. Comparison of spectra of the isobaric compounds, diisopropyl methylphosphonate and theophylline, acquired by 4.7 T FT‐ICR MS and TOF MS, demonstrates that the TOF resolving power can be insufficient for compositionally complex samples. 9.4 T FT‐ICR MS yielded the highest mass resolving power yet reported with DART ionization for 1,2‐benzanthracene and 9,10‐diphenylanthracene. Polycyclic aromatic hydrocarbons exhibit a spatial dependence in ionization mechanisms between the DART source and the mass spectrometer. The feasibility of analyzing a variety of samples was established with the introduction and analysis of food products and crude oil samples. DART FT‐ICR MS provides complex sample analysis that is rapid, highly selective and information‐rich, but limited to relatively low‐mass analytes. Copyright © 2010 John Wiley & Sons, Ltd.     

DART-Orbitrap MS: a novel mass spectrometric approach for the identification of phenolic compounds in propolis

This is the first direct analysis in real-time mass spectrometry (DART-MS) study of propolis and a first study on the analysis of bee products using high-resolution DART-MS (DART-HRMS). Identification of flavonoids and other phenolic compounds in propolis using direct analysis in real-time coupling with Orbitrap mass spectrometry (DART-Orbitrap MS) was performed in the negative ion mode for minimizing the matrix effects, while the positive ion mode was used for the confirmation of selected compounds. Possible elemental formulae were suggested for marker components. The duration of one sample analysis by DART-MS analysis lasted ca. 30 s, and all benefits of high-resolution mass spectrometry were used upon data processing using the coupling of DART with the Orbitrap mass spectrometer. The possibility for scanning analysis of dried propolis extract spots on a planar porous surface was investigated in the heated gas flow of the DART ion source with adjustable angle. As an independent method, the approach of scanning analysis is of high interest and of future potential for confirmation of the results obtained from liquid sample analysis. Scanning analysis is highly promising for further development in the bioanalytical field due to the convenience of the storage and transportation of dried sample spots.     

Surface swabbing technique for the rapid screening for pesticides using ambient pressure desorption ionization with high-resolution mass spectrometry

A rapid screening method for pesticides has been developed to promote more efficient processing of produce entering the United States. Foam swabs were used to recover a multiclass mixture of 132 pesticides from the surfaces of grapes, apples, and oranges. The swabs were analyzed using direct analysis in real time (DART) ionization coupled with a high‐resolution Exactive Orbitrap? mass spectrometer. By using a DART helium temperature gradient from 100–350°C over 3 min, a minimal separation of analytes based on volatility differences was achieved. This, combined with the Exactive's mass resolution of 100 000, allowed the chromatographic step, along with the typical compositing and extraction steps associated with gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS) approaches, to be eliminated. Detection of 86% of the analytes present was consistently achieved at levels of 2 ng/g (per each apple or orange) and 10 ng/g (per grape). A resolution study was conducted with four pairs of isobaric compounds analyzed at a mass resolution of 100 000. Baseline separation was achieved with analyte ions differing in mass by 25 ppm and analyte ions with a mass difference of 10 ppm were partially resolved. In addition, field samples that had undergone traditional sample preparation using QuEChERS (quick, easy, cheap, rugged, and safe) were analyzed using both LC/MS and DART‐MS and the results from the two techniques were found to be comparable in terms of identification of the pesticides present. The use of swabs greatly increased sample throughput by reducing sample preparation and analysis time. Published in 2010 by John Wiley & Sons, Ltd.     

Some new features of Direct Analysis in Real Time mass spectrometry utilizing the desorption at an angle option

The present study is a first step towards the unexplored capabilities of Direct Analysis in Real Time (DART) mass spectrometry (MS) arising from the possibility of the desorption at an angle: scanning analysis of surfaces, including the coupling of thin‐layer chromatography (TLC) with DART‐MS, and a more sensitive analysis due to the preliminary concentration of analytes dissolved in large volumes of liquids on glass surfaces. In order to select the most favorable conditions for DART‐MS analysis, proper positioning of samples is important. Therefore, a simple and cheap technique for the visualization of the impact region of the DART gas stream onto a substrate was developed. A filter paper or TLC plate, previously loaded with the analyte, was immersed in a derivatization solution. On this substrate, owing to the impact of the hot DART gas, reaction of the analyte to a colored product occurred. An improved capability of detection of DART‐MS for the analysis of liquids was demonstrated by applying large volumes of model solutions of coumaphos into small glass vessels and drying these solutions prior to DART‐MS analysis under ambient conditions. This allowed the introduction of, by up to more than two orders of magnitude, increased quantities of analyte compared with the conventional DART‐MS analysis of liquids. Through this improved detectability, the capabilities of DART‐MS in trace analysis could be strengthened. Copyright © 2011 John Wiley & Sons, Ltd.     

Increasing the rate of sample vaporization in an open air desorption ionization source by using a heated metal screen as a sample holder

Rapid vaporization of sample into the ionizing gas exiting a direct analysis in real time (DART®) source has been enabled by directing a high electrical current through a metal wire screen to which sample has been applied. This direct heating of the screen enables rapid vaporization of sample as the wire temperature rises from room temperature to greater than 400°C in less than 20 s. Positioning the screen between the DART source and atmospheric pressure inlet of the mass spectrometer ensures that the ionizing gas is in close proximity to the sample molecules, resulting in efficient ionization while significantly reducing the time required for mass spectrometric analysis. The capability to modulate the electrical current flow through the wires facilitates either rapid desorption for the determination of single component samples or slower desorption where analysis of mixtures might be desired. The technology also enables deployment of strategies for the determination of chemicals present as powders that might otherwise require dissolution prior to analysis. Results from the use of this thermally assisted DART (‘TA‐DART’) system for the analysis of pure compounds, simple mixtures, solids and low vapor pressure samples are presented. Copyright © 2011 John Wiley & Sons, Ltd.     

Release kinetics of actives from chewing gums into saliva monitored by direct analysis in real time mass spectrometry

Direct analysis in real time mass spectrometry (DART‐MS) was used to monitor the release kinetics of a taste‐refreshing compound from chewing gums into the saliva of subjects. A new DART‐MS sample probe was designed which was about four times more sensitive than the current benchmark probe. This decreased the impact of the dilution of the saliva samples that was required to minimize ion suppression effects and make quantitative analyses without an internal standard possible. The new probe was also about three times more reproducible, which allowed quantitative measurements to be conducted manually without requiring the enhanced precision provided by an automatic sample positioner. The accuracy of analyses performed by DART‐MS was verified by comparing the results obtained from saliva samples analyzed both by DART‐MS and by a more classical liquid chromatography/mass spectrometry (LC/MS) method. This investigation showed good agreement between the two techniques. DART‐MS could then be used to objectively demonstrate the efficiency of a granular carbohydrate‐based delivery system to boost for a few minutes the release of a lipophilic flavor raw material with a high octanol/water partition coefficient, cyclohexanecarboxamide, N‐ethyl‐5‐methyl‐2‐(1‐methylethyl) (WS‐3), from chewing gum into saliva. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid screening for synthetic antidiabetic drug adulteration in herbal dietary supplements using direct analysis in real time mass spectrometry

Adulteration of herbal supplements with synthetic drugs is illegal. A rapid and reliable method which utilizes direct analysis in real time mass spectrometry (DART-MS) was developed for the identification of seven synthetic antidiabetic drugs used as adulterants in herbal dietary supplements. The supplement sample was simply extracted with methanol/water by manually shaking several times and directly analyzed using DART-MS. The presence of synthetic drug adulterants was confirmed through the accurate m/z values and MS/MS data obtained via quadruple time of flight mass spectrometry (QTOF MS). Parameters for the DART source were systematically optimized, and the limits of detection (LODs) in herbal supplement matrices were measured. This method was successfully applied to examine five commercial herbal dietary supplements, and two of them proved to be adulterated with metformin without labeling.     

Coupling of planar chromatography with Direct Analysis in Real Time mass spectrometry

Direct Analysis in Real Time mass spectrometry (DART-MS) is an emerging and rapidly developing area of ambient desorption ionization mass spectrometric techniques. Its coupling with planar chromatography is especially promising, as compared to other ambient desorption ionization techniques, because it does not require the use of liquids that may distort the shape of a spot by diffusion effects. In the first publications on TLC/HPTLC-DART-MS, due to the fixed, horizontally aligned supply of the gas flow from the DART ionization source to the MS inlet, the introduction of HPTLC/TLC plates as cut strips was inconvenient for quantitation, and the repeatability was very low due to the manual positioning. Recently a new version of the DART ion source was suggested, which allows adjusting the angle of the DART gas stream and the use of a motorized rail, thereby, improving highly the capabilities of TLC/HPTLC-DART-MS. This comprehensive review describes the development and analytical capabilities of TLC/HPTLC-DART-MS, and the general DART-MS perspectives for surface analysis or imaging MS.      

Analysis of hairy root culture of Rauvolfia serpentina using direct analysis in real time mass spectrometric technique

The applicability of a new mass spectrometric technique, DART (direct analysis in real time) has been studied in the analysis of the hairy root culture of Rauvolfia serpentina. The intact hairy roots were analyzed by holding them in the gap between the DART source and the mass spectrometer for measurements. Two nitrogen-containing compounds, vomilenine and reserpine, were characterized from the analysis of the hairy roots almost instantaneously. The confirmation of the structures of the identified compounds was made through their accurate molecular formula determinations. This is the first report of the application of DART technique for the characterization of compounds that are expressed in the hairy root cultures of Rauvolfia serpentina. Moreover, this also constitutes the first report of expression of reserpine in the hairy root culture of Rauvolfia serpentina.     

Self-aspirating atmospheric pressure chemical ionization source for direct sampling of analytes on surfaces and in liquid solutions

A self-aspirating heated nebulizer probe is described and demonstrated for use in the direct analysis of analytes on surfaces and in liquid samples by atmospheric pressure chemical ionization (APCI) mass spectrometry. Functionality and performance of the probe as a self-aspirating APCI source is demonstrated using reserpine and progesterone as test compounds. The utility of the probe to sample analytes directly from surfaces was demonstrated first by scanning development lanes of a reversed-phase thin-layer chromatography plate in which a three-component dye mixture, viz., Fat Red 7B, Solvent Green 3, and Solvent Blue 35, was spotted and the components were separated. Development lanes were scanned by the sampling probe operated under computer control (x, y plane) while full-scan mass spectra were recorded using a quadrupole ion trap mass spectrometer. In addition, the ability to sample the surface of pharmaceutical tablets (viz., Extra Strength Tylenol and Evista tablets) and to detect the active ingredients (acetaminophen and raloxifene, respectively) selectively was demonstrated using tandem mass spectrometry (MS/MS). Finally, the capability to sample analyte solutions from the wells of a 384-well microtiter plate and to perform quantitative analyses using MS/MS detection was illustrated with cotinine standards spiked with cotinine-d3 as an internal standard.     

Optimization of a direct analysis in real time/time-of-flight mass spectrometry method for rapid serum metabolomic fingerprinting

Metabolomic fingerprinting of bodily fluids can reveal the underlying causes of metabolic disorders associated with many diseases, and has thus been recognized as a potential tool for disease diagnosis and prognosis following therapy. Here we report a rapid approach in which direct analysis in real time (DART) coupled with time-of-flight (TOF) mass spectrometry (MS) and hybrid quadrupole TOF (Q-TOF) MS is used as a means for metabolomic fingerprinting of human serum. In this approach, serum samples are first treated to precipitate proteins, and the volatility of the remaining metabolites increased by derivatization, followed by DART MS analysis. Maximum DART MS performance was obtained by optimizing instrumental parameters such as ionizing gas temperature and flow rate for the analysis of identical aliquots of a healthy human serum samples. These variables were observed to have a significant effect on the overall mass range of the metabolites detected as well as the signal-to-noise ratios in DART mass spectra. Each DART run requires only 1.2 min, during which more than 1500 different spectral features are observed in a time-dependent fashion. A repeatability of 4.1% to 4.5% was obtained for the total ion signal using a manual sampling arm. With the appealing features of high-throughput, lack of memory effects, and simplicity, DART MS has shown potential to become an invaluable tool for metabolomic fingerprinting.     

Newborn screening of phenylketonuria using direct analysis in real time (DART) mass spectrometry

Phenylketonuria (PKU) is commonly included in the newborn screening panel of most countries, with various techniques being used for quantification of l-phenylalanine (Phe). To diagnose PKU as early as possible in newborn screening, a rapid and simple method of analysis was developed. Using direct analysis in real time (DART) ionization coupled with triple-quadrupole tandem mass spectrometry (TQ-MS/MS) and with use of a 12 DIP-it tip scanner autosampler in positive ion mode, we analyzed dried blood spot (DBS) samples from PKU newborns. The concentration of Phe was determined using multiple reaction monitoring mode with the nondeuterated internal standard N,N-dimethylphenylalanine. The results of the analysis of DBS samples from newborns indicated that the DART-TQ-MS/MS method is fast, accurate, and reproducible. The results prove that this assay as a newborn screen for PKU can be performed in 18 s per sample for the quantification of Phe in DBS samples. DART-TQ-MS/MS analysis of the Phe concentration in DBS samples allowed us to screen newborns for PKU. This innovative protocol is rapid and can be effectively applied on a routine basis to analyze a large number of samples in PKU newborn screening and PKU patient monitoring.

Rapid detection of bacterial endotoxins in ophthalmic viscosurgical device materials by direct analysis in real time mass spectrometry

Bacterial endotoxins are lipopolysaccharides bound to the bacterial cell wall and released when bacteria rupture or disintegrate. Possible contamination of endotoxin in ophthalmic devices can cause a painful eye inflammation or result in toxic anterior segment syndrome after cataract surgery. Measurement of bacterial endotoxin in medical device materials is difficult since endotoxin binds with polymer matrix and some of the materials are very viscous and non-water soluble, where traditional enzyme-based Limulus amebocyte lysate (LAL) assay cannot be applied. Here we propose a rapid and high throughput ambient ionization mass spectrometric (MS) method using direct analysis in real time (DART) for the evaluation of endotoxin contamination in medical device materials. Large and structurally complex endotoxin instantaneously breaks down into low-mass characteristic fragment ions using DART and is detected by MS in both positive and negative ion modes. This method enables the identification and separation of endotoxin from medical materials with a detection limit of 0.03 ng mL⁻¹ endotoxins in aqueous solution. Ophthalmic viscosurgical device materials including sodium hyaluronate (NaHA), non-water soluble perfluoro-n-octane (PFO) and silicone oil (SO) were spiked with different known concentrations of endotoxin and analyzed by DART MS, where the presence of endotoxin was successfully detected and featured small mass fragment ions were generated for NaHA, PFO and SO as well. Current findings showed the feasibility of measuring endotoxin contamination in medical device materials using DART-MS, which can lead to a one-step analysis of endotoxins in different matrices, avoiding any potential contamination during sample pre-treatment steps.     

Analysis of isoflavones in soybeans employing direct analysis in real-time ionization-high-resolution mass spectrometry

A direct analysis in real‐time (DART) ion source coupled to a high‐resolution orbitrap mass spectrometer was used for the quantitative analysis of isoflavones isolated from soybeans. For the isolation of genistein, daidzein, glycitein, and their respective acetyl, malonyl, and glucoside forms, an extraction employing 80% aqueous MeOH enhanced by sonication was used. As far as the total isoflavones (expressed as aglycones) were to be determined, an acid hydrolysis with 80% aqueous EtOH and refluxing had to be employed, while in the latter case a good agreement of the results with the data generated by the UHPLC‐orbitrap MS method was achieved, in the case of the analysis of non‐hydrolyzed extracts, some overestimation of the results as compared with those generated by UHPLC‐orbitrap MS was observed. A careful investigation of this phenomenon showed that the free aglycones originated from the conjugated forms of isoflavones in the DART ion source, thus contributing significantly to the “free” genistein/daidzein/glycitein signals during the DART analysis. Good recoveries (95–102%) and repeatabilities (RSD: 7–15%) were obtained at the spiking levels of 0.5, 1, and 0.05 g/kg, for daidzein, genistein, and glycitein, respectively. The limits of detection estimated for the respective analytes were 5 mg/kg.     

Analysis of Geting Bituminous Coal by Electrospray Ionization and Direct Analysis in Real Time Mass Spectrometry

Understanding the structure and composition of coals is important for effective, clean, and value-added utilization. In addition to gas chromatography/mass spectrometry which is commonly used to analyze coal, mass spectrometry (MS) may be used with other ion sources such as electrospray ionization (ESI) and direct analysis in real time (DART) for characterization. In this work, Geting bituminous coal was extracted sequentially and exhaustively with petroleum ether, carbon disulfide, methanol, acetone, an isometric acetone/carbon disulfide mixture, tetrahydrofuran, and an isometric tetrahydrofuran/carbon disulfide mixture. Raw coal, extracts, and the extraction residue were analyzed using MS equipped with ESI or DART. Organic heteroatomic species in the extracts were determined by liquid chromatography-mass spectrometry equipped with ESI. Molecular weight distributions of organic species in raw coal, extracts, and extraction residue were characterized by ESI-MS and DART-MS. Associated molecules and homologous compounds in coal extracts were identified.     

Comparison of tandem and conventional mass spectrometry using electron capture negative ionization in the detection of chemically oxidized dexamethasone in human plasma

Chemistry and Biochemistry, Michigan State University, East Lansing, Michigan, USA Dexamethasone, a synthetic steroid, can be oxidized chemically to a ketonic steroid structure that is readily detected by electron capture negative ionization mass spectrometry (ECNI/MS). Previous work from this laboratory has demonstrated that the chemical oxidation procedure provides advantages of low detection limits and high selectivity for detection of oxidized dexamethasone against chemical background that would otherwise interfere with detection of this steroidal drug in biological samples using more conventional methodology. This report describes the extent to which tandem mass spectrometry (MS/MS) can further enhance the selectivity of the oxidation/ECNI methodology for the detection of dexamethasone during the analysis of human plasma and presents evidence that sample introduction by direct inlet probe (DIP) can be used successfully under ECNI conditions. For purposes of comparing the methodologies, the same human plasma samples are analyzed by ECNI, first with detection by conventional mass spectrometry using selected ion monitoring (SIM) and then by MSIMS using selected reaction monitoring (SRM) with sample introduction by the gas chromatographic (GC) inlet and by the DIP. The results indicate that use of the DIP is a viable means of sample introduction for ECNI when sample processing involves the specialized oxidation procedure described herein because the sample matrix does not compete significantly for the thermal electrons in the ion source. Whereas SIM and SRM provide comparable results when sample introduction is achieved by the GC inlet, the MS/MS approach offers the possibility for sample introduction using the DIP, which significantly simplifies and shortens the analysis.     

Ultra‐performance hydrophilic interaction liquid chromatography/tandem mass spectrometry for the determination of everolimus in mouse plasma

Ultra‐performance hydrophilic interaction liquid chromatography (UPHILIC) interfaced with the electrospray ionization (ESI) source of a tandem mass spectrometer (MS/MS) was developed for the simultaneous determination of everolimus in mouse plasma samples. UPHILIC was performed on a sub‐2 µm bare silica particle packing with the column pressure under traditional high‐performance liquid chromatography (HPLC) to allow fast separation of pharmaceutical compounds within a chromatographic analysis time of 1 min. This UPHILIC technology is comparable with reversed‐phase ultra‐performance liquid chromatography (RPUPLC) in terms of chromatographic efficiency but demands neither expensive ultra‐high‐pressure instrumentation nor new laboratory protocols. With the ESI source, multiple reaction monitoring (MRM) of the ammoniated adduct ions of the analyte was used for tandem mass spectrometric detection. The retention mechanism profiles of the test compounds under HILIC conditions were explored. The influences of experimental factors such as the compositions of mobile phases on the chromatographic performance and the ionization efficiency of the test compounds in positive ion mode were investigated. A UPHILIC/MS/MS approach following a protein precipitation procedure was applied for the quantitative determination of everolimus at the low ng/mL region in support of a pharmacodynamic study. The analytical results obtained by the UPHILIC/MS/MS approach were fond to be in good agreement with those obtained by the RPUPLC/MS/MS method in terms of assay sample throughput, sensitivity and accuracy. Copyright © 2009 John Wiley & Sons, Ltd.     

实时直接分析高分辨质谱技术研究碳硼烷类化合物

该文运用高分辨质谱技术对实时直接分析(Direct analysis in real time,DART)离子化条件下碳硼烷化合物的质谱行为进行了研究,对碳硼烷化合物DART高分辨质谱中所得到的同位素峰簇进行了表征与归属。研究结果表明,选取的碳硼烷化合物在DART负离子条件下均能得到较好的质谱信号,这可能与硼笼结构的“缺电性”有关。含10个B原子的碳硼烷化合物形成的离子同位素峰簇信号中,通常情况下相对丰度最高的同位素峰中含2个10B以及8个11B。将碳硼烷化合物高分辨质谱分析的精确m/z数据信息与图谱中同位素峰轮廓分析相结合,是碳硼烷化合物有效的质谱定性分析与表征策略。     

Rapid qualitative analysis of 2 flavonoids,rutin and silybin,in medical pills by direct analysis in real‐time mass spectrometry (DART‐MS) combined with in situ derivatization

Direct analysis in real‐time mass spectrometry (DART‐MS) with in situ silylation was used for the rapid analysis of the flavonoids silybin ((2R,3R)‐3,5,7‐trihydroxy‐2‐[3‐(4‐hydroxy‐3‐methoxyphenyl)‐2‐hydroxymethyl‐2,3‐dihydrobenzo[1,4]dioxin‐6‐yl]chroman‐4‐one) and rutin (quercetin‐3‐O‐rutinoside). Three different derivatization reagents, hexamethyldisilazane/trimethylchlorosilane/pyridine (HMDS/TMCS/pyridine), N,O‐bis(trimethylsilyl)acetamide/trimethylchlorosilane/N‐trimethylsilyimidazole (BSA/TMCS/TMSI), and N,O‐bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (BSTFA/TMCS), were applied. Silybin and rutin were detected with various degrees of silylation, and the formation of dimers with pyridine and imidazole was also observed. HMDS/TMCS/pyridine was the best choice for the DART‐MS analysis of silybin, and BSA/TMCS/TMSI was the most effective for the detection of rutin. The effects of the DART source temperature on desorption, ionization, in‐source fragmentation, dimer formation, and hydrolysis of the trimethylsilyl groups were also studied. In addition, the collision‐induced dissociation properties of the derivatized silybin and rutin were explored. With our in situ silylation method, the derivatized bioactive compounds in intact medical pills could also be detected by DART‐MS.