Direct Analysis In Real Time Ion Source Research Articles

Ambient mass spectrometry imaging of food natural products by angled direct analysis in real time high-resolution mass spectrometry

Direct surface analysis in ambient conditions provides information on the position and chemical composition of an object at the time of investigation. An angled sampling probe is developed in this work for direct analysis in real time (DART) ionization high-resolution mass spectrometry. The DART ion source and the interface were modified for improved surface resolution, increased ion transfer efficiency, as well as enabling two-dimensional surface scanning. The angled probe DART-MS system was used for investigating a variety of food samples including fruit peels, ginseng root, plant leaves and sections of radish. Abundant signals and distinct chemical profiles are obtained in seconds, and spatial distribution of different molecules across the sample surfaces can be observed. In addition, the developed system can quickly identify the chemical changes when the surfaces were treated. The method is capable of directly evaluating food sample surfaces with different shapes, hardness, and conditions, without any sample pretreatments.

Online mass spectrometry of exhaled breath with a modified ambient ion source

Exhaled breath (EB) may contain metabolites that are closely related to human health conditions. Real time analysis of EB is important to study its true composition, however, it has been difficult. A robust ambient ionization mass spectrometry method using a modified direct analysis in real time (DART) ion source was developed for the online analysis of breath volatiles. The modified DART ion source can provide a confined region for direct sampling, rapid transmission and efficient ionization of exhaled breath. With different sampling methods, offline analysis and near real-time evaluation of exhaled breath were also achieved, and their unique molecular features were characterized. High resolution MS data aided the putative metabolite identification in breath samples, resulting in hundreds of volatile organic compounds being identified in the exhalome. The method was sensitive enough to be used for monitoring the breath feature changes after taking different food and over-the-counter medicine. Quantification was performed for pyridine and valeric acid with fasting and after ingesting different food. The developed method is fast, simple, versatile, and potentially useful for evaluating the true state of human exhaled breath.

Characterization of ginsenoside structural isomers from mixtures using in situ methylation with direct analysis in real-time ionization tandem mass spectrometry

Characterization of structural isomers of bioactive molecules is important for recognizing their functions, but it has been challenging due to their highly similar structures. As the main bioactive constituents of Panax ginseng, ginsenosides have different structural isomers attributed to the aglycone structure and glycosylation sites as well as stereochemistry of sugar groups attached. This work demonstrated a simple and robust in situ methylation reaction with tetramethylammonium hydroxide (TMAH) using ambient ionization source of direct analysis in real time (DART) to characterize saponin structural isomers. The DART ion source provides favorable conditions to methylate hydroxyl groups of ginsenoside instantaneously with TMAH, and it can ionize the methylated products at the same time. Methylated ginsenoside stereoisomers even with subtle structure differences generated very different mass signals from full-scan MS and tandem MS. High-resolution mass spectrometry aided the assignment of molecular structures of the various precursor and fragment ions from different ginsenosides, which provided structural information for both the aglycone skeleton and the sugar moieties in ginsenosides. The presented method was successfully used for the identification of ginsenosides in Panax ginseng, and saponin isomers were characterized without the need for chromatographic separation and/or tedious offline sample pretreatment.

Evaluation of direct analysis in real time for the determination of highly polar pesticides in lettuce and celery using modified Quick Polar Pesticides Extraction method

Direct analysis in real time (DART) was evaluated for the determination of a number of highly polar pesticides using the Quick Polar Pesticides Extraction (QuPPe) method. DART was hyphenated to high resolution mass spectrometry (HRMS) in order to get the required selectivity that allows the determination of these compounds in complex samples such as lettuce and celery. Experimental parameters such as desorption temperature, scanning speed, and distances between the DART ion source and MS inlet were optimized. Two different mass analyzers (Orbitrap and QTOF) and two accessories for sample introduction (Dip-it® tips and QuickStrip™ sample cards) were evaluated. An extra clean-up step using primary-secondary amine (PSA) was included in the QuPPe method to improve sensitivity. The main limitation found was in-source fragmentation, nevertheless QuPPe-DART-HRMS proved to be a fast and reliable tool with quantitative capabilities for at least seven compounds: amitrole, cyromazine, propamocarb, melamine, diethanolamine, triethanolamine and 1,2,4-triazole. The limits of detection ranged from 20 to 60μg/kg. Recoveries for fortified samples ranged from 71 to 115%, with relative standard deviations <18%.

Internal Energy Deposition in Dielectric Barrier Discharge Ionization is Significantly Lower than in Direct Analysis in Real-Time Mass Spectrometry

The extent of internal energy deposition using three different plasma-based ionization mass spectrometry (MS) methods, atmospheric pressure chemical ionization (APCI), direct analysis in real time (DART), and active capillary dielectric barrier discharge ionization (DBDI), was investigated using benzylammonium ‘thermometer’ ions. Ions formed by DBDI were activated significantly less than those that were formed by DART and APCI under these conditions. Thermal ion activation by DART can be reduced slightly by positioning the DART source further from the capillary entrance to the MS and reducing the heat that is applied to metastable atoms exiting the DART source. For example, the average ion internal energy distribution decreased by less than 10 % (166.9 ± 0.3 to 152.2 ± 1.0 kJ mol−1) when the distance between the DART source and the MS was increased by 250 % (10 to 25 mm). By lowering the DART temperature from 350 to 150°C, the internal energy distributions of the thermometer ions decreased by ~15 % (169.93 ± 0.83 to 150.21 ± 0.52 kJ mol−1). Positioning the DART source nozzle more than 25 mm from the entrance to the MS and decreasing the DART temperature further resulted in a significant decrease in ion signal. Thus, varying the major DART ion source parameters had minimal impact on the ‘softness’ of the DART ion source under these conditions. Overall, these data indicate that DBDI can be a significantly ‘softer’ ion source than two of the most widely used plasma-based ion sources that are commercially available.

Application of direct analysis in real time (DART) and direct inlet probe (DIP-APCI) mass spectrometry in the context of industrial sample analysis

In today’s industry, quick and reliable analytical methods play an important role for quality control. On that account, two emerging techniques, namely direct inlet probe-atmospheric pressure chemical ionization (DIP-APCI) and direct analysis in real time (DART) mass spectrometry, are particularly promising. In case of a DIP-APCI source, small amounts of solid or liquid samples can be studied without sample pre-treatment. A similar system is the DART ion source. In addition to the analysis of solid and liquid samples without pre-separation, this ion source offers the possibility to scan the surface of a sample. A method for industrial sample analysis focusing on the study of delamination of coatings from a panel was developed using DIP-APCI-Q-TOF-MS and DART-Q-TOF-MS, respectively. Comparative studies based on the conventional pyrolysis–GC–MS were carried out.

A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures.

A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. Here, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required.

Extraction‐free In situ Derivatisation of Timosaponin AIII Using Direct Analysis in Real Time TOF/MS

Direct analysis in real time (DART) TOF/MS has been used for mass information of various non-polar phytochemicals in raw material with no sample preparation. However, low ionisation efficiency for polar compounds including glycosides limits its extensive use in the field of phytochemical analysis. In order to develop a direct analysis method for polar glycosides using in situ derivatisation, which improves ionisation efficiency of hydrophilic glycosides. Anemarrhena Rhizoma was used as a model plant targeting on Timosaponin AIII utilising a Dip-It module. Permethylation was applied to the powdered raw material with tetramethylammonium hydroxide in front of a DART ion source. Also, DART TOF/MS combined with permethylation was applied to timosaponin AIII standard solution to obtain the limit of detection (LOD). In situ methylation of timosaponin AIII and Anemarrhena Rhizoma raw material were successfully used to ionise the glycoside. The LOD was found to be in the range of 2.4-4.8 ng for permethylated timosaponin AIII and this level is four times higher than the range of the underivatisation analysis. Direct analysis of permethylated timosaponin from Anemarrhena Rhizoma was also successfully performed. A simple and quick derivatisation method with tetramethylammonium hydroxide was developed for the direct identification of a hydrophilic saponin from the plant tissue. Better ionisation efficiency conferred by in situ permethylation enabled ionisation of whole molecules of timosaponin AIII from the plant tissue. This simple analytical method will provide a solution to reduce tedious sample preparation steps, not only for non-polar but also hydrophilic natural products directly from the tissue.

Direct Analysis in Real Time Mass Spectrometry (DART-MS) Analysis of Skin Metabolome Changes in the Ultraviolet B-Induced Mice

Ultraviolet (UV) radiation is a major environmental factor that leads to acute and chronic reactions in the human skin. UV exposure induces wrinkle formation, DNA damage, and generation of reactive oxygen species (ROS). Most mechanistic studies of skin physiology and pharmacology related with UV-irradiated skin have focused on proteins and their related gene expression or single- targeted small molecules. The present study identified and analyzed the alteration of skin metabolites following UVB irradiation and topical retinyl palmitate (RP, 5%) treatment in hairless mice using direct analysis in real time (DART) time-of-flight mass spectrometry (TOF-MS) with multivariate analysis. Under the negative ion mode, the DART ion source successfully ionized various fatty acids including palmitoleic and linolenic acid. From DART-TOF-MS fingerprints measured in positive mode, the prominent dehydrated ion peak (m/z: 369, M+H-H2O) of cholesterol was characterized in all three groups. In positive mode, the discrimination among three groups was much clearer than that in negative mode by using multivariate analysis of orthogonal partial-least squares-discriminant analysis (OPLS-DA). DART-TOF-MS can ionize various small organic molecules in living tissues and is an efficient alternative analytical tool for acquiring full chemical fingerprints from living tissues without requiring sample preparation. DART-MS measurement of skin tissue with multivariate analysis proved to be a powerful method to discriminate between experimental groups and to find biomarkers for various experiment models in skin dermatological research.

Identification of saccharides by using direct analysis in real time (DART) mass spectrometry

Direct analysis in real time (DART) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF-MS) was applied to investigate the different types of saccharides including ginseng oligosaccharide extract with minimal sample pretreatment. Helium gas temperature into the DART ion source was adjusted to find optimal ionization temperature for glucose in increments of 50°C from 50°C to 450°C. It was observed that gas temperature had a significant effect on signal intensity in DART mass spectra. The temperature for the glucose ionization should be at least 150°C, and the signal intensity reached optimal ionization state at 250°C. With the increase of a sugar chain, a higher gas temperature was needed for saccharide ionization. Interestingly, sugar cluster formation can be observed at 450°C for disaccharides and trisaccharides. It is found that the [M+NH4]+ ions of trisaccharide and ginseng oligosaccharides can be rarely produced without any derivatization even at 450°C. All the samples except five carbon monosaccharides can generate the m/z 198 ion, so it is difficult to determine whether a m/z 198 ion is the [M+NH4]+ ion of glucose or merely a sample fragment ion most likely an ammoniated monosaccharide fragment.

Fragmentation Behavior Studies of Chalcones Employing Direct Analysis in Real Time (DART)

Chalcones are naturally occurring, biologically active molecules generating interest from a wide range of researchapplications including synthetic methodology development, biological activity investigation and studying fragmentation patterns. Inthis article, a series of chalcones has been synthesized and their fragmentation behavior was studied using modern ambient ionizationtechnique Direct Analysis in Real Time (DART). DART ion source connected with an ion trap mass spectrometer wasused for the fragmentation of various substituted chalcones. The chalcones were introduced to the DART source using a glasscapillary without sample preparation step. All the chalcones showed prominent molecular ion peaks [M]•+ corresponding to thestructures. Multistage mass spectral data MSn (MS2 and MS3) were collected for all the chalcones studied. The chalcones withsubstitutions at 3, 4 or 5 positions gave product ion peaks with the loss of a phenyl radical (Ph•) by radical initiated α-cleavage,while substitution at 2 position of chalcone in the A-ring gave a product ion peak with the loss of substituted styryl radical(PhCH = CH•). In case of the chalcones with the substituent at 4 positions in A and B rings gave both types of fragmentation patterns. In conclusion, chalcones can be easily characterized using modern DART interface in very short time and efficientlywithout any cumbersome sample pretreatment.

Applications of a confined DART (direct analysis in real time) ion source for online in vivo analysis of human breath

Human breath is composed of many volatile metabolites that can potentially provide information about a person's health status. A sensitive assay to identify these volatile organic compounds as biomarkers is ideal and important for early detection of many diseases. However, the diagnosis through online breath analysis based on mass spectrometry is difficult due to the low concentration of pertinent compounds in breath. The DART (direct analysis in real time) ion source is a novel atmospheric pressure ionization technique, which can efficiently ionize gaseous, liquid and solid samples with high throughput. A major problem in analysis of gaseous samples is its poor detection sensitivity due to open-air sampling. Recently, a confined DART ion source (cDART) was developed, where the plasma generated by the atmospheric pressure glow discharge ionizes gas-phase molecules in a Tee-shaped flow tube instead of open air. It leads to a significant increase of the ionization efficiency of gaseous samples. In this study, the cDART source was modified and applied in online analysis of exhaled breath. The limit of detection of the improved cDART source was determined to be at the level of low ppb. The cDART source was used in the real-time monitoring of an oral anesthetic drug. The concentration variations of two active ingredients in exhaled breath, menthol and benzocaine, and their metabolites with time were obtained. The results show that the cDART ion source is a powerful analytical tool to provide high sensitivity and high throughput analysis for volatile organic compounds in human breath, and can find potential applications in clinical diagnosis and therapy.

Confined direct analysis in real time ion source and its applications in analysis of volatile organic compounds of Citrus limon (lemon) and Allium cepa (onion)

The DART (direct analysis in real time) ion source is a novel atmospheric pressure ionization technique that enables efficient ionization of gases, liquids and solids with high throughput. A major limit to its wider application in the analysis of gases is its poor detection sensitivity caused by open-air sampling. In this study, a confined interface between the DART ion source outlet and mass spectrometer sampling orifice was developed, where the plasma generated by the atmospheric pressure glow discharge collides and ionizes gas-phase molecules in a Tee-shaped flow tube instead of in open air. It leads to significant increase of collision reaction probability between high energy metastable molecules and analytes. The experimental results show that the ionization efficiency was increased at least by two orders of magnitude. This technique was then applied in the real time analysis of volatile organic compounds (VOCs) of Citrus Limon (lemon) and wounded Allium Cepa (onion). The confined DART ion source was proved to be a powerful tool for the studies of plant metabolomics.

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.

ケミカルズ覚え書き（機器編）リアルタイム直接質量分析イオン源ＤＡＲＴ（Ｄｉｒｅｃｔ Ａｎａｌｙｓｉｓ ｉｎ Ｒｅａｌ Ｔｉｍｅ）質量分析法の応用

We introduce applications of Direct Analysis in Real Time (DART) ion source, used in open-air at ground potential under ambient conditions, coupled with time-of-flight or quadrupole mass spectrometer.DART was developed in 2005 as a new type of atmospheric pressure ion source. One of the most interesting features of DART is that researchers can access the place of ionizing point directly. It means we can observe real-time spectra of a sample at ground potential under ambient condition. In this paper, we will introduce the principles and features of DART ion source and some applications of DART mass spectrometry.

Ambient mass spectrometry employing a DART ion source for metabolomic fingerprinting/profiling: a powerful tool for beer origin recognition

A metabolomic fingerprinting/profiling generated by ambient mass spectrometry (MS) employing a direct analysis in real time (DART) ion source coupled to high-resolution time-of-flight mass spectrometry (TOFMS) was employed as a tool for beer origin recognition. In a first step, the DART–TOFMS instrumental conditions were optimized to obtain the broadest possible representation of ionizable compounds occurring in beer samples (direct measurement, no sample preparation). In the next step, metabolomic profiles (mass spectra) of a large set of different beer brands (Trappist and non-Trappist specialty beers) were acquired. In the final phase, the experimental data were analyzed using partial least squares discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and artificial neural networks with multilayer perceptrons (ANN-MLP) with the aim of distinguishing (i) the beers labeled as Rochefort 8; (ii) a group consisting of Rochefort 6, 8, 10 beers; and (iii) Trappist beers. The best prediction ability was obtained for the model that distinguished the group of Rochefort 6, 8, 10 beers from the rest of beers. In this case, all chemometric tools employed provided ≥95% correct classification. The current study showed that DART–TOFMS metabolomic fingerprinting/profiling is a powerful analytical strategy enabling quality monitoring/authenticity assessment to be conducted in real time.

Quantitative analysis of major dibenzocyclooctane lignans in schisandrae fructus by online TLC‐DART‐MS

Direct analysis in real time (DART) ion source is a powerful ionising technique for the quick and easy detection of various organic molecules without any sample preparation steps, but the lack of quantitation capacity limits its extensive use in the field of phytochemical analysis. To improvise a new system which utilize DART-MS as a hyphenated detector for quantitation. A total extract of Schisandra chinensis fruit was analyzed on a TLC plate and three major lignan compounds were quantitated by three different methods of UV densitometry, TLC-DART-MS and HPLC-UV to compare the efficiency of each method. To introduce the TLC plate into the DART ion source at a constant velocity, a syringe pump was employed. The DART-MS total ion current chromatogram was recorded for the entire TLC plate. The concentration of each lignan compound was calculated from the calibration curve established with standard compound. Gomisin A, gomisin N and schisandrin were well separated on a silica-coated TLC plate and the specific ion current chromatograms were successfully acquired from the TLC-DART-MS system. The TLC-DART-MS system for the quantitation of natural products showed better linearity and specificity than TLC densitometry, and consumed less time and solvent than conventional HPLC method. A hyphenated system for the quantitation of phytochemicals from crude herbal drugs was successfully established. This system was shown to have a powerful analytical capacity for the prompt and efficient quantitation of natural products from crude drugs.

Identification of marker compounds in herbal drugs on TLC with DART-MS

This study was conducted to provide a more versatile and specific information on Thin Layer Chromatographic (TLC) analysis of medicinal plants. TLC plates developed with the extract of herbal medicines were analyzed with direct analysis in real time (DART) ion source. Three well known herbal drugs were extracted and developed on a silica-coated TLC plate with the conditions pre-established in Korean Pharmacopoeia IX. The developed plate was placed between the DART ion source and TOF-MS analyzer to get real time mass spectra from the bands on the TLC plate directly. The marker coumarin compounds, decursin and decursinol were successfully identified from the TLC plate developed with Angelicae gigantis radix, along with alkaloid compounds of rutaecarpine and evodiamine from Evodiae fructus, and lignan molecules of gomisin A, N, and schisandrin from Schisandrae fructus. This hyphenation system of TLC and DART-MS could provide unique and specific information on the major constituents of crude plant drug on TLC through uncovering high resolution mass number of each band on the TLC plate directly in real time.

Direct Analysis in Real Time (DART) Mass Spectrometry of Nucleotides and Nucleosides: Elucidation of a Novel Fragment [C 5H 5O] + and Its In-Source Adducts

Direct analysis in real time (DART) mass spectrometry is a recently developed innovative technology, which has shown broad applications for fast and convenient analysis of complex samples. Due to the ease of sample preparation, we have recently initiated an investigation of the feasibility of detecting nucleotides and nucleosides using the DART-AccuTOF instrument, which we will refer to as the DART mass spectrometer. Our experimental results reveal that the ions representing the intact molecules of nucleotides are not detectable in either positive-ion or negative-ion mode. Instead, all four natural nucleotides fragment in the DART ion source, and a common fragment ion, [C(5)H(5)O](+) (1), is observed, which is probably formed via multiple-elimination reactions. Interestingly, 1 can form adducts with nucleobases in different molar ratios in the DART ion source. In contrast to nucleotides, the ions representing the intact molecules of nucleosides are detected in both positive-ion and negative-ion mode using DART mass spectrometry. Surprisingly, the fragmentation pattern of nucleosides is different from that of nucleotides in the DART ion source. In the cases of nucleosides (under positive-ion conditions), the production of 1 is not observed, indicating that the phosphate group plays an important role for the multiple eliminations observed in the spectra of nucleotides. The in-source reactions described in the present work show the complexity of the conditions in the DART ion source, and we hope that our results illustrate a better understanding about DART mass spectrometry.

Detection of Gamma‐Hydroxybutyric Acid in Various Drink Matrices via AccuTOF‐DART*

A new screening method for detecting gamma-hydroxybutyric acid (GHB) in drink matrices, using the IonSense, Inc. (Saugus, MA) direct analysis in real time (DART) ion source coupled to a JEOL exact mass time-of-flight mass spectrometer (AccuTOF), was validated and compared with the current screening methodology. The DART ion source allows for analysis of samples under ambient conditions with little to no sample preparation. Fifty drink specimens were spiked at levels of 1, 2, 3, and 4 mg/mL GHB, and analyzed on the AccuTOF-DART. Positive detection of GHB occurred for each of the samples at each concentration level, giving 100% accuracy for the samples tested. Twenty-five of the 50 drink specimens were spiked at 1 mg/mL GHB and tested using a color test known as the GHB Color Test #3. Only two of these 25 specimens tested positive for the presence of GHB, giving only 8% accuracy. Implementation of this new methodology as a screening tool for GHB analysis will quickly eliminate negative specimens allowing the examiner to focus analysis time on those that screened positive.