Article pubs.acs.org/ac
Solvent Jet Desorption Capillary Photoionization-Mass Spectrometry Markus Haapala, Jaakko Teppo, Elisa Ollikainen, Iiro Kiiski, Anu Vaikkinen, Tiina J. Kauppila, and Risto Kostiainen* Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland S Supporting Information *
ABSTRACT: A new ambient mass spectrometry method, solvent jet desorption capillary photoionization (DCPI), is described. The method uses a solvent jet generated by a coaxial nebulizer operated at ambient conditions with nitrogen as nebulizer gas. The solvent jet is directed onto a sample surface, from which analytes are extracted into the solvent and ejected from the surface in secondary droplets formed in collisions between the jet and the sample surface. The secondary droplets are directed into the heated capillary photoionization (CPI) device, where the droplets are vaporized and the gaseous analytes are ionized by 10 eV photons generated by a vacuum ultraviolet (VUV) krypton discharge lamp. As the CPI device is directly connected to the extended capillary inlet of the MS, high ion transfer efficiency to the vacuum of MS is achieved. The solvent jet DCPI provides several advantages: high sensitivity for nonpolar and polar compounds with limit of detection down to low fmol levels, capability of analyzing small and large molecules, and good spatial resolution (250 μm). Two ionization mechanisms are involved in DCPI: atmospheric pressure photoionization, capable of ionizing polar and nonpolar compounds, and solvent assisted inlet ionization capable of ionizing larger molecules like peptides. The feasibility of DCPI was successfully tested in the analysis of polar and nonpolar compounds in sage leaves and chili pepper.
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namely, desorption APPI (DAPPI)7 in 2007 and laser ablation APPI (LAAPPI)8 in 2012. In DAPPI, the analytes are thermally desorbed to gas-phase by directing a narrow hot solvent vapor jet to the surface of a solid sample.7,9 The analytes in the gas-phase are photoionized similarly as in APPI by a vacuum ultraviolet (VUV) lamp emitting 10.0 and 10.6 eV photons. In order to achieve high ionization efficiency, the ionization energy of the solvent must be below the energy of the photons. Suitable solvents are, for example, toluene, chlorobenzene, anisole, and acetone. DAPPI is a very simple, stable, and sensitive method for the analysis of nonpolar and polar compounds from surfaces, and it has successfully been applied, for example, in drug,7 environmental,10 food,10,11 pyrogenic black carbon,12 and forensic analysis.13,14 MS imaging of plant leaves and mouse brain tissue sections has also been demonstrated by DAPPI.15 However, the spatial resolution in DAPPI-MS imaging is limited to about 1 mm due to the thermal desorption process. The thermal desorption also limits the range of analyzable compounds to small and thermostable analytes.7,11 In the recently introduced LAAPPI, the analytes are ablated from water-rich surfaces with an infrared (IR) laser.8 Above the sample surface, the ablated sample plume is intercepted with an orthogonal hot solvent vapor jet (e.g., toluene or anisole).
mbient mass spectrometry (MS), which enables rapid, direct analysis of biological sample surfaces in their native states without any pretreatment, is an important new technique in bioanalysis. In ambient MS, compounds are desorbed directly from surfaces and ionized outside a mass spectrometer using atmospheric pressure ionization techniques. The first and still most commonly used ambient MS techniques are desorption electrospray ionization (DESI)1 and direct analysis in real time (DART)2 introduced in 2004 and 2005, respectively. After this, over 30 variations have been introduced with different combinations of desorption and ionization methods. The sampling can be achieved by liquid spray based techniques, thermal desorption, plasma based techniques, lasers, or a solvent bridge method, while the most common ionization methods are electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), sonic spray ionization (SSI), and different kinds of plasma based methods.3−5 Ambient MS methods have mostly been applied to the analysis of polar and ionic compounds, as most ambient ionization techniques provide high ionization efficiency for these types of compounds via the ion evaporation process (e.g., DESI, desorption sonic spray ionization) or via gas-phase reactions (e.g., DAPCI, DART, plasma based methods). Among ionization methods, APPI is an exception, as it provides high ionization efficiency not only for polar compounds via proton transfer but also for nonpolar compounds via charge exchange.6 Two ambient MS methods based on APPI have been presented, © 2015 American Chemical Society
Received: November 11, 2014 Accepted: February 25, 2015 Published: February 25, 2015 3280
DOI: 10.1021/ac504220v Anal. Chem. 2015, 87, 3280−3285
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Analytical Chemistry The ablated analytes are desolvated, ionized in the gas-phase by APPI, and transferred into the MS. The unique feature of LAAPPI over other ambient MS techniques is that it provides high sensitivity for nonpolar compounds with good, about 300 μm lateral resolution comparable to, for example, matrixassisted laser desorption ionization. LAAPPI has successfully been applied in imaging of nonpolar compounds in sage leaves.16 However, the experimental setup is relatively complex and requires the use of an expensive IR laser. The aim of this work was to develop a new universal ambient MS method, solvent jet desorption capillary photoionization (DCPI), capable of ionizing efficiently nonpolar and polar, as well as small and large molecules, with high lateral resolution and a simple and cheap setup. In order to achieve this goal, we used a solvent jet at ambient temperature and pressure for the desorption of the analytes from the sample surface and our recently introduced capillary photoionization (CPI)17 source for ionization. The feasibility of DCPI in the analysis of polar and nonpolar compounds from sage leaves and chili pepper is demonstrated.
Figure 1. Schematic of the solvent jet DCPI setup (not to scale).
Cambridge, UK) emitting 10.0 eV (124 nm) and 10.6 eV (117 nm) photons was used to initiate the ionization. The CPI capillary was heated using a Biltema 20-3917 soldering iron (Biltema Finland Ltd., Helsinki, Finland), and the temperature was set to 300 °C with a temperature controller (Omega CN7500, OMEGA Engineering Ltd., Manchester, UK). In addition, the outer end of the CPI capillary was heated with a power of 3.0 W using a resistance wire heater and an external power source (Iso-Tech IPS-603, RS Components Ltd., Northants, UK). The mass spectrometer used in the experiments was an Agilent 6330 ion trap (Agilent Technologies, Santa Clara, CA) with a commercial capillary extension (KR Analytical Ltd., Sandbach, UK) connected to the front of the glass inlet capillary. The CPI device was connected to the capillary extension by inserting the extension a few millimeters into the CPI capillary. The capillary extension piece was heated with nitrogen (AGA Ltd., Espoo, Finland) from the drying gas line of the MS with a flow rate of 4.0 L min−1 and a temperature setting of 285 °C. The mass spectrometer was controlled, and the data was analyzed with Agilent 6300 Series TrapControl Version 6.1 (Build 92) and DataAnalysis for 6300 Series Ion Trap LC/MS Version 3.4 (Build 192) software, respectively. The MS measurements were done in positive ion mode. The solvent jet nebulizer for the desorption of the analytes was made using a syringe pump (Harvard Apparatus, Holliston, MA) connected to a fused silica solvent capillary (50 μm i.d., 150 μm o.d., Polymicro Technologies Inc., Phoenix, AZ), which was inserted coaxially inside a nebulizing gas PEEK capillary (250 μm i.d., 1/16 in. o.d., VICI AG International, Schenkon, Switzerland) connected to a PEEK t-piece (VICI AG International). Toluene, chlorobenzene, methanol, and water in different compositions were used as spray solvents at 10 μL min−1 flow rate. The detailed solvent compositions are presented separately for each experiment in the Results and Discussion section. Nebulizing gas (nitrogen) was delivered coaxially between the solvent and nebulizing gas capillaries with a pressure of 10 bar. The nebulizing gas and solvent produced a solvent jet, which was directed to the sample spot on the surface. xyz stages (Märzhäuser Wetzlar GmbH & Co. KG, Wetzlar, Germany) were used to adjust the positions of the spraying capillary and the sample plate. The sprayer for testing the spatial resolution was similar to the one used for MS measurements, but several combinations of outer and inner capillary dimensions were tested. The i.d.’s of the outer capillary were 180, 220, or 250 μm; the o.d.’s of the inner capillary were 147 and 105 μm, and respective i.d.’s of the inner capillary were 50 and 40 μm. The test surface was black
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EXPERIMENTAL SECTION Chemicals and Samples. Water was purified with a Milli-Q water purification system (Millipore, Molsheim, France). Testosterone, angiotensin II, LC-MS grade methanol, and HPLC grade toluene and chlorobenzene were from SigmaAldrich (Steinheim, Germany). Verapamil hydrochloride was purchased from Aldrich Chem. Co. (Milwaukee, WI), benzo[a]pyrene (B[a]P) from Tamro (Vantaa, Finland), and caffeine from University Pharmacy (Helsinki, Finland). Fresh sage (Salvia of f icinalis) herb in a growing pot was purchased from a local supermarket, and chili peppers were home-cultivated by M.H. Stock solutions containing 10 mmol L−1 verapamil, testosterone, and caffeine were prepared in methanol, and a stock solution containing 10 mmol L−1 benzo[a]pyrene was prepared in toluene. From these, 10 μmol L−1 working solutions containing all four standard compounds were prepared in methanol/water (1/1, v/v) and further diluted to appropriate concentrations with methanol/water (1/1, v/v). A standard solution of angiotensin II at a concentration of 100 μmol L−1 was prepared in methanol/water (1/1, v/v) including 0.1% of formic acid. For each sample spot, one microliter of sample solution was applied on a poly(methyl methacrylate) (PMMA) or poly(tetrafluoroethylene) (PTFE) sample plate and left to dry at ambient temperature. Sage leaves and pieces of chili pepper were attached on microscope glass slides with tape. The PMMA and PTFE sample plates were from Vink Finland Ltd. (Kerava, Finland), and microscope glass slides were from Gerhard Menzel GmbH (Braunschweig, Germany). PMMA plates and microscope glass slides were used as such, whereas pieces of PTFE were attached on PMMA plates with doublesided tape (Scotch, Cergy-Pontoise, France). Instrumentation. The solvent jet DCPI device consisted of a sprayer, a sampling plate, and a capillary photoionization (CPI) interface (Figure 1). The CPI interface consisted of a 1.5 mm i.d. stainless steel (SS) capillary (CPI capillary) between the atmosphere and the vacuum of the MS with a 1 mm wide and 15 mm long opening, a flat SS plate with a similar opening hard-soldered on the capillary, a 3 mm thick MgF2 window (Thorlabs Sweden AB, Gothenburg, Sweden), a top plate with an 18 mm circular opening, and Kalrez o-rings (Tiivistekeskus Ltd., Vantaa, Finland) used as seals to hold the MgF2 window in place. An rf krypton VUV lamp (Heraeus Noblelight Analytics Ltd., 3281
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The effect of desorption solvent composition on the signal intensities of the standard compounds was studied by comparing methanol/toluene as the spray solvent with 100/0, 75/25, 50/50, 25/75, and 0/100 (v/v) ratios. The results in Figure S-3, Supporting Information, show that all test compounds were efficiently ionized with methanol/toluene ratios of 75/25, 50/50, 25/75, and 0/100 so that the best sensitivity was achieved with the ratio of 25/75. With 100% methanol, only verapamil was substantially ionized, albeit less efficiently than with the addition of toluene. This indicates that the presence of a solvent with ionization energy below 10 eV was necessary for achieving high sensitivity. Verapamil and testosterone having relatively high proton affinities produced intense protonated molecules by proton transfer reactions. Caffeine and benzo[a]pyrene having relatively low ionization energies produced protonated molecules and molecular ions formed by charge exchange reactions (Figure 2). (See also
printing wax printed with a Xerox Phaser 8560 (Xerox Corporation, Wilsonville, OR). The sprayer was moved by a computer controlled xyz stage (Thorlabs Sweden AB) using an in-house developed software. Speeds of 0.5 and 1.0 mm/s and directions perpendicular and parallel to the spray direction were tested. The sprayer was situated either 0.5 or 1.0 mm above the test surface. The image of the resulting track on the wax surface was captured with an optical microscope, and spatial resolution was measured using Motic Images Plus 2.0 software (Motic Deutschland GmbH, Wetzlar, Germany).
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RESULTS AND DISCUSSION The desorption process in DCPI is suggested to be similar to the droplet pick-up mechanism presented earlier for DESI.18 The narrow solvent jet generated at ambient temperature by a coaxial nebulizer of the DCPI device (Figure 1) is directed onto the sample surface, which is first prewetted by forming a solvent film, to which the surface analytes are dissolved. After this, secondary droplets containing the analytes are formed in collisions between the solvent jet and the solvent film. The secondary droplets ejected from the surface are directed into the heated CPI device, where the droplets are vaporized and gaseous analytes are ionized by VUV photons. The ionization may also take place similarly as in the solvent assisted inlet ionization presented earlier by Pagnotti et al.19 As the CPI device is directly connected to the extended capillary of the MS, high ion transfer efficiency to the MS and therefore improved sensitivity can be achieved. However, operation parameters, which have a significant effect on the performance of DCPI, must be carefully optimized. The positions of the nebulizer nozzle and sample spot in relation to the CPI capillary inlet were adjusted for maximum sensitivity and stability. The optimal performance of the system was achieved with approximately 1 mm distance between the nebulizer nozzle and the sampling spot. The short nebulizer-tosurface distance provides a more efficient desorption process than longer distances. The sampling spot was positioned onaxis as close to the CPI inlet as possible in order to ensure maximum transfer of secondary droplets into CPI. The effect of the angle of the nebulizer in relation to the surface was tested with angles of 15°, 25°, 35°, 45°, 55°, and 65°. (See Figure S-1 in the Supporting Information.) The highest sensitivity was achieved with the lowest angles (15° and 25°), which provide improved transfer of secondary droplets into CPI and desorption of the analytes from a larger area than the larger nebulizer-surface angles. The effect of solvent (methanol/ toluene, 70/30, v/v) flow rate was tested with flow rates of 1, 2, 4, 6, 8, 10, 14, and 20 μL min−1. (See Figure S-2 in the Supporting Information.) With all the test compounds (verapamil, testosterone, caffeine, and benzo[a]pyrene), the signal intensities increased when the solvent flow rate was increased from 1 to 4 μL min−1 and maximum sensitivity was achieved at flow rates of 4−10 μL min−1. At higher flow rates (14 and 20 μL min−1), the signal intensities started to decrease. The increase of signal intensity is partly because at higher flow rates the solvent molecules spread to a larger area at the sample surface resulting in a larger number of desorbed analyte molecules. Another reason may be that the partial pressure of the dopant at lower flow rates is not sufficient for efficient ionization as presented earlier for a low flow rate APPI system.20 The nebulizing gas pressure (and flow rate) had no significant effect on the signal intensity at pressures of 5, 7, and 10 bar.
Figure 2. Solvent jet DCPI spectra of standards (10 pmol (caffeine 30 pmol) on the sample plate) using methanol/toluene (75/25, v/v) as spray solvent.
Figure S-3 in the Supporting Information.) According to the earlier presented APPI mechanism,6 the radical cation of toluene reacts with methanol or with residual water (in the case of 100% toluene) producing protonated methanol or water based clusters, which can react further with analytes via proton transfer reactions. The formation of molecular ions of analytes occurs most likely via the radical cation of toluene formed in the photoionization process or directly by 10 eV photons. The feasibility of the solvent jet DCPI method for larger molecules was tested using angiotensin II (MW 1045) as a standard compound and methanol/water/formic acid (50/50/0.1, v/v/v) as the spray solvent. The MS spectrum of angiotensin II showed protonated and doubly protonated molecules at m/z 1046.5 and m/z 523.9, respectively (Figure 3). Angiotensin was ionized with similar efficiency with and without the VUV lamp on, indicating that the ionization process is not photoionization. Furthermore, the use of dopant did not improve the ionization efficiency of angiotensin II, although its use in CPI is necessary for achieving high ionization efficiency for nonpolar compounds.17 In this case, the ionization of angiotensin II obviously takes place inside the heated CPI capillary similarly to the solvent assisted inlet ionization presented earlier by Pagnotti et al.,19 who showed that efficient ionization for large molecules can be achieved simply by injecting a sample directly into the heated capillary between atmosphere and the vacuum of MS. The ionization mechanism in our experiments is most likely based on statistical distribution of charges in aerosol droplets followed by the ion evaporation mechanism as suggested also for laser spray ionization by 3282
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Figure 3. DCPI spectrum of angiotensin II (100 pmol on the sample plate) measured by solvent jet DCPI with methanol/water/formic acid (50/50/0.1, v/v/v) as the spray solvent.
Trimpin et al.21 This could explain why peptides can be analyzed by DCPI. This is a clear advantage over ambient MS methods based on thermal desorption, such as DART, DAPCI, and DAPPI, which are not capable of analyzing large biomolecules. The limits of detection (LOD) were tested in full scan MS and MS/MS modes using verapamil and testosterone as model compounds. Transitions at m/z 455 → 303 and m/z 298 → 97 were used for determination of signal-to-noise (S/N) ratios for verapamil and testosterone, respectively. The LODs were measured with increasing sample amounts applied (1 μL) to the sample plate with intervals of 1 order of magnitude starting from 10 fmol in MS mode and from 1 fmol in MS/MS mode. The LODs were estimated to have an S/N ratio of 5. In MS mode, both 1 pmol of verapamil and 10 pmol of testosterone produced an S/N ratio of ∼20 (Figure 4A,B). In MS/MS mode, 10 fmol of verapamil produced an S/N ratio of 15 and 100 fmol of testosterone had an S/N ratio of 10 (Figure 4C,D). Thus, the estimated LODs were 0.25 pmol and 2.5 pmol in full scan MS mode and 3 fmol and 50 fmol in MS/MS mode, for verapamil and testosterone, respectively, indicating similar sensitivity as achieved with other ambient MS techniques.4,5 The spatial resolution of DCPI was studied by spraying methanol/chlorobenzene (70/30, v/v) on a printed wax surface. Wax was visibly (but not completely) removed from the surface when solvent was sprayed on it with a constantly moving sprayer. Figure 5 shows an example of the track caused by a perpendicular spray. The width of the track was measured at several spots from an image taken with an optical microscope, and the average width was used as the result. The maximal resolution was achieved by using small spray capillaries (outer capillary i.d. 180 μm and inner capillary i.d. 40 μm and o.d. 105 μm), spraying height of 0.5 mm, solvent flow rate of 3 μL mL−1, angle of 55°, and sample moving speed of 0.5 mm/s. On average, spatial resolution of 250 and 300 μm was achieved in perpendicular and parallel directions, respectively, showing the potential of DCPI for MS imaging. The feasibility of the solvent jet DCPI in analysis of polar and nonpolar compounds in sage (Salvia of f icinalis) leaves and chili pepper was tested. The DCPI spectrum of a sage leaf (Figure 6) showed an intense ion at m/z 286, which we suspect to be due to the [M − HCOOH]+ ion of carnosic acid and/or the [M − CO2]+ ion of carnosol. The ions at m/z 333 and m/z 331 may represent the respective [M + H]+ ions. The m/z 205 ion is most likely due to nonpolar sesquiterpene [M + H]+ ions (e.g., humulene or caryophyllene). According to a previous report, concentrations of carnosic acid and carnosol are high in
Figure 4. DCPI scan spectra of 1 pmol of verapamil (A) and 10 pmol of testosterone (B) and DPCI MS/MS spectra of 10 fmol of verapamil (C) and 100 fmol of testosterone (D).
Figure 5. Spatial resolution of solvent jet DCPI measured by wax removal technique. Methanol/chlorobenzene (70/30) was used as the spray solvent.
fresh sage leaves: 12.4 and 1.66 mg/g, respectively.22 Similarly, the total amount of hydrocarbon sesquiterpenes in sage leaves 3283
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DART and DAPPI are suitable for analysis of polar and nonpolar compounds, they are not suitable for the analysis of larger biomolecules. Furthermore, their spatial resolution is limited due to a thermal desorption process. Recently introduced LAAPPI provides high spatial resolution and high sensitivity for nonpolar compounds but uses an expensive infrared laser.8 The solvent jet DCPI provides a new and efficient ambient MS method combining several advantages, which all cannot be achieved simultaneously with current ambient MS methods: high sensitivity for nonpolar and polar compounds with LODs down to the low fmol level, capability of analyzing small and large molecules, good spatial resolution (250 μm), and easy setup. In DCPI, polar and nonpolar compounds are ionized via APPI,6 while larger compounds such as peptides can be ionized similarly as in solvent assisted inlet ionization.19 These advantages of DCPI make it a potential new and universal method not only for ambient MS but also for MS imaging.
Figure 6. DCPI spectrum of sage (Salvia of f icinalis) leaf. Methanol/ chlorobenzene (70/30) was used as the spray solvent.
is in the range of mg/g.23 When compared to recently reported LAAPPI-MS spectra of sage leaves,16 DCPI shows the same main species as LAAPPI-MS. The sage leaf analysis shows that DCPI is suited for the analysis of low polarity compounds from plant tissue, and the results correlate well with a previously applied ambient MS method. The DCPI spectrum of chili pepper (Capsicum chinense var. Bonda ma Jacques) measured from the placental part shows clearly all the major capsaicinoids as their protonated molecules: capsaicin (m/z 306), dihydrocapsaicin (m/z 308), nordihydrocapsaicin, nonivamide, or both (m/z 294), homocapsaicin (m/z 320), and homodihydrocapsaicin (m/z 322) (Figure 7). The ion at m/z 137 is a typical vanillyl moiety
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org/.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: risto.kostiainen@helsinki.fi. Tel: +358-29 4159134. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the Academy of Finland (Projects 218150, 255559, 257316, and 251575) for financial support. REFERENCES
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Figure 7. DCPI spectrum of Capsicum chinense chili pepper (var. Bonda ma Jacques) measured from the placental part. Methanol/ chlorobenzene (70/30) was used as the spray solvent.
fragment of capsaicinoids.24,25 The capsaicinoids detected by DCPI are in accordance with the earlier studies made by LC-MS using ESI24 and APCI.25 Of the ambient methods, DART2 and low-temperature plasma (LTP) ionization26 have been applied to the analysis of capsaicinoids in chili peppers. DART was capable of detecting the major capsaicinoids (capsaicin, dihydrocapsaicin, and nonivamide) in a red pepper pot, and LTP was capable of detecting capsaicin in a Jalapeño chili pepper indicating that DCPI is at least as efficient as DART in the detection of capsaicinoids directly from chili pepper.
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CONCLUSIONS The previously introduced ambient MS methods have shown their potential in many applications; however, at the same time, they are limited to certain types of compounds, or their constructions may be complex and expensive. For example, DESI is capable of analyzing polar and ionic, as well as small and large, molecules with high sensitivity, but the ionization efficiency for nonpolar compounds tends to be poor. Although 3284
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Analytical Chemistry (14) Kauppila, T. J.; Arvola, V.; Haapala, M.; Pól, J.; Aalberg, L.; Saarela, V.; Franssila, S.; Kotiaho, T.; Kostiainen, R. Rapid Commun. Mass Spectrom. 2008, 22, 979−985. (15) Pól, J.; Vidová, V.; Kruppa, G.; Kobliha, V.; Novák, P.; Lemr, K.; Kotiaho, T.; Kostiainen, R.; Havlíček, V.; Volný, M. Anal. Chem. 2009, 81, 8479−8487. (16) Vaikkinen, A.; Shrestha, B.; Koivisto, J.; Kostiainen, R.; Vertes, A.; Kauppila, T. J. Rapid Commun. Mass Spectrom. 2014, 28, 2490− 2496. (17) Haapala, M.; Suominen, T.; Kostiainen, R. Anal. Chem. 2013, 85, 5715−5719. (18) Costa, A. B.; Cooks, R. G. Chem. Phys. Lett. 2008, 464, 1−8. (19) Pagnotti, V. S.; Chubatyi, N. D.; McEwen, C. N. Anal. Chem. 2011, 83, 3981−3985. (20) Kauppila, T. J.; Ö stman, P.; Marttila, S.; Ketola, R. A.; Kotiaho, T.; Franssila, S.; Kostiainen, R. Anal. Chem. 2004, 76, 6797−6801. (21) Trimpin, S.; Wang, B.; Inutan, E. D.; Li, J.; Lietz, C. B.; Harron, A.; Pagnotti, V. S.; Sardelis, D.; McEwen, C. N. J. Am. Soc. Mass Spectrom. 2012, 23, 1644−1660. (22) Okamura, N.; Fujimoto, Y.; Kuwabara, S.; Yagi, A. J. Chromatogr. A 1994, 679, 381−386. (23) Santos-Gomes, P. C.; Fernandes-Ferreira, M. J. Agric. Food Chem. 2001, 49, 2908−2916. (24) Bijttebier, S.; Zhani, K.; D’Hondt, E.; Noten, B.; Hermans, N.; Apers, S.; Voorspoels, S. J. Agric. Food Chem. 2004, 62, 4812−4831. (25) Schweiggert, U.; Carle, R.; Schieber, A. Anal. Chim. Acta 2006, 557, 236−244. (26) Maldonado-Torres, M.; López-Hernández, J. F.; JiménezSandoval, P.; Winkler, R. J. Proteomics 2014, 102, 60−65.
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Solvent Jet Desorption Capillary Photoionization-Mass Spectrometry Markus Haapala, Jaakko Teppo, Elisa Ollikainen, Iiro Kiiski, Anu Vaikkinen, Tiina J. Kauppila, and Risto Kostiainen* Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland S Supporting Information *
ABSTRACT: A new ambient mass spectrometry method, solvent jet desorption capillary photoionization (DCPI), is described. The method uses a solvent jet generated by a coaxial nebulizer operated at ambient conditions with nitrogen as nebulizer gas. The solvent jet is directed onto a sample surface, from which analytes are extracted into the solvent and ejected from the surface in secondary droplets formed in collisions between the jet and the sample surface. The secondary droplets are directed into the heated capillary photoionization (CPI) device, where the droplets are vaporized and the gaseous analytes are ionized by 10 eV photons generated by a vacuum ultraviolet (VUV) krypton discharge lamp. As the CPI device is directly connected to the extended capillary inlet of the MS, high ion transfer efficiency to the vacuum of MS is achieved. The solvent jet DCPI provides several advantages: high sensitivity for nonpolar and polar compounds with limit of detection down to low fmol levels, capability of analyzing small and large molecules, and good spatial resolution (250 μm). Two ionization mechanisms are involved in DCPI: atmospheric pressure photoionization, capable of ionizing polar and nonpolar compounds, and solvent assisted inlet ionization capable of ionizing larger molecules like peptides. The feasibility of DCPI was successfully tested in the analysis of polar and nonpolar compounds in sage leaves and chili pepper.
A
namely, desorption APPI (DAPPI)7 in 2007 and laser ablation APPI (LAAPPI)8 in 2012. In DAPPI, the analytes are thermally desorbed to gas-phase by directing a narrow hot solvent vapor jet to the surface of a solid sample.7,9 The analytes in the gas-phase are photoionized similarly as in APPI by a vacuum ultraviolet (VUV) lamp emitting 10.0 and 10.6 eV photons. In order to achieve high ionization efficiency, the ionization energy of the solvent must be below the energy of the photons. Suitable solvents are, for example, toluene, chlorobenzene, anisole, and acetone. DAPPI is a very simple, stable, and sensitive method for the analysis of nonpolar and polar compounds from surfaces, and it has successfully been applied, for example, in drug,7 environmental,10 food,10,11 pyrogenic black carbon,12 and forensic analysis.13,14 MS imaging of plant leaves and mouse brain tissue sections has also been demonstrated by DAPPI.15 However, the spatial resolution in DAPPI-MS imaging is limited to about 1 mm due to the thermal desorption process. The thermal desorption also limits the range of analyzable compounds to small and thermostable analytes.7,11 In the recently introduced LAAPPI, the analytes are ablated from water-rich surfaces with an infrared (IR) laser.8 Above the sample surface, the ablated sample plume is intercepted with an orthogonal hot solvent vapor jet (e.g., toluene or anisole).
mbient mass spectrometry (MS), which enables rapid, direct analysis of biological sample surfaces in their native states without any pretreatment, is an important new technique in bioanalysis. In ambient MS, compounds are desorbed directly from surfaces and ionized outside a mass spectrometer using atmospheric pressure ionization techniques. The first and still most commonly used ambient MS techniques are desorption electrospray ionization (DESI)1 and direct analysis in real time (DART)2 introduced in 2004 and 2005, respectively. After this, over 30 variations have been introduced with different combinations of desorption and ionization methods. The sampling can be achieved by liquid spray based techniques, thermal desorption, plasma based techniques, lasers, or a solvent bridge method, while the most common ionization methods are electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), sonic spray ionization (SSI), and different kinds of plasma based methods.3−5 Ambient MS methods have mostly been applied to the analysis of polar and ionic compounds, as most ambient ionization techniques provide high ionization efficiency for these types of compounds via the ion evaporation process (e.g., DESI, desorption sonic spray ionization) or via gas-phase reactions (e.g., DAPCI, DART, plasma based methods). Among ionization methods, APPI is an exception, as it provides high ionization efficiency not only for polar compounds via proton transfer but also for nonpolar compounds via charge exchange.6 Two ambient MS methods based on APPI have been presented, © 2015 American Chemical Society
Received: November 11, 2014 Accepted: February 25, 2015 Published: February 25, 2015 3280
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Analytical Chemistry The ablated analytes are desolvated, ionized in the gas-phase by APPI, and transferred into the MS. The unique feature of LAAPPI over other ambient MS techniques is that it provides high sensitivity for nonpolar compounds with good, about 300 μm lateral resolution comparable to, for example, matrixassisted laser desorption ionization. LAAPPI has successfully been applied in imaging of nonpolar compounds in sage leaves.16 However, the experimental setup is relatively complex and requires the use of an expensive IR laser. The aim of this work was to develop a new universal ambient MS method, solvent jet desorption capillary photoionization (DCPI), capable of ionizing efficiently nonpolar and polar, as well as small and large molecules, with high lateral resolution and a simple and cheap setup. In order to achieve this goal, we used a solvent jet at ambient temperature and pressure for the desorption of the analytes from the sample surface and our recently introduced capillary photoionization (CPI)17 source for ionization. The feasibility of DCPI in the analysis of polar and nonpolar compounds from sage leaves and chili pepper is demonstrated.
Figure 1. Schematic of the solvent jet DCPI setup (not to scale).
Cambridge, UK) emitting 10.0 eV (124 nm) and 10.6 eV (117 nm) photons was used to initiate the ionization. The CPI capillary was heated using a Biltema 20-3917 soldering iron (Biltema Finland Ltd., Helsinki, Finland), and the temperature was set to 300 °C with a temperature controller (Omega CN7500, OMEGA Engineering Ltd., Manchester, UK). In addition, the outer end of the CPI capillary was heated with a power of 3.0 W using a resistance wire heater and an external power source (Iso-Tech IPS-603, RS Components Ltd., Northants, UK). The mass spectrometer used in the experiments was an Agilent 6330 ion trap (Agilent Technologies, Santa Clara, CA) with a commercial capillary extension (KR Analytical Ltd., Sandbach, UK) connected to the front of the glass inlet capillary. The CPI device was connected to the capillary extension by inserting the extension a few millimeters into the CPI capillary. The capillary extension piece was heated with nitrogen (AGA Ltd., Espoo, Finland) from the drying gas line of the MS with a flow rate of 4.0 L min−1 and a temperature setting of 285 °C. The mass spectrometer was controlled, and the data was analyzed with Agilent 6300 Series TrapControl Version 6.1 (Build 92) and DataAnalysis for 6300 Series Ion Trap LC/MS Version 3.4 (Build 192) software, respectively. The MS measurements were done in positive ion mode. The solvent jet nebulizer for the desorption of the analytes was made using a syringe pump (Harvard Apparatus, Holliston, MA) connected to a fused silica solvent capillary (50 μm i.d., 150 μm o.d., Polymicro Technologies Inc., Phoenix, AZ), which was inserted coaxially inside a nebulizing gas PEEK capillary (250 μm i.d., 1/16 in. o.d., VICI AG International, Schenkon, Switzerland) connected to a PEEK t-piece (VICI AG International). Toluene, chlorobenzene, methanol, and water in different compositions were used as spray solvents at 10 μL min−1 flow rate. The detailed solvent compositions are presented separately for each experiment in the Results and Discussion section. Nebulizing gas (nitrogen) was delivered coaxially between the solvent and nebulizing gas capillaries with a pressure of 10 bar. The nebulizing gas and solvent produced a solvent jet, which was directed to the sample spot on the surface. xyz stages (Märzhäuser Wetzlar GmbH & Co. KG, Wetzlar, Germany) were used to adjust the positions of the spraying capillary and the sample plate. The sprayer for testing the spatial resolution was similar to the one used for MS measurements, but several combinations of outer and inner capillary dimensions were tested. The i.d.’s of the outer capillary were 180, 220, or 250 μm; the o.d.’s of the inner capillary were 147 and 105 μm, and respective i.d.’s of the inner capillary were 50 and 40 μm. The test surface was black
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EXPERIMENTAL SECTION Chemicals and Samples. Water was purified with a Milli-Q water purification system (Millipore, Molsheim, France). Testosterone, angiotensin II, LC-MS grade methanol, and HPLC grade toluene and chlorobenzene were from SigmaAldrich (Steinheim, Germany). Verapamil hydrochloride was purchased from Aldrich Chem. Co. (Milwaukee, WI), benzo[a]pyrene (B[a]P) from Tamro (Vantaa, Finland), and caffeine from University Pharmacy (Helsinki, Finland). Fresh sage (Salvia of f icinalis) herb in a growing pot was purchased from a local supermarket, and chili peppers were home-cultivated by M.H. Stock solutions containing 10 mmol L−1 verapamil, testosterone, and caffeine were prepared in methanol, and a stock solution containing 10 mmol L−1 benzo[a]pyrene was prepared in toluene. From these, 10 μmol L−1 working solutions containing all four standard compounds were prepared in methanol/water (1/1, v/v) and further diluted to appropriate concentrations with methanol/water (1/1, v/v). A standard solution of angiotensin II at a concentration of 100 μmol L−1 was prepared in methanol/water (1/1, v/v) including 0.1% of formic acid. For each sample spot, one microliter of sample solution was applied on a poly(methyl methacrylate) (PMMA) or poly(tetrafluoroethylene) (PTFE) sample plate and left to dry at ambient temperature. Sage leaves and pieces of chili pepper were attached on microscope glass slides with tape. The PMMA and PTFE sample plates were from Vink Finland Ltd. (Kerava, Finland), and microscope glass slides were from Gerhard Menzel GmbH (Braunschweig, Germany). PMMA plates and microscope glass slides were used as such, whereas pieces of PTFE were attached on PMMA plates with doublesided tape (Scotch, Cergy-Pontoise, France). Instrumentation. The solvent jet DCPI device consisted of a sprayer, a sampling plate, and a capillary photoionization (CPI) interface (Figure 1). The CPI interface consisted of a 1.5 mm i.d. stainless steel (SS) capillary (CPI capillary) between the atmosphere and the vacuum of the MS with a 1 mm wide and 15 mm long opening, a flat SS plate with a similar opening hard-soldered on the capillary, a 3 mm thick MgF2 window (Thorlabs Sweden AB, Gothenburg, Sweden), a top plate with an 18 mm circular opening, and Kalrez o-rings (Tiivistekeskus Ltd., Vantaa, Finland) used as seals to hold the MgF2 window in place. An rf krypton VUV lamp (Heraeus Noblelight Analytics Ltd., 3281
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The effect of desorption solvent composition on the signal intensities of the standard compounds was studied by comparing methanol/toluene as the spray solvent with 100/0, 75/25, 50/50, 25/75, and 0/100 (v/v) ratios. The results in Figure S-3, Supporting Information, show that all test compounds were efficiently ionized with methanol/toluene ratios of 75/25, 50/50, 25/75, and 0/100 so that the best sensitivity was achieved with the ratio of 25/75. With 100% methanol, only verapamil was substantially ionized, albeit less efficiently than with the addition of toluene. This indicates that the presence of a solvent with ionization energy below 10 eV was necessary for achieving high sensitivity. Verapamil and testosterone having relatively high proton affinities produced intense protonated molecules by proton transfer reactions. Caffeine and benzo[a]pyrene having relatively low ionization energies produced protonated molecules and molecular ions formed by charge exchange reactions (Figure 2). (See also
printing wax printed with a Xerox Phaser 8560 (Xerox Corporation, Wilsonville, OR). The sprayer was moved by a computer controlled xyz stage (Thorlabs Sweden AB) using an in-house developed software. Speeds of 0.5 and 1.0 mm/s and directions perpendicular and parallel to the spray direction were tested. The sprayer was situated either 0.5 or 1.0 mm above the test surface. The image of the resulting track on the wax surface was captured with an optical microscope, and spatial resolution was measured using Motic Images Plus 2.0 software (Motic Deutschland GmbH, Wetzlar, Germany).
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RESULTS AND DISCUSSION The desorption process in DCPI is suggested to be similar to the droplet pick-up mechanism presented earlier for DESI.18 The narrow solvent jet generated at ambient temperature by a coaxial nebulizer of the DCPI device (Figure 1) is directed onto the sample surface, which is first prewetted by forming a solvent film, to which the surface analytes are dissolved. After this, secondary droplets containing the analytes are formed in collisions between the solvent jet and the solvent film. The secondary droplets ejected from the surface are directed into the heated CPI device, where the droplets are vaporized and gaseous analytes are ionized by VUV photons. The ionization may also take place similarly as in the solvent assisted inlet ionization presented earlier by Pagnotti et al.19 As the CPI device is directly connected to the extended capillary of the MS, high ion transfer efficiency to the MS and therefore improved sensitivity can be achieved. However, operation parameters, which have a significant effect on the performance of DCPI, must be carefully optimized. The positions of the nebulizer nozzle and sample spot in relation to the CPI capillary inlet were adjusted for maximum sensitivity and stability. The optimal performance of the system was achieved with approximately 1 mm distance between the nebulizer nozzle and the sampling spot. The short nebulizer-tosurface distance provides a more efficient desorption process than longer distances. The sampling spot was positioned onaxis as close to the CPI inlet as possible in order to ensure maximum transfer of secondary droplets into CPI. The effect of the angle of the nebulizer in relation to the surface was tested with angles of 15°, 25°, 35°, 45°, 55°, and 65°. (See Figure S-1 in the Supporting Information.) The highest sensitivity was achieved with the lowest angles (15° and 25°), which provide improved transfer of secondary droplets into CPI and desorption of the analytes from a larger area than the larger nebulizer-surface angles. The effect of solvent (methanol/ toluene, 70/30, v/v) flow rate was tested with flow rates of 1, 2, 4, 6, 8, 10, 14, and 20 μL min−1. (See Figure S-2 in the Supporting Information.) With all the test compounds (verapamil, testosterone, caffeine, and benzo[a]pyrene), the signal intensities increased when the solvent flow rate was increased from 1 to 4 μL min−1 and maximum sensitivity was achieved at flow rates of 4−10 μL min−1. At higher flow rates (14 and 20 μL min−1), the signal intensities started to decrease. The increase of signal intensity is partly because at higher flow rates the solvent molecules spread to a larger area at the sample surface resulting in a larger number of desorbed analyte molecules. Another reason may be that the partial pressure of the dopant at lower flow rates is not sufficient for efficient ionization as presented earlier for a low flow rate APPI system.20 The nebulizing gas pressure (and flow rate) had no significant effect on the signal intensity at pressures of 5, 7, and 10 bar.
Figure 2. Solvent jet DCPI spectra of standards (10 pmol (caffeine 30 pmol) on the sample plate) using methanol/toluene (75/25, v/v) as spray solvent.
Figure S-3 in the Supporting Information.) According to the earlier presented APPI mechanism,6 the radical cation of toluene reacts with methanol or with residual water (in the case of 100% toluene) producing protonated methanol or water based clusters, which can react further with analytes via proton transfer reactions. The formation of molecular ions of analytes occurs most likely via the radical cation of toluene formed in the photoionization process or directly by 10 eV photons. The feasibility of the solvent jet DCPI method for larger molecules was tested using angiotensin II (MW 1045) as a standard compound and methanol/water/formic acid (50/50/0.1, v/v/v) as the spray solvent. The MS spectrum of angiotensin II showed protonated and doubly protonated molecules at m/z 1046.5 and m/z 523.9, respectively (Figure 3). Angiotensin was ionized with similar efficiency with and without the VUV lamp on, indicating that the ionization process is not photoionization. Furthermore, the use of dopant did not improve the ionization efficiency of angiotensin II, although its use in CPI is necessary for achieving high ionization efficiency for nonpolar compounds.17 In this case, the ionization of angiotensin II obviously takes place inside the heated CPI capillary similarly to the solvent assisted inlet ionization presented earlier by Pagnotti et al.,19 who showed that efficient ionization for large molecules can be achieved simply by injecting a sample directly into the heated capillary between atmosphere and the vacuum of MS. The ionization mechanism in our experiments is most likely based on statistical distribution of charges in aerosol droplets followed by the ion evaporation mechanism as suggested also for laser spray ionization by 3282
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Figure 3. DCPI spectrum of angiotensin II (100 pmol on the sample plate) measured by solvent jet DCPI with methanol/water/formic acid (50/50/0.1, v/v/v) as the spray solvent.
Trimpin et al.21 This could explain why peptides can be analyzed by DCPI. This is a clear advantage over ambient MS methods based on thermal desorption, such as DART, DAPCI, and DAPPI, which are not capable of analyzing large biomolecules. The limits of detection (LOD) were tested in full scan MS and MS/MS modes using verapamil and testosterone as model compounds. Transitions at m/z 455 → 303 and m/z 298 → 97 were used for determination of signal-to-noise (S/N) ratios for verapamil and testosterone, respectively. The LODs were measured with increasing sample amounts applied (1 μL) to the sample plate with intervals of 1 order of magnitude starting from 10 fmol in MS mode and from 1 fmol in MS/MS mode. The LODs were estimated to have an S/N ratio of 5. In MS mode, both 1 pmol of verapamil and 10 pmol of testosterone produced an S/N ratio of ∼20 (Figure 4A,B). In MS/MS mode, 10 fmol of verapamil produced an S/N ratio of 15 and 100 fmol of testosterone had an S/N ratio of 10 (Figure 4C,D). Thus, the estimated LODs were 0.25 pmol and 2.5 pmol in full scan MS mode and 3 fmol and 50 fmol in MS/MS mode, for verapamil and testosterone, respectively, indicating similar sensitivity as achieved with other ambient MS techniques.4,5 The spatial resolution of DCPI was studied by spraying methanol/chlorobenzene (70/30, v/v) on a printed wax surface. Wax was visibly (but not completely) removed from the surface when solvent was sprayed on it with a constantly moving sprayer. Figure 5 shows an example of the track caused by a perpendicular spray. The width of the track was measured at several spots from an image taken with an optical microscope, and the average width was used as the result. The maximal resolution was achieved by using small spray capillaries (outer capillary i.d. 180 μm and inner capillary i.d. 40 μm and o.d. 105 μm), spraying height of 0.5 mm, solvent flow rate of 3 μL mL−1, angle of 55°, and sample moving speed of 0.5 mm/s. On average, spatial resolution of 250 and 300 μm was achieved in perpendicular and parallel directions, respectively, showing the potential of DCPI for MS imaging. The feasibility of the solvent jet DCPI in analysis of polar and nonpolar compounds in sage (Salvia of f icinalis) leaves and chili pepper was tested. The DCPI spectrum of a sage leaf (Figure 6) showed an intense ion at m/z 286, which we suspect to be due to the [M − HCOOH]+ ion of carnosic acid and/or the [M − CO2]+ ion of carnosol. The ions at m/z 333 and m/z 331 may represent the respective [M + H]+ ions. The m/z 205 ion is most likely due to nonpolar sesquiterpene [M + H]+ ions (e.g., humulene or caryophyllene). According to a previous report, concentrations of carnosic acid and carnosol are high in
Figure 4. DCPI scan spectra of 1 pmol of verapamil (A) and 10 pmol of testosterone (B) and DPCI MS/MS spectra of 10 fmol of verapamil (C) and 100 fmol of testosterone (D).
Figure 5. Spatial resolution of solvent jet DCPI measured by wax removal technique. Methanol/chlorobenzene (70/30) was used as the spray solvent.
fresh sage leaves: 12.4 and 1.66 mg/g, respectively.22 Similarly, the total amount of hydrocarbon sesquiterpenes in sage leaves 3283
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DART and DAPPI are suitable for analysis of polar and nonpolar compounds, they are not suitable for the analysis of larger biomolecules. Furthermore, their spatial resolution is limited due to a thermal desorption process. Recently introduced LAAPPI provides high spatial resolution and high sensitivity for nonpolar compounds but uses an expensive infrared laser.8 The solvent jet DCPI provides a new and efficient ambient MS method combining several advantages, which all cannot be achieved simultaneously with current ambient MS methods: high sensitivity for nonpolar and polar compounds with LODs down to the low fmol level, capability of analyzing small and large molecules, good spatial resolution (250 μm), and easy setup. In DCPI, polar and nonpolar compounds are ionized via APPI,6 while larger compounds such as peptides can be ionized similarly as in solvent assisted inlet ionization.19 These advantages of DCPI make it a potential new and universal method not only for ambient MS but also for MS imaging.
Figure 6. DCPI spectrum of sage (Salvia of f icinalis) leaf. Methanol/ chlorobenzene (70/30) was used as the spray solvent.
is in the range of mg/g.23 When compared to recently reported LAAPPI-MS spectra of sage leaves,16 DCPI shows the same main species as LAAPPI-MS. The sage leaf analysis shows that DCPI is suited for the analysis of low polarity compounds from plant tissue, and the results correlate well with a previously applied ambient MS method. The DCPI spectrum of chili pepper (Capsicum chinense var. Bonda ma Jacques) measured from the placental part shows clearly all the major capsaicinoids as their protonated molecules: capsaicin (m/z 306), dihydrocapsaicin (m/z 308), nordihydrocapsaicin, nonivamide, or both (m/z 294), homocapsaicin (m/z 320), and homodihydrocapsaicin (m/z 322) (Figure 7). The ion at m/z 137 is a typical vanillyl moiety
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org/.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: risto.kostiainen@helsinki.fi. Tel: +358-29 4159134. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the Academy of Finland (Projects 218150, 255559, 257316, and 251575) for financial support. REFERENCES
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Figure 7. DCPI spectrum of Capsicum chinense chili pepper (var. Bonda ma Jacques) measured from the placental part. Methanol/ chlorobenzene (70/30) was used as the spray solvent.
fragment of capsaicinoids.24,25 The capsaicinoids detected by DCPI are in accordance with the earlier studies made by LC-MS using ESI24 and APCI.25 Of the ambient methods, DART2 and low-temperature plasma (LTP) ionization26 have been applied to the analysis of capsaicinoids in chili peppers. DART was capable of detecting the major capsaicinoids (capsaicin, dihydrocapsaicin, and nonivamide) in a red pepper pot, and LTP was capable of detecting capsaicin in a Jalapeño chili pepper indicating that DCPI is at least as efficient as DART in the detection of capsaicinoids directly from chili pepper.
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CONCLUSIONS The previously introduced ambient MS methods have shown their potential in many applications; however, at the same time, they are limited to certain types of compounds, or their constructions may be complex and expensive. For example, DESI is capable of analyzing polar and ionic, as well as small and large, molecules with high sensitivity, but the ionization efficiency for nonpolar compounds tends to be poor. Although 3284
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