- Email: [email protected]
Negative ion laser ablation glow discharge-time of flight mass spectrometry of organic molecules
International Journal of Mass Spectrometry 315 (2012) 60–65
Contents lists available at SciVerse ScienceDirect
International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Negative ion laser ablation glow discharge-time of flight mass spectrometry of organic molecules G. Lotito, D. Günther ∗ Laboratory of Inorganic Chemistry, ETH Zurich, 8093 Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 20 December 2011 Received in revised form 28 February 2012 Accepted 28 February 2012 Available online 7 March 2012 Keywords: Negative ion Pulsed glow discharge Afterglow Laser desorption MALDI
a b s t r a c t Laser ablation glow discharge-time of flight-mass spectrometry (LAGD-TOFMS) provides structural and molecular information when analyzing organic molecules. This study focused on negative ion detection of alpha-cyano-4-hydroxycinnamic acid, phosphatidylethanolamine, and reserpine using LAGD-TOFMS. A laser energy of few tens of J, suggesting a desorption process, was used to introduce the material into the glow discharge. It was found that phosphatidylethanolamine and reserpine provided a higher sensitivity in negative ion mode compared to positive ion mode. For comparison, the same compounds were analyzed using matrix assisted laser desorption ionization (MALDI). The results also indicate that LAGD-TOFMS has a less matrix dependency behavior than MALDI. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Analytical GD has been extensively used for trace element analysis at concentrations as low as ng/g in conducting samples [1]. By using a radiofrequency (rf) GD it is also possible to analyze nonconductive samples [2,3]. Recently, the analysis of polymers using an rf-GD has been reported [4] which demonstrated the possibility of molecular depth profiling of thin films made of polymers. Pulsed GD has also been reported and offers different temporal regimes characterized by different ionization processes and thus different information about the analytes [5,6]. The majority of mass spectrometric analyses have been carried out using positive ions, especially in argon plasmas where most of the ions are positive and the negative charges are carried almost totally by electrons [7]. Since electrons have a higher mobility than the larger positive ions, this gives rise to a sheath near grounded surfaces (see Fig. 1) [8]. This sheath has a potential gradient which accelerates positive ions and decelerates negative charge carriers through the sheath. Because of this gradient and due to the small number of negative ions present, measurements with a mass spectrometer through a grounded orifice are not expected to provide high signal intensities for negative ions. In case of a pulsed GD, during the afterglow (or afterpeak), which is the time following the end of the applied power pulse, the electron temperature drops rapidly along with the electron number density. This allows the
∗ Corresponding author. Tel.: +41 44 633 45 23; fax: +41 44 633 10 71. E-mail address: [email protected] (D. Günther). 1387-3806/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijms.2012.02.027
formation of negative ions which can be transported to a grounded sampling orifice [9–11]. Negative ions in a pulsed GD are primarily formed through electron attachment, dissociative electron attachment, and ion pair formation [12]. In pulsed rf GD-MS the detection of negative ions has been shown to provide additional information, in particular in measurements of halogen-containing compounds and complementary structural information for molecular ions [9]. This study was focused on the formation of negative ions in laser ablation coupled to a pulsed GD and a time-of-flight mass spectrometer [13]. A laser ablation system equipped with a nitrogen laser emitting light with a wavelength of 337 nm was used to introduce alpha-cyano-4-hydroxycinnamic acid, phosphatidylethanolamine, and reserpine into a pulsed glow discharge. As previously mentioned, a laser pulse energy of a few tens of J was used, suggesting a desorption rather than an ablation process. Following the laser desorption the neutral analytes were introduced into the pulsed GD plasma for post-ionization. Desorption and ionization were thus separated in time and space. To demonstrate the performance of LAGD-TOFMS, the spectra were compared to measurements of the same samples using MALDI. 2. Experimental 2.1. Instrumentation The details of the setup and operating conditions used have been described elsewhere [14]. Here only a few of the important parameters are reported. For the LAGD setup, the pressure used ranged between 36 and 45 Pa and the voltage applied between the
G. Lotito, D. Günther / International Journal of Mass Spectrometry 315 (2012) 60–65
61
Fig. 1. Schematics is shown displaying different spatial regions of a pulsed GD when the voltage is applied to the electrodes. The sheath near the grounded anode and the negative glow, where the majority of exciting and ionizing processes occur, are depicted. After the end of the applied voltage, during the afterglow, the drop of the electron temperature and density causes the collapse of the anode sheath favoring the transport of negative ions in the sampling region.
electrodes was 1 kV. The measurements reported here were acquired in negative ion mode, which was accomplished by switching the polarity of the power supply (TPS, Tofwerk AG, Thun). 2.2. Sample preparation The samples were prepared according to common MALDI protocols, with alpha-cyano-4-hydroxycinnamic acid (CHCA) (Sigma–Aldrich, St. Louis, MO), which has formula of C10 H7 O3 N and a monoisotopic molecular mass of 189.04 u serving as the matrix. Solutions of CHCA were prepared by diluting 20 mg in 700 L of acetonitrile and 100 L of 0.1% trifluoroacetic acid, giving a concentration of few hundreds nmol/L. Phosphatidylethanolamine (PE) (Sigma–Aldrich, St. Louis, MO), molecular formula of C41 H78 NO8 P and monoisotopic molecular mass of 743.55 u (see its structural formula in Fig. 2, and for other two compounds see [14]), was mixed with CHCA at a concentration of 50% (mol/mol). Reserpine (Sigma–Aldrich, St. Louis, MO), formula of C33 H40 N2 O9 and
monoisotopic molecular mass of 608.27 u, was mixed at 1, 10, and 50% (mol/mol) with CHCA. The concentrations of these compounds ranged between few tens to a few hundreds of nmol/L.
3. Results and discussion The analytes phosphatidylethanolamine and reserpine were measured using LAGD and MALDI. It is important to note that the high concentrations of the analytes were used in order to provide a high signal to background ratio when using LAGD. For comparison, the samples at the same concentrations were analyzed using MALDI, noting that these analyte concentrations were unusually high. However, the present work was focused on negative ion detection. Beside the formation of negative ions, matrix effects were investigated to confirm the reduced matrix dependent ionization in LAGD-TOFMS, which has been reported [14].
3.1. Phosphatidylethanolamine (PE)
Fig. 2. Molecular structure of PE.
Phosphatidylethanolamine in CHCA was analyzed in both negative-ion (NI) and positive-ion (PI) mode using LAGD and MALDI. This molecule contains a phosphate group, which is an oxoanion and therefore negatively charged and should be preferentially detectable in NI mode. To confirm this, measurements in PI mode provided no significant analyte signal in either LAGD or MALDI. In contrast, the measurements in LAGD NI mode provided a peak at m/z 743.55 assignable to the molecular ion of PE which appeared during the afterglow (Fig. 3). The GD only and CHCA only measurements provide evidence that the molecular information is not affected by interferences. Peaks assignable to the molecular ion of the matrix and to its deprotonated form are also shown in Fig. 3 (m/z 189.04). MALDI measurements of the same compound at 50% (mol/mol) with CHCA were performed in both PI and NI mode. However, the molecular ion was detected in NI mode only. Fig. 4 shows spectra of the analyte mixed with the matrix (PE in CHCA) and the matrix only (CHCA only). The enlarged figure shows the mass range close to the molecular ion of the analyte.
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G. Lotito, D. Günther / International Journal of Mass Spectrometry 315 (2012) 60–65
Fig. 3. Comparison between different spectra in the afterglow acquired by switching on the pulsed GD (labeled as GD only), by switching on the pulsed GD and introducing the matrix only using laser desorption (CHCA only), by switching on the pulsed GD and introducing the matrix mixed with the analyte by laser desorption (PE). The zoomed window shows the matrix molecular and deprotonated ion for all three modes of operation.
No interferences from the matrix affected the PE analyte signal. However, a pronounced matrix suppression effect (MSE) [15] was observed in the analyte spectrum (PE in CHCA), which is common for high concentrations of analytes. 3.2. Reserpine A series of mixtures containing reserpine in CHCA at 1%, 10%, and 50% (mol/mol) ratios were also analyzed in both PI and NI mode using LAGD and MALDI. In PI mode in LAGD a concentration of the analyte of 50% with CHCA was required for signal detection. However, in NI mode, detection of the molecular ion was observed at all concentrations. To illustrate the detection capabilities, the
afterglow spectra of the 10% (mol/mol) analyte concentration in CHCA are shown in Fig. 5. The figure also shows that the signal is not influenced by interferences from the GD or CHCA. The spectra show a peak assignable to the deprotonated molecule and other peaks assignable to other isotopologues which are all related to the sample containing the analyte. The spectral region between m/z 187 and 191 in Fig. 5 shows two peaks which can be assigned to the deprotonated molecule and the molecular ion of the matrix. MALDI detection of reserpine in PI mode was possible at all three concentrations, and MSE was also observed at all of these concentrations. However, in NI mode the molecule was not detected (Fig. 6).
Fig. 4. NI-MALDI spectra of PE 50% with CHCA (PE in CHCA) and CHCA only; the small box is an enlargement of the molecular ion region.
G. Lotito, D. Günther / International Journal of Mass Spectrometry 315 (2012) 60–65
63
Fig. 5. Comparison between different spectra in the afterglow acquired by turning only the pulsed GD (labeled as GD only), the pulsed GD and a sample containing only the matrix (CHCA only), the pulsed GD and the sample containing the matrix and the analyte (reserpine). The smaller window shows the mass region of the matrix molecular ion.
In both spectra the dominant peaks are from the matrix. The enlargement of the molecular ion region does not exhibit any peak assignable to the molecular ion or the deprotonated molecule. In MALDI it is common that in the opposite polarity to that in which MSE is observed, analyte signals are very weak or almost absent. This depends on the acidity of the neutral molecule which is strongly correlated to the acidity of the corresponding protonated or deprotonated species [16]. The results shown in this study indicate that in LAGD measurements no or minor MSE were observed, as previously reported [14]. This indicates that the ionization is less matrix dependent when compared to MALDI, since desorption and ionization in LAGD are physically and temporally separated, as shown in Fig. 7. The
signal intensities of the deprotonated and molecular ion of CHCA and deprotonated molecular ion of reserpine along with its 13 C isotopologue are plotted as a function of time (Fig. 7). At the beginning only the laser is turned on and no detection of those species is observed. This could be explained by the fact that at the experimental conditions used (pulsed laser energy at ca. 19 J and spot size of ca. 30 m) ionization is not taking place through laser desorption or if it does, then the ions generated do not reach the sampling area. As the pulsed GD is switched on (with the laser in operation) detection of those species is clearly visible. This could be due to the fact that the desorbed neutral species, whose ratio to the ion species (ions/neutrals) ranges between 10−5 and 10−3 [17], are post-ionized in the pulsed GD plasma. As the laser is turned off
Fig. 6. Spectrum of reserpine 50% with CHCA and spectrum of CHCA alone measured using MALDI-TOFMS in NI mode.
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G. Lotito, D. Günther / International Journal of Mass Spectrometry 315 (2012) 60–65
Fig. 7. Signal intensities of the deprotonated and molecular ion of matrix (CHCA) and deprotonated molecular ion and its 13 C isotopologue of reserpine as a function of time. During the first 9 s only the laser was in operation (LA), then the pulsed GD was additionally turned on until 23 s ca. and then the laser was turned off with only the pulsed GD in operation (GD).
Table 1 Comparison between intensities of peak area representing the deprotonated molecule of reserpine for different concentrations in LAGD and MALDI. Concentration reserpine in matrix (%)
LAGD signal intensity normalized to matrix (a.u.)
MALDI signal intensity (a.u.)
1 10 50
2.25 × 10−4 1.69 × 10−3 1.75 × 10−2
1.19 × 10+4 9.77 × 10+3 6.69 × 10+3
(at ca. 23 s) and only the pulsed GD is in operation, all the signal intensities decrease by several orders of magnitude because no introduction of those species to the plasma was taking place. A similar behavior was also observed for PE in CHCA (data not shown). To demonstrate the reduced matrix dependence three different concentrations of reserpine in CHCA were measured in both LAGD NI mode and MALDI PI mode (since in NI mode no detection was possible) and the area of the peak assigned to the deprotonated molecule of reserpine was normalized to the area of the peak assigned to the molecular ion of the matrix; such normalization was not possible in MALDI due to the MSE. Table 1 contains the intensities and relative analyte to matrix concentrations for both techniques. The qualitative trend observed in LAGD-TOFMS indicates a lower matrix dependent ionization behavior and potentially allow some quantification. This is achievable because desorption and ionization can be optimized separately. However, the current LAGD prototype requires more fundamental studies on improving the sensitivity, which is currently orders of magnitude lower than currently reported for MALDI [18,19].
4. Conclusions Negative mode ionization of matrix-assisted laser desorbed PE and reserpine analytes in CHCA during the afterglow of a pulsed GD was investigated. It was shown that the NI mode allows the detection of some molecular ions, which were not detectable in PI mode. Furthermore, indications of a reduced matrix dependent ionization behavior was observed in LAGD when compared to MALDI. The reduced matrix dependence is probably due to the separation of the desorption and ionization process. Therefore, the potential for a semi-quantitative analysis of organic compounds is very high, especially if the current low sensitivity can be improved.
Acknowledgments The authors would like to thank the European Community for financial support through GLADNET, a Marie Curie-RTN within the FP 6, and TOFWERK AG for providing the instrumentation used. The authors also would like to thank Dr. R. Knochenmuss for his advice and support and L. Bertschi of the MS service of ETH for having assisted in performing the MALDI measurements. The authors would also like to thank Dr. Henry Longerich for advice and revision of the manuscript.
References [1] V. Hoffmann, M. Kasik, P.K. Robinson, C. Venzago, Glow discharge mass spectrometry, Anal. Bioanal. Chem. 381 (2005) 173–188. [2] D.C. Duckworth, R. Kenneth Marcus, Radio frequency powered glow discharge atomization/ionization source for solids mass spectrometry, Anal. Chem. 61 (1989) 1879–1886. [3] R. Kenneth Marcus, Glow Discharge Spectroscopies, Plenum Press, New York, 1993. [4] N. Tuccitto, L. Lobo, A. Tempez, I. Delfanti, P. Chapon, S. Canulescu, N. Bordel, J. Michler, A. Licciardello, Pulsed radiofrequency glow discharge time-of-flight mass spectrometry for molecular depth profiling of polymer-based films, Rapid Commun. Mass Spectrom. 23 (2009) 549–556. [5] C.K. Pan, F.L. King, Ion formation processes in the afterpeak time regime of pulsed glow discharge plasmas, J. Am. Soc. Mass Spectrom. 4 (1993) 727–732. [6] V. Majidi, M. Moser, C. Lewis, W. Hang, F.L. King, Explicit chemical speciation by microsecond pulsed glow discharge time-of-flight mass spectrometry: concurrent acquisition of structural, molecular and elemental information, J. Anal. At. Spectrom. 15 (2000) 19–25. [7] B.L. Bent, W.W. Harrison, Negative ions of sputtered cathode species in a glow discharge, Int. J. Mass Spectrom. 37 (1981) 167–176. [8] R.A. Gottscho, Glow-discharge sheath electric fields: negative-ion, power, and frequency effects, Phys. Rev. A 36 (1987) 2233–2243. [9] S. Canulescu, J. Whitby, K. Fuhrer, M. Hohl, M. Gonin, T. Horvath, J. Michler, Potential analytical applications of negative ions from a pulsed radiofrequency glow discharge in argon, J. Anal. At. Spectrom. 24 (2009) 178–180. [10] L.J. Overzet, Y. Lin, L. Luo, Modeling and measurements of the negative ion flux from amplitude modulated RF discharges, J. Appl. Phys. 72 (1992) 5579–5592. [11] S. Canulescu, I. Molchan, C. Tauziede, A. Tempez, J.A. Whitby, G.E. Thompson, P. Skeldon, P. Chapon, J. Michler, Detection of negative ions in glow discharge mass spectrometry for analysis of solid specimens, Anal. Bioanal. Chem. 396 (2010) 2871–2879. [12] E. Stoffels, W.W. Stoffels, D. Vender, M. Haverlag, G.M.W. Kroesen, F.J. De Hoog, Negative ions in low pressure discharges, Contrib. Plasma Phys. 35 (1995) 331–357. [13] M. Tarik, G. Lotito, J. Whitby, J. Koch, K. Fuhrer, M. Gonin, J. Michler, J. Bolli, D. Günther, Development and fundamental investigation of laser ablation glow discharge time-of-flight mass spectrometry (LA-GD-TOFMS), Spectrochim. Acta B 64 (2009) 262–270. [14] G. Lotito, D. Günther, Tunable fragmentation of organic molecules in laser ablation glow discharge-time of flight mass spectrometry, Anal. Bioanal. Chem. (2011), doi:10.1007/s00216-011-5498-x.
G. Lotito, D. Günther / International Journal of Mass Spectrometry 315 (2012) 60–65 [15] R. Knochenmuss, Ion formation mechanisms in UV-MALDI, Analyst 131 (2006) 966–986. [16] R. Knochenmuss, R. Zenobi, MALDI ionization: the role of in-plume processes, Chem. Rev. 103 (2003) 441–452. [17] K. Dreisewerd, The desorption process in MALDI, Chem. Rev. 103 (2003) 395–425.
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[18] E. Nordhoff, H. Lehrach, J. Gobom, Exploring the limits and losses in MALDI sample preparation of attomole amounts of peptide mixtures, Int. J. Mass Spectrom. 268 (2007) 139–146. [19] J.H. Gross, Mass Spectrometry: A Textbook, second ed., Springer-Verlag, Berlin, 2004.
Contents lists available at SciVerse ScienceDirect
International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Negative ion laser ablation glow discharge-time of flight mass spectrometry of organic molecules G. Lotito, D. Günther ∗ Laboratory of Inorganic Chemistry, ETH Zurich, 8093 Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 20 December 2011 Received in revised form 28 February 2012 Accepted 28 February 2012 Available online 7 March 2012 Keywords: Negative ion Pulsed glow discharge Afterglow Laser desorption MALDI
a b s t r a c t Laser ablation glow discharge-time of flight-mass spectrometry (LAGD-TOFMS) provides structural and molecular information when analyzing organic molecules. This study focused on negative ion detection of alpha-cyano-4-hydroxycinnamic acid, phosphatidylethanolamine, and reserpine using LAGD-TOFMS. A laser energy of few tens of J, suggesting a desorption process, was used to introduce the material into the glow discharge. It was found that phosphatidylethanolamine and reserpine provided a higher sensitivity in negative ion mode compared to positive ion mode. For comparison, the same compounds were analyzed using matrix assisted laser desorption ionization (MALDI). The results also indicate that LAGD-TOFMS has a less matrix dependency behavior than MALDI. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Analytical GD has been extensively used for trace element analysis at concentrations as low as ng/g in conducting samples [1]. By using a radiofrequency (rf) GD it is also possible to analyze nonconductive samples [2,3]. Recently, the analysis of polymers using an rf-GD has been reported [4] which demonstrated the possibility of molecular depth profiling of thin films made of polymers. Pulsed GD has also been reported and offers different temporal regimes characterized by different ionization processes and thus different information about the analytes [5,6]. The majority of mass spectrometric analyses have been carried out using positive ions, especially in argon plasmas where most of the ions are positive and the negative charges are carried almost totally by electrons [7]. Since electrons have a higher mobility than the larger positive ions, this gives rise to a sheath near grounded surfaces (see Fig. 1) [8]. This sheath has a potential gradient which accelerates positive ions and decelerates negative charge carriers through the sheath. Because of this gradient and due to the small number of negative ions present, measurements with a mass spectrometer through a grounded orifice are not expected to provide high signal intensities for negative ions. In case of a pulsed GD, during the afterglow (or afterpeak), which is the time following the end of the applied power pulse, the electron temperature drops rapidly along with the electron number density. This allows the
∗ Corresponding author. Tel.: +41 44 633 45 23; fax: +41 44 633 10 71. E-mail address: [email protected] (D. Günther). 1387-3806/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijms.2012.02.027
formation of negative ions which can be transported to a grounded sampling orifice [9–11]. Negative ions in a pulsed GD are primarily formed through electron attachment, dissociative electron attachment, and ion pair formation [12]. In pulsed rf GD-MS the detection of negative ions has been shown to provide additional information, in particular in measurements of halogen-containing compounds and complementary structural information for molecular ions [9]. This study was focused on the formation of negative ions in laser ablation coupled to a pulsed GD and a time-of-flight mass spectrometer [13]. A laser ablation system equipped with a nitrogen laser emitting light with a wavelength of 337 nm was used to introduce alpha-cyano-4-hydroxycinnamic acid, phosphatidylethanolamine, and reserpine into a pulsed glow discharge. As previously mentioned, a laser pulse energy of a few tens of J was used, suggesting a desorption rather than an ablation process. Following the laser desorption the neutral analytes were introduced into the pulsed GD plasma for post-ionization. Desorption and ionization were thus separated in time and space. To demonstrate the performance of LAGD-TOFMS, the spectra were compared to measurements of the same samples using MALDI. 2. Experimental 2.1. Instrumentation The details of the setup and operating conditions used have been described elsewhere [14]. Here only a few of the important parameters are reported. For the LAGD setup, the pressure used ranged between 36 and 45 Pa and the voltage applied between the
G. Lotito, D. Günther / International Journal of Mass Spectrometry 315 (2012) 60–65
61
Fig. 1. Schematics is shown displaying different spatial regions of a pulsed GD when the voltage is applied to the electrodes. The sheath near the grounded anode and the negative glow, where the majority of exciting and ionizing processes occur, are depicted. After the end of the applied voltage, during the afterglow, the drop of the electron temperature and density causes the collapse of the anode sheath favoring the transport of negative ions in the sampling region.
electrodes was 1 kV. The measurements reported here were acquired in negative ion mode, which was accomplished by switching the polarity of the power supply (TPS, Tofwerk AG, Thun). 2.2. Sample preparation The samples were prepared according to common MALDI protocols, with alpha-cyano-4-hydroxycinnamic acid (CHCA) (Sigma–Aldrich, St. Louis, MO), which has formula of C10 H7 O3 N and a monoisotopic molecular mass of 189.04 u serving as the matrix. Solutions of CHCA were prepared by diluting 20 mg in 700 L of acetonitrile and 100 L of 0.1% trifluoroacetic acid, giving a concentration of few hundreds nmol/L. Phosphatidylethanolamine (PE) (Sigma–Aldrich, St. Louis, MO), molecular formula of C41 H78 NO8 P and monoisotopic molecular mass of 743.55 u (see its structural formula in Fig. 2, and for other two compounds see [14]), was mixed with CHCA at a concentration of 50% (mol/mol). Reserpine (Sigma–Aldrich, St. Louis, MO), formula of C33 H40 N2 O9 and
monoisotopic molecular mass of 608.27 u, was mixed at 1, 10, and 50% (mol/mol) with CHCA. The concentrations of these compounds ranged between few tens to a few hundreds of nmol/L.
3. Results and discussion The analytes phosphatidylethanolamine and reserpine were measured using LAGD and MALDI. It is important to note that the high concentrations of the analytes were used in order to provide a high signal to background ratio when using LAGD. For comparison, the samples at the same concentrations were analyzed using MALDI, noting that these analyte concentrations were unusually high. However, the present work was focused on negative ion detection. Beside the formation of negative ions, matrix effects were investigated to confirm the reduced matrix dependent ionization in LAGD-TOFMS, which has been reported [14].
3.1. Phosphatidylethanolamine (PE)
Fig. 2. Molecular structure of PE.
Phosphatidylethanolamine in CHCA was analyzed in both negative-ion (NI) and positive-ion (PI) mode using LAGD and MALDI. This molecule contains a phosphate group, which is an oxoanion and therefore negatively charged and should be preferentially detectable in NI mode. To confirm this, measurements in PI mode provided no significant analyte signal in either LAGD or MALDI. In contrast, the measurements in LAGD NI mode provided a peak at m/z 743.55 assignable to the molecular ion of PE which appeared during the afterglow (Fig. 3). The GD only and CHCA only measurements provide evidence that the molecular information is not affected by interferences. Peaks assignable to the molecular ion of the matrix and to its deprotonated form are also shown in Fig. 3 (m/z 189.04). MALDI measurements of the same compound at 50% (mol/mol) with CHCA were performed in both PI and NI mode. However, the molecular ion was detected in NI mode only. Fig. 4 shows spectra of the analyte mixed with the matrix (PE in CHCA) and the matrix only (CHCA only). The enlarged figure shows the mass range close to the molecular ion of the analyte.
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Fig. 3. Comparison between different spectra in the afterglow acquired by switching on the pulsed GD (labeled as GD only), by switching on the pulsed GD and introducing the matrix only using laser desorption (CHCA only), by switching on the pulsed GD and introducing the matrix mixed with the analyte by laser desorption (PE). The zoomed window shows the matrix molecular and deprotonated ion for all three modes of operation.
No interferences from the matrix affected the PE analyte signal. However, a pronounced matrix suppression effect (MSE) [15] was observed in the analyte spectrum (PE in CHCA), which is common for high concentrations of analytes. 3.2. Reserpine A series of mixtures containing reserpine in CHCA at 1%, 10%, and 50% (mol/mol) ratios were also analyzed in both PI and NI mode using LAGD and MALDI. In PI mode in LAGD a concentration of the analyte of 50% with CHCA was required for signal detection. However, in NI mode, detection of the molecular ion was observed at all concentrations. To illustrate the detection capabilities, the
afterglow spectra of the 10% (mol/mol) analyte concentration in CHCA are shown in Fig. 5. The figure also shows that the signal is not influenced by interferences from the GD or CHCA. The spectra show a peak assignable to the deprotonated molecule and other peaks assignable to other isotopologues which are all related to the sample containing the analyte. The spectral region between m/z 187 and 191 in Fig. 5 shows two peaks which can be assigned to the deprotonated molecule and the molecular ion of the matrix. MALDI detection of reserpine in PI mode was possible at all three concentrations, and MSE was also observed at all of these concentrations. However, in NI mode the molecule was not detected (Fig. 6).
Fig. 4. NI-MALDI spectra of PE 50% with CHCA (PE in CHCA) and CHCA only; the small box is an enlargement of the molecular ion region.
G. Lotito, D. Günther / International Journal of Mass Spectrometry 315 (2012) 60–65
63
Fig. 5. Comparison between different spectra in the afterglow acquired by turning only the pulsed GD (labeled as GD only), the pulsed GD and a sample containing only the matrix (CHCA only), the pulsed GD and the sample containing the matrix and the analyte (reserpine). The smaller window shows the mass region of the matrix molecular ion.
In both spectra the dominant peaks are from the matrix. The enlargement of the molecular ion region does not exhibit any peak assignable to the molecular ion or the deprotonated molecule. In MALDI it is common that in the opposite polarity to that in which MSE is observed, analyte signals are very weak or almost absent. This depends on the acidity of the neutral molecule which is strongly correlated to the acidity of the corresponding protonated or deprotonated species [16]. The results shown in this study indicate that in LAGD measurements no or minor MSE were observed, as previously reported [14]. This indicates that the ionization is less matrix dependent when compared to MALDI, since desorption and ionization in LAGD are physically and temporally separated, as shown in Fig. 7. The
signal intensities of the deprotonated and molecular ion of CHCA and deprotonated molecular ion of reserpine along with its 13 C isotopologue are plotted as a function of time (Fig. 7). At the beginning only the laser is turned on and no detection of those species is observed. This could be explained by the fact that at the experimental conditions used (pulsed laser energy at ca. 19 J and spot size of ca. 30 m) ionization is not taking place through laser desorption or if it does, then the ions generated do not reach the sampling area. As the pulsed GD is switched on (with the laser in operation) detection of those species is clearly visible. This could be due to the fact that the desorbed neutral species, whose ratio to the ion species (ions/neutrals) ranges between 10−5 and 10−3 [17], are post-ionized in the pulsed GD plasma. As the laser is turned off
Fig. 6. Spectrum of reserpine 50% with CHCA and spectrum of CHCA alone measured using MALDI-TOFMS in NI mode.
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G. Lotito, D. Günther / International Journal of Mass Spectrometry 315 (2012) 60–65
Fig. 7. Signal intensities of the deprotonated and molecular ion of matrix (CHCA) and deprotonated molecular ion and its 13 C isotopologue of reserpine as a function of time. During the first 9 s only the laser was in operation (LA), then the pulsed GD was additionally turned on until 23 s ca. and then the laser was turned off with only the pulsed GD in operation (GD).
Table 1 Comparison between intensities of peak area representing the deprotonated molecule of reserpine for different concentrations in LAGD and MALDI. Concentration reserpine in matrix (%)
LAGD signal intensity normalized to matrix (a.u.)
MALDI signal intensity (a.u.)
1 10 50
2.25 × 10−4 1.69 × 10−3 1.75 × 10−2
1.19 × 10+4 9.77 × 10+3 6.69 × 10+3
(at ca. 23 s) and only the pulsed GD is in operation, all the signal intensities decrease by several orders of magnitude because no introduction of those species to the plasma was taking place. A similar behavior was also observed for PE in CHCA (data not shown). To demonstrate the reduced matrix dependence three different concentrations of reserpine in CHCA were measured in both LAGD NI mode and MALDI PI mode (since in NI mode no detection was possible) and the area of the peak assigned to the deprotonated molecule of reserpine was normalized to the area of the peak assigned to the molecular ion of the matrix; such normalization was not possible in MALDI due to the MSE. Table 1 contains the intensities and relative analyte to matrix concentrations for both techniques. The qualitative trend observed in LAGD-TOFMS indicates a lower matrix dependent ionization behavior and potentially allow some quantification. This is achievable because desorption and ionization can be optimized separately. However, the current LAGD prototype requires more fundamental studies on improving the sensitivity, which is currently orders of magnitude lower than currently reported for MALDI [18,19].
4. Conclusions Negative mode ionization of matrix-assisted laser desorbed PE and reserpine analytes in CHCA during the afterglow of a pulsed GD was investigated. It was shown that the NI mode allows the detection of some molecular ions, which were not detectable in PI mode. Furthermore, indications of a reduced matrix dependent ionization behavior was observed in LAGD when compared to MALDI. The reduced matrix dependence is probably due to the separation of the desorption and ionization process. Therefore, the potential for a semi-quantitative analysis of organic compounds is very high, especially if the current low sensitivity can be improved.
Acknowledgments The authors would like to thank the European Community for financial support through GLADNET, a Marie Curie-RTN within the FP 6, and TOFWERK AG for providing the instrumentation used. The authors also would like to thank Dr. R. Knochenmuss for his advice and support and L. Bertschi of the MS service of ETH for having assisted in performing the MALDI measurements. The authors would also like to thank Dr. Henry Longerich for advice and revision of the manuscript.
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