Surface-Assisted Laser Desorption Ionization

Dec 24, 2008 - Prokhorov General Physics Institute of the Russian Academy of Sciences, Vavilov str. 38, 119991, Moscow, Russia, Vernadsky Institute of...
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Anal. Chem. 2009, 81, 1255–1261

Gas Chromatography/Surface-Assisted Laser Desorption Ionization Mass Spectrometry of Amphetamine-like Compounds S. Alimpiev,† A. Grechnikov,‡ J. Sunner,*,§ A. Borodkov,‡ V. Karavanskii,† Ya. Simanovsky,† and S. Nikiforov† Prokhorov General Physics Institute of the Russian Academy of Sciences, Vavilov str. 38, 119991, Moscow, Russia, Vernadsky Institute of Geochemistry and Analytical Chemistry of the Russian Academy of Sciences, Kosygin str. 19, 119991, Moscow, Russia, and School of Pharmacy and Biomedical Sciences, University of Portsmouth, PO1 2DT, Portsmouth, U.K. A variety of amphetamine-like compounds were analyzed by gas chromatography/surface-assisted laser desorption ionization mass spectrometry, GC/SALDI-MS. In the SALDI method, compounds are adsorbed on a solid SALDI substrate and directly ionized from the substrate by means of a laser pulse. The interfacing of a SALDI ion source with a gas chromatograph is presented here for the first time. The end of the GC column is situated 20 mm from the silicon substrate in the vacuum of the ion source of a time-of-flight mass spectrometer, and the compounds eluted from the GC capillary are adsorbed onto the nanostructured silicon surface. The mass spectra show very low levels of background noise and no reagent ions. GC/SALDI-MS detection limits are several orders of magnitude lower than those previously reported for GC/MS analysis of amphetamine-like compounds. The extent of fragmentation is under experimental control by changing the laser fluence. Arylalkylamines are a diverse group of compounds. In particular, substituted phenylalkylamines include many pharmacological and psychoactive compounds. Amphetamine and methamphetamine are prominent examples, but the group also includes hormones, neurotransmitters, stimulants, hallucinogens, antidepressants, and many other drugs. Methylenedioxy derivatives of amphetamine or methamphetamine are common as designer drugs, and N-substituted derivatives are therapeutically used as anorectics, antiparkinsonians, and vasodilators. Because of their medical and forensic importance, there is great interest in, and need for, rapid, sensitive, and quantitative analyses of arylalkylamines from a variety of matrixes. Publications devoted to this subject number in the hundreds. The analysis of amphetamines and related compounds was reviewed in 1998.1 With complex matrixes, the compounds of interest must first be isolated. With aqueous matrixes like blood and urine, this can be * To whom correspondence should be addressed. E-mail: [email protected]. † Prokhorov General Physics Institute of the Russian Academy of Sciences. ‡ Vernadsky Institute of Geochemistry and Analytical Chemistry of the Russian Academy of Sciences. § University of Portsmouth. (1) Kraemer, T.; Maurer, H. H. J. Chromatogr., B 1998, 713, 163–187. 10.1021/ac802176j CCC: $40.75  2009 American Chemical Society Published on Web 12/24/2008

achieved by liquid-liquid extraction or by solid-phase microextraction (SPME). The extracted compounds are generally separated by gas chromatography (GC) or liquid chromatography (LC) and detected and identified by mass spectrometry (MS). Amphetamine-like compounds are commonly derivatized in order to improve GC performance, to form characteristic fragment ions, or to improve ionization properties, particularly for the formation of negative ions. For the mass spectrometric analysis, both electron ionization (EI) and chemical ionization (CI) are used. For maximum sensitivity, the mass spectrometer is often operated in single-ion monitoring mode. The reported limits of detection for GC/MS, after extraction and preconcentration procedures, are commonly in the 0.1-10 ng/mL of blood or urine.1 The focus of the present work is on the identification and quantitation of arylalkylamines by GC/MS using surface-assisted laser desorption ionization (SALDI). In SALDI, analyte ions are desorbed directly from a solid substrate surface in a process that critically depends on the physical and chemical properties of the solid surface. The use of graphite surfaces, in the form of a powder, was introduced in 1995, and it was reported that carbon was unique, among many materials tried, in its ability to generate ions under pulsed laser irradiation.2 Subsequently, other materials have been shown to be “SALDI-active”, for example, activated carbon,3 carbon nanotubes,4 and metal oxides.5 Many forms of silicon materials are also SALDI-active.6 In particular, porous silicon is used in DIOS (desorption/ionization on silicon).7 Silicon is almost always used as a monolithic substrate with analytes deposited as liquid solutions. DIOS has been used to detect amphetamines, deposited as liquid solutions in atmospheric air.8 (2) Sunner, J.; Dratz, E.; Chen, Y. C. Anal. Chem. 1995, 67, 4335–4342. (3) Chen, Y.-C. Surface Assisted Laser Desorption Ionization (SALDI) Mass Spectrometry. Ph.D. Dissertation, Montana State University, 1997. (4) Xu, S. Y.; Li, Y. F.; Zou, H. F.; Qiu, J. S.; Guo, Z.; Guo, B. C. Anal. Chem. 2003, 75, 6191–6195. (5) Kinumi, T.; Saisu, T.; Takayama, M.; Niwa, H. J. Mass Spectrom. 2000, 35, 417–422. (6) Alimpiev, S.; Grechnikov, A.; Sunner, J.; Karavanskii, V.; Simanovsky, Y.; Zhabin, S.; Nikiforov, S. J. Chem. Phys. 2008, 128, 014711. (7) Wei, J.; Buriak, J. M.; Siuzdak, G. Nature 1999, 399, 243–246. (8) Pihlainen, K.; Grigoras, K.; Franssila, S.; Ketola, R.; Kotiaho, T.; Kostiainen, R. J. Mass Spectrom. 2005, 40, 539–545.

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In 2001, gas-phase analyte introduction onto SALDI substrates was introduced.9 In this method, individual, gas-phase analyte molecules are adsorbed on the SALDI substrate and subsequently ionized and desorbed by the laser pulse. Both porous and nonporous silicon substrates can be used, in particular, both porous vapor-etched and nonporous amorphous silicon are excellent substrates.9 With the use of gas-phase SALDI, it was possible to make careful studies of the mechanism of ion generation from amine analytes.9 It was concluded that amine analytes were first adsorbed on the surface with hydrogen bonding of the amine to a surface Si-OH group. The laser pulse causes excitation of electrons from the conduction band, and subsequent relaxation causes the trapping of positive charges in the near-surface. This increases the acidity of the surface and enables proton transfer to the adsorbed amine. The observation that the proton affinity, and not the pKa, of the analyte that determines whether protonated analytes are observed, shows that any solvent (water) present on the surface has limited influence on the desorption/ionization process. In particular, in this implementation of SALDI, there is no requirement of an entrainment action of a matrix compound. Gas-phase introduction of analytes in SALDI was shown to be associated with a very high efficiency of ionization, up to 1%.9 For this reason, analytes with vapor pressures as low as 10-15 Torr in the ion source can be detected. The extremely high sensitivity of gas-phase SALDI, the ability to generate both molecular and fragment ions, the use of a lowcost time-of-flight (TOF) mass spectrometer, and the absence of reagent ions in the mass spectra means that this method has an outstanding potential for GC/MS applications. Here, we present the first implementation of GC/SALDI and present results for the analyses of amphetamine-like compounds. EXPERIMENTAL SECTION Mass Spectrometer. The GC/SALDI instrument is a laboratory-built, linear, turbo-pumped (Varian, V 250) TOF with a 0.70 m flight tube. The ion acceleration voltage was 16.0 kV, and the ions were detected by a 40 mm diameter, Chevron microchannel plate (MCP) assembly FTD-2003 (Galileo Electro-Optics Corp., Sturbridge, MA). The signal from the MCP was recorded by an ADC digitizer (Detek Electrix, Manchester, U.K.) with 1 Gs/s sampling rate and ability to store mass spectra at a rate of up to 1 kHz. Thus, the mass spectrum obtained with every laser pulse was recorded and stored on a PC. The mass resolution, m/∆m, was approximately 1000, and the accuracy of m/z determinations was approximately 0.1 Da. Laser, Optics, and Beam Scanning. A diode-pumped YAG laser (ELS Co., Moscow, Russia, model RL-1.0/355) with neardiffraction-limited beam quality, 0.5 ns pulse duration, and a maximum pulse repetition frequency of 1 kHz was operated at 300 Hz. A frequency-tripling crystal yielded a 355 nm laser pulse of about 100 µJ. The 355 nm laser beam was attenuated as required and focused on the Si substrate by a 20 cm focal-length lens. Laser pulse energy was measured with a laser power/energy calorimeter (Solo PE; pyroelectric Joulemeter ED-100 UV, Gentec, Quebec, Canada) and surface absorber sensor (PS-310, Gentec). The profile of the near-Gaussian laser beam was monitored, in the lens focal (9) Alimpiev, S.; Nikiforov, S.; Karavanskii, V.; Minton, T.; Sunner, J. J. Chem. Phys. 2001, 115, 1891–1901.

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plane, by a WinCamD (Gentec) instrument with a 1 µm spatial resolution. The fluence in the laser focal point was calculated from the laser pulse energy, measured with the Gentec ED-100 UV, and beam profile contour was obtained with Gentec WinCamD. The analyte was adsorbed over the surface of the SALDI substrate, about 5 × 5 mm2. In contrast, the size of the laser focus within the 1/e intensity contour is approximately 7 × 10-5 cm2. In order to more efficiently utilize the analyte, the laser focus was scanned over a 1 × 2 mm2 sample adsorption area using a computer-controlled, dual-mirror scanner (USL-03, IMCES, Tomsk, Russia). The laser focus moved approximately 0.08 mm between successive laser shots, and about 300 individual mass spectra were obtained from different points on the probed area of the SALDI substrate and were accumulated by the ADC digitizer during one “scanning cycle”. Since the laser repetition rate was 300 Hz, any point on the surface was sampled approximately once every second. This mode of laser focus scanning also increased the ion signal by about 300 times over the signal obtained with a stationary focus. The multiplier supply voltage was adjusted to provide a single ion pulse amplitude that was equal to one in the digitizer output signal. Thus, the integrated output signal from the ADC digitizer equaled the number of ions per scanning cycle. Similarly, the total number of detected ions for any ion peak in mass spectrum, over any specified time interval of the GC run, was obtained from the ADC digitizer software. Gas Chromatography. The GC instrument (ThermoQuest, GC8000 series, Italy) was equipped with an 15 m long, 250 µm i.d. silica capillary column with a fused-silica (Rtx-5MS) stationary phase (Restek cat. no. 12620). Analytes were dissolved in methanol at a concentration of 0.01-100 ng/mL and a 0.1-1 µL volume was injected in either split (1:10) or splitless mode with the injector temperature at 250 °C. The column temperature was programmed to increase from 80 to 110 °C, with a rate of 2 °C/min, and then to 170 °C with a rate of 6.0 °C/min, unless otherwise specified. The carrier gas was nitrogen with a flow rate of 1 mL/min. The interface between the GC and the SALDI ion source is critical to the present work, and a schematic representation is seen in the inset in Figure 1. The GC capillary passes through a short heated transfer line to extend into the ion source. Inside the ion source, the capillary is covered with a stainless steel capillary, heated to 200 °C. The end of the GC capillary was positioned so as to direct the gas flow toward the silicon substrate, see Figure 1. The distance between the end of the GC capillary and the laser sampling area on the silicon substrate could be varied from 20 to 55 mm. To avoid excessive interference with ion extraction, the GC capillary was positioned at a 45° angle to the SALDI substrate. It was confirmed that the ion signal was nearly inversely proportional to the square of the distance. The background pressure in the ion source during GC operation was about 10-5 Torr, mainly due to the nitrogen carrier gas. The water background pressure was approximately 3 × 10-6 Torr. For selected experiments, additional water at about 10-5 Torr was added through an electromagnetically controlled valve. Silicon Substrates. The SALDI substrates were approximately 5 × 5 mm2, and they were attached to the instrument‘s stainless steel insertion probe. Two different silicon substrates were used: vapor-etched silicon and amorphous silicon.

Figure 1. Total ion chromatogram of a GC/SALDI separation of seven different phenylethylamines: (1) N-methyl-1-phenylethylamine, (2) N,Ndimethyl-1-phenylethylamine, (3) N-isopropyl-1-phenylethylamine, (4) N-methyl-1-(4-methylphenyl)-ethylamine, (5) N,N-dipropyl-1-phenylethylamine, (6) N-benzyl-1-phenylethylamine, and (7) N-methyl,N-benzyl-1-phenylethylamine. “Ion signal” refers to the number of ion counts during each 1 s long scan of 300 laser pulses. The duration of the GC separation was about 26 min, and the SALDI substrate was amorphous Si. The mass spectra of the individual compounds are shown in Figure 2.

Vapor-etched silicon substrates were produced in-house by etching of commercially available n- and p-type, (100)-oriented Si wafers (ELMA, Russia) of 0.01 ohm · cm resistivity. The substrates were etched for 15-20 h at room temperature in a closed chamber containing saturated vapors of an HF-water solution (1:1) and I2 powder. The substrates were very stable under atmospheric conditions, and there was no significant decrease in ionization efficiency after a few months storage in air. Substrates were used for 1 or occasionally several weeks of GC/MS experiments without significant decrease in sensitivity. The substrates remained inside the instrument during this time and were not “cleaned” in any way, except by laser irradiation during the experiments. Amorphous Si substrates, R-Si, were deposited on monocrystalline silicon by standard rf sputtering of Si in a low-pressure (10-3 Torr) Ar atmosphere, using a turbo-molecular-pumped, laboratory-built apparatus. The thickness of the deposited film was approximately 0.5 µm. Care was taken to use ultrapure chemicals and clean procedures in the sputtering deposition, and the produced substrates were of high purity. The amorphous Si substrates were found to be unstable in ambient air, with a practical storage time of a few days. Therefore, these substrates were always used “fresh”, i.e., within a couple of hours of manufacture. No ion signals were obtained without the SALDI substrates in place. Chemicals. N-Isopropyl-1-phenylethylamine was synthesized by standard Leuckart synthesis.10 N,N-Dipropyl-1-phenylethylamine was obtained from 1-phenylethylamine by alkylation with n-propyl iodide, and N-methyl,N-benzyl-1-phenylethylamine was obtained from N-benzyl-1-phenylethylamine by alkylation with methyl iodide. The structures of all synthesized compounds were verified by NMR spectrometry. All other chemicals were obtained commercially and used without further purification. (10) Novelli, A. J. Am. Chem. Soc. 2008, 61, 520–521.

RESULTS AND DISCUSSION Figure 1 shows a typical GC/SALDI total ion chromatogram (TIC) for a separation of phenylethylamines. Amorphous silicon was used as the SALDI substrate. In contrast to CI, no reagent ions are present in the mass spectra, and the total ion signal was obtained by integrating each mass spectrum from m/z ) 1 to 250. The sharp peaks in the chromatogram illustrates that memory effects are not a serious problem in SALDI/GC. The injected solution contained 2 × 10-14 mol of each of seven compounds. The differently sized peaks, numbered nos. 1-7 in Figure 1, reflect differences in sensitivity. Compound “5” had the highest sensitivity and is here assigned a relative sensitivity, S, of 100. The relative sensitivities of the other compounds were obtained by simply dividing the respective peak areas. The numbered peaks in Figure 1 refer to the following compounds, with the respective relative sensitivity: (no. 1) N-methyl-1-phenylethylamine, S ) 9; (no. 2) N,N-dimethyl-1-phenylethylamine, S ) 32; (no. 3) N-isopropyl-1-phenylethylamine, S ) 33; (no. 4) N-methyl-1-(4-methylphenyl)-ethylamine, S ) 11; (no. 5) N,Ndipropyl-1-phenylethylamine, S ) 100; (no. 6) N-benzyl-1-phenylethylamine, S ) 18; (no. 7) N-methyl,N-benzyl-1-phenylethylamine, S ) 60. The respective molecular structures are shown in Figure 2. The reasons for the variation in sensitivity between different arylalkylamines are of significant interest. A compound‘s proton affinity (PA) is one of the most important factors for its SALDI sensitivity. The proton affinities of all studied arylalkylamines are higher than 900 kJ/mol. The sensitivity of gas-phase SALDI is near zero at PA < 850 kJ/mol but increases rapidly with increasing PA above 850 kJ/mol.6 In particular, the secondary amines (nos. 1 and 6) have lower PA than their homologous, N-methylsubstituted tertiary amines (nos. 2 and 7) and, indeed, have lower SALDI sensitivities. Similarly, the SALDI sensitivity of primary amines is usually several times lower than for homologous, N-methyl-substituted, secondary amines (results not shown). Analytical Chemistry, Vol. 81, No. 3, February 1, 2009

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Figure 2. SALDI mass spectra of seven phenylethylamines obtained during the GC separation in Figure 1. The SALDI substrate was amorphous silicon, and it was irradiated with 30 mJ/cm2 of 355 nm laser pulses. 1258

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Overall, the tested arylalkyamines demonstrate a strong positive correlation between SALDI sensitivity and PA. However, other factors also play a role. There is a tendency for SALDI sensitivity to decrease with increasing molecular weight and with changes to the molecular structure that is expected to increase the adsorption energy on the silicon surface. For example, the sensitivities of a number of substituted 1-phenylpropyl- and 1-phenylbutyl-amines (results not shown) were slightly lower than for the substituted 1-phenylethylamines in Figure 1. Mass Spectra. Figure 2 shows the mass spectra of the compounds separated in the GC/SALDI run depicted in Figure 1. These were obtained by integrating the mass spectra over the respective TIC peaks. The laser fluence used for these mass spectra was 30 mJ/cm2 and, as discussed below, significant fragmentation is observed at this fluence. The seven compounds include both secondary (nos. 1, 3, 4, 6) and tertiary (nos. 2, 5, 7) arylalkyl-amines. At this fluence, an abundant MH+ ion was observed for all tertiary and two of the secondary amines (nos. 3 and 6). When the alkyl group bound to the nitrogen of the secondary amine is a methyl group (nos. 1 and 4), MH+ ions fragment to yield (M - H)+, due to facile loss of H2 to form a stable immonium ion. At a high enough fluence, (M - H)+ ions are observed for most of studied amines, and the ratio of abundances of the (M - H)+ to MH+ ions increases with increasing fluence, as shown below. In addition to (M - H)+, major fragment ions are formed by heterolytic cleavage of the bond between nitrogen and the carbon of the aromatic ring-containing substituent: Ra1N+HR2R3 f (Ra1)+ + NHR2R3

(1)

where Ra1 represents an aromatic ring-containing substituent, R2 is either an alkyl group or a hydrogen atom, and R3 is either an alkyl group or a benzyl group. This process yields the ions at m/z ) 91 (tropylium ion, nos. 6 and 7), 105 (nos. 1, 2, 3, 5, 6, 7), and 119 (no. 4). These ions are marked with “R” in Figure 2. The same bond cleavage, accompanied by hydrogen transfer from the arylalkyl group to the nitrogen, yields a protonated amine and a neutral alkene: Ra1N+HR2R3 f [Ra1 - H] + [NH2R2R3]+

(2)

These fragment ions include m/z ) 32 (nos. 1 and 4), 46 (no. 2), 60 (no. 3), 102 (no. 5), 108 (no. 6), and 122 (no. 7). These ions are marked with a “β” in Figure 2. Finally, when the cleavage of the arylalkyl-nitrogen bond is accompanied by loss of H2 from the nitrogen moiety, an immonium fragment ion is formed. Such ions were observed in mass spectra of benzylamines. An example is the ion at m/z ) 44 for N,N-dimethylbenzylamine in Figure 3. Chemical ionization mass spectra of amines typically suffer from a lack of structure-specific fragment ions.11,12 As shown in this work, SALDI mass spectra of arylalkylamines, in contrast, yield an abundance of such fragment ions. This is similar to what is observed in collision-induced fragmentation of CI-formed MH+ ions,13 but achieved without the use of a more complex MS/MS instrument. The ability to increase the internal excitation energy of the formed MH+ ions by simply increasing the

Figure 3. SALDI mass spectrum of 10 fg of N,N-dimethylbenzylamine obtained by splitless GC injection of 1 µL of a 10 pg/mL solution of the amine in methanol at laser fluence of 30 mJ/cm2. The mass spectrum was accumulated over the elution peak. “Ion signal” refers to the total number of ion counts during 30 1 s long scans of 300 laser pulses each.

laser fluence constitutes a very important advantage of the SALDI method. It should be noted that the mass spectra in Figure 2 contain minor impurity peaks, notably K+ at m/z ) 39 and Na+ at m/z ) 23. Clusters of minor background peaks throughout the mass range are mainly due to bleeding from the column. In general, however, the spectra are very “clean” with very low chemical background and no reagent ions. This property of SALDI minimizes the need for sample cleanup. Limit of Detection. Sensitivity and limit of detection are critical issues for any method for trace drug detection. Figure 3 shows a SALDI mass spectrum of 8 × 10-17 mol (10 fg) of N,Ndimethylbenzylamine injected into the GC in splitless mode. The spectrum was obtained from amorphous silicon by integrating over the elution peak. N,N-Dimethylbenzylamine gives three main peaks due to the protonated molecule, MH+, at m/z ) 136, the benzyl cation (tropylium ion) at m/z ) 91, and the immonium fragment ion, mentioned above, at m/z ) 44. If the three most abundant ions are used to confirm the identity of the compound, the least abundant ion, here the m/z ) 44 ion, determines the limit of detection. Using the criterion of a signal-to-noise ratio of 3:1 (peak-to-peak noise definition) for this ion, the limit of detection of N,N-dimethylbenzylamine is about 6 × 10-17 mol. If only the two most abundant ions are used to identify the benzylamine, the detection limit is three times lower at 2 × 10-17 mol or 2.5 fg per GC injection. Quantitation is achieved using the most abundant ion at m/z 91, and the limit of quantitation is equal to the limit of detection. On the scale given in connection with Figure 1, N,N-dimethylbenzylamine has a sensitivity of S ) 27. Some other amines have higher sensitivities. For example, N,N-dipropyl-1-phenylethylamine has a sensitivity of S ) 100, and its detection limit in splitless mode is approximately 5 × 10-18 mol. (11) Hunt, D. F.; McEwen, C. N.; Upham, R. A. Tetrahedron Lett. 1971, 4539– 4542. (12) Milne, G. W. A.; Fales, H. M.; Colburn, R. W. Anal. Chem. 1973, 45, 1952– 1954. (13) Borth, S.; Hansel, W.; Rosner, P.; Junge, T. J. Mass Spectrom. 2000, 35, 705–710.

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Figure 4. Mass spectra of N,N-dimethyl-1-phenylethylamine at three different values of laser fluence: (a) 12, (b) 25, and (c) 40 mJ/cm2.

Comparisons between porous, vapor-etched and amorphous vapor-deposited silicon showed that these SALDI substrates have similar sensitivities for phenylethylamines. However, in the mass range of 20-150 Da, amorphous silicon has significantly lower background noise, at least 3 times higher signal-to-noise ratio, and correspondingly lower limits of detection. The GC/SALDI detection limits obtained in this work are in the 1-10 fg range and several orders of magnitude lower than those reported for GC/MS using electron impact ionization, such as 32 ng per GC injection for 3,4-methylenedioxy-N-methylam1260

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phetamine (MDMA).14 Much lower GC/MS detection limits have been reported when using CI after extraction and preconcentration procedures. These are commonly in the 0.1-10 ng/mL of blood or urine.1 These are still 2-4 magnitudes higher than for GC/ SALDI-MS. These results confirm the unequalled sensitivity of SALDI for gas-phase analysis. The extremely low detection limits reported here for phenylethylamines were obtained with a first-generation GC/SALDI (14) O’Connell, D.; Heffron, J. J. A. Analyst 1999, 125, 119–121.

instrumental setup. It is expected that further improvement in GC/ SALDI interface design will substantially lower the detection limits. In GC/SALDI-MS, sensitivity and limit of detection depend critically on the location and distance between the GC capillary and the irradiated area of the SALDI substrate. In the experimental setup, shown in the inset in Figure 1, the molecular jet emerging from the capillary has a large divergence. Assuming Knudsen’s cosine law for vapor flux distribution, the fraction of the GC eluent that made contact with the laser-irradiated area of the SALDI substrate is calculated to be only 10-3. It is evident that increasing the laser spot size and optimizing the GC capillary position has the potential of further lowering detection limit, possibly to the high-zeptomole range. Linearity. The SALDI ions signal was measured for different amounts of analytes over a range of 5 orders of magnitude by successive dilutions of the injected solutions and by decreasing the volume of injected analyte solution from 1 to 0.1 µL. The linearity was better than 0.995 for all studied analytes and for both vapor-etched and amorphous silicon. The calibration curves were not forced through origin, and any small positive residuals were included in calculations. At the lower limit of the dynamic range, about 0.01 ions were detected per laser pulse, i.e., of the order of three ions per “focal track cycle” (or per second). With a laser-irradiated area of approximately 10-4 cm2 and an ionization efficiency of 1%, it is estimated that 1 × 105 analyte molecules were adsorbed per square centimeter prior to the laser pulse. At the upper limit of the dynamic range, the number of adsorbed molecules would thus be 1 × 109 cm-2. This means that the surface coverage was about 0.01% of a monolayer. At the upper end of the linear dynamic range, the number of ions detected is about 1000. Saturation of the MCP detector is maybe the most common cause for nonlinearity in TOF mass spectrometric measurements. It indeed occurs when the number of ions striking the detector is above about 103. However, in GC/ SALDI, the number of detected ions is easily decreased by decreasing the size of the laser focus. Reproducibility. Seven replicate analyses were carried out over a 1 day period for each of the seven compounds used for Figure 1, using the same experimental conditions. The relative standard deviation was less than 5% for all seven compounds. However, a slow degradation of the SALDI substrates was observed as a decrease in their SALDI activity, even when the substrates were stored in vacuum. The sensitivity typically decreased by 50% after about 150-200 injections for amorphous silicon and after more than 500 injections for vapor-etched silicon. Fluence Dependence of Fragmentation. In gas-phase SALDI, the extent of fragmentation increases with increasing laser fluence. This is illustrated in Figure 4, which shows mass spectra of N,N-dimethyl-1-phenylethylamine obtained from amorphous silicon. The spectrum in panel a of Figure 4 was obtained at 12 mJ/cm2, which is slightly above the threshold for ion generation from amorphous silicon. The MH+ ion at m/z ) 150 dominates the spectrum. The two main fragment ions, at m/z ) 46 and 105, are both obtained by breaking the bond between the nitrogen and phenylethyl group, see discussion above. It is seen that, at this low fluence, the abundances of the fragment ions are quite low. For example, I(105+)/I(150+) ) 0.09, where I is

obtained by integration of the respective mass peaks. At the threshold fluence, no fragment ions are observed. The mass spectrum in panel b of Figure 4, obtained at an intermediate fluence of 25 mJ/cm2, shows a good balance between molecule ion and fragment ions and the ratio of I(105+) to I(150+) is 2.5. Finally, panel c of Figure 4 shows the mass spectrum at a high fluence of 40 mJ/cm2. Here, the MH+ ion has a very small relative abundance, and the spectrum is dominated by fragment ions, including the (M - H)+ ion at m/z ) 148. The ratio I(105+)/I(150+) has increased to 20. The reason that ion fragmentation in gas-phase SALDI increases with laser fluence is easily explained. A detailed model for SALDI ion formation was presented in a previous research report.6 According to this model, analyte molecules adsorb on the surface and form a hydrogen bond with a Si-OH surface group. As a result of laser irradiation of the substrate, positive charges are trapped in the surface, leading the proton transfer to the bound analyte: dSi+ - OH ··· NR3 f d Si - O• + H - N+R3

(3)

This is followed by a dissociation of the analyte ion from the surface. This process is relatively slow and is expected to be mainly thermally induced. As the laser fluence increases, the peak temperature of the surface increases as well, approaching the melting point of the silicon substrate at the highest fluence levels. For crystalline silicon, the melting point is 1410 °C. It is apparent from results, such as those in Figure 4, that at least partial thermalization occurs between the hot substrate surface and analyte molecules before the latter leaves the surface. The ability to control the extent of fragmentation in SALDI is extremely useful in practical mass spectrometric analysis. The use of low fluence, where essentially only protonated molecule ions are detected, greatly simplifies the analysis of complex mixtures, while the use of high fluence yields structural information and allows for the identification of individual compounds. CONCLUSIONS (1) The first implementation of GC/SALDI-MS has been demonstrated. (2) The detection limits for amphetamine-like compounds are in the 10 amol range, with a potential for improvement by 1-3 orders of magnitude. (3) The linear dynamic range extends over at least 5 orders of magnitude. (4) The SALDI mass spectra are very “clean” with very low chemical background and no reagent ions, minimizing the need for sample cleanup. (5) SALDI offers facile control over the extent of molecule ion fragmentation by changing the laser fluence. ACKNOWLEDGMENT The authors are grateful for financial support from the Russian Academy of Sciences Program “Nanostructural Materials for Photonics Applications” and to the Russian Foundation for Basic Research Programs, Grant No. 08-02-01237-a. Received for review December 2, 2008.

October

14,

2008.

Accepted

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