IR-MALDI-MS Analysis of HPTLC-Separated ... - ACS Publications

Jun 23, 2007 - Institute of Medical Physics and Biophysics, Westfälische Wilhelms-Universität Münster, Robert-Koch-Strasse 31, 48149. Münster, Ger...
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Anal. Chem. 2007, 79, 5793-5808

IR-MALDI-MS Analysis of HPTLC-Separated Phospholipid Mixtures Directly from the TLC Plate Andreas Rohlfing,† Johannes Mu 1 thing,† Gottfried Pohlentz,† Ute Distler,† Jasna Peter-Katalinic´,† ‡ Stefan Berkenkamp, and Klaus Dreisewerd*,†

Institute of Medical Physics and Biophysics, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Robert-Koch-Strasse 31, 48149 Mu¨nster, Germany, and Sequenom GmbH, Mendelssohnstrasse 15d, 22761 Hamburg, Germany

The application of a recently developed direct coupling of high-performance thin-layer chromatography (HPTLC) and infrared matrix-assisted laser desorption/ionization orthogonal extracting time-of-flight mass spectrometry (Dreisewerd, K.; Mu1 thing, J.; Rohlfing, A.; Meisen, I.; Vukelic, Z.; Peter-Katalinic, J.; Hillenkamp, F.; Berkenkamp, S. Anal. Chem. 2005, 77, 4098-4107) to the analysis of phospholipid mixtures is demonstrated. Mixtures of six phospholipid types were exemplarily analyzed. The sensitivity was found to be in the range between about 10 and 150 pmol of material spotted for HPTLC, depending on phospholipid acidity, Rf value, and ion polarity. The lateral resolution of the analysis is on the order of the laser focus diameter of about 220 × 300 µm2, allowing differentiation between phospholipid species of different acyl chain composition within one single HPTLC band, which were undistiguishable by a mere visual assessment. Analyte diffusion due to the addition of glycerol to the HPTLC plate was found to besif at all notablesof only minor importance. Phospholipids (PLs) are the major components of biological membranes. They usually consist of a polar headgroup, a phosphate group, a glycerol backbone, and two acyl chains of variable length and saturation. PL analysis is routinely carried out by high-performance thin-layer chromatography (HPTLC), which, compared to other analytical techniques, constitutes a particularly inexpensive and robust method.1 However, even under optimal conditions, the information available from HPTLC remains limited, since species of similar structure and mobility can frequently not be differentiated unless additional methods, e.g., immunostaining, are employed. Mass spectrometry (MS) allows us to obtain detailed structural information by means of fragmentation experiments.2,3 Nevertheless, the analysis of complex mixtures is often difficult since isobaric species cannot be differentiated in the initial MS1 spectrum, and ions of a particular type may be suppressed in the presence of another.4 Efficient mapping of complex * To whom correspondence should be addressed. Tel.: +49-251-8356726. Fax: +49-251-8355121. E-mail: [email protected]. † Westfa ¨lische Wilhelms-Universita¨t Mu ¨ nster. ‡ Sequenom GmbH. (1) Peterson, B. L.; Cummings, B. S. Biomed. Chromatogr. 2006, 20, 227243. (2) Griffiths, W. J. Mass Spectrom. Rev. 2003, 22, 81-152. (3) Pulfer, M.; Murphy, R. C. Mass Spectrom. Rev. 2003, 22, 332-364. 10.1021/ac070633x CCC: $37.00 Published on Web 06/23/2007

© 2007 American Chemical Society

mixtures, as typical for phospholipid samples, is therefore greatly facilitated if chromatographic separation precedes the MS analysis. Various strategies for coupling HPTLC and MS have been reported. Roughly, these can be divided into more direct (see below) and indirect methods, the latter involving the scraping-off of identified HPTLC bands from developed plates and extraction of analyte molecules from the silica gel.5,6 Although this procedure is widely compatible with state-of-the-art electrospray ionization (ESI)3,7 and matrix-assisted laser desorption/ionization (MALDI)8,9 mass spectrometry, it has some clear drawbacks concerning the additional preparation steps and a potential loss of sample material. Moreover, closely migrating HPTLC bands may be difficult to scrape off separately. Direct coupling of HPTLC and ESI-MS has been realized by eluting the analyte molecules from single bands via a liquid microjunction,10 by employing an “extractor” that flushes extraction solvent through a defined part of the analyte-loaded silica gel,11 and by using desorption electrospray ionization.12 The lateral resolution of these techniques is restricted to ∼1 mm. Potentially, a high salt concentration in the eluent can hamper the ESI-MS analysis, and this will eventually apply restrictions on the use of developing solvent for HPTLC. MALDI-MS forms an alternative approach for a direct coupling of planar HPTLC and mass spectrometry.13 Although, in principle, a suitable matrix must only be applied to the developed HPTLC plate, in practice this approach is also associated with several drawbacks. One of them is related to the fact that mostly solid matrixes are employed for MALDI, which require a proper cocrystallization with the analyte. This may be hampered by the (4) Schiller, J.; Su ¨ β, R.; Petkovic, M.; Zscho ¨rnig, O.; Arnold, K. Anal. Biochem. 2002, 309, 311-314. (5) Hildebrandt, H.; Jonas, U.; Ohashi, M.; Klaiber, I.; Rahmann, H. Comp. Biochem. Physiol. B 1999, 122, 83-88. (6) Meisen, I.; Peter-Katalinic, J.; Mu ¨ thing, J. Anal. Chem. 2004, 76, 22482255. (7) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (8) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (9) Schiller, J.; Su ¨ β, R.; Arnhold, J.; Fuchs, B.; Leβig, J.; Mu ¨ ller, M.; Petkovic, M.; Spalteholz, H.; Zscho ¨rnig, O.; Arnold, K. Prog. Lipid Res. 2004, 43, 449-488. (10) Van Berkel, G. J.; Sanchez, A. D.; Quirke, J. M. E. Anal. Chem. 2002, 74, 6216-6223. (11) Luftmann, H. Anal. Bioanal. Chem. 2004, 378, 964-968. (12) Van Berkel, G. J.; Ford, M. J.; Deibel, M. A. Anal. Chem. 2005, 77, 12071215. (13) Gusev, A. I. Fresenius J. Anal. Chem. 2000, 366, 691-700.

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presence of the silica gel or residual components of the developing solvent. Apart from that, application of matrix solution to the HPTLC plate may induce an undesirable lateral analyte spread and, hence, a reduction of the achievable spatial resolution. Several preparation protocols have been implemented in order to address these issues, including brushing of a matrix slurry onto the HPTLC plate,14 pressing a preprepared matrix layer on top of the silica gel,14,15 and controlled spraying of matrix solution over the plate, either with a pneumatic sprayer16 or by electrospray.14 However, most of these methods still require an additional wetting of the HPTLC plate with an extraction solvent in order to resolvate the analyte molecules from the silica gel and to enable their incorporation into matrix crystals. This again carries the risk of unwanted spread of analyte across the plate. In an alternative approach, entire chromatograms have been blotted onto polymer membranes that were then coated with matrix and analyzed by MALDI-MS.17 This procedure also requires the application of extraction solvent. If common MALDI with ultraviolet lasers (UV-MALDI) is employed for the MS analysis, another problem emerges from the rather shallow volume ablated per single laser pulse. While silica gel layers of HPTLC plates usually exhibit thicknesses of a few hundred micrometers, typically sample layers of less than 100nm thickness are ablated per single laser pulse in UV-MALDI.18 Only analyte molecules incorporated in matrix crystals on top of the silica gel are thus desorbed, resulting in a rapid decline of ion signal with the number of applied laser pulses. This problem can be overcome by using an infrared (IR) laser for desorption/ ionization. In IR-MALDI, sample layers with thicknesses on the micrometer scale are typically ablated,19 facilitating an efficient release of analyte molecules even from within the silica gel. Furthermore, a wider range of potential matrixes is available for IR-MALDI, including liquids like, for example, glycerol. These matrixes do not require a cocrystallization with the analyte, which completely circumvents the problems associated with matrix crystallization stated above. We have recently applied direct HPTLC-IR-MALDI-MS with a glycerol matrix to the analysis of gangliosides20 and oligosaccharides.21 An important improvement in these studies was the employment of an orthogonal extracting time-of-flight mass spectrometer (oTOF-MS) in which the desorption/ionization process is decoupled from the mass determination.22 Previous problems regarding the mass resolution and accuracy, which are imminent if analysis is performed from uneven and electrically nonconduct(14) Mowthorpe, S.; Clench, M. R.; Cricelius, A.; Richards, D. S.; Parr, V.; Tetler, L. W. Rapid Commun. Mass Spectrom. 1999, 13, 264-270. (15) Gusev, A. I.; Vasseur, O. J.; Proctor, A.; Sharkey, A. G.; Hercules, D. M. Anal. Chem. 1995, 67, 4565-4570. (16) Bristow, A. W. T.; Creaser, C. S. Rapid Commun. Mass Spectrom. 1995, 9, 1465-1469. (17) Guittard, J.; Hronowski, X. P. L.; Costello, C. E. Rapid Commun. Mass Spectrom. 1999, 13, 1838-1849. (18) Dreisewerd, K. Chem. Rev. 2003, 103, 395-425. (19) Dreisewerd, K.; Berkenkamp, S.; Leisner, A.; Rohlfing, A.; Menzel, C. Int. J. Mass Spectrom. 2003, 226, 189-209. (20) Dreisewerd, K.; Mu ¨ thing, J.; Rohlfing, A.; Meisen, I.; Vukelic, Z.; PeterKatalinic, J.; Hillenkamp, F.; Berkenkamp, S. Anal. Chem. 2005, 77, 40984107. (21) Dreisewerd, K.; Ko ¨lbl, S.; Peter-Katalinic, J.; Berkenkamp, S.; Pohlentz, G. J. Am. Soc. Mass Spectrom. 2006, 17, 139-150. (22) Ivleva, V. B.; Sapp, L. M.; O’Connor, P. B.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2005, 16, 1552-1560.

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ing HPTLC plates with a still more common axial-TOF mass spectrometer,14,15,17 have effectively been solved. The standard high mass accuracy of the oTOF instrument significantly facilitates a less ambiguous ion signal assignment. Similar features can in principle be expected if Fourier transform ion cyclotron resonance (FT-ICR)23 or Paul ion trap mass spectrometers are used. In the present article, we describe the application of direct HPTLC-IR-MALDI-oTOF-MS for the analysis of PL mixtures. Glycerol is employed as liquid matrix. An efficient sample volume ablation and particularly soft desorption/ionization is achieved with a Q-switched Er:YAG laser emitting pulses of ∼100-ns duration at a wavelength of 2.94 µm. An oTOF mass spectrometer with collisional cooling24 is utilized for the mass analysis, providing a mass accuracy of ∼20 ppm. Mixtures of five biologically relevant PL types purified from bovine tissue and synthetic phosphatidic acid were separated by HPTLC. The individual bands were analyzed by IR-MALDI-MS in positive and negative ion mode. Limits of detection (LODs) were assessed by performing a dilution series. The lateral resolution was investigated by scanning the laser spot over selected bands. To our knowledge, this is the first time that PLs have been analyzed by a direct HPTLC-MALDI method. EXPERIMENTAL SECTION Chemicals. Glycerol (p.a.), triethylamine (>99%), and KCl were purchased from Sigma (Deisenhofen, Germany), as well as the calibrant peptides bradykinin fragment 1-7 and substance P. 2-Propanol (p.a.) was from Merck (Darmstadt, Germany), as well as methanol and chloroform, which were distilled before use. HPTLC plates (10 × 10 cm2, glass-backed, coated with 0.2-mm silica gel 60) were obtained from Merck (Article No. 1.05633.0001). The plates were activated before use by heating for 40 min in an oven at 110 °C and then stored over silica gel with auxiliary P2O5 in an exsiccator. Molybdenium(VI) oxide (p.a.) and molybdenum powder (>99%) were purchased from Merck. Phospholipids. Phospholipid samples were purchased from Sigma: phosphatidylcholine (PC, from bovine heart; P9513), phosphatidylethanolamine (PE, from bovine brain; P9137), phosphatidylglycerol (PG, from bovine heart; P9399), phosphatidic acid (PA, synthetic; P2767), cardiolipin (CL, from bovine heart; C5646), and sphingomyelin (SM, from bovine brain; S7004). They were used without further processing or purification. Except for the synthesized PA, the samples may vary with respect to the acyl chain composition of the PLs. In order to account for this diversity, PLs will, throughout this article, be designated by their headgroup and the total number of carbon atoms at both the sn-1 and sn-2 positions, followed by the total number of double bonds. For instance, PC(36:2) denotes a phosphatidylcholine molecule with a total of 36 carbon atoms and two double bonds anywhere in the acyl chains. It should be noted that a determination of the exact composition of either of the two acyl chains is usually not possible on the basis of plain molecular weight information. However, this information might be obtainable by tandem MS analysis, which was, however, not available with the current setup. (23) Ivleva, V. B.; Elkin, Y. N.; Budnik, B. A.; Moyer, S. C.; O’Connor, P. B.; Costello, C. E. Anal. Chem. 2004, 76, 6484-6491. (24) Loboda, A. V.; Ackloo, S.; Chernushevich, I. V. Rapid Commun. Mass Spectrom. 2003, 17, 2508-2516.

A major difference between sphingomyelin and the other five investigated lipid classes is that the former contains a sphingoid base instead of the glycerol backbone and only one additional acyl chain. Since the exact compositions of the sphingosine backbone and the acyl chain cannot be determined by MS,1 sphingomyelinspecieswillsinanalogytotheglycerophospholipidss be designated by the total number of carbon atoms in the sphingoid backbone and the acyl chain, followed by the total number of double bonds. For instance, sphingomyelin containing a d18:1 sphingoid base and a 16:0 acyl chain will be referred to as SM (34:1). High-Performance Thin-Layer Chromatography. A volume of 50 µL of a mixture of the above phospholipids, dissolved at a concentration of 0.1 g/L per single PL sample in chloroform/ methanol (2:1, v/v), was applied to an HPTLC plate with an automatic TLC applicator (Linomat IV, CAMAG, Muttenz, Switzerland), yielding a sample load of 5 µg per single PL sample in bands of 5-mm width. In order to evaluate the sensitivity of the HPTLC-IR-MALDI-oTOF-MS method, a dilution series was prepared. Mixtures containing 5, 2, and 1 µg and 500, 200, 100, 50, 20, and 10 ng per single PL sample were applied to parallel HPTLC lanes. Plates were developed in a standard glass tank lined with filter paper under saturated atmosphere with chloroform/methanol/ 2-propanol/triethylamine/0.25% KCl (30:9:25:18:6 by volume) for 45 min. For each experiment, two identical HPTLC plates were developed. One of them was subsequently stained with molybdenium blue reagent according to the procedure of Dittmer and Lester,25 with minor modifications as described in detail previously.26 After chromatography, the plates were dipped for 1 s in molybdenium blue reagent and dried at ambient temperature in a fume hood. The phosphate derivatives appear as blue stains on the silica gel layer, reaching their maximum intensity after ∼20 min, while the gel itself remains mostly white. Stained chromatograms served as references for the following MALDI preparation (see below). Color images of stained chromatograms were taken with a commercial scanner (hp scanjet 7400c). Optical intensity profiles (densitograms) were obtained by plotting line scans across selected bands using public license image analysis software (ImageJ Version 1.33u, URL http://rsb.info.nih.gov/ij/). Orthogonal-TOF Mass Spectrometer. The oTOF mass spectrometer employed is a modified prototype, similar to the one described by Loboda et al.24 In the meantime, a commercial version of this instrument (prOTOF 2000) has become available from Perkin-Elmer. In the mass range relevant for PL analysis, the employed prototype provides a mass resolution of ∼10 000 and a mass accuracy of ∼20 ppm. The mass spectrometer is by default equipped with a nitrogen laser for UV desorption/ ionization. For IR-MALDI-MS, an Er:YAG infrared laser (Bioscope, BiOptics Laser Systems AG, Berlin, Germany) has been added. This laser emits Q-switched pulses of ∼100-ns duration at a wavelength of 2.94 µm. The laser beam is coupled into the ion source via an additional vacuum port and focused onto the sample by a planoconvex lens of 200-mm nominal focal length, yielding a focal spot size of ∼220 × 300 µm2. A CCD camera serves for sample observation with a resolution of ∼20 µm. (25) Dittmer, J. C.; Lester, R. L. J. Lipid Res. 1964, 5, 126-127. (26) Mu ¨ thing, J.; Radloff, M. Anal. Biochem. 1998, 257, 67-70.

The ion source is operated at an elevated pressure of 0.1-1 mbar nitrogen. Ions desorbed from the sample are vibrationally de-excited by collisional cooling and accelerated into a quadrupole ion guide by a low extraction field of ∼25 V mm-1. The pressure in the ion guide is maintained at ∼10-3 mbar in order to ensure a sufficient degree of collisional focusing. The lower cutoff of the quadrupole was set to an m/z of 400 throughout the experiments. After passing the ion guide, ions are extracted into a differentially pumped region containing electrostatic lenses for shaping and steering of the ion beam. Subsequently, ions enter the highvacuum TOF section of the instrument maintained at a residual gas pressure of 10-7 mbar, where they are accelerated by a pulsed pusher voltage in orthogonal direction with respect to their original movement. Pusher voltages were set to 10 and -10 kV for positive and negative ion modes, respectively. After passing through a first field-free drift region, ions are reflected by a dual-stage reflectron, providing second-order time focusing, and are detected by a microchannel plate detector (MCP) in a chevron configuration. After amplification by the MCP, the secondary electron signal is collected by four independent anodes and forwarded to a multichannel time-to-digital converter (TDC × 8; Ionwerks, Houston, TX), which records the arrival time of each ion. In order to accommodate 1.8-mm-thick cut-out HPTLC plates, a custom-made sample plate based on the standard 100-well format of the instrument was used. A central region of 32 × 45 mm2 was milled out of an in-house-reproduced brass sample plate to a depth of 2 mm. Sections of HPTLC plates were attached in the cutout with double-sided adhesive pads of 200-µm thickness. The oTOF instrument was calibrated with a two-point calibration using molecular ions of bradykinin fragment 1-7 and substance P, desorbed from a standard glycerol preparation applied next to the cutout. Mass spectra were processed and evaluated with MoverZ3 software (version 2002.02.20, Genomics Solutions, Ann Arbor, MI). Peak assignment is based on the criterion that experimental and theoretical molecular weights match within the instrumental accuracy of 20 ppm. Sample Preparation for MALDI-MS. HPTLC-IR-MALDI-MS was exclusively performed from unstained plates. For the matrix application, a stained reference chromatogram was placed next to the corresponding unstained one, and three to five drops of ∼0.3 µL of glycerol were applied next to each other across each identified band using an Eppendorf pipet with 0.1-10-µL tips. The glycerol was rapidly soaked up by the porous silica gel and spread out to wetted spots of ∼2 mm in diameter, which were easily identifiable on the basis of their optical contrast. As an alternative to this “spotting” protocol, it has recently been shown that a preparation is also possible by spraying a glycerol/alcohol mixture across the entire plate, yielding essentially the same analytical results.20 For the sake of convenience, the spraying protocol was, however, omitted in the present study. Developed HPTLC plates were cut to pieces of ∼10 × 40 mm2 in order to fit into the sample plate. Acquisition of mass spectra was started directly after introducing the sample plate into the ion source of the mass spectrometer. Typically, between 50 and 120 single laser shots were applied on different positions of single HPTLC bands. For standard IR-MALDI-MS, 2 µL of the individual PL samples was dissolved in chloroform/methanol (2:1, v/v) at a concentration Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

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of 1 g/L and mixed with 0.5 µL of glycerol on a standard sample plate.

RESULTS AND DISCUSSION Standard IR-MALDI-oTOF-MS Analysis of PL Samples. In order to assess the general IR-MALDI-oTOF-MS performance, purified phospholipid samples containing only one distinct PL type were prepared as described above and analyzed in positive and negative ion modes. The corresponding mass spectra are shown in Figures 1a-f and 2a-f, respectively. Positive Ion Mode. Proposed identities of the ion species observed in the positive ion mode mass spectra are listed in Table 1, together with the calculated and experimental mass values for the 12C isotopomer. For each individual PL sample, several ion species are observed. The mass spectrum of PG, displayed in Figure 1b, is a typical example of a phospholipid with only one distinct type of acyl chain composition, namely, 36:2. PG molecular ions are detected as a series of sodiated and potassiated species, including singly and doubly sodiated and potassiated PG, respectively, and a species containing both sodium and potassium. In addition, adduct ions carrying one or more glycerol molecules are observed at high abundance. This is not surprising, since it is well-known that MALDI ion sources operated at elevated pressure tend to enhance matrix adduct formation.27,28 Finally, adduct formation with NaCl is also detected, albeit with lower abundance; these adducts are easily identified by the characteristic isotopic pattern of chlorine. Accordingly, almost all signals found in the mass spectrum obtained from the PG sample can be attributed to molecular ions with varying adduct content. Fragments of PG were not observed in the covered m/z range. Except for the NaCl adducts, which are clearly visible only for CL, PG, and SM (Figure 1a, b, and f), the above stated ion species are, in principle, also found in all other positive ion mode spectra. The mass spectrum of PE, plotted in Figure 1c, illustrates the example of a phospholipid with varying acyl chain composition (34:1, 34:2, 36:1, 36:2, and 38:4). Sodiated and potassiated molecular ions, along with their glycerol adducts, are also observed in this case, but since the adduct pattern is convoluted with the pattern of the acyl chain distribution, the PE spectrum is more complex than that of PG. PC and SM contain choline as a headgroup. Besides the acyl chain and cation/glycerol adduct patterns, the corresponding mass spectra, shown in Figure 1e and f, respectively, exhibit an additional series of signals that can be attributed to fragment ions emerging from a neutral loss of trimethylamine (N(CH3)3) from the choline headgroup. These fragment ions are likewise observed with varying cation or glycerol adduct content. Negative Ion Mode. For the negative ion mode, the proposed structures of the detected ion species are listed in Table 2, together with calculated and experimental m/z values for the 12C isotopomer. As in the positive ion mode, a variety of ion species is observed for each single PL sample. A second typical example of a phospholipid containing only one distinct acyl chain composition, which produces high signal intensities in the negative ion (27) Loboda, A. V.; Krutchinsky, A. N.; Bromirski, M.; Ens, W.; Standing, K. G. Rapid Commun. Mass Spectrom. 2000, 14, 1047-1057. (28) O’Connor, P. B.; Costello, C. E. Rapid Commun. Mass Spectrom. 2001, 15, 1862-1868.

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mode, is given by PA. Its mass spectrum obtained from a standard preparation is displayed in Figure 2d. PA is detected as [M H]- but also in form of deprotonated adduct complexes containing glycerol, NaCl, or both, as already noted for the positive ion mode. Generally, the mass accuracy of ∼20 ppm provided by the mass spectrometer helps substantially in the tentative compositional assignment of species, even if a tandem-MS option is unfortunately not available on the prOTOF. In some cases, a small but notable offset between the calculated and detected mass values prevented a false identification. As an example, one could attribute the signals marked with an asterisk in Figure 2d by their “nominal” mass to deprotonated PA (44:2) (calculated monoisotopic mass, 811.6216 Da) and the corresponding glycerol adduct (calculated monoisotopic mass, 903.6690 Da). However, the detected m/z values of 811.51 and 903.55 Da, respectively, are both ∼110 mDa too low to confirm this assignment. The origin of the two ion signals could as yet not be clarified. Possible explanations could include fragmentation pathways or adduct formation not taken into account by the authors or the presence of impurities contained in the original phospholipid samples. A typical negative ion mode spectrum of a phospholipid with varying acyl chain content is given by that of PE, plotted in Figure 2c. Again, different PE species are observed as deprotonated molecular ions and their glycerol adducts. NaCl salt adducts are not clearly visible, probably because their signal intensity is too low to exceed the level of chemical noise. An interesting case is the spectrum of CL, displayed in Figure 2a. Unlike the other investigated phospholipids, CL contains two phosphate groups and, hence, two acidic moieties.29 Therefore, in negative ion mode, CL is detected as doubly deprotonated and singly sodiated species, exhibiting a net charge of -1. Besides the adduct ion with glycerol, minor ion signals are observed, which can be attributed to a successive loss of acyl chains, starting from the loss of one and two linoleyl moieties, respectively, and going up to the complete loss of two acyl chains and one of the two glycerol backbones of CL. Acyl chain losses are also observed in the PG spectrum shown in Figure 2b. In this case, an oleyl moiety is cleaved from the deprotonated molecular ion. In addition, the typical glycerol and NaCl adducts are detected for PG. The mass spectra of PC and SM, presented in Figure 2e and f, contain intense signals that correspond to fragment ions representing a loss of either a methyl cation (CH3+), trimethylammonium (HN(CH3)3+), or vinyltrimethylammonium (CH2CHN(CH3)3+) from the choline headgroup. These fragments are also detected in the form of adducts with glycerol. The data do not allow us to differentiate whether the glycerol adduct formation happened after fragmentation or whether fragmentation occurred from a molecule complexed with glycerol. Simple deprotonated molecular ions ([M - H]-) are not detected for PC and SM. HPTLC of Phospholipid Mixtures. Mixtures of the six investigated phospholipid samples (CL, PG, PE, PA, PC, SM) were prepared and separated by HPTLC according to the procedure described above. Developed plates were subsequently stained with molybdenium blue. A typical chromatogram is shown in Figure 3. For the employed nonpolar liquid and polar stationary-phase combination, PLs are essentially separated according to their (29) Beckedorf, A. I.; Scha¨ffer, C.; Messner, P.; Peter-Katalinic, J. J. Mass Spectrom. 2002, 37, 1086-1094.

Figure 1. IR-MALDI-oTOF mass spectra of the six investigated phospholipid samples acquired from standard preparations with a glycerol matrix in positive ion mode: (a) cardiolipin, (b) phosphatidylglycerol, (c) phosphatidylethanolamine, (d) phosphatidic acid, (e) phosphatidylcholine, and (f) sphingomyelin. Labels indicate proposed ion structures (see Table 1 for a comprehensive list). Gro, glycerol.

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Figure 2. IR-MALDI-oTOF mass spectra of the six investigated phospholipid samples acquired from standard preparations with a glycerol matrix in negative ion mode: (a) cardiolipin, (b) phosphatidylglycerol, (c) phosphatidylethanolamine, (d) phosphatidic acid, (e) phosphatidylcholine, and (f) sphingomyelin. Labels indicate proposed ion structures (see Table 2 for a comprehensive list). Asterisks in (d) denote ion signals that have the same nominal mass as [PA (44:2) - H]- and its glycerol adduct, respectively, but exhibit an exact m/z value ∼110 mDa too high. 5798 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

Table 1. Proposed Structures of Molecular Ions Detected by Positive Ion Mode IR-MALDI-oTOF-MS of Phospholipid Samples from Standard Preparations Using Glycerol as Matrix (Standard MALDI) and from Identified Bands after HPTLC Separation and Wetting with Glycerol (HPTLC-MALDI). phospholipid ion species

m/z calcd

std MALDI

HPTLCMALDI

CL

m/z calcd

std MALDI

PA (36:2) + Na PA (36:2) - H + 2Na PA (36:2) - H + Na + K PA (36:2) - 2H + 3Na PA (36:2) - 2H + 2Na + K PA (36:2) + Na + Gro PA (36:2) - H + 2Na + Gro PA (36:2) - H + Na + K + Gro PA (36:2) - 2H + 3Na + Gro PA (36:2) - 2H + 2Na + K + Gro PA (36:2) - H + 2Na + 2Gro PA (36:2) - 2H + 3Na + 2Gro PA (36:2) - 2H + 2Na + K + 2Gro PA (36:2) - 2H + 3Na + 3Gro PA (36:2) - 2H + 2Na + K + 3Gro

723.4941 745.4760 761.4499 767.4580 783.4319 815.5414 837.5234 853.4973 859.5053 875.4793 929.5707 951.5527 967.5266 1043.6000 1059.5739

723.50 745.48 761.45 767.46 783.43 815.54 837.53 853.50 859.50 875.48 929.56 951.55 967.52 1043.60 1059.58

721.4784 723.4941 737.4523 739.4680 749.5097 751.5254 758.5699 760.5856 780.5519 782.5676 796.5258 798.5415 808.5832 810.5989 815.5414 824.5571 826.5728 872.5992 874.6149 888.5732 890.5888 900.6305 902.6462

721.49 723.50 737.46 739.47 749.52 751.53

HPTLCMALDI

PA

CL (72:8) - H + 2Na CL (72:8) - 2H + 3Na CL (72:8) - 2H + 2Na + K CL (72:8) - 2H + 3Na + NaCl CL (72:8) - H + 2Na + Gro CL (72:8) - 2H + 3Na + Gro CL (72:8) - 2H + 2Na + K + Gro CL (72:8) - 2H + 3Na + Gro + NaCl CL (72:8) - 2H + 3Na + 2Gro

1493.9439 1515.9259 1531.8998 1573.8845 1585.9912 1607.9732 1623.9471 1665.9319 1700.0206

1515.93 1531.90 1573.88 1607.98 1623.94 1665.93

1493.94 1515.92 1573.89 1586.00 1607.97 1665.94 1700.02

PG PG (36:2) + Na PG (36:2) + K PG (36:2) - H + 2Na PG (36:2) - H + Na + K PG (36:2) - H + 2K PG (36:2) - H + 2Na + NaCl PG (36:2) - H + Na + K + NaCl PG (36:2) - H + 2Na + Gro PG (36:2) - H + Na + K + Gro PG (36:2) - H + 2K + Gro PG (36:2) - H + 2Na + Gro + NaCl PG (36:2) - H + Na + K + Gro + NaCl PG (36:2) - H + 2Na + 2Gro

797.5308 813.5047 819.5128 835.4867 851.4606 877.4715 893.4454 911.5601 927.5341 943.5080 969.5188 985.4927 1003.6075

797.53 813.51 819.52 835.48 851.46 877.47 893.44 911.56 927.54 943.50 969.52 985.49 1003.61

797.52 819.51

PC 877.47 911.55

1003.62

PE PE (34:2) + Na PE (34:1) + Na PE (34:2) - H + 2Na PE (34:1) - H + 2Na PE (36:2) + Na PE (36:1) + Na PE (36:2) - H + 2Na PE (36:1) - H + 2Na PE (38:4) + Na PE (36:2) - H + Na + K PE (36:1) - H + Na + K PE (36:2) - 2H + 3Na PE (36:1) - 2H + 3Na PE (36:1) - 2H + 2Na + K PE (34:1) + Na + Gro PE (34:2) - H + 2Na + Gro PE (34:1) - H + 2Na + Gro PE (34:1) - H + Na + K + Gro PE (36:2) - H + 2Na + Gro PE (36:1) - H + 2Na + Gro PE (36:1) - 2H + 3Na + Gro PE (34:1) - 2H + 2Na + K + Gro PE (34:1) - H + 2Na + 2Gro PE (36:2) - H + 2Na + 2Gro PE (36:1) - H + 2Na + 2Gro PE (36:1) - H + Na + K + 2Gro

a

phospholipid ion species

738.5050 740.5206 760.4869 762.5026 766.5363 768.5519 788.5182 790.5339 790.5363 804.4921 806.5078 810.5002 812.5159 828.4898 832.5679 852.5343 854.5499 870.5238 880.5656 882.5812 904.5632 920.5371 946.5972 972.6129 974.6285 996.6105

738.51 740.52 762.50 766.54 768.55 788.53 790.53 790.53 804.50 806.51 --812.52 828.50 832.57 852.53 854.56 870.52 880.58 882.59 904.58 920.54 946.60 972.61 974.63 996.61

740.52 760.49 762.51 766.53 768.55 788.52 790.53

810.51 812.52

PC (34:2) - N(CH3)3 + Na PC (34:1) - N(CH3)3 + Na PC (34:2) - N(CH3)3 + K PC (34:1) - N(CH3)3 + K PC (36:2) - N(CH3)3 + Na PC (36:1) - N(CH3)3 + Na PC (34:2) + H PC (34:1) + H PC (34:2) + Na PC (34:1) + Na PC (34:2) + K PC (34:1) + K PC (36:2) + Na PC (36:1) + Na PC (34:1) - N(CH3)3 + Na + Gro PC (36:2) + K PC (36:1) + K PC (34:2) + Na + Gro PC (34:1) + Na + Gro PC (34:2) + K + Gro PC (34:1) + K + Gro PC (36:2) + Na + Gro PC (36:1) + Na + Gro

780.56 782.57 796.53 798.55 808.59 810.60 815.55 824.56 826.58 872.60 874.61 888.59 890.59 900.64 902.65

721.48 723.49 749.51 751.52 758.56 760.60 780.55 782.57 808.58 810.60

872.60 874.61 900.63 902.65

SM 852.53 854.55 880.56 882.57 904.57 946.59 972.60 974.62

SM (36:1) - N(CH3)3 + Na SM (36:1) - N(CH3)3 + K SM (34:1) + Na SM (36:1) + Na SM (36:1) + K SM (42:2) - N(CH3)3 + Na SM (36:1) - N(CH3)3 + Na + Gro SM (36:1) + Na + NaCl SM (34:1) + Na + Gro SM (42:2) + Na SM (36:1) + Na + Gro SM (42:2) + K SM (36:1) + K + Gro SM (42:2) + Na + NaCl SM (36:1) + Na + Gro + NaCl SM (42:2) + Na + Gro SM (42:2) + K + Gro SM (42:2) + Na + Gro + NaCl

694.5151 710.4891 725.5573 753.5886 769.5625 776.5934 786.5625 811.5473 817.6047 835.6669 845.6360 851.6408 861.6099 893.6255 903.5946 927.7142 943.6881 985.6729

694.52 710.49 725.56 753.59 769.57 776.59 786.56 817.61 835.66 845.64 851.64 861.61 927.71 943.70 985.68

694.52a 725.55a 753.60a 776.59b 786.56a 811.55a 817.61a 835.67b 845.63a 893.62b 903.59a 927.71b 985.67 b

Detected in the lower region of the SM band. b Detected in the upper region of the SM band.

polarity, with the least polar species (CL, PG) exhibiting the highest Rf values. The polarity of a phospholipid is primarily determined by its headgroup. Therefore, the individual PL types are separated into discrete bands. Though of minor effect, another

criterion affecting the polarity is the acyl chain composition. Phospholipids with identical headgroups but acyl chains of sufficiently different lengths may be split up into closely migrating multiple bands, as will be demonstrated for SM below. Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

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Table 2. Proposed Structures of Molecular Ions Detected by Negative Ion Mode IR-MALDI-oTOF-MS of Phospholipid Samples from Standard Preparations Using Glycerol as Matrix (Standard MALDI) and from Identified Bands after HPTLC Separation and Wetting with Glycerol (HPTLC-MALDI). phospholipid ion species

m/z calcd

std MALDI

HPTLCMALDI

CL

phospholipid ion species

m/z calcd

std MALDI

HPTLCMALDI

699.4964 757.4551 791.5438 849.5024

699.50 757.46 791.54 849.50

699.49 757.46 791.54 849.50

673.4808 697.4808 699.4964 725.5121 727.5277 742.5386 744.5543 791.5438 834.5860 836.6016

673.49 697.47 699.49 725.52 727.53 742.54 744.56 791.54 834.58 836.59

673.49

PA

CL (72:8) - 2 linoleyl + 2H - Gro + Na CL (72:8) - 2 linoleyl + 2H + Na CL (72:8) - linoleyl + H + Na CL (72:8) - 2H + Na CL (72:8) - 2H + Na + Gro

853.4396 945.4870 1207.7166 1469.9463 1561.9936

PA (36:2) - H PA (36:2) - H + NaCl PA (36:2) - H + Gro PA (36:2) - H + Gro + NaCl

853.44 945.50 1207.73 1469.95 1561.99

PC PG PG (36:2) - oleyl + H PG (36:2) - oleyl + H + NaCl PG (36:2) - H PG (36:2) - H + NaCl PG (36:2) - H + Gro PG (36:2) - H + Gro + NaCl 2 PG (36:2) - oleyl + Na 2 PG (36:2) - 2H + Na

509.2879 567.2466 773.5332 831.4919 865.5805 923.5392 1305.8109 1570.0562

509.29 567.25 773.53 831.50 865.59 923.55

509.29 773.53 831.49 865.58 923.54 1305.81 1570.06

PC (34:1) - CH2CHN(CH3)3 PC (34:2) - HN(CH3)3 PC (34:1) - HN(CH3)3 PC (36:2) - HN(CH3)3 PC (36:1) - HN(CH3)3 PC (34:2) - CH3 PC (34:1) - CH3 PC (34:1) - HN(CH3)3 + Gro PC (34:2) - CH3 + Gro PC (34:1) - CH3 + Gro

699.49

744.56 791.53 836.60

PE SM PE (34:2) - H PE (34:1) - H PE (36:2) - H PE (36:1) - H PE (38:4) - H PE (34:2) - H + Gro PE (34:1) - H + Gro PE (36:2) - H + Gro PE (36:1) - H + Gro PE (38:4) - H + Gro

a

714.5073 716.5230 742.5386 744.5543 766.5386 806.5547 808.5703 834.5860 836.6016 858.5860

714.51 716.54 742.53 744.55 766.54 806.56 808.58 834.59 836.61 858.59

SM (34:1) - CH2CHN(CH3)3 SM (34:1) - HCN(CH3)3 SM (34:1) - HN(CH3)3 SM (36:1) - CH2CHN(CH3)3 SM (36:1) - HCN(CH3)3 SM (36:1) - HN(CH3)3 SM (34:1) - CH3 SM (36:1) - CH3 SM (42:2) - CH2CHN(CH3)3 SM (42:2) - HCN(CH3)3 SM (42:2) - HN(CH3)3 SM (36:1) - CH3 + NaCl SM (42:2) - CH3 SM (36:1) - CH3 + Gro SM (42:2) - H SM (42:2) - CH3 + Gro

616.4706 630.4862 642.4862 644.5019 658.5175 670.5175 687.5441 715.5754 726.5801 740.5958 752.5958 773.5340 797.6536 807.6227 811.6693 889.7009

642.49 644.51 658.52 670.52 715.58 726.58 740.60 752.59 797.65 807.63 889.70

616.47a 630.48a 642.49a 644.50a 658.52a 670.52a 687.55a 715.57a 726.58b 740.60b 752.60b 773.53a 797.65b 807.62a 811.66b 889.71b

Detected in the lower region of the SM band. b Detected in the upper region of the SM band.

IR-MALDI-oTOF-MS of HPTLC-Separated Phospholipids. Positive Ion Mode. Positive ion mode mass spectra recorded from the bands of five of the six HPTLC-separated phospholipids are shown in Figure 4; see Table 1 for the experimental m/z values and proposed ion structures. The mass spectra obtained from the SM band are not shown here but will be discussed below. Although no detailed analysis of the “reproducibility” has been carried out so far, several separately developed plates were analyzed in both ion modes. Relevant changes concerning the obtained ion yields and patterns were not notable. The inset in the CL spectrum (Figure 4a) displays an enlargement of the signal corresponding to triply sodiated CL (72:8) at 1515.92 Da for the 12C isotopomer, demonstrating the high resolving power of the oTOF instrument, providing baseline isotopic resolution. When compared to the corresponding mass spectra acquired from the standard preparations (Figure 1), one striking feature of the positive ion mode HPTLC-MALDI spectra is the complete absence of signals related to potassiated ions. This effect, facilitating data interpretation, may be related to a certain desalination of the phospholipid samples during the HPTLC separation but, however, remains surprising considering the high concentration of KCl in the HPTLC developing solvent. Furthermore, the extent of protonation versus sodiation appears to be slightly enhanced, 5800 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

as can, for example, be seen from the appearance of additional peaks in the HPTLC-MALDI spectrum of PC (Figure 4e) at 758.56 and 760.60 Da, corresponding to protonated molecular ions of PC (34:2) and PC (34:1), respectively, which were not observed in the standard MALDI mass spectrum. The same holds for the ion signal at 1493.94 Da in the HPTLC-MALDI spectrum of CL (Figure 4a), attributable to only singly deprotonated and doubly sodiated CL (72:8). This discrimination of sodiation versus protonation may likewise be related to a certain sample desalination during the HPTLC separation. Except for the absence of all potassium-related signals, the HPTLC-MALDI spectrum of PG, displayed in Figure 4b, is qualitatively identical to the one measured from the standard preparations (Figure 1b). However, in contrast to PC and SM, cationization by sodium appears to be favored over protonation in this case, as the relative signal intensity of singly sodiated PG at 797.52 Da is slightly suppressed in comparison with the doubly sodiated species at 819.51 Da. For PE, which constitutes a phospholipid sample with varying acyl chain content, almost all acyl chain species that were recorded from the standard preparation are also found in the HPTLCMALDI spectrum (Figure 4c). However, no evidence was found for the presence of PE (38:4), which was clearly visible in the

Figure 3. HPTLC chromatogram of a mixture of the six investigated phospholipids stained with molybdenum blue. 5 µg per single PL sample was applied. Separation of the single PL types is based on the difference in polarity, with the most unpolar species exhibiting the largest Rf values.

negative ion mode spectrum obtained from the standard preparation (Figure 2c), and was also indicated in positive ion mode by a disproportionally high ion signal at 790.53 Da, most probably due to a superposition of [PE (36:1) - H + 2Na]+ and [PE (38:4) + Na]+. It appears likely that PE (38:4) was eluted into a weaker separate band during the HPTLC run due to the distinct difference in acyl chain length and that it could hence not be detected in the HPTLC-MALDI analysis of the stronger band inclosing the PE species adding up a total of 34 and 36 carbon atoms. By their “nominal” mass, the signals marked in the figure with one and two asterisks could, in principle, be attributed to protonated glycerol adducts of PE (34:2) and PE (34:1), respectively. Their exact detected m/z values of 808,48 and 810,50 Da are, however, each ∼90 mDa too low to affirm this assumption when compared to the theoretical masses of 808.5703 and 810.5860 Da, respectively. Most likely, the latter ion signal, therefore, represents triply sodiated PE (36:2) (calculated m/z value, 810.5002). The origin of the other ion signal could not finally be clarified. Except for the potassium-related signals, all species found in the standard MALDI spectrum of PC (Figure 1e) are also observed in the HPTLC-MALDI spectrum, plotted in Figure 4e, including the fragment ions derived from a loss of trimethylamine from the choline headgroup. As stated above, protonated molecular ions of PC (34:2) and PC (34:1) are found in the HPTLC-MADLI spectrum, which were not detected in the standard MALDI spectrum. One important exception is given by the spectrum acquired from the PA band, displayed in Figure 4d. In this case, mass spectra recorded from the HPTLC band and the standard

preparation look entirely different. None of the observed lowintensity ion signals obtained from the HPTLC band was found back in the spectrum from the standard preparation. Likewise, none of them could be matched satisfactorily with any PA ion species. The signal marked with three asterisks in Figure 4d, having an m/z value of 929.51 Da, could by its nominal mass be attributed to [PA (36:2) - H + 2Na + 2Gro]+, where Gro represents a glycerol molecule. However, the detected m/z value is ∼60 mDa too low to adequately match the theoretical mass of 929.5707 Da. The same holds for the signal marked with four asterisks located at an m/z value of 931.53 Da, which is ∼70 mDa too high to be assigned to [PA (36:2) + Na + Gro + 2NaCl]+ (calculated mass, 931.4587 Da). The origin of the observed ion signals is not clear. However, similar signal patterns were found neither in the HPTLC-MALDI spectra from other PL bands nor in spectra acquired from positions off the bands. Therefore, it appears reasonable to assume that the observed ion signals are, nevertheless, somehow related to PA, possibly via fragmentation pathways or the formation of adducts not taken into account by the authors so far. Negative Ion Mode. Figure 5 displays negative ion mode HPTLC-MALDI spectra acquired from five of the six PL bands. Proposed molecular ion structures are listed in Table 2, together with theoretical and detected monoisotopic mass values. The mass spectra obtained from the SM band will be discussed below. For PG, all ion species observed in the mass spectrum of the standard preparation are also found in the HPTLC-MALDI spectrum, displayed in Figure 5b, including deprotonated PG, the fragment ion derived from loss of an oleyl chain, and adduct complexes with glycerol and NaCl. An extended mass range is shown in Figure 5b, in order to illustrate the presence of gas-phase dimers and of a mixed complex ion species composed of one intact PG molecule and one PG having lost an oleyl moiety. Both species are singly sodiated, hence exhibiting a total net charge of -1. Likewise, all ion species that were identified in the standard MALDI spectrum of PA (Figure 2d) are also found in the HPTLCMALDI spectrum plotted in Figure 5d. It is interesting to note that the signals that were observed from the standard preparation and could not be matched with any PA ion species (asterisks in Figure 2d) are completely absent in the HPTLC-MALDI spectrum. This observation substantiates the suspicion that these signals originate from sample impurites, which were eliminated from the PA band during the HPTLC separation. The HPTLC-MALDI mass spectrum of PC, displayed in Figure 5e, exhibits a considerably lower signal-to-noise (S/N) ratio than that acquired from the standard preparation. Hence, only major ion signals corresponding to the most abundant PC (34:1) species are detected with sufficient S/N ratio. As for the standard preparation, essentially fragment ions derived from successive losses of parts of the choline headgroup and their glycerol adducts are observed. Ion signals other than the labeled ones could not be matched satisfactorily with any PC ion species. Possibly they stem from sample impurities eluted into the PC band or adhesives from the silica gel. For CL and PE (Figure 5a and c), no clear signals assignable to the corresponding phospholipid ion species could be found in the negative ion mode. In the spectrum acquired from the PE band, only a few distinct ion signals of very low intensity are Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

5801

Figure 4. HPTLC-IR-MALDI-oTOF mass spectra acquired directly from individual glycerol-wetted phospholipid bands in positive ion mode: (a) cardiolipin, (b) phosphatidylglycerol, (c) phosphatidylethanolamine, (d) phosphatidic acid, and (e) phosphatidylcholine. Labels indicate proposed ion structures (see Table 1 for a comprehensive list). The inset in (a) shows a magnification of the [CL (72:8) - 2H + 3Na]+ ion signal at 1516 Da, demonstrating the high resolving power of the oTOF instrument of ∼10 000. Single and double asterisks in (c) denote ion signals which have the same nominal mass as protonated glycerol adducts of PE (34:2) and PE (34:1), respectively, but exhibit exact m/z values ∼90 mDa too low. The three asterisks in (d) denote an ion signal that has the same nominal mass as [PA (36:2) - H + 2Na + 2Gro]+, but exhibits an exact m/z value ∼60 mDa too low. 5802

Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

Figure 5. HPTLC-IR-MALDI-oTOF mass spectra acquired directly from individual glycerol-wetted phospholipid bands in negative ion mode: (a) cardiolipin, (b) phosphatidylglycerol, (c) phosphatidylethanolamine, (d) phosphatidic acid, (e) phosphatidylcholine. Labels indicate proposed ion structures (see Table 2 for a comprehensive list). The asterisk in (a) denotes an ion signal attributable to deprotonated PG (36:2), most probably stemming from an overlap of the CL band with the neighboring PG band. Gro: glycerol.

Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

5803

observed. None of them could satisfactorily be matched with appropriate PE ion species. The spectrum acquired from the CL band does not exhibit any differentiated features in the mass range from 1400 to 1600 Da, where CL ion signals would be expected to occur. In contrast, a collection of lower mass background ions is observed, with m/z values up to 800 Da. The signal marked with an asterisk is located at an m/z value of 773.55 Da. It can straighforwardly be attributed to deprotonated PG (36:2) and is, therefore, most probably due to a partial overlap with the neighboring PG band. Discussion of Phospholipid Spectra. For both the standard preparations and direct HPTLC-MALDI-MS, the different kinds of phospholipids are detected as different ion species and with strongly varying S/N ratios. Partly, this observation can be explained by the differences in acidity of the phospholipids, which determines their deprotonation state under the conditions in the MALDI sample. Where applicable, effects of the acyl chain length variability have furthermore to be taken into account. Since glycerol does not exhibit any acidic or basic moieties, it is reasonable to assume a neutral pH of ∼7 inside the MALDI sample. Under these conditions, different PL types will exhibit different deprotonation states and will, therefore, accumulate different net charges.30 For instance, PC and PE both form zwitterions due to the combination of a deprotonated and, hence, negatively charged phosphate group with a positively charged headgroup, holding a total net charge of zero. In contrast, PG and PA possess neutral headgroups and are thus more acidic. They must be expected to hold a net charge of -1 in the glycerol solution. Finally, CL exhibits two deprotonated phosphate moieties, making this species especially acidic with a net charge of -2. For detection in the positive ion mode, PLs must accumulate a positive net charge. As visible from the corresponding spectra (Figures 1 and 4), this is essentially achieved by adduct formation with an adequate number of cations (sodium for HPTLC-MALDI spectra; sodium and potassium for the standard preparations), yielding a net charge of exclusively +1 as typical for MALDI ionization.31 Apparently, protonation of phospholipids is of minor importance: Ion species that have received an additional proton with respect to their expected solution charge state are, for example, [CL (72:8) - H + 2Na]+, [PG (36:2) + Na]+, [PC (34: 2) + H]+, and [PC (34:1) + H]+, respectively. As can be seen from the corresponding mass spectra (Figure 4a, b, and e), these species are detected at only rather low abundances as compared to the exclusively alkali-cationized species. The different ion yields observed in the spectra of the six PL samples are to some extent also related to the variation in acyl chain composition, splitting the prepared molar amounts of individual species according to the relative composition of the sample. In the negative ion mode, the different S/N ratios can, in most of the cases, directly be related to the solution charge states of the different phospholipid species. For example, it is not surprising that PE and PC generally exhibit comparatively low signal intensities and PE is not even observed in the negative ion mode (30) Lipid Metabolism. URL http://web.indstate.edu/thcme/mwking/lipid-synthesis.html. (31) Karas, M.; Glu ¨ ckmann, M.; Scha¨fer, J. J. Am. Soc. Mass Spectrom. 2000, 35, 1-12.

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Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

HPTLC-MALDI spectra, since both species hold a net charge of zero in solution and do not possess any further acidic moieties. In addition, PE and PC are both phospholipid types with varying acyl chain content, further reducing the intensity of individual ion signals as discussed above. In contrast, PG and PA constitute phospholipids with only one distinct acyl chain composition and are readily deprotonated in their solution state, which promotes the generation of mass spectra with high signal intensities. It is, however, still rather surprising, that no ion signals attributable to CL could be recorded from the corresponding HPTLC band. Since CL can be expected to be doubly deprotonated in solution, it should, in principle, be detectable as a singly sodiated species as in the negative ion mode spectrum acquired from the standard preparation. In an additional experiment, 2 µL of a CL solution (1 g/L in chloroform/methanol, 2:1, v/v) was applied to an HPTLC plate, which had been predeveloped with the solvent system described in the Experimental Section but without any PLs on the plate. The CL solution was subsequently applied to that region, where CL would have migrated. After evaporation of the solvent, glycerol was added as in the HPTLC-MALDI experiments, and negative ion mode spectra were acquired from the glycerol-wetted area. Intense ion signals corresponding to [CL (72: 8) - 2H + Na]+ and its glycerol adduct complex were observed in this experiment (spectrum not shown). The reason why, in contrast, no CL ion signals were observable in the negative mode HPTLC-MALDI spectra could not finally be clarified. Fine Structure of the SM Band and Lateral Resolution. SM is another example of a phospholipid with variable acyl chain or sphingoid base length and saturation, ranging from 34:1 to 42: 2. The corresponding variation in polarity can be expected to result in different Rf values for the individual species. However, optically such a fine structure is not discernible from the chromatogram (see Figure 3). Also, the optical intensity profile (densitogram) of the SM band obtained by a line scan (see inset of Figure 7a) does not indicate any substructure. Instead the profile is approximately Gaussian in shape with a width at the 1/e2 level (∼13.5%) of ∼2.8 mm. In contrast, distinctly different MS patterns are recorded at different positions of the SM band. This is demonstrated in Figure 6, showing spectra acquired from the upper and the lower part of the band in positive and negative ion modes, respectively. It should be noted that positive ion mode spectra were recorded directly after applying the glycerol matrix to the SM band, whereas for this experiment, the negative ion mode spectra were acquired from the plate after it was stored at 5 °C for 3 months and then rewetted with glycerol. Generally, SM ions are detected by HPTLC-MALDI with ion patterns similar to those in the standard MALDI measurements (see Figures 1f and 2f and Tables 1 and 2). However, the SM (42:2) species, containing the longest fatty acid residues and having the lowest polarity, is exclusively found at higher Rf values in the upper part of the band (Figure 6a and c), whereas SM (34: 1) and SM (36:1), containing short-chain acyl moieties, are observed in the lower region of the SM band (Figure 6b and d). Signals marked with an asterisk in the negative ion mode spectrum in Figure 6c most likely stem from a slight overlap of the lower and the upper band and can be attributed to residual SM (36:1) ions.

Figure 6. HPTLC-IR-MALDI-oTOF mass spectra acquired directly from two positions on the glycerol-wetted SM band with higher and lower Rf value: (a) higher Rf value, positive mode; (b) lower Rf value, positive mode; (c) higher Rf value, negative mode; (d) lower Rf value, negative mode. Labels indicate proposed ion structures (see Tables 1 and 2 for a comprehensive list).

The substructure of the SM “double band” can be used to roughly assess the lateral resolution of the HPTLC-MALDI method. An inherent limitation of the lateral resolution is given by the laser spot size of (currently) ∼220 × 300 µm2, approximately defining the extent of the region from which ions are generated by each single desorption laser pulse. We are currently modifying the laser stage to allow for variable laser foci and smaller spot sizes. An MS intensity profile of the SM band recorded in positive ion mode is given in Figure 7b. The laser was scanned in steps of

200 µm across the band in chromatographic direction. For each step, 40 single spectra were summed, with the laser being moved across the band transversally to the chromatographic direction. From the recorded set of spectra, the ion signal intensities of the 12C isotopomers of [SM (34:1) + Na]+, [SM (36:1) + Na]+, and [SM (42:2) + Na]+ were determined by signal integration. The three profiles have been normalized for better comparability. Figure 7b reveals that SM does actually not only form a double but rather a triple band. The center of the SM (34:1) and SM (36:1) profiles are clearly separated by ∼0.5 mm, and the center Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

5805

Figure 7. Spatially resolved profiles of the SM band: (a) optical densitogram, (b) positive mode ion signal intensity profiles of the sodium adducts of SM (34:1), SM (36:1), and SM (42:2), and (c) negative mode ion signal intensity profiles of the demethylated fragments of SM (34:1), SM (36:1), and SM (42:2). Profile widths were derived by assessing the full widths at a fixed relative intensity value of 1/e2 (∼13.5%). The dashed lines in (b) and (c) indicate profiles of additional ion signals occurring at m/z values ∼50 mDa lower than that of [SM (34:1) + Na]+ and ∼40 mDa lower than that of [SM (34: 1) - CH3]-, respectively. Most probably these signals arise from an oxidative cleavage of the double bond in the 24:1 acyl chain of SM (42:2) (see text for details). Note that the profile in (c) was acquired after storing the glycerol-wetted HPTLC plate at 5 °C for 3 months.

of the SM (42:2) profile is separated by another 1.5 mm. This observation demonstrates a clear advantage of the combined method over a mere HPTLC analysis: The discovery of three different SM species within one band would not have been possible using a simple staining procedure. 5806 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

If one considers the rising edge of the SM (34:1) MS profile and the trailing edge of the SM (42:2) profile, an overall width at the 1/e2 peak level of ∼3.5 mm can be derived for the full SM band. Comparing this value to the width of the SM band of 2.8 mm as determined from the densitogram, the ion profiles would appear considerably broadened at first glance, even if one takes the dimensions of the laser spot size into account. However, it should be noted that this is probably not an effect of analyte diffusion due to the application of the glycerol matrix but rather reflecting the higher sensitivity of the MS analysis (see discussion below). An interesting feature can be found in the SM (34:1) profile displayed in Figure 7b. In addition to the principal maximum centered around 1.5 mm, a secondary maximum arises around 3.3 mm (dashed line in Figure 7b), stemming from an ion signal appearing in the corresponding spectra at almost the same mass as [SM (34:1) + Na]+ (spectra not shown). However, at second glance, it turns out that its exact detected mass of 725.50 Da is ∼50 mDa too low to be identified with [SM (34:1) + Na]+ (theoretical mass, 725.5573 Da). As can be seen from the profiles, the Rf value of this species is apparently very similar or even equal to that of SM (42:2). Therefore, it is reasonable to assume that the observed ion signal originates from the latter compound. Supposing a common d18:1 sphingoid backbone, a possible explanation can be given by oxidative cleavage of the double bond in the 24:1 fatty acid residue of SM (42:2), yielding an aldehyde moiety. The prevalent 24:1 fatty acid in vertebrates is nervonic acid, with the double bond located at position 15. An oxidative cleavage of this double bond would thus give rise to an ion containing a truncated C15 acyl residue with an ω-aldehyde group instead of the complete 24:1 acyl chain. The theoretical mass of the sodium adduct of this species amounts to 725.5209 Da, which is indeed ∼40 mDa lower than that of [SM (34:1) + Na]+. We note that obviously the same effect also appeared in our recent study on the analysis of gangliosides by HPTLC-IR-MALDIoTOF-MS during the recording of ion intensity profiles from a GM3 double band (see Figure 5 and Table 1 in ref 20), but was, however, overlooked at that time. The exact mechanisms leading to the oxidative cleavage of the double bond were not further investigated in the scope of the present study. Nevertheless, it is interesting to note that this type of fragmentation provides the opportunity to determine the positions of double bonds in an acyl chain, even though the relative intensity of the fragment ion signal is on the order of only a few percent in comparison to the [SM (42:2) + Na]+ parent ion. In negative ion mode, the signals of the demethylated fragment ions [SM - CH3]- of the three SM species were evaluated to obtain the ion intensity profiles shown in Figure 7c. Qualitatively, these profiles are similar to those recorded in positive ion mode with respect to the clear separation of the three SM species. Again, the SM (34:1) profile exhibits a principal maximum and the indication of a secondary maximum, with the latter showing approximately the same mass shift of, in this case, ∼40 mDa toward lower m/z values as in positive mode, confirming the results obtained from the positive ion mode profile and the conclusions drawn thereon. Considering again the rising and trailing edges of SM (34:1) and SM (42:2), respectively, the negative ion mode profile of the SM band exhibits an overall width

at 1/e2 peak intensity of 2.9 mm, slightly less than the 3.5 mm measured from the positive ion mode profile. This difference can be accounted for by the ∼5-8 times higher signal intensities obtained in positive ion mode. From the fact that the single SM species could still be detected as clearly separated profiles, even after the 3-months storage of the SM band coated with glycerol, we conclude that analyte diffusion in the glycerol matrix is only of minor importance for the spatial resolution. Sensitivity. In order to obtain a rough estimate of the analytical sensitivity of the direct HPTLC-MALDI coupling, a dilution series was prepared, with sample amounts ranging from 5 µg down to 10 ng per single PL type, which were applied to parallel HPTLC lanes. For each individual band, 80 single-shot spectra were averaged. PG and SM were selected as test analytes for the sensitivity study, since both generally yield comparatively intense ion signals in both positive and negative ion modes and can thus be expected to be detectable even for rather low sample loads. Sensitivities are, hence, to be understood as best estimates, not as certainties. A very rough estimate of the sensitivity for the other four species can be obtained by comparing their S/N ratios with those of PG and SM in the spectra obtained from the HPTLCseparated mixture (Figures 4 and 5). The mass spectrum displayed in Figure 8a was acquired in positive ion mode from the lower SM band of the 10-ng lane. Ion signals of sodiated SM (36:1) and its glycerol adduct can still be clearly distinguished from the chemical background noise. As a second example, two positive ion mode spectra acquired from the PG bands of the 50-ng and the 20-ng lane, respectively, are shown in Figure 8b. In this case, the 50-ng spectrum still reveals a detectable ion signal of [PG (36:2) - H + 2Na]+, whose intensity is, however, well within that of the chemical background. The 20-ng spectrum of PG exhibits only background ions, most probably originating from the silica gel, the glycerol, or both (note that essentially the same background ions are found in the SM spectrum). Hence, for PG, a LOD of ∼50 ng can straightforwardly be assumed in positive in mode, corresponding to a molar sample amount of ∼65 pmol. (Note that the LOD is mainly limited by the level of chemical background noise and can, hence, not be improved significantly by averaging over a larger number of laser exposures.) To calculate the LOD for the SM sample, the varying acyl chain content has to be taken into account. On the basis of the relative ion signal intensities in Figure 1f and Figure 6a and b, one can estimate that the percentage of SM (36:1) in the overall SM sample amounts to ∼50%. Thus, an actual amount of ∼5 ng of the SM (36:1) species was applied to the 10-ng lane, and the LOD can hence be estimated to be better than 5 ng in this case, corresponding to a molar sample amount of as little as 7 pmol applied for HTPLC. Similarly, LODs of 130 pmol for PG and 70 pmol for individual SM species can be estimated in the negative ion mode (spectra not shown). Compared to the “optical” LOD, which was estimated from the corresponding molybdenum-stained chromatograms shown in Figure 8c to be on the order of ∼100-200 ng, roughly corresponding to ∼200 pmol, the sensitivity is a factor of ∼2 better for negative ion mode HPTLC-MALDI and ∼1 order of magnitude better in the positive ion mode. (32) Schiller, J.; Su ¨ β, R.; Fuchs, B.; Mu ¨ ller, M.; Zscho¨rnig, O.; Arnold, K. Chromatographia 2003, 57, S297-S302.

Figure 8. Sensitivity of HPTLC-IR-MALDI-oTOF-MS of phospholipids. (a, b) Mass spectra acquired directly from glycerol-wetted single phospholipid bands on HPTLC lanes loaded with different sample amounts: (a) lower SM band at the lowest investigated sample load of 10 ng; (b) PG band at two different sample loads of 50 and 20 ng, demonstrating the approximate limit of detection for PG. Note that background ion signal patterns are similar for both phospholipid species. (c) Image of the corresponding HPTLC lanes stained with molybdenum blue.

In order to roughly assess the potential of HPTLC-MALDIMS for quantitative measurements, PG ion signal intensities from positive and negative ion mode measurements have been plotted as a function of the applied sample load in Figure 9a and b, Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

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Figure 9. HPTLC-MALDI ion signal intensities of PG ions as a function of the sample amount applied to the HPTLC plate: (a) positive ion mode, [PG (36:2) - H + 2Na ]+; (b) negative ion mode, [PG (36:2) - H]-. The solid lines display the results of linear regression analyses, yielding correlation coefficients as given in the figures.

respectively. The resulting graphs both appear slightly S-shaped. Despite a high statistical error, which may be due to the simple data evaluation protocol or, eventually, slight variations of the exact irradiated positions on the unstained plates, the data can, however, be fit in good approximation with a linear regression, yielding correlation coefficients of 0.972 and 0.931 for positive and negative ion modes, respectively. Although these values are not sufficient for a highly accurate quantitation, a rough estimate of the applied sample load can nevertheless be obtained by a comparison of the respective ion signal intensity with that of standards applied to separate HPTLC lanes. CONCLUSION We have demonstrated the application of the recently developed HPTLC-IR-MALDI-oTOF-MS method to the analysis of complex phospholipid mixtures. To the best of our knowledge, this constitutes the first example of phospholipids being analyzed by direct HPTLC-MALDI-MS. All six investigated phospholipid samples produced abundant molecular ion signals in either the positive or negative ion mode, largely depending on their acidity.

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Fragmentation of PLs was generally low, in contrast to standard UV-MALDI-TOF-MS analysis of standard preparations prepared with 2,5-dihydroxybenzoic acid as matrix.32 Partially, the low degree of fragmentation can be attributed to the collisional cooling provided by the oTOF instrument. The mass spectrometric LOD was found to range from a few picomoles to ∼100 pmol of material spotted for HPTLC, again depending on the chemical nature of the PLs. In most of the cases, the LOD was substantialy lower than the sensitivity of a mere optical assessment after staining with molybdenum blue. Summation over a higher number of applied laser pulses (instead of the 50-120 used here) may only slightly improve the sensitivity further, since the level of unspecific chemical background noise forms the limiting criterion. The lateral resolution of the analysis is on the order of the laser spot diameter of currently ∼250 µm. Analyte diffusion in the matrix was found to be negligible. Together, this allowed for the observation of substructures in the HPTLC band of sphingomyelin that were not discernible from the optical densitogram. Technically, the liquid glycerol provides a well homogeneous preparation that would, for exampe, facilitate an automated analysis. A drawback of the current protocol is the abundant adduct formation with matrix, NaCl molecules, and alkali cations. This complicates the mass spectra substantially for measurements from both standard preparations and the glycerol-coated HPTLC plate. Potassium adducts were, however, dramatically supressed after HPTLC. Enhanced preparation protocols or instrumental modifications, e.g., by installation of a heating coil in the quadrupole region of the oTOF, which is currently being realized in our laboratory, may help to reduce some of the adducts. Another focus of our current research is the coupling of an IR-MALDI source to an FT-ICR mass spectrometer. Besides the unmatched resolving power and mass accuracy of this type of mass analyzer, MS/MS and even MSn experiments will eventually become possible and may allow for an even more unambiguous identification of ion signals. ACKNOWLEDGMENT The authors thank Sequenom GmbH (Hamburg, Germany) for providing use of their oTOF instrument, and Ewald Kalthoff for his expert technical assistance with the HPTLC. We also thank Franz Hillenkamp for support of the project. Financial support by the Deutsche Forschungsgemeinschaft under grant DR 416/5-1 is gratefully acknowledged.

Received for review March 30, 2007. Accepted May 16, 2007. AC070633X