On-Line Overpressure Thin-Layer Chromatographic Separation and

Anal. Chem. 2003, 75, 118-125

On-Line Overpressure Thin-Layer Chromatographic Separation and Electrospray Mass Spectrometric Detection of Glycolipids Wengang Chai,* Christine Leteux, Alexander M. Lawson, and Mark S. Stoll

MRC Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Hospital, Watford Road, Harrow, Middlesex HA1 3UJ, U.K.

On-line thin-layer chromatographic separation and electrospray mass spectrometry (TLC/ESI-MS) has been accomplished by direct linking of a commercial overpressure TLC instrument, OPLC 50, and a Q-TOF mass spectrometer. Mass spectrometric detection sensitivity and chromatographic resolution achieved by this configuration were assessed using acidic glycolipids as examples. Under the optimized conditions, a sensitivity of 5 pmol of glycosphingolipid was readily demonstrated for TLC/ESI-MS and 20 pmol for TLC/ESI-MS/MS production scanning to derive the saccharide sequence and long chain base/fatty acid composition of the ceramide. Initial preconditioning of TLC plates is necessary to achieve high sensitivity detection by reducing chemical background noise. Plates can be used repeatedly (at least 10 times) for analysis, although this may result in a minor reduction in TLC resolution. Following solvent development, separated components on the TLC plates can be detected in the conventional way by nondestructive staining or UV absorption or fluorescence and can be stored for on-line TLC/ESI-MS analysis at a later stage without reduction in mass spectrometric detection sensitivity and chromatographic resolution. Aspects for further improvement of OPLC instrumentation include use of narrower TLC plate dimensions and refined design of the eluate exit system. Thin-layer chromatography (TLC) has been used extensively for the separation, purification, and qualitative and quantitative examination of virtually all types of compounds in mixtures.1 The greater spatial resolution achieved in high-performance TLC (HPTLC) and continuing improvements in the variety of stationary phases available complement the basic simplicity and versatility of planar chromatography. Several forced-flow TLC methods have been introduced in recent years, and a compact and simple instrument for overpressure layer chromatography (OPLC)2,3 is now commercially available. In OPLC, the TLC plate is covered with an inert membrane sheet under external pressure, and the mobile phase is pumped through the sealed layer of stationary * Corresponding author: Phone: 44-20-8869 3252. Fax: 44-20-8869 3253. E-mail: [email protected]. (1) Sherma, J. Anal. Chem. 2000, 72, 9R-25R. (2) Tyiha´k, E.; Mincsovics, E.; Sze´kely, T. J. J. Chromatogr. 1979, 174, 75-81. (3) Nyiredy, S. Trends Anal. Chem. 2001, 20, 91-101.

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phase, such as silica gel. The plate equates to a planar column and results in a substantially shorter analysis time, higher efficiency, and lower consumption of solvent as compared with conventional TLC. Analysis can be carried out in the conventional way with UV detection or staining of the TLC plate after solvent development. Additional methods are often required for unambiguous identification of fractionated components by TLC, and mass spectrometry (MS) has been the primary choice due to its detection sensitivity and speed of analysis. Early success in coupling TLC with MS has been off-line with in situ analysis of TLC bands by either liquid secondary ion mass spectrometry (LSIMS) or fast atom bombardment mass spectrometry. The liquid matrix used serves as an extraction solvent for the adsorbed sample molecules that are ionized directly from the condensed phase. Chang et al.4,5 first demonstrated direct analysis of TLC spots by this technique. Several other workers employed similar methods using double-sided adhesive tape to retain either the removed sorbent (silica gel)6 or a section of the TLC plate7-11 on a normal or modified probe tip. In addition, the development of an automated TLC target stage allowed mass spectra to be acquired from the silica surface over a continuous area of the TLC lane of interest.12-19 We have successfully used in situ HPTLC/ (4) Chang, T. T.; Jackson, O. L., Jr.; Francel, R. J. Anal. Chem. 1984, 56, 111113. (5) Chang, T. T.; Lee, T. M.; Borders, D. B. J. Antibiot. 1984, 37, 1098-1100. (6) Bare, K. J.; Read, H. Analyst 1987, 112, 433-436. (7) Kushi, Y.; Handa, S. J. Biochem. 1985, 98, 265-268. (8) Iwabushi, H.; Nakagawa, A.; Nakamura, K.-I. J. Chromatogr. Biomed. Appl. 1987, 414, 139-148. (9) Kushi, Y.; Rokukawa, C.; Handa, S. Anal. Biochem. 1988, 175, 167-176. (10) Nakaishi, H.; Sanai, Y.; Shibuya, M.; Iwamori, M.; Nagai, Y. Cancer Res. 1988, 48, 1753-1758. (11) Chai, W.; Cashmore, G. C.; Carruthers, R. A.; Stoll, M. S.; Lawson, A. M. Biol. Mass Spectrom. 1991, 20, 169-178. (12) Tamura, J.; Sakamoto, S.; Kubota, E. Analusis, 1988, 16, 64-69. (13) Nagashima, Y.; Nishio, S.; Noguchi, T.; Arakawa, O.; Kanoh, S.; Hashimoto, K. Anal. Biochem. 1988, 175, 258-262. (14) Masuda, K.; Harada, K.-I.; Susuki, M.; Oka, H.; Kawamura, N.; Yamada, M. Org. Mass Spectrom. 1989, 24, 74-75. (15) Lawson, A. M.; Chai, W.; Stoll, M.; Bateman, R. H.; Curtis, J.; Hounsell, E. F.; Feizi, T. Proceedings of the 37th ASMS Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, May, 1989; p 790. (16) Karlsson, K.-A.; Lanne, B.; Pimlott, W.; Teneberg, S. Carbohydr. Res. 1991, 221, 49-61. (17) Fiola, J. W., DiDonato, G. C.; Busch, K. L. Rev. Sci. Instrum. 1986, 57, 2294-2302. (18) DiDonato, G. C.; Busch, K. L. Anal. Chem. 1986, 54, 3231-3232. 10.1021/ac025833h CCC: $25.00

© 2003 American Chemical Society Published on Web 11/27/2002

Figure 1. TLC/ESI-MS of a glycolipid mixture containing 500 pmol each of GM3, GD3, and GT1b using an HTSorb silica gel plate: (a-c) extracted ion chromatograms; (d) total ion chromatogram; and (e-g) mass spectra obtained from the peaks of GM3, GD3, and GT1b, respectively. Intensities of ion peaks are indicated in brackets as ion counts.

LSI-MS for determination of oligosaccharide structures as neoglycolipid derivatives. Typical examples include the isolation and structural determination of a novel type of E-selectin ligand, sulfated Lewisa/x tetrasaccharides20,21 and the HNK-1 epitope on a new class of O-linked mannosyl glycans, and also the assignment of oligosaccharide sequences present in complex mixtures.22-24 Matrix-assisted laser desorption ionization (MALDI) and electrospray ionization mass spectrometry (ESI-MS) techniques have found wide application for nonvolatile and thermally labile compounds. Although MALDI has been attempted for in situ detection of various types of analytes on TLC plates,25-27 linking TLC with ESI-MS has been particularly difficult, mainly because of incompatibility between the dynamic liquid-phase sample introduction of ESI-MS and the static nature of TLC. However, some effort has been made to address this problem.28,29 (19) Dunphy, J. C.; Busch, K. L. Biomed. Environ. Mass Spectrom. 1988, 17, 405-410. (20) Yuen, C.-T.; Lawson, A. M.; Chai, W.; Larkin, M.; Stoll, M. S.; Stuart, A. C.; Sullivan, F. X.; Ahern, T. J.; Feizi, T. Biochemistry 1992, 31, 9126-9131. (21) Chai, W.; Feizi, T.; Yuen, C.-T.; Lawson, A. M. Glycobiology 1997, 7, 861872. (22) Lawson, A. M.; Hounsell, E. F.; Stoll, M. S.; Feeney, J.; Chai, W.; Rosankiewicz, J. R.; Feizi, T. Carbohydr. Res. 1991, 221, 191-208. (23) Chai, W.; Stoll, M. S.; Cashmore, G. C.; Lawson, A. M. Carbohydr. Res. 1993, 239, 107-115. (24) Chai, W.; Yuen, C.-T.; Feizi, T.; Lawson, A. M. Anal. Biochem. 1999, 270, 314-322. (25) Kubis, A. J.; Somayajula, K. V.; Sharkey, A. G.; Hercules, D. M. Anal. Chem. 1989, 61, 2516. (26) Guittard, J.; Hronowski, X. L.; Costello, C. E. Rapid Commun. Mass Spectrom. 1999, 13, 1838-1849. (27) Mehl, J. T.; Hercules, D. M. Anal. Chem. 2000, 72, 68-73.

Previous experiments with direct TLC/MS have been limited to off-line in situ analysis. Here, we have attempted on-line TLC/ ESI-MS separation and detection, taking advantage of the continuous elution of components separated by HPTLC using a commercial system, OPLC 50, that can be linked to a Q-TOF mass spectrometer. We now report preliminary results of the assessment of the mass spectrometric detection sensitivity and chromatographic resolution achievable by on-line TLC/ESI-MS of acidic glycolipids and neoglycolipids30 as examples. For many years, the identification of mixtures of glycolipids has relied on TLC for separation and MS for structural determination, for example, saccharide sequence and long-chain base (LCB) and fatty acid (FC) composition of the ceramide.31,32 In addition, the development of a solid-phase assaying method for investigating biological activities of glycolipids33 and neoglycolipids34 on TLC plates has further strengthened the central role of TLC in (28) Anderson, R. M.; Busch, K. L. J. Planar Chromatogr. - Mod. TLC 1998, 11, 336-341. (29) Hsu, F.-L.; Yuan, C.-H.; Shiea, J. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 27-31, 2001, TPA005 (A011241). (30) Feizi, T.; Stoll, M. S.; Yuen, C.-T.; Chai, W.; Lawson, A. M. Methods Enzymol. 1994, 230, 484-519. (31) Nudelman, E. D.; Levery, S. B.; Igarashi, Y.; Halomori, S. J. Biol. Chem. 1992, 267, 11007-11016. (32) Reinhold: V. N.; Reinhold: B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784. (33) Magnani, J. L.; Brockhaus, M.; Smith, D. F., Ginsburg, V.; Blaszczyk, M.; Mitchell, K. F.; Steplewski, Z.; Koprowski, H. Science 1981, 212, 55-56. (34) Tang, P. W.; Gooi, H. C.; Hardy, M.; Lee, Y. C.; Feizi, T. Biochem. Biophys. Res. Commun. 1985, 132, 474-480.

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glycoconjugate research. In addition, an integrated strategy for fractionation, binding assay and structural determination by in situ MS of glycolipids on TLC plates has facilitated the assignment of structure/function relationships.30 The on-line TLC/ESI-MS method developed in the present study will not only facilitate glycoconjugate research but will find also application in other areas. EXPERIMENTAL SECTION Materials. Gangliosides GM3, GD3, and GT1b, and chondroitin sulfate disaccharides 0S, 4S, diSD, and triS were purchased from Sigma (Poole, England). A heparan sulfate tetrasaccharide fraction was obtained from nitrous acid hydrolysis, which resulted in an anhydrous mannose residue (anMan) at each reducing terminus, followed by gel filtration (sample preparation by courtesy of Camilla Westling and Ulf Lindahl, University of Uppsala). Neoglycolipids of the heparan sulfate tetrasaccharide fraction and a mixture of the chondroitin sulfate disaccharides were prepared by conjugation with the fluorescent amino phospholipid, N-aminoacetyl-N-(9-anthracenylmethyl)-1,2-dihexadecyl-sn-glycero-3phosphoethanolamine (ADHP)35 as described. All solvents used are of either analytical or HPLC grade. Caution should be taken when handling chloroform because of its potential carcinogenicity (Aldrich). OPLC and TLC. Aluminum-backed silica gel TLC plates with perimeter seal (HTSorb, 5-µm fluorescent 254-nm and 11-µm nonfluorescent, 5 cm × 20 cm, from Bionisis SA) were used for OPLC and aluminum-backed silica gel HPTLC plates (5 µm, Merck) for conventional TLC. A Linomat IV (Camag, Muttenz, Switzerland) was employed for sample application and development and elution was with CHCl3/CH3OH/H2O (60:35:8, by volume). Overpressure TLC was performed using an OPLC 50 system (Bionisis SA, Le Plessis Robinson, France) consisting of separation and solvent delivery units. The plate was placed on a cassette covered by a sheet of Teflon membrane and inserted into the separation unit. The membrane sheet was subjected to a pressure of 50 bar and eluent was pumped through the sealed thin layer silica gel at a constant flow rate of 100 or 125 µL/min. Conventional TLC was carried out in a glass tank at room temperature with upward development for a distance of 17.5 cm. The solvent was equilibrated in the tank for 30 min before placing the plate, with sample loaded, directly into the solvent for development. The tank was kept in a draft-free environment to minimize solvent evaporation during development. Electrospray Mass Spectrometry. Electrospray mass spectrometry was carried out on a Micromass Q-TOF instrument (Micromass, Manchester, U.K.) in negative ion mode. Nitrogen was used as the desolvation and nebulizer gas at a flow rate of 250 and 15 L/h, respectively. Source temperature was 80°C, and the desolvation temperature was 150°C. Capillary voltage was maintained at 3 kV. Collision-induced dissociation (CID) MS/MS product ion scanning was carried out using argon as the collision gas at a pressure of 1.7 bar (measured in the gas line prior to the gas cell). The collision energy was adjusted between 33 and 55 eV for optimal fragmentation. A scan rate of 1.5 s/scan for the (35) Stoll, M. S.; Feizi, T.; Loveless, R. W.; Chai, W.; Lawson, A. M.; Yuen, C.-T. Eur. J. Biochem. 2000, 267, 1795-1804.

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Figure 2. Comparison of chemical background noise in the mass spectra obtained using HTSorb silica gel plates with and without prewashing: (a) a HTSorb plate without prewashing; (b) after washing with 10 bed volumes of methanol; and (c) after overnight washing with a mixture of chloroform, methanol, and water. Intensities of the base peaks are indicated in brackets as ion counts.

mass range of 200-1500 was used for both ESI-MS and CID MS/ MS experiments with the MassLynx data system. Mass spectra scanned across the chromatographic peak were summed, with or without background subtraction, for presentation. For on-line TLC/ESI-MS, a short piece of capillary PEEK tubing (130-µm i.d. × 1.6-mm o.d., from Upchurch Scientific, Oak Harbor, WA) was connected between the outlet of the OPLC and the inlet of the standard electrospray probe. A flow rate between 60 and 125 µL/min was used for TLC/ESI-MS experiments. RESULTS AND DISCUSSION Direct Coupling of OPLC 50 with Q-TOF for TLC/ESIMS. The optimum flow rate for the OPLC 50 with a 5 × 20-cm plate is ∼100 µL/min (data from Bionisis SA) and is within the range of typical flow rates for conventional electrospray, allowing direct connection of the outlet of the OPLC 50 to the inlet of the ion source of the Q-TOF instrument. A typical result obtained with this configuration using a mixture of GM3, GD3, and GT1b (500 pmol of each) is shown in Figure 1. The total ion chromatogram (TIC) was dominated by ion current from a high chemical

Figure 3. TLC/ESI-MS of a glycolipid mixture containing 500 pmol each of GM3, GD3, and GT1b using a thoroughly washed HTSorb plate: (a-c) extracted ion chromatograms; (d) total ion chromatogram; and (e-g) mass spectra obtained from the peaks of GM3, GD3 and GT1b, respectively. Intensities of ion peaks are indicated in brackets as ion counts.

background noise (Figure 1d), and no sample peaks were apparent. The good separation and presence of the three components could be demonstrated only by the extracted ion chromatograms, where [M - H]- at m/z 1262 was plotted for the monosialylated GM3 (Figure 1a), [M - 2H]2- at m/z 749 for the disialylated GD3 (Figure 1b), and [M - 2H]2- at m/z 718 for the trisialylated GT1b (Figure 1c). Weak signals from the sample molecules were indicated by the ion counts obtained (170 for m/z 1262, 581 for m/z 749, and 170 for m/z 718). Not surprisingly, mass spectra obtained by summing across each of the three chromatographic peaks were also dominated by the chemical background noise. In the spectrum of GM3 (Figure 1e), [M H]- was present at m/z 1261.9 as a minor peak in the spectrum. GD3 gave a better spectrum (Figure 1f) with [M - H]2- at m/z 749.0, clearly above the background. Similarly, the spectrum of GT1b was complicated by presence of the chemical noise, and assignment of the doubly and triply charged molecular ions at m/z 1077.9 and m/z 718.1, respectively, was difficult. An electrospray mass spectrum of the chemical noise background acquired from a new TLC plate over a period of 1 min, which represents a typical chromatographic peak width in TLC/ ESI-MS, is shown in Figure 2a. The ion series with a mass increment of 74 Da dominated the spectrum, and the base peak m/z 1017 had an intensity of 2.37 × 103 ion counts (Figure 2a). These ions are similar to the background obtained by MALDI of

a silica gel TLC plate36 and are likely to be derived from the organic binder used for preparation of the plates. They were not related to the fluorescent additives, because a nonfluorescent plate (11 µm silica gel) showed the same contaminant ion series (data not shown). Improvement in ESI-MS Detection Sensitivity by Reducing Chemical Background Noise by Extensive Washing of TLC Plates. Prewashing of plates is generally required for highsensitivity detection with TLC. Conventional washing by a single development of the plate with methanol followed by immersion in the same solvent37 proved inadequate for ESI-MS detection, as it failed to remove the contaminants (data not shown). For online UV detection in OPLC, it has been suggested by the manufacturer that washing with 10 bed volumes (∼16 mL for the 5 × 20-cm plates) of methanol can remove the background contaminants from the TLC plate. However, in our hands, the chemical background in the mass spectrum obtained after this prolonged washing was still not satisfactory. As demonstrated by the spectrum (Figure 2b) acquired from the washed plate, the contaminant peak intensities in the mass range of 500-1500 were very high (e.g., m/z 571, 1.26 × 103 counts, Figure 2b). Extensive washing of a plate with at least of 100 bed volumes of a solvent mixture containing chloroform, methanol, and water used for (36) Gusev, A. I.; Vasseur, O. J.; Proctor, A.; Sharkey, A. G.; Hercules, D. M. Anal. Chem. 1995, 67, 4565-4570. (37) Maxwell, R. J. J. Planar Chromatogr. - Mod TLC 1995, 12, 109-113.

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Figure 4. TLC/ESI-MS of a glycolipid mixture containing 5 pmol each of GM3, GD3, and GT1b using a thoroughly washed HTSorb silica gel plate: (a-c) extracted ion chromatograms; (d) total ion chromatogram; and (e-g) mass spectra obtained from the peaks of GM3, GD3, and GT1b, respectively. Intensities of ion peaks are indicated in brackets as ion counts.

TLC/MS was necessary to improve the ESI-MS detection sensitivity. This can be carried out conveniently by overnight washing prior to analysis, and the chemical background was then reduced by almost an order of magnitude (e.g., m/z 581, 367 counts, Figure 2c). However, it was apparent that the residual ion series after the two washing methods were different, for example, m/z 571, 645, 719, ... (Figure 2b) and m/z 581, 655, 729, ... (Figure 2c), although the mass increment of 74 Da remained the same for all of the ion series. The chemical background noise could not be completely eliminated, even after repeated use of the plate. TLC/ESI-MS was then carried out with the same glycolipid mixture containing 500 pmol of each component on a plate prewashed overnight with the elution solvent. The chromatograms and mass spectra obtained were much cleaner (Figure 3), and analyte ions predominated in all spectra. The weak chemical noise was almost undetectable in the spectra of GD3 and GT1b (Figure 3f and g). The ion signals from GM3, GD3, and GT1b (Figure 3ac) were 7, 27, and 71-fold greater, respectively, than those from the unwashed TLC plate (Figure 1a-c) and confirmed the earlier serious ion suppression by the chemical background. To assess the sensitivity achievable by TLC/ESI-MS, the same sample mixture was tested at reduced concentration. With as little as 5 pmol of each glycolipid, molecular ion signals were still detectable (Figure 4). In this case, background subtraction was necessary to achieve unambiguous assignment. The reduction of the high chemical background noise by washing the plate overnight made CID MS/MS product ion 122

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scanning possible. A slower flow rate (60 µL/min) for TLC was applied just before elution of the first component to acquire more scans for improved signal-to-electrical noise ratio. The base peak in the spectrum obtained with 20 pmol of GM3 was the fragment ion of NeuAc at m/z 290 (Figure 5). Other fragment ions, although weak, clearly identified the saccharide sequence of GM3, with losses of NeuAc (m/z 970), NeuAc-Hex (m/z 808), and NeuAcHex-Hex (m/z 646). A unique ceramide fragment (m/z 408) was deduced to arise by double cleavage and, together with the ion for the intact lipid moiety (m/z 646), indicated the d18:1/24:1 (LCB/FA) components of the ceramide. Chromatographic Resolution. As shown by comparison of the ion chromatograms (Figures 1 and 3), extensive washing can compromise, to a minor degree, the resolution that can be achieved by TLC. Using an unwashed plate, baseline resolution was obtained for the three components (Figure 1a-c), while the plate subjected to extensive washing exhibited a reduced resolution, as exemplified by the earlier elution of GT1b (Figure 3c). The resolution could not be restored, even after reactivation of the silica gel plate by equilibration with anhydrous solvent or overnight heating to 110 °C (data not shown). The slight loss of resolution is related to reduced capacity, since at the 5 pmol level (Figure 4), good resolution could be achieved on a plate after prewashing and repeated use. TLC/ESI-MS Analysis after Development and Storage of TLC Plates. One of the advantages of TLC is versatility. For example plates can be developed and stored for future analysis.

Figure 5. Product-ion spectrum of GM3 acquired by TLC-CID ESI-MS/MS scanning. The structure indicates the position of proposed fragmentation using the nomenclature based on that introduced by Domon and Costello.38

Figure 6. TLC/ESI-MS of a fluorescent neoglycolipid mixture of chondroitin sulfate disaccharides: (a-d) extracted ion chromatograms; (e) total ion chromatogram; (f) fluorescent detection of conventional HPTLC; and (g-j) mass spectra from the peaks of neoglycolipids of 0S, 4S, diSD, and triS, respectively.

This can be achieved by the present OPLC/ESI-MS system. The separated components can be detected by nondestructive staining or UV in the conventional way prior to elution and then stored. On-line TLC/ESI-MS can be carried out with the stored plate at a later stage. However, during storage, a portion of material may be irreversibly adsorbed and may not be recovered from the silica gel matrix. The resolution may also be influenced by the second solvent development. (38) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

Using a mixture of fluorescent neoglycolipids derived from chondroitin sulfate disaccharides, we have assessed the sensitivity and resolution that can be obtained with a prewashed plate after storage. Following separation, the plate was removed and dried, and fluorescence detection was performed. The plate was stored in the dark for 16 h and then replaced in the OPLC instrument, where TLC/MS analysis was resumed. The neoglycolipid mixture consisted of 100 pmol each of the nonsulfated and mono- to trisulfated disaccharides 0S, 4S, diSD, and triS, respectively. The Analytical Chemistry, Vol. 75, No. 1, January 1, 2003

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Figure 7. TLC/ESI-MS of a fluorescent neoglycolipid mixture of a heparan sulfate tetrasaccharide fraction: (a-d) extracted ion chromatograms; (e) total ion chromatogram; (f) fluorescent detection of conventional HPTLC; and (g-j) mass spectra from the chromatographic peaks eluting at 14.3, 16.2, 17.1, and 17.6 min, respectively.

extracted ion chromatograms (Figure 6a-d) from TLC/MS clearly showed that good separation was maintained after the second solvent development and that this resolution could be compared with that achieved from the first chromatography, as shown by the conventional fluorescent detection (Figure 6f) of the plate before storage. The detection sensitivity for ESI-MS (Figure 6gj) increased for the disaccharides with increasing sulfate content, with a weak ion signal for 0S (Figure 6g) and very intense ion signals for triS (Figure 6j). Ion signals obtained from the double development of the stored plate were slightly weaker than those from a fresh plate with a single development (data not shown). The minor reduction in sensitivity is probably due to partial degradation of the fluorescent label on the silica gel surface (unpublished result, M.S.S.), rather than the retention of the material on the TLC plate, because the sample bands, after development, were not detectable by fluorescence. This degradation is reduced if the plates are stored dry and in the dark. TLC/ESI-MS of a Heparan Sulfate Tetrasaccharide Mixture as Neoglycolipids. An unknown mixture of fluorescent neoglycolipids, derived from a tetrasaccharide fraction isolated from aortic heparan sulfate following nitrous acid treatment, was analyzed by the TLC/ESI-MS method. The ion chromatograms (Figure 7a-d) clearly indicated four major components present in the mixture comparable to fluorescent detection by conventional HPTLC (Figure 7f). Although the chromatographic peaks were overlapping, the spectra of all four components were of good quality after background subtraction, and the structures could be readily assigned. The early eluting peak (Figure 7a) with a 124

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retention time of 14.3 min exhibited a [M - 2H]2- at m/z 804.9 and a [M - 3H]3- at m/z 536.3 (Figure 7g), corresponding to a molecule of 1611.9 Da and consistent with a nonsulfated tetrasaccharide GlcA-GlcNAc-GlcA-anMan from heparan sulfate. The second peak (retention time 16.2 min, Figure 7b) had a [M 2H]2- at m/z 826.9 and a [M - 3H]3- at m/z 551.0 corresponding to a molecule of 1655.9 Da and consistent with a doubly dehydrated tetrasaccharide containing one sulfate. The last two peaks (Figure 7c and d) were barely resolved, but their respective structures could still be assigned from their distinctive mass spectra following background subtraction. The [M - 2H]2- at m/z 844.8 and [M - 3H]3- at m/z 562.9 for the peak at 17.1 min (Figure 7i) and the [M - 3H]3- at m/z 577.6 and [M - 4H]4- at m/z 433.0 for the peak at 17.6 min (Figure 7j) are consistent with a tetramer with one sulfate and a dehydrated tetramer with two sulfates, respectively. CONCLUSIONS On-line TLC/ESI-MS separation and detection has been achieved by directly linking an OPLC 50 instrument and a Q-TOF mass spectrometer. High chemical background noise was a major factor affecting the detection sensitivity of ESI-MS. Although electrospray can be relatively tolerant toward contamination, sample ion signals were suppressed to a major degree in this case. These contaminants are likely to be from the fragments of polymeric plate binder, and a silica gel plate with a different inorganic binder should be considered for future development. Precleaning is a prerequisite to achieve high sensitivity detection

in TLC/ESI-MS, and a solvent wash of at least 100 bed volumes was necessary to reduce background substantially. Sequential use of methanol and a mixture of chloroform, methanol, and water may provide a better means of reducing background, as different solvents were found to differentially remove contaminants. Typically, in the OPLC 50 system, 20 × 20-cm plates are used for multiple sample application, whereas for single samples, as in the present investigation of on-line TLC/ESI-MS, plates of 5 × 20 cm are commercially available. A narrower plate (e.g., 3 cm) will improve sensitivity and resolution still further. The high spatial resolution achieved by the OPLC instrument does not translate directly into resolution on-line because of the current simple design of the solvent exit system of the OPLC. Repeated use of a TLC plate can be made, and we have found no adverse effects with reuse at least 10 times. Repeated solvent development cannot completely eliminate the chemical background noise and can reduce resolution with time. Methods to reactivate the silica gel absorbent need to be further investigated. The on-line TLC/ESI-MS system should hold considerable promise for the analysis of many types of compounds for which molecular weight and structural information are required. It will be particularly valuable for the glycolipid field, in which TLC has been in use for many years. With the present configuration, a

detection sensitivity of 5 pmol has been readily achieved for TLC/ ESI-MS of acidic glycolipids, and 20 pmol for TLC-CID ESI-MS/ MS. These analyses have allowed unambiguous characterization of the ganglioside by defining the saccharide sequence and LCB/ FA composition of the ceramide. Neoglycolipids of chondroitin and heparan sulfate oligosaccharides have proved difficult in the past for in situ TLC/LSIMS analysis mainly as a result of their highly acidic nature and sulfate lability. Their solvent elution from TLC and on-line ESI-MS detection in the present work markedly reduces sulfate loss, whereas their ionic character is highly compatible with this ionization method. ACKNOWLEDGMENT We are grateful to Bionisis SA for the short-term loan of the OPLC 50 instrument, Drs. Camilla and Ulf Lindahl for the heparan sulfate tetrasaccharide sample, and Dr. Ten Feizi for valuable discussions. This work was supported by Program Grant G9601454 from the U.K. Medical Research Council.

Received for review June 6, 2002. Accepted October 16, 2002. AC025833H

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