Identification of Natural Products Using HPLC-SPE Combined with

Anal. Chem. , 2007, 79 (2), pp 727–735. DOI: 10.1021/ac0616963. Publication Date (Web): November 17, 2006. Copyright © 2007 American Chemical Socie...
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Anal. Chem. 2007, 79, 727-735

Identification of Natural Products Using HPLC-SPE Combined with CapNMR Maja Lambert,† Jean-Luc Wolfender,‡ Dan Stærk,† S. Brøgger Christensen,† Kurt Hostettmann,‡ and Jerzy W. Jaroszewski*,†

Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark, and Laboratory of Pharmacognosy and Phytochemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland

Two major development areas in HPLC-NMR hyphenation are postcolumn solid-phase extraction (HPLC-SPE-NMR) and capillary separations with NMR detection by means of solenoidal microcoils (CapNMR). These two techniques were combined off-line into HPLC-SPE-CapNMR, which combines the advantage of high loadability of normal-bore HPLC columns with high mass sensitivity of capillary NMR probes with an active volume of 1.5 µL. The technique was used for rapid identification of complex sesquiterpene lactones and esterified phenylpropanoids present in an essentially crude plant extract (toluene fraction of an ethanolic extract of Thapsia garganica fruits). Elution profiles of 10 × 1 mm i.d. SPE cartridges filled with poly(divinylbenzene) resin were found to be only marginally broader than those observed upon direct injection of 6-µL samples into the probe. Thus, the technique focuses analytes emerging in the HPLC elution bands of 0.5-1 mL into volumes of ∼10 µL, compatible with the CapNMR probe. Using this technique, nine natural products (1-9) present in the plant extract in amounts varying from 0.1 to 20% were identified by means of 1D and 2D NMR spectra, supported by parallel HPLC-ESIMS measurements. Therefore, HPLC-SPE-CapNMR should be regarded as an attractive alternative to other applications of CapNMR for mixture analysis. There are currently two major trends in the development of hyphenated NMR techniques.1-6 The first is the use of a solidphase extraction (SPE) interface between the NMR spectrometer and the chromatograph,7-20 which enables analyte focusing, change from a nondeuterated HPLC solvent to a deuterated NMR * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +45 3530 6040. † The Danish University of Pharmaceutical Sciences. ‡ University of Geneva. (1) Albert, K., Ed. On-line NMR and Related Techniques; Wiley: Chichester, England, 2002. (2) Jaroszewski, J. W. Planta Med. 2005, 71, 691-700. (3) Jaroszewski, J. W. Planta Med. 2005, 71, 795-802. (4) Exarchou, V.; Krucker, M.; van Beek, T. A.; Vervoort, J.; Gerothanassis, I. P.; Albert, K. Magn. Reson. Chem. 2005, 43, 681-687. (5) Webb, A. G. Magn. Reson. Chem. 2005, 43, 688-696. (6) Lewis, R. J.; Bernstein, M. A.; Duncan, S. J.; Sleigh, C. J. Magn. Reson. Chem. 2005, 43, 783-789. (7) Exarchou, V.; Godejohann, M.; van Beek, T. A.; Gerothanassis, I. P.; Vervoort, J. Anal. Chem. 2003, 75, 6288-6294. 10.1021/ac0616963 CCC: $37.00 Published on Web 11/17/2006

© 2007 American Chemical Society

solvent, and multiple SPE trapping for increasing sensitivity. The other is miniaturization,21-24 the major facets of which are speed, the ability to work with very small samples, and increased mass sensitivity of solenoidal NMR microcoils. Capillary HPLC separations (CapHPLC) normally lead to increased analyte/solvent ratios in the chromatographic elution bands as compared to standardsize column, which makes the solvent suppression easier. Moreover, the low solvent consumption of CapHPLC makes chromatographic separations in deuterated solvents economically feasible. However, the CapHPLC-CapNMR combination suffers from potential drawbacks inherent to direct HPLC-NMR methods,2 particularly the necessity to work in stopped-flow mode. Because peak elution volumes (typically 5-10 µL) are larger than the active volume of the NMR probe (usually 1.5 µL), only a fraction of the analyte injected onto the CapHPLC column contributes to the NMR signal, a problem that is amplified by imperfections of the separation. Even more significantly, severe (8) Godejohann, M.; Tseng, L.-H.; Braumann, U.; Fuchser, J.; Spraul, M. J. Chromatogr., A 2004, 1058, 191-196. (9) Seger, C.; Godejohann, M.; Tseng, L.-H.; Spraul, M.; Girtler, A.; Sturm, S.; Stuppner, H. Anal. Chem. 2005, 77, 878-885. (10) Clarkson, C.; Stærk, D.; Hansen, S. H.; Jaroszewski, J. W. Anal. Chem. 2005, 77, 3547-3553. (11) Miliauskas, G.; van Beek, T. A.; de Waard, P.; Venskutonis, R. P.; Sudho¨lter, E. J. R. J. Nat. Prod. 2005, 68, 168-172. (12) Lambert, M.; Stærk, D.; Hansen, S. H.; Sairafianpour, M.; Jaroszewski, J. W. J. Nat. Prod. 2005, 68, 1500-1509. (13) Sandvoss, M.; Bardsley, B.; Beck, T. L.; Lee-Smith, E.; North, S. E.; Moore, P. J.; Edwards, A. J.; Smith, R. J. Magn. Reson. Chem. 2005, 43, 762-770. (14) Lambert, M.; Stærk, D.; Hansen, S. H.; Jaroszewski, J. W. Magn. Reson. Chem. 2005, 43, 771-777. (15) Christophoridou, S.; Dais, P.; Tseng, L.-H.; Spraul, M. J. Agric. Food Chem. 2005, 53, 4667-4679. (16) Wang, C.-Y.; Lee, S.-S. Phytochem. Anal. 2005, 16, 120-126. (17) Pukalskas, A.; van Beek, T. A.; de Waard, P. J. Chromatogr., A 2005, 1074, 81-88. (18) Nicholls, A. W.; Wilson, I. D.; Godejohann, M.; Nicholson, J. K.; Shockcor, J. P. Xenobiotica 2006, 36, 615-629. (19) Clarkson, C.; Stærk, D.; Hansen, S. H.; Smith, P. J.; Jaroszewski, J. W. J. Nat. Prod. 2006, 69, 527-530. (20) Clarkson, C.; Stærk, D.; Hansen, S. H.; Smith, P. J.; Jaroszewski, J. W. J. Nat. Prod. 2006, 69, 1280-1288. (21) Lacey, M. E.; Subramanian, R.; Olson, D. L.; Webb, A. G.; Sweedler, J. V. Chem. Rev. 1999, 99, 3133-3152. (22) Wolters, A. M.; Jayawickrama, D. A.; Sweedler, J. V. Curr. Opin. Chem. Biol. 2002, 6, 711-716. (23) Jayawickrama, D. A.; Sweedler, J. V. J. Chromatogr., A 2003, 1000, 819840. (24) Olson, D. L.; Norcross, J. A.; O’Neil-Johnson, M.; Molitor, P. F.; Detlefsen, D. J.; Wilson, A. G.; Peck, T. L. Anal. Chem. 2004, 76, 2966-2974.

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column loading limitations make the analysis of minor constituents of mixtures difficult. Although a number of applications of CapHPLC-CapNMR hyphenation have been reported, usually 1D 1H NMR spectra and only rarely homonuclear 2D correlations have been obtained.25-32 Thus, flow injection mode employing pure samples as concentrated as 50 mM appears to be the main current application area of CapNMR.33,34 Moreover, purified natural products have been analyzed by CapNMR injecting concentrated solutions directly into the probe.35-40 Thus, in spite of the high mass sensitivity of microcoils, analyte amounts necessary to obtain comprehensive sets of 2D NMR spectra from individual components of complex mixtures are not immediately achievable in the hyphenated CapHPLC-CapNMR operation mode. A possible solution to this problem is analyte focusing and multiple trapping through an SPE interface. In this work, we explore the combination of HPLC-SPE with CapNMR. The two techniques were combined off-line as an introduction to possible future automation. One of the most important areas of application of HPLC-NMR is plant extract analysis.1-4 Crude plant extracts constitute particularly complex analyte mixtures, and the complexity of natural products requires a very high quality and versatility of the NMR measurements, including the ability to acquire heteronuclear correlations.2 In this work, an essentially crude extract of Thapsia garganica L., a plant that produces complex, pharmacologically interesting sesquiterpene lactones,41 was used as a test case of the combination of HPLC-SPE with CapNMR. EXPERIMENTAL SECTION Instrumentation. Chromatographic separations were performed on a HPLC system consisting of a quaternary pump, degasser, column oven, and UV-visible PDA detector (Agilent (25) Behnke, B.; Schlotterbeck, G.; Tallarek, U.; Strohschein, S.; Tseng, L.-H.; Keller, T.; Albert, K.; Bayer, E. Anal. Chem. 1996, 68, 1110-1115. (26) Schlotterbeck, G.; Tseng, L.-H.; Ha¨ndel, H.; Braumann, U.; Albert, K. Anal. Chem. 1997, 69, 1421-1425. (27) Krucker, M.; Lienau, A.; Putzbach, K.; Grynbaum, M. D.; Schuler, P.; Albert, K. Anal. Chem. 2004, 76, 2623-2628. (28) Sandvoss, M.; Roberts, A. D.; Ismail, I. M.; North, S. E. J. Chromatogr., A 2004, 1028, 259-266. (29) Xiao, H. B.; Krucker, M.; Putzbach, K.; Albert, K. J. Chromatogr., A 2005, 1067, 135-143. (30) Putzbach, K.; Krucker, M.; Grynbaum, M. D.; Hentschel, P.; Webb, A. G.; Albert, K. J. Pharm. Biomed. Anal. 2005, 38, 910-917. (31) Hentschel, P.; Krucker, M.; Grynbaum, M. D.; Putzbach, K.; Bischoff, R.; Albert, K. Magn. Reson. Chem. 2005, 43, 747-754. (32) Hentschel, P.; Grynbaum, M. D.; Molnar, P.; Putzbach, K.; Rehbein, J.; Deli, J.; Albert, K. J. Chromatogr., A 2006, 1112, 285-292. (33) Bailey, N. J. C.; Marshall, I. R. Anal. Chem. 2005, 77, 3947-3953. (34) Jansma, A.; Chuan, T.; Albrecht, R. W.; Olson, D. L.; Peck, T. L.; Geierstanger, B. H. Anal. Chem. 2005, 77, 6509-6515. (35) Gronquist, M.; Meinwald, J.; Eisner, T.; Schroeder, F. C. J. Am. Chem. Soc. 2005, 127, 10810-10811. (36) Hu, J.-F.; Yoo, H.-D.; Williams, C. T.; Garo, E.; Cremin, P. A.; Zeng, L.; Vervoort, H. C.; Lee, C. M.; Hart, S. M.; Goering, M. G.; O’Neil-Johnson, M.; Eldridge, G. R. Planta Med. 2005, 71, 176-180. (37) Hu, J.-F.; Garo, E.; Yoo, H.-D.; Cremin, P. A.; Zeng, L.; Goering, M. G.; O’NeilJohnson, M.; Eldridge, G. R. Phytochem. Anal. 2005, 16, 127-133. (38) Yoo, H.-D.; Cremin, P. A.; Zeng, L.; Garo, E.; Williams, C. T.; Lee, C. M.; Goering, M. G.; O’Neil-Johnson, M.; Eldridge, G. R.; Hu, J.-F. J. Nat. Prod. 2005, 68, 122-124. (39) Hu, J.-F.; Garo, E.; Yoo, H.-D.; Cremin, P. A.; Goering, M. G.; O’Neil-Johnson, M.; Eldridge, G. R. Phytochemistry 2005, 66, 1077-1082. (40) Wolfender, J.-L.; Queiroz, E. F.; Hostettmann, K. Magn. Reson. Chem. 2005, 43, 697-709. (41) Christensen, S. B.; Andersen, A.; Smitt, U. W. Prog. Chem. Org. Nat. Prod. 1997, 71, 129-167.

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1100 series). Separation was optimized on a 150 × 4.6 mm i.d., C18(2) Phenomenex Luna column (3 µm, 100 Å) operated at 40 °C. A K100 Wellchrom makeup pump (Knauer, Berlin, Germany) diluted the flow from the PDA detector before analyte trapping on HySphere GP [poly(divinylbenzene) resin] SPE cartridges (10 × 1 mm i.d., Spark Holland, Emmen, Netherlands), using a Prospekt-2 SPE unit (Spark Holland). HPLC separations and SPE peak trapping were controlled by Bruker HyStar version 2.3 software. A hand clamp (Spark Holland) was connected to a 5-µL capillary microcoil NMR probe (CapNMR probe) with 1.5-µL active volume (Magnetic Resonance Microsensors, Savoy, IL) through a 50-µm-i.d. fused-silica capillary and used to elute dried SPE cartridges. The CapNMR probe was installed in a Varian Unity Inova 500-MHz NMR instrument, controlled by VNMR version 6.1c software. The NMR data were processed and analyzed with Bruker TopSpin version 1.2 software. Direct injection of samples into the CapNMR probe was done using a 25-µL syringe from Hamilton (Reno, NV), also connected through 50-µm-i.d fusedsilica capillaries. HPLC-MS analysis was performed separately on an Agilent 1100 series HPLC system interfaced with an Agilent 1100 MSD trap mass spectrometer with ESI source. Chemicals. HPLC grade solvents and water purified by deionization and 0.22-µm membrane filtration (Millipore) were used. Methanol-d4 (99.8 atom % deuterium) was obtained from Armar Chemicals (Do¨ttingen, Switzerland). Pure, authentic thapsigargin (1) and compound 6 were available from previous studies. Sample Preparation. Fruits of T. garganica L. (Apiaceae) were collected on Ibiza, Spain, in July 2002; a voucher specimen (DFHSBC1) was deposited in Herbarium C (Botanical Museum, University of Copenhagen, Denmark). The powdered fruits (20 g) were extracted with 200 mL of 96% ethanol at ambient temperature for 6 h. Half of the extract was evaporated in vacuo and partitioned between water and toluene; evaporation of the toluene phase gave 374 mg of the extract, dissolved in acetonitrile (31 mg/mL) for HPLC-SPE analysis. Authentic thapsigargin (1) in acetonitrile (48 mg/mL) and 6 (2 mg/mL) were used as standards. HPLC-SPE Experiments. A two-step linear gradient of acetonitrile in water (containing 0.1% formic acid) was delivered at a flow rate of 0.5 mL/min, rising from 54.5 to 68% in 30 min and further to 95% after 60 min. Injection volumes were 10 and 1 µL of the purified extract sample and reference compounds, respectively. The UV absorption at 230 nm was used to monitor the separation and for setting an absorbance threshold for triggering the SPE trapping. A makeup flow of water at 1.0 mL/ min was added to the eluate before SPE trapping in order to lower the eluotropic strength. SPE cartridges were conditioned before use with 500 µL of acetonitrile at 6 mL/min and equilibrated with 500 µL of water at 1 mL/min. For each of the chromatographic peaks selected for analysis of the plant extract, four repeated trappings were performed per cartridge, whereas only one injection of the thapsigargin standard was done. Chromatographic solvents were removed from the SPE cartridges with a flow of nitrogen gas for 30 min. Quantitative Analysis. Estimation of amounts of constituents of the plant extract was performed by comparison of HPLC peak areas obtained after injection of standard solutions of 1 and 6 with those obtained with the extract.

CapNMR Experiments. Elution of the dried cartridges was done manually with a hand clamp connected to the CapNMR probe through fused-silica capillaries. The probe was filled with methanol-d4, and prior to connecting the hand clamp to the probe, methanol-d4 was injected to the cartridge and the transfer capillary to fill up their dead volume with the solvent, i.e., until the solvent appeared at the disconnection point (∼10 µL). This was done in order to avoid air in the probe. After connecting the capillary to the probe, an additional 12 µL of methanol-d4 was injected to complete the elution and to position the sample optimally in the probe. The latter push volume was determined experimentally in an initial calibration experiment, where a cartridge loaded with 48 µg of thapsigargin was eluted with methanol-d4, recording 1H NMR spectra for every 1 µL of the injected solvent. Manual injections of thapsigargin standard to the CapNMR probe were performed by the following procedure. Aliquots of acetonitrile solution containing 48 µg of thapsigargin were transferred to Eppendorf tubes, evaporated, and dissolved in 6 µL of methanold4. The solutions were injected to the CapNMR probe, followed by a calibrated push volume of 8.5 µL of methanol-d4 to position the sample optimally in the probe. Between each experiment, the probe was rinsed with 50 µL of methanol-d4 to avoid sample carryover. NMR Experiments. All NMR spectra were acquired at 30 °C in methanol-d4. 1D 1H NMR spectra with 32k data points, sweep width of 8.5 ppm, and pulse repetition time of 4.3 s were recorded using 8-2048 transients depending on sample concentration and without solvent peak suppression. Magnitude-mode, gradientselected COSY experiments were performed collecting 1604 × 512 data points with 4-64 transients, applying sine-bell apodization in both directions prior to Fourier transformation. Phase-sensitive TOCSY spectra (80-ms spin lock) were recorded with 1024 × 512 data points (4 transients), and the data were Fourier-transformed using a π/2-shifted sine-bell in both dimensions. HSQC spectra were acquired using gradient selection and adiabatic pulses,42 collecting 1276 × 512 data points (8-128 transients/increment), and the data were Fourier-transformed using π/2-shifted squared sine-bell in both dimensions. HMBC data (acquired using gradient selection and adiabatic pulses42) contained 1024 × 512 data points (24 transients/increment) and were Fourier-transformed using a nonshifted sine-bell in both dimensions. The heteronuclear correlations were optimized for 1JC,H ) 140 Hz and nJC,H ) 8 Hz. Spectral widths were adjusted to the individual samples, and linear prediction was applied in all 2D experiments to improve resolution in the indirect direction. 1H and 13C chemical shifts were referenced to solvent signals of methanol-d4, δ(1H) ) 3.31 and δ(13C) ) 49.1. HPLC-MS Measurements. HPLC-MS measurements were performed using the HPLC method used for HPLC-SPE. ESI mass spectra were recorded in both positive- and negative-ion modes with a drying temperature of 350 °C, nebulizer pressure of 50 psi, drying gas flow of 10 L/min, spray voltage of 3500 V, and trap drive of 50 (arbitrary units).

Figure 1. Flow diagram for experimental design used in this work.

RESULTS AND DISCUSSION The experimental design used in this work is illustrated in Figure 1. Crude ethanolic extract of T. garganica fruits was

preprocessed by toluene-water partitioning in order to remove unwanted polar constituents such as sugars. Following development of an appropriate reversed-phase HPLC separation system, extract constituents eluted as individual HPLC peaks were trapped on solid-phase extraction cartridges using a Prospekt-2 SPE device (Spark Holland), which handles two trays with 96 cartridges each and thus enables trapping of a large number of individual HPLC peaks in automation. The SPE cartridges were subsequently dried, and the analytes were eluted into the CapNMR probe for NMR analysis. Since total volume of the CapNMR probe is 5 µL, with an additional volume needed to fill up feed lines, analyte elution profiles from the SPE cartridges are crucial for success of the experiment. Moreover, solvent volumes used for cartridge elution and analyte transfer must be calibrated for optimal sample centering in the probe. Since thapsigargin (1, Table 1) was expected to be a major constituent of the extract of T. garganica fruits,41,43 determination of SPE elution parameters was performed with a purified sample of this compound. Using the same procedure as for the HPLC-SPE trapping of extract constituents (see below), 10 × 1 mm i.d. Hysphere GP SPE cartridges were loaded with 1 by injecting 48 µg into the HPLC column. The dried cartridge containing 48 µg of 1 (assuming that no loss occurred during HPLC separation and SPE trapping) was placed in a hand clamp, connected to the probe, and eluted with the dead volume of the cartridge plus a total of 40 µL of methanol-d4 in 1-µL intervals. 1H NMR spectra were recorded for each volume increment. The obtained signal-to-noise ratios plotted as a function of the solvent volume are shown in Figure 2. It can be seen that the maximum concentration of the desorbed compound reaches the active probe region after injecting 12 µL of the solvent (following the 10 µL of the solvent used to fill up the dead volume of the cartridge and connecting capillaries). The total elution volume of 1 from the SPE cartridge was roughly 10 µL. Figure 2 also shows the corresponding profile after direct injection of an

(42) Boyer, R. D.; Johnson, R.; Krishnamurthy, K. J. Magn. Reson. 2003, 165, 253-259.

(43) Christensen, S. B.; Larsen, I. K.; Rasmussen, U.; Christophersen, K. J. Org. Chem. 1982, 47, 649-652.

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Table 1. Structures of Thapsia garganica Constituents with 1H NMR Data Obtained in HPLC-SPE-CapNMR Experiments and HPLC-MS Dataa

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Table 1 (Continued)

a

Ang ) angelic acid ) (Z)-CH3CHdC(CH3)COOH.

equivalent amount of 1 in 6 µL of the solvent directly into the probe (according to the literature examples,35-40 direct injections usually employ sample volumes of 5-6.5 µL). Because of a different length of connecting capillaries, 1 reaches the probe

earlier, but the elution profile is only slightly narrower, resulting in a somewhat higher maximal concentration of 1 in the probe (Figure 2). This experiment demonstrates that the elution profile of a 10 × 1 mm i.d. SPE cartridge is comparable to that obtained Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

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Figure 2. Elution profile of 1 from a 10 × 1 mm i.d. GP resin SPE cartridge with methanol-d4 and after direct injection of the compound dissolved in 6 µL of methanol-d4 into CapNMR probe, obtained by determination of signal-to-noise ratios in 1H NMR spectra recorded for each 1-µL increment of the eluting solvent/push solvent volume (signal region δ 0.50-1.50, noise region δ 6.50-8.50). The SPE elution profile was corrected for the dead volume of the cartridge and the clamp (10 µL).

Figure 3. HPLC trace for toluene fraction of ethanolic extract of T. garganica fruits (150 × 4.6 mm i.d., C18(2) Phenomenex Luna column, 3-µm particle size, acetonitrile gradient in water at 0.5 mL/ min, 40 °C, 0.31 mg of extract injected). Peaks selected for HPLCSPE trappings are labeled 1-12.

under direct injection conditions. Therefore, SPE cartridge elution directly into the CapNMR probe is expected to give NMR spectra with practically the same signal-to-noise ratio as upon direct injection of the same analyte amount dissolved in 6 µL of a solvent. Reversed-phase HPLC with a two-step linear gradient of acetonitrile in water showed the presence of numerous constituents of the toluene fraction of the ethanolic extract of T. garganica fruits (Figure 3). All well-defined peaks (12 in total) were selected for the automated HPLC-SPE trapping based on the absorbance thresholds defined at 230 nm, as described elsewhere.10,14,19,20 However, 10 × 2 mm i.d. SPE cartridges normally used in HPLCSPE-NMR experiments were replaced with 10 × 1 mm i.d. cartridges, which have four times smaller bed volume and thus elution volumes compatible with CapNMR (Figure 2). A postcolumn makeup flow of water was added to the eluate in a ratio of 1:2 in order to improve retention of compounds on the cartridges. Four cumulative trappings with 0.31 mg of the extract per injection were performed. After drying, the cartridges were eluted and the 732 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

eluates transferred to the CapNMR probe, as described above, for NMR data acquisition at 500 MHz. Parallel HPLC-ESIMS measurements in positive as well as negative ion mode were performed in order to assist structure elucidation. In the following, structure elucidation of compounds eluted with peaks 1, 4-7, and 9-12 is described; the quantity of material eluted in peaks 2, 3, and 8 was too low to obtain satisfactory NMR data under conditions used. 1H NMR spectra of compounds eluted with peaks 1, 4, 5, and 8 were characteristic of polyoxygenated guaianolides found in the genus Thapsia.41 The 1H NMR spectrum recorded with the main peak 11 (Figure 3) was identical to that obtained with 1 in the calibration experiments described above. COSY, TOCSY, HSQC, and HMBC spectra allowed assignments in agreement with literature data.43 The signals characteristic of the thapsigargin skeleton were mainly five methine hydrogens at δ 4.36 (H-1), 5.51 (H-2), 5.59 (H-8), 5.67 (H-3), and 5.71 (H-6) and a pair of diastereotopic methylene hydrogens at δ 2.31 (H-9A) and 2.99 (H-9B). Three methyl group signals at δ 1.36 (H-13), 1.41 (H-14), and 1.86 (H-15) were observed. The remaining resonances found in the 1H NMR spectrum belong to the angelic acid residue and to aliphatic acyl side chains attached to O-2, O-8, and O-10 (Table 1). The presence of thapsigargin in peak 11 was in agreement with negative-ion mode ESIMS (m/z ) 649.3, [M - H]-). The 1H NMR spectrum recorded with peak 9 was very similar to that of 1, but an overlay of the two spectra revealed minor differences.Thus, the triplet at δ 0.91 assigned to the terminal methyl group in the octanoyl side chain (H-8′′′′) of 1 was shifted to δ 0.93, and the integral of the methylene envelope was reduced from eight to four hydrogen atoms, suggesting replacement of the octanoyl group with a hexanoyl group. This observation was corroborated by negative-ion mode ESIMS data (m/z ) 621.3, [M - H]-), leading to identification of the compound as thapsigargicin (2).43 The molecular ion obtained with peak 5 (m/z ) 607.3, [M - H]-) was 14 amu lower than for 2, which is compatible with the presence of a pentanoyl, 2-methylbutyryl, 3-methylbutyryl, or 2,2-dimethylpropionyl side chain.41 All but the 3-methylbutyryl residue could be ruled out by observation of two doublets (J ) 6.3 Hz) of the diastereotopic methyl groups δ 1.00 and 1.01. The presence of the 3-methylbutyryl side chain was supported by the intensity of the multiplet at δ 2.15-2.35, which in addition to H-2′′′ and H-9A contained H-2′′′′ and H-3′′′′, in agreement with literature data.43 The compound eluted with peak 5 is therefore thapsivillosin J (3). The 1H NMR spectrum recorded with peak 4 revealed the presence of an angeloyl moiety attached to O-2, as supported by ESIMS (m/z ) 605.3, [M - H]-). The remaining signals could be easily assigned using a COSY spectrum and by comparison with the spectra of 1-3. The compound eluted with peak 4 is therefore thapsivillosin I (4).44 The most polar compound in the chromatogram (peak 1, m/z ) 507.2, [M - H]-) was identified as nortrilobolide (5).45 Thus, only three methine hydrogens attached to oxygenated carbons were observed in the spectrum (δ 5.5-5.8 region), and these could be assigned to H-6, H-3, and H-8 using a COSY spectrum. The signal of H-3 was much broader than that observed in 1-4 as result of coupling to a diastereotopic hydrogen pair at δ 1.67 and 2.51, as evident from the COSY (44) Christensen, S. B.; Norup, E.; Rasmussen, U.; Madsen, J. Ø. Phytochemistry 1984, 23, 1659-1663. (45) Smitt, U. W.; Christensen, S. B. Planta Med. 1991, 57, 196-197.

Figure 4. (A) 1H NMR spectrum (256 transients) acquired with peak 9 (Figure 3) containing thapsigargicin (2). Shown is a difference spectrum obtained by subtraction of a spectrum obtained by eluting an empty cartridge from the actual spectrum in order to eliminate solvent resonances (both spectra were acquired without solvent peak suppression). (B) COSY spectrum acquired with peak 9 (containing 2); 8 transients, 512 increments, total acquisition time 1.5 h. (C) HSQC spectrum acquired with peak 11 containing thapsigargin (1); 4 transients, 512 increments, total acquisition time 43 min. (D) NOESY spectrum acquired with peak 11 (1); 16 transients, 1024 increments, total acquisition time 12 h. (E) Aliphatic region of an HMBC spectrum acquired with peak 11 (1); 24 transients, 512 increments, total acquisition time 4 h.

spectrum. As also H-1 was coupled to the same hydrogen pair, the absence of oxygenation at C-2 was proved. 1H NMR spectra of compounds eluted with peaks 6, 7, 10, and 12 exhibited features characteristic of phenylpropanoids previously isolated from Thapsia species.46,47 Thus, all spectra exhibited the

presence of a methoxy group (δ 3.91), a methylenedioxy group (δ 5.96), and two meta-coupled hydrogen resonances (δ 6.57(46) Saouf, A.; Guerra, F. M.; Rubal, J. J.; Jorge, Z. D.; Akssira, M.; Mellouki, F.; Moreno-Doradoo, F. J.; Massanet, G. M. Phytochemistry 2006, 67, 800804.

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Figure 5. Fragments of 1H NMR spectra of minor constituents. (A) Spectrum acquired with peak 7 (7); 2000 transients. (B) Spectrum acquired with peak 4 (thapsivillosin I, 4); 256 transients. (C) Spectrum acquired with peak 5 (thapsivillosin J, 3); 2000 transients.

6.64) attributable to a tetrasubstituted aromatic ring, as well as a spin system corresponding to CH(O)-CH(O)-CH3 and CH(O)CH3 moieties. The remaining 1H NMR resonances were attributable to acyl residues: two angeloyl residues in the compound eluted as peak 6, one angeloyl and one 2-methylbutyryl residue in the compound eluted as peak 7, one angeloyl and one hexanoyl residue in the compound eluted as peak 10, and an angeloyl along with an octanoyl residue in the compound eluted as peak 12. These data, together with ESIMS data, allowed identification of the four phenylpropanoids as 6-9, respectively (Table 1). The positiveion mode ESIMS spectra of all compounds showed loss of angelic acid (Table 1). All four compounds have previously been isolated from Thapsia species,46,47 but 7 is reported from T. garganica for the first time. The stereochemistry of Thapsia sesquiterpenes and phenylpropanoids has previously been investigated in detail41,43,47 and is specified in Table 1 even though no information about absolute configuration of 1-9 was obtained in the present work. Examples of 1H NMR spectra obtained in this work are shown in Figures 4 and 5. Excellent quality 2D spectra could be obtained rapidly with more abundant extract constituents, including the least sensitive experiments such as HMBC and NOESY (Figure 4), rarely reported in studies employing direct HPLC-NMR methods.2 Good or satisfactory 1D 1H NMR spectra could be obtained with minor extract constituents (Figure 5), including peak 5, which contains thapsivillosin J (3). The failure to obtain (47) Liu, H.; Jensen, K. G.; Trann, L. M.; Chen, M.; Zhai, L.; Olsen, C. E.; Søhoel, H.; Denmeade, S. R.; Isaacs, J. T.; Christensen, S. B. Phytochemistry, in press.

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useful 1H NMR data with peaks 2, 3, and 8 is apparently due to the presence of multiple coeluting constituents or very small amounts of compounds with strong UV absorption. These results show that rapid accumulation of NMR data, including efficient acquisition of 2D NMR spectra, is possible using the combination of HPLC-SPE with CapNMR. Comparison of the chromatogram shown in Figure 3 with those of standard solutions of 1 shows that a single injection of the extract (0.31 mg) corresponds to ∼70 µg (110 nmol) of 1 (22.5% of the toluene extract or 0.84% of the fruits). The amount of 2, which has the same chromophores as 1, is ∼30 µg (50 nmol, 10% of the toluene extract or 0.35% of the fruits). Thus, in order to elute some 5 µg of 1 from a CapHPLC chromatographic system to the CapNMR probe, 22 µg of the actual extract would have to be injected to the capillary column, and this amount would have to be doubled in order to achieve a comparable amount of 2. Since the injection valve of a CapHPLC system is usually smaller than 1 µL, such amounts of the extract are not deliverable, and we conclude that determination of major components of T. garganica extract, straightforward by the present technique, may be difficult using capillary HPLC with CapNMR detection. The advantage of HPLCSPE combined with CapNMR is even more apparent in the case of the phenylpropanoids 6-9. By use of a reference standard of 6, the amount of this compound is roughly 2 µg (4 nmol) per injection or 0.6% of the toluene extract (0.02% of the fruits). Even for the minor phenylpropanoids 7 (peak 7) and 8 (peak 10), usable 1H NMR data could be obtained (Table 1). The amount of 8 can be estimated as roughly 0.4 µg/injection (0.7 nmol), or 0.1% of the extract (0.003% of the fruits). A sufficient amount of 8 could nevertheless be accumulated in the HPLC-SPE step, leading to the elucidation of its structure (Table 1). Therefore, the HPLC-SPE-CapNMR hyphenation with multiple trapping, employing normal-bore columns for increased capacity, allows structure elucidation of truly minor extract constituents and may be generally superior to CapHPLC-CapNMR for analysis of natural products mixtures. Previous applications of the CapNMR probe to natural products35-40 involved microfractionations, i.e., collecting and drying HPLC fractions followed by redissolving the resulting samples in extremely small solvent volumes (single-digit microliter amounts) for CapNMR analysis. In addition to automation, the HPLC-SPE procedure described in the present work offers analyte accumulation by repeated peak trapping, with the possibility of using a different number of cumulative trappings for major and minor extract components. Analyte isolation by HPLC-SPE, in contrast to microfractionation procedures that involve evaporation, is in principle compatible with the presence of nonvolatile buffers. Furthermore, SPE cartridge elution directly into the CapNMR probe is perhaps easier than direct handling of microliter amounts of solvents and circumvents possible problems with dissolution of isolated compounds. Further developments should therefore lead to a fully automated HPLC-SPE-CapNMR system, integrating a mass spectrometer for on-line determination of molecular masses or SPE trapping according to the MS signal for compounds not detectable by UV detectors. CONCLUSIONS The reported proof-of-concept study demonstrated that combination of HPLC-SPE with CapNMR is an attractive technique for analysis of complex mixtures such as natural product extracts.

A successful investigation of a plant extract using off-line combination of automated HPLC-SPE with CapNMR was performed, allowing structure determination of complex natural products constituting less than 0.5% of the extract. The HPLC-SPE-CapNMR technique combines the high mass sensitivity of the CapNMR probe with the high loadability of standard HPLC columns and the advantages of the SPE interface. HPLC-SPE-CapNMR hyphenation is therefore regarded an important application of CapNMR technology, supplementing its use in conjunction with flow injection analysis and separations by capillary HPLC.

ACKNOWLEDGMENT We thank Ms. Huizhen Liu (The Danish University of Pharmaceutical Sciences) for preparation of the extract of T. garganica and Dr. Karin Ndjoko (University of Geneva) for assistance with the CapNMR equipment.

Received for review September 8, 2006. Accepted October 6, 2006. AC0616963

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