Anal. Chem. 2004, 76, 2623-2628
Hyphenation of Capillary HPLC to Microcoil 1H NMR Spectroscopy for the Determination of Tocopherol Homologues Manfred Krucker, Annette Lienau, Karsten Putzbach, Marc David Grynbaum, Paul Schuler, and Klaus Albert*
Institute of Organic Chemistry, University of Tu¨bingen, Auf der Morgenstelle 18, D-72076 Tu¨bingen, Germany
Highly selective reversed phases (C30 phases) are selfpacked in 250 µm inner diameter fused-silica capillaries and employed for capillary HPLC separation of shapeconstrained natural compounds (tocopherol homologues, vitamin E). Miniaturized hyphenated systems such as capillary HPLC-ESI-MS (positive ionization mode) and, with special emphasis, continuous-flow capillary HPLCNMR are used for structural determination of the separated compounds. Despite the small amount of sample available (1.33 µg of each tocopherol), the authors have been able to monitor the capillary HPLC separation under continuous-flow 1H NMR conditions, thus allowing an immediate peak identification. Further structural assignment was carried out in the stopped-flow NMR mode as shown, for example, by a 2D 1H,1H COSY NMR spectrum of r-tocopherol. We demonstrate in this paper the considerable potential of hyphenated capillary separations coupled to MS and NMR for the investigation of restricted amounts of sample. To fulfill the need for fast and efficient identification of analytes from complex mixtures, hyphenation of chromatographic separation techniques with spectroscopic or spectrometric detection methods has taken an increased importance. Mostly used today is the direct on-line coupling of high-performance liquid chromatography (HPLC) to mass spectrometry (MS). Such LC-MS systems with specially developed interfaces for efficient spray formation of the LC effluent and soft ionization of the analytes, e.g., electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), yield valuable information for a wide range of compounds in a fast, sensitive, and selective way. Despite all the advantages of the LC-MS coupling shown in numerous applications,1-3 nuclear magnetic resonance spectroscopy (NMR) is considered to be one of the most powerful spectroscopic techniques for the unambiguous structural elucidation of unknown compounds. In particular, NMR spectroscopy has the capability * To whom correspondence should be addressed. Phone: (+49) 7071-2975335. Fax: (+49) 7071-29-5875. E-mail:
[email protected]; http://www.uni-tuebingen.de/uni/cok. (1) Wilson, I. D.; Morgan, E. D.; Lafont, R.; Schockcor, J. P.; Lindon, J. C.; Nicholson, J. K.; Wright, B. Chromatographia 1999, 49, 374-378. (2) Gelpi, E. J. Chromatogr., A 2003, 1000, 567-581. (3) Konstiainen, R.; Kotiaho, T.; Kuuranne, T.; Auriola, S. J. Mass Spectrom. 2003, 38, 357-372. 10.1021/ac030379i CCC: $27.50 Published on Web 03/27/2004
© 2004 American Chemical Society
to distinguish between structural and conformational isomers. Therefore, on-line HPLC-NMR in the stopped-flow as well as continuous-flow mode is now an established analytical tool demonstrated by many successful applications4 showing the feasibility and benefits of this technique. However, a prerequisite for successful NMR detection is a much greater amount of sample than for other detection methods, and NMR sensitivity still remains a major challenge. Many efforts have been made to overcome this limitation. The homogenicity and field strengths have been increased using superconducting magnets, but it has become increasingly difficult and expensive to extend these parameters any further. Novel polarization transfer methods,5 spin-lattice relaxation time reduction techniques,6 and cryogenic NMR probes7 have also been utilized to address this issue. Another approach is the reduction of the radio frequency (rf) coil diameter,8 as mass-limited samples can be dissolved in smaller solvent volumes, thus enhancing their concentration. These microcoils (1.5 µL active volume) are also well size-matched to the eluting peak volumes (∼2 µL) of a capillary separation, which also increases NMR sensitivity. Two different geometrical probe designs have been employed: the saddle-type coil,9 in analogy to conventional NMR probes, and the solenoidal-type, which is constructed by directly wrapping the rf coil around a capillary column,10 immersing the coil into a susceptibility-matching fluid, and placing it transversal to the magnetic field. Therefore, the solenoidal probe design shows severalfold better sensitivity than saddle-shaped coils.11 Both probe types have successfully been utilized for on-line coupled HPLC-NMR measurements,12 but only the solenoidal-type microprobes are commercially available at (4) Albert, K., Ed. On-line LC NMR and Related Techniques; John Wiley & Sons Ltd.: Chichester, U.K., 2002. (5) Dorn, H. C.; Glass, T. E.; Gitti, R.; Tsai, K. H. Appl. Magn. Reson. 1991, 2, 9-27. (6) Fischer, H. H.; Seiler, M.; Ertl, T. S.; Eberhardinger, U.; Bertagnolli, H.; Schmitt-Willich, H.; Albert, K. J. Phys. Chem. B 2003, 10, 4879-4886. (7) Spraul, M.; Freund, A. S.; Nast, R. E.; Withers, R. S.; Mass, W. E.; Corcoran, O. Anal. Chem. 2003, 75, 1546-1551. (8) Olson, D. L.; Lacey, M. E.; Sweedler, J. V. Anal. Chem. 1998, 70, 257A264A. (9) Behnke, B.; Schlotterbeck, G.; Tallarek, U.; Strohschein, S.; Tseng, L.-H.; Keller, T.; Albert, K.; Bayer, E. Anal. Chem. 1996, 68, 1110-1115. (10) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849-3857. (11) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-1970. (12) Jayawickrama, D. A.; Sweedler, J. V. J. Chromatogr., A 2003, 1000, 819840.
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present.13 Among these improvements toward a more sensitive NMR detection, there has been much significant progress enhancing the overall performance of the HPLC-NMR systems. One approach is to use miniaturized separation techniques since they show higher efficiencies and therefore result in elevated concentrations at the eluting peak maximums. Capillary separation techniques hyphenated to microcoil NMR detection may therefore be the best suited configuration. An additional benefit of microseparations is the low solvent consumption, which makes the use of fully deuterated solvents economically feasible. The commonly used flow rate in capillary HPLC of 5 µL min-1 corresponds to a mobile-phase reduction of a factor of 200 in comparison to the 1 mL min-1 used in classical HPLC. At an overall daily solvent consumption in capillary HPLC of ∼7 mL, not only water is exchanged with deuterium oxide, as usually done in the HPLC-NMR coupling, but fully deuterated organic modifiers can also be used. NMR solvent suppression techniques may not be necessary any more. Various capillary separation techniques are coupled to NMR detection, i.e., capillary electrophoresis (CENMR),14,15 capillary electrochromatography (CEC-NMR),16 capillary isotachophoresis (capillary ITP-NMR),17 and capillary highperformance liquid chromatography (capillary HPLC-NMR).18,19 Such miniaturized hyphenated systems coupling capillary separation techniques to microcoil 1H NMR detection are wellsuited for analyzing mass-limited samples, e.g., the identification of carotenoid stereoisomers from chicken, bovine, or even rat and mouse single retina to study the prevention and treatment of agerelated macula degeneration as well as their bioavailability.20 This has been already demonstrated with LC-APCI-MS21 down to the picomole range, but these measurements could not unequivocally distinguish between the stereoisomers of a single carotenoid, e.g., zeaxanthin stereoisomers. NMR could, but the sensitivity (continuous-flow 1H NMR 240 µg, stopped-flow mode 800 ng) is insufficient to apply the system to retina analysis. The same is true for tocopherol analysis utilizing HPLC-NMR22 (continuousflow 1H NMR 286 µg). New solenoidal microprobes enable the measurement of samples in the low-nanogram range for flow injection analysis with a single transient. The hyphenated system sensitivity performance is slightly weaker; still, 200 ng can be easily detected in the stopped-flow mode. This paper deals with an application of miniaturized hyphenated systems, i.e., the separation of an artificial standard solution of shape-constrained tocopherol homologues (vitamin E derivatives) utilizing capillary HPLC with self-packed 250 µm inner diameter, (13) http://www.protasis.com. (14) Schewitz, J.; Pusecker, K.; Gfo ¨rer, P.; Go ¨tz, U.; Tseng, L.-H.; Albert, K.; Bayer, E. Chromatographia 1999, 50, 333-337. (15) Wolters, A. M.; Jayawickrama, D. A.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 2002, 74, 5550-5555. (16) Rapp, E.; Jakob, A.; Schefer, A. B.; Bayer, E.; Albert, K. Anal. Bioanal. Chem. 2003, 376, 1053-1061. (17) Wolters, A. M.; Jayawickrama, D. A.; Larive, C. K.; Sweedler, J. V. Anal. Chem. 2002, 74, 2306-2313. (18) Lacey, M. E.; Tan, Z. J.; Webb, A. G.; Sweedler, J. V. J. Chromatogr., A 2001, 922, 139-149. (19) Schlotterbeck, G.; Tseng, L.-H.; Ha¨ndel, H.; Braumann, U.; Albert, K. Anal. Chem. 1997, 69, 1421-1425. (20) Lienau, A.; Glaser, G.; Tang, G.; Dolnikowski, G. G.; Grusak, M. A.; Albert, K. J. Nutr. Biochem. 2003, 16, 663-670. (21) Dachtler, M.; Glaser, T.; Kohler, K.; Albert, K. Anal. Chem. 2001, 73, 667674. (22) Lienau, A.; Glaser, T.; Krucker, M.; Zeeb, D.; Ley, F.; Curro, F.; Albert, K. Anal. Chem. 2001, 74, 5192-5198.
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highly shape-selective C30 capillaries. The miniaturized chromatographic separation is on-line coupled to ESI-MS and solenoidal microcoil 1H NMR detection, mainly focusing on continuous-flow 1H NMR detection, where an absolute amount of 1.33 µg of each eluting tocopherol was monitored, allowing clear differentiation and structure determination of all homologues. In the pharmaceutical and food industries, the importance of tocopherols has dramatically increased due to their antioxidative properties.23 Epidemological studies indicate beneficial effects against many diseases, e.g., certain types of cancer and the aging process in general.24,25 Vitamin E consists of four tocopherol homologues, named R-, β-, γ-, and δ-tocopherol, which possess different bioavailabilities. In addition to these four naturally occurring tocopherols, R-tocopherol acetate is mostly used due to its greater stability in industrial applications. Tocopherol homologues are easily oxidizable in the presence of light and air. Therefore, the on-line hyphenated capillary HPLC-NMR system is ideal for their analysis as it prevents the analytes from degrading.26 EXPERIMENTAL SECTION Materials. Methanol (LiChrosolv, gradient grade), methanold4 (Uvasol, 99,8%), and D2O (Uvasol, 99,8%) were purchased from Merck (Darmstadt, Germany). Water was purified using a Milli-Q water purification system (Millipore, Billerica, MA). The tocopherol homologues and R-tocopherol acetate were obtained from Calbiochem (San Diego, CA). Tocol was donated by Eisai Inc. (Teaneck, NJ). The tocopherol standards were prepared by dissolving the tocopherols in methanol-d4 until the desired concentration was reached, i.e., 1.67 mg mL-1 for capillary HPLCESI-MS and 6.65 mg mL-1 (each tocopherol) for continuous-flow capillary HPLC-NMR. Packing Capillaries. Twenty milligrams of the C30 stationary phase (Bischoff ProntoSil 200 Å, 3 µm, C30) were suspended in 300 µL of carbon tetrachloride and put in an ultrasonic bath for 10 min. Afterward, the gained slurry was transferred into a slurry chamber (empty 1 mm × 12.5 cm HPLC column) and forced downward into a 250 µm i.d. × 12 cm fused-silica capillary with a pneumatic HPLC pump (Knauer GmbH, Berlin-Zehlendorf, Germany). Initially a pressure of 400 bar was used and increased to 650 bar within 5 min. This final pressure was maintained for 30 min. The capillary end fittings consisted of zero dead volume unions ZU1C, steal screens 2SR1, and ferrules FS1.4-5 (Vici AG Valco Int., Schenkon, Switzerland). Chromatography. Two capillary HPLC systems were utilized, an Eldex MicroPro dual-syringe pump (Eldex Laboratories, Napa, CA) equipped with on-column (100 µm i.d.) UV detection performed at 285 nm on a Knauer UV detector K-2500 (Knauer GmbH) and a microinjection valve kit (Upchurch Scientific, Oak Harbor, WA) with a 200 nL fused-silica injection loop for the capillary HPLC-NMR measurements. The second system used was a ternary modular capillary HPLC pump (Waters, Milford, MA) equipped with on-column (75 µm i.d.) UV detection per(23) Pryor, W. A., Bowman, B. A., Russel, R. M., Eds. Present Knowledge in Nutrition; International Life Sciences Institute Press: Washington, DC, 2001. (24) Marchioli, R. Pharmacol. Res. 1999, 40, 227-238. (25) Flynn, B. L.; Ranno, A. E. Ann. Pharmacother. 1999, 33, 188-197. (26) Sto ¨ggl, M.; Huck, C. W.; Scherz, H.; Popp, M.; Bonn, G. K. Chromatographia 2001, 54, 179-185.
Table 1. Sensitivity-Optimized Settings for the Capillary HPLC-ESI+/MS Measurements segment (analyte)
time (min)
scan range (m/z)
averages
target
1 (tocol) 2 (δ-tocopherol) 3 (γ-, β-tocopherol) 4 (R-tocopherol) 5 (R-tocopherol acetate)
0.0-4.2 4.3-5.2 5.3-6.2 6.3-8.5 8.6-14.0
380-420 395-435 400-450 425-460 460-500
25 25 30 45 60
15 000 15 000 15 000 15 000 12 000
formed at 285 nm on a Bischoff Lambda 1010 UV detector (Bischoff Chromatography, Leonberg, Germany) and a Vici Cheminert 1004-.1 (Vici AG) injection valve (100 nL internal loop) for the capillary HPLC-ESI-MS (positive ionization mode) measurements. The chromatographic separation utilized isocratic mixtures of methanol and water or methanol-d4 and deuterium oxide, respectively, at a flow rate of 5 µL min-1. All transfer capillaries were fused silica with a dimension of 50 µm i.d./360 µm o.d. Capillary HPLC-ESI-MS. To couple the capillary HPLC system to the esquire 3000plus ion trap mass spectrometer (Bruker Daltonik, Bremen, Germany), a 75 µm i.d. capillary was used for on-column UV detection as well as transfer capillary to the microflow nebulizer needle of the ESI ionization source. The detection was performed in positive ion mode with nitrogen as nebulizer (11.0 psi) and drying gas (5 L min-1) at a temperature of 325 °C. The capillary voltage was set to 3.5 kV, compound stability and trap drive level to 75%, respectively. Maximum detection sensitivity was achieved by assigning five time segments with optimized parameter settings (Table 1). Capillary HPLC-NMR. For coupling the capillary HPLC system to the NMR instrument (AMX 600, Bruker BioSpin GmbH, Rheinstetten, Germany), the outlet of the UV detector was connected with the 1.5 µL active volume 1H selective capillary NMR probe (Protasis Corp., Marlboro, MA) inlet using a 3 m fused-silica transfer capillary (50 µm i.d.). For stopped-flow measurements, an additional peak parking valve had to be inserted prior to the injection valve. As mobile phase for the continuous-flow experiment, fully deuterated solvents and an isocratic solvent mixture of methanold4: D2O ) 90:10 (v/v) was applied. The continuous-flow experiment was recorded with the pulse program lc2pnps, suppressing the solvent signals via shaped pulses. In this way, 16 transients with 4K complex data points and a spectral width of 9615 Hz were accumulated with a relaxation delay of 1 s. The pulse angle was set to 30°. During the separation, 256 rows with an acquisition time of 36 s per row were recorded. Prior to Fourier transformation, a squared sine bell function was applied to the FID. For the stopped-flow 2D NMR measurement of R-tocopherol, the chromatographic conditions of the separation shown in Figure 2 were applied (methanol-d4:D2O ) 96:4 (v/v)). The stopped-flow experiment was recorded with the pulse program cosypnps, a homonuclear 2D-COSY 45 experiment with a solvent presaturation using shaped pulses. Hereby, 2K transients with 1K complex data points and a spectral width of 6024 Hz were accumulated in the F2 dimension and 243 transients with 512 complex data points were accumulated in the F1 dimension, leading to an overall measurement time of 19 h. The pulse angle was set to 30°. Prior
Figure 1. Chemical structure of tocol, the tocopherol homologues, and R-tocopherol acetate.
Figure 2. HPLC-UV (A), capillary HPLC-UV (B), and capillary HPLC-ESI-MS (C) chromatograms of the tocopherol separation.
to Fourier transformation, a squared sine bell function was applied to the FID. The 1H spectra (along the F1 and F2 dimensions) were measured with direct flow injection of a 10 mg mL-1 standard solution utilizing the pulse program zgcpprsp. This program is a 1D sequence that uses solvent presaturation with shaped pulses Analytical Chemistry, Vol. 76, No. 9, May 1, 2004
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Table 2. Comparison of HPLC and Capillary HPLC Separation of Tocopherols
tocol δ-tocopherol γ-tocopherol β-tocopherol R-tocopherol R-tocopherol acetate a
HETP (10-5 m)
R
k′ HPLC
cHPLC
HPLC
cHPLK
HPLC
cHPLC
2.35 3.11 3.94 4.16 5.08 8.25
1.76 2.36 2.36 3.24 3.86 6.21
1.32 1.32 1.06 1.22 1.62
1.34 1.34 1.05 1.19 1.61
3.57 3.95 a a 9./19 9.98
2.46 3.52 a a 2.92 b
Not separated to baseline. b Below limit of detection.
accumulating 32K transients with 32K complex data points and a spectral line width of 9091 Hz. RESULTS AND DISCUSSION The first step of the analysis was to transfer the analytical HPLC separation of tocopherols to capillary-scale HPLC. With selfmade slurry-packed C30 capillaries, the authors were able to separate all homologues including tocol, often used as internal standard and R-tocopherol acetate (structures are depicted in Figure 1). A 100 nL aliquot of a standard solution containing 1.67 mg mL-1 of each tocopherol was injected. Although this concentration may not appear challenging for any modern analytical technique, however, the absolute amount of sample injected is relatively small (100 nL injection volume corresponds to an absolute amount of 167 ng of each tocopherol). The chromatogram obtained on a home-packed (250 µm × 12 cm) C30 capillary column using a mobile phase of methanol-d4:D2O ) 96:4 (v/v) at a flow rate of 5 µL min-1 is shown in Figure 2B in comparison to a chromatogram achieved using a classical (4.6 mm × 25 cm) HPLC column (Figure 2A) packed with the same stationary phase using the same isocratic mobile phase at a flow rate of 1 mL min-1. The capillary HPLC separation shows narrow and symmetrical peaks representing a high-quality packing and good system performance with less dead volumes that often lead to unprepossessing chromatograms. Even the constitution isomers β- and γ-tocopherol were slightly separated, a separation of these two homologues to baseline could not be achieved in classical HPLC, and is completely impossible on C18 stationary phases,27 as tailored C30 stationary phases exhibit a much higher shape selectivity for shape-constrained natural compounds such as carotenoid stereoisomers28 and tocopherol homologues29 and enhanced sample loading capacity30 in comparison to conventionally used C18 materials. The separation is completed after 11 min in comparison to 25 min in classical HPLC with comparable results. These are summarized in Table 2. The capillary separation shows greatly reduced retention factors k′ but the separation factors R for all homologues stay almost equal. More important for the hyphen(27) Abidi, S. L. J. Chromatogr., A 2000, 881, 197-216. (28) Sander, L. C.; Epler Sharpless, K.; Craft, N. E.; Wise, S. A. Anal. Chem. 1994, 66, 1667-1674. (29) Strohschein, S.; Pursch, M.; Lubda, D.; Albert, K. Anal. Chem. 1998, 70, 13-18. (30) Dachtler, M.; Glaser, T.; Ha¨ndel, H.; Lacker, T.; Tseng, L.-H.; Albert, K. In Encyclopedia of Separation Science; Wilson, I. D., Adlard, E. R., Cooke, M., Poole, C. F., Eds.; Academic Press: London, 2000; Level II, pp 747-760.
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ation to the NMR is that the height equivalents of a theoretical plate (HETP) are much smaller in comparison to classical HPLC, leading to higher concentrations in the eluting peak maximums. Hence, drastically shortened retention times, low consumption of solvents, and the small amount of sample needed clearly show the benefits of the capillary separation technique. In addition to UV detection, hyphenated systems coupling capillary separation techniques to mass spectrometry (capillary HPLC-MS) and nuclear magnetic resonance spectroscopy (capillary HPLC-NMR) are becoming increasingly important. Therefore, the tocopherol separation was coupled to positive mode ESI-MS detection first, as electrospray ionization is the most commonly used interface in coupled LC-MS systems today. Former studies added AgClO4 for better ionization of nonpolar analytes, e.g., tocopherols and carotenoids, in ESI-MS mode;31 however, this was not necessary in this work. A sensitivity-optimized extracted ion chromatogram for all tocopherols is shown in Figure 2C. One can easily distinguish between the tocopherols, but the constitution isomers β- and γ-tocopherol, which have the same mass, cannot be differentiated from one another. Often, characteristic fragment ions allow unambiguous identification of analytes in highthroughput screening multiple-stage MS(n) analysis. For tocopherols, a thermally allowed retro-Diels-Alder reaction leads to such a characteristic chroman ring parent ion.32 It should be noted, however, that this parent ion for the β- and γ-tocopherol isobars differs in the substitution of the aromatic ring but has the same mass for the two isomers, and a differentiation cannot be achieved. The authors could only detect a minor qualitative difference among these isomers, i.e., the affinity to form sodium adducts, which seems to be more pronounced for β-tocopherol (31.6% [M + Na]+ in contrast to 26.9% [M + Na]+ for γ-tocopherol). It is interesting to note that the formation of sodium adducts decreases with an increase in the number of methyl groups attached to the aromatic ring and therefore higher electron density within the ring from tocol (51.9% [M + Na]+) to R-tocopherol (18.4% [M + Na]+). Even so sodium adducts may give the chemist an idea about the nature of the isomer, it cannot be considered as a good tool for the differentiation. NMR is the detector that is able to derive this kind of information from the data acquired. For the capillary HPLC-NMR hyphenation experiments, the separation had to be adjusted to the continuous-flow 1H NMR detection requirements. Most important is to guarantee an adequate residence time of the analytes in the NMR probe. Second, one should also take care that the separation of the analytes is good enough to ensure the measurement of a single analyte within one row (in this case 36 s), which was especially difficult to achieve for the β- and γ-tocopherol isomers. Both requirements could be fulfilled using a mobile phase of methanold4:D2O ) 90:10 (v/v) at a flow rate of 5 µL min-1 in a C30 capillary column of 20 cm length. This is due to the fact that the high amount of deuterium oxide in the mobile phase increased the retention times of all tocopherols drastically, guaranteeing improved separation. This is especially beneficial for measuring βand γ-tocopherol, as their retention times differ now ∼2 min (selectivity factor R could be improved to 1.08). Third, the (31) Rentel, C.; Strohschein, S.; Albert, K.; Bayer, E. Anal. Chem. 1998, 70, 4394-4400. (32) Perri, E.; Mazzotti, F.; Raffaelli, A.; Sindona, G. J. Mass Spectrom. 2000, 35, 1360-1361.
Figure 3. Continuous-flow capillary HPLC-NMR (600 MHz, 1.5 µL solenoidal microprobe) measurement of tocopherols.
concentration of the tocopherol homologues had to be increased to 6.65 mg mL-1 (each tocopherol); 200 nL of the standard solution was injected resulting in an absolute amount of 1.33 µg of each tocopherol, which is ∼8-fold more than injected in the capillary HPLC separation coupled to UV and ESI-MS detection shown in Figure 2. The high concentration leads to overloaded conditions for capillary HPLC, making the separation of the isomers β- and γ-tocopherol even more challenging. However, this concentration was the best compromise between the capillary separation and the NMR detection requirements. Comparable result matching former studies22 (266.8 µg of each tocopherol standard utilizing classical HPLC-NMR coupling in the continuous-flow mode) could be derived from the capillary HPLC-NMR data shown in this paper, only the signal-to-noise ratios are slightly poorer. However, a sample reduction by a factor of 200 demonstrates major advances in the sensitivity of capillary HPLC-NMR systems. The continuous-flow capillary HPLC-NMR spectrum is shown in Figure 3. On the x axis (F2 dimension), the proton chemical shift is plotted, while the y axis (F1 dimension) shows the retention time scale of the adjusted capillary separation, resulting in a pseudo-2D NMR contour map. All tocopherol derivatives can unambiguously be identified. The isomer pair β- and γ-tocopherol could be detected separately and easily differentiated from each other in contrast to the capillary HPLC-ESI-MS measurements. This can be noticed at the aromatic protons (protons 5,7; light gray box). The aromatic proton chemical shift region also distinguishes between all tocopherols, additionally assisted by the characteristic differences of the methyl group signals bound to the aromatic ring between 2.0 and 2.2 ppm (gray box). For the last eluting R-tocopherol acetate, one can also detect the methylene protons of the acetate group at ∼2.3 ppm (dark gray box). Aromatic proton signals occur only for the δ-, β-, and γ-tocopherol, while δ-tocopherol shows two signals corresponding to the protons 5 and 7. β- and γ-tocopherol have only one aromatic proton signal representing proton 5 or 7, respectively. The other important group of signals are the methyl groups attached to the aromatic ring, which are present for all tocopherols. δ-Tocopherol shows one, β- and γ-tocopherol show two, and R-tocopherol as well as R-tocopherol acetate shows three of these methyl signals. For a more detailed interpretation of the 1H NMR spectra, the contour plot of the continuous-flow NMR measurement is ill-suited.
Figure 4. Extracted 1H NMR spectra (600 MHz, 1.5 µL solenoidal microprobe) of the tocopherol homologues at the corresponding peak maximums; * residual solvent signals.
Therefore, the extracted proton NMR spectra of the tocopherols at the corresponding peak maximums of the continuous-flow measurement are depicted in Figure 4, reflecting the elution order from bottom to top. Although the signal-to-noise ratio is relatively poor and some remaining signals of (only partially) deuterated solvents are still visible due to incomplete solvent suppression, unequivocal structural assignment of the tocopherol homologues was carried out. Therefore, each NMR spectrum is divided into three sections, an aromatic region at ∼6.5 ppm, a region at ∼2.1 ppm corresponding to the protons of the methyl groups bound to the aromatic ring, and a high-field region between 0.8 and 1.7 ppm showing the signals of protons in the saturated phytyl side chain. Two important additional peaks are visible at ∼2.8 ppm representing the protons at position 4 and at ∼1.9 ppm representing the protons at position 3. The characteristic differences already derived from the continuous-flow data shall be discussed in detail in order to interpret these extracted proton spectra. In the aromatic region, two signals occur for δ-tocopherol, representing proton 7 at 6.5 ppm and proton 5 at 6.4 ppm. γ-Tocopherol only shows one small signal at 6.4 ppm (proton 5), while β-tocopherol has one signal at 6.5 ppm (proton 7) and a minor superimposed signal at 6.4 ppm arising from the previously eluting γ-tocopherol. Such superimposed signals are only visible for the difficult to separate isomers γ- and β-tocopherol. All other spectra are free from coeluting substance signals. However, the incomplete Analytical Chemistry, Vol. 76, No. 9, May 1, 2004
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separation of γ- and β-tocopherol can be monitored in the NMR spectra, especially at the signal of proton 4 at ∼2.7 ppm, as a methyl group attached to the aromatic ring at position 5 causes a slight upfield shift of this signal. For example, the spectrum of γ-tocopherol exhibits two signals for proton 4, the one with a minor upfield shift arises from β-tocopherol. The other important signal region making the differentiation of the homologues possible is the group of signals corresponding to the methyl groups attached to the aromatic ring. These signals are present for all tocopherols. δ-Tocopherol shows one signal representing the 8-CH3 at ∼2.1 ppm, β- and γ-tocopherol show two signals of 5-, 8-CH3 or 7-, 8-CH3, respectively, and R-tocopherol and R-tocopherol acetate show three of these methyl signals corresponding to 5-, 7-, 8-CH3. Due to the influence of the acetate group, the methyl signals of the R-tocopherol acetate are shifted to higher field compared to the signals of R-tocopherol. For complete structure elucidation of complex analytes, 1H 1D NMR spectra often contain an insufficient amount of information. Hence, 2D NMR measurements were employed to accomplish unambiguous peak assignment, as 2D homonuclear correlation NMR spectroscopy offers a way to identify spin-coupled pairs of nuclei. These data can be measured in hyphenated HPLC-NMR systems by stopping the chromatographic run when the peak maximum of the analyte of interest reaches the NMR probe detection volume (stopped-flow NMR). After data acquisition has been carried out, the separation can be resumed, allowing one to trap a subsequent analyte in the NMR probe (multiple stoppedflow NMR within a single chromatographic run). A 2D 1H,1H COSY 45 stopped-flow NMR spectrum of R-tocopherol is shown in Figure 5. All off-diagonal peaks, so-called cross-peaks, indicate homonuclear spin coupling between the protons. Major crosspeaks can be detected between protons 3 and 4 and in the phytyl side chain between 0.8 and 1.6 ppm. In this region, one can observe the couplings between the methyl, methylene, and methine protons, confirming their assignment done in the 1H NMR spectra (Figure 4). The two methyl groups at the end of the phytyl chain (12’-CH3) at a slightly lower field as well as the methyl groups within the chain (4’-, 8’-CH3) at ∼0.9 ppm show crosspeaks to the methine protons (4’, 8’, 12’). The cross-peaks among the methylene protons in the phytyl side chain can also clearly be seen. CONCLUSION The results obtained demonstrate that the hyphenation of capillary HPLC to soleniodal-type microprobe 1H NMR detection supplies adequate information for full structure determination of pharmaceutically active compounds from mass-limited samples. Although sensitivity, in contrast to capillary HPLC-MS, is still a
2628 Analytical Chemistry, Vol. 76, No. 9, May 1, 2004
Figure 5. 2D homonuclear 1H,1H COSY stopped-flow NMR spectrum (600 MHz, 1.5 µL solenoidal microprobe) of R-tocopherol; * residual solvent signals.
major challenge when coupling capillary separation techniques to the relatively insensitive NMR, continuous-flow 1H NMR measurements of a small amount of sample were successfully carried out, allowing immediate structure identification of the eluting analytes, as demonstrated in this paper with tocopherol homologues including the constitution isomers β- and γ-tocopherol. However, the continuous-flow NMR mode can only hardly be applied to real life applications, since ∼1 µg is still need for successful NMR detection. Utilizing stopped-flow NMR hyphenated to capillary HPLC, limits of detection in the low-nanogram range for proton NMR can be achieved, therefore allowing identification of analytes from biological samples. Additionally, 2D NMR experiments, which are often indispensable for unambiguous structure elucidation of unknown compounds, can be performed in the stopped-flow NMR mode. ACKNOWLEDGMENT This work was supported by the European Union project HPRICT-1999-50018 and the Deutsche Forschungsgemeinschaft (AL 298/10-1). Received for review November 3, 2003. Accepted February 5, 2004. AC030379I
