HPLC-NMR Techniques for Plant Extract Analysis

Medicinal Plant Biotechnology: From Basic Research to Industrial Applications

Volumn , Issue , 2008, Pages 29-48

  Stærk, Dan ^a   Lambert, Maja ^a   Jaroszewski, Jerzy W ^a  

^a UNIVERSITY OF COPENHAGEN   (Denmark)

Author keywords

Chemistry; Investigations; Isolation; Mankind; Spectroscopic methods

Indexed keywords

EID: 84889328847     PISSN: None     EISSN: None     Source Type: Book    
DOI: 10.1002/9783527619771.ch3     Document Type: Chapter

References (95)

1. Cragg, G.M., Newmann, D.J., Snader, K.M. Natural products in drug discovery and development. J. Nat. Prod. 1997, 60, 52-60. [Useful review of the role of natural products in drug discovery.]
2. Newmann, D.J., Cragg, G.M., Snader, K.M. Natural products as sources of new drugs over the period 1981-2002. J. Nat. Prod. 2003, 66, 1022-1037. [Useful review of the role of natural products in drug discovery.]
3. Pitman, N.C.A., Jørgensen, P.M. Estimating the size of the world's threatened flora. Science 2002, 298, 989.
4. Chadwick, D.J., Marsh, J. Ethnobotany and the Search for New Drugs. John Wiley & Sons, Chichester, 1994.
5. Iwu, M.M., Wootton, J.C. Ethnomedicine and Drug Discovery. Elsevier, Amsterdam, 2002.
6. Polya, G. Biochemical Targets of Plant Bioactive Compounds. Taylor and Francis, London, 2003.
7. Zhang, L., Demain, A.L. Natural Products-Drug Discovery and Therapeutic Medicine. Humana Press, Totowa, 2005.
8. Bindseil, K.U., Jakupovic, J., Wolf, S.D., Lavayre, J., Leboul, J., van der Pyl, D. Pure compound libraries: a new perspective for natural product based drug discovery. Drug Discov. Today 2001, 4, 206-220. [An important review of pure compound libraries based on natural products.]
9. Koehn, F.E., Carter, G.T. The evolving role of natural products in drug discovery. Nature Rev. Drug Discov. 2005, 4, 206-220. [An important review of the renewed interest of natural products in drug discovery.]
10. Larsson, J., Gottfries, J., Bohlin, L., Backlund, A. Expanding the ChemGPS chemical space with natural products. J. Nat. Prod. 2005, 68, 985-991.
11. Feher, M., Jonathan, M. Schmidt, J.M. Property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry. J. Chem. Inf. Comput. Sci. 2003, 43, 218-227.
12. Romeo, J.T., Dixon, R.A. (Eds.), Phytochemistry in the Genomics and Post-Genomics Eras. Pergamon, Amsterdam, 2002.
13. Sumner, L.W., Mendes, P., Dixon, R.A. Plant metabolomics: large-scale phytochemistry in the functional genomics era. Phytochemistry 2003, 62, 817-836.
14. Zhang, W., Franco, C., Curtin, C., Conn, S. To stretch the boundary of secondary metabolite production in plant cell-based bioprocessing: anthocyanin as a case study. J. Biomed. Biotechnol. 2004, 5, 264-271.
15. Sweetlove, L.J., Fernie, A.R. Regulation of metabolic networks: understanding metabolic complexity in the systems biology era. New Phytologist 2005, 168, 9-24.
16. Huber, L., George, S.A. Diode Array Detection in HPLC. Marcel Dekker, New York, 1993.
17. Niessen, W.M.A. Liquid Chromatography-Mass Spectrometry, 2nd edn. Marcel Dekker, New York, 1999.
18. Bringmann, G., Messer, K., Wohlfarth, M., Kraus, J., Dumbuya, K., Rückert, M. HPLC-CD on-line coupling in combination with HPLC-NMR and HPLC-MS/MS for the determination of the full absolute stereostructure of new metabolites in plant extracts. Anal. Chem. 1999, 71, 2678-2686.
19. Somsen, G.W., Gooijer, C., Brinkmann, U.A.Th. Liquid chromatography-Fouriertransform infrared spectrometry. J. Chromatogr. A 1999, 856, 213-243.
20. Watanabe, N., Niki, E. Direct-coupling of FT-NMR to high-performance liquid chromatography. Proc. Jpn. Acad. Ser. B 1978, 54, 194-199.
21. Bayer, E., Albert, K., Nieder, M., Grom, E., Keller, T. On-line coupling of highperformance liquid chromatography and nuclear magnetic resonance. J. Chromatogr. 1979, 186, 497-507.
22. Haw, J.F., Glass, T.E., Hausler, D.W., Dorn, H.C. Direct coupling of a liquid chromatograph to a continuous flow hydrogen nuclear magnetic resonance detector for analysis of petroleum and synthetic fuels. Anal. Chem. 1980, 52, 1135-1140.
23. Bayer, E., Albert, K., Nieder, M., Grom, E., An, Z. Examples of application of direct HPLC-NMR-coupling with high resolution. Fresenius Z. Anal. Chem. 1980, 304, 111-116.
24. Buddrus, J., Herzog, H. Coupling of HPLC and NMR. 1. Analysis of flowing liquid-chromatographic fractions by proton magnetic resonance. Org. Magn. Res. 1980, 13, 153-155.
25. Haw, J.F., Glass, T.E., Dorn, H.C. Continuous flow high field nuclear magnetic resonance detector for liquid chromatographic analysis of fuel samples. Anal. Chem. 1981, 53, 2327-2332.
26. Bayer, E., Albert, K., Nieder, M., Grom, E., Wolff, G., Rindlisbacher, M. On-line coupling of liquid chromatography and high-field nuclear magnetic resonance spectrometry. Anal. Chem. 1982, 54, 1747-1750.
27. Albert, K. On-line use of NMR detection in separation chemistry. J. Chromatogr. A 1995, 703, 123-147.
28. Jaroszewski, J.W. Hyphenated NMR methods in natural products research, part 1: direct hyphenation. Planta Med. 2005, 71, 691-700. [A comprehensive review of direct HPLC-NMR methods in natural products research.]
29. Jaroszewski, J.W. Hyphenated NMR methods in natural products research, part 2: HPLC-SPE-NMR and other new trends in NMR hyphenation. Planta Med. 2005, 71, 795-802. [A comprehensive review of HPLC-SPE-NMR methods in natural products research.]
30. Nicholson, J.K., Foxal, P.J.D., Spraul, M., Farrant, R.D., Lindon, J.C. 750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma. Anal. Chem. 1995, 67, 793-811.
31. Smallcombe, S.H., Patt, S.L., Keifer, P.A. WET solvent suppression and its applications to LC NMR and high-resolution NMR spectroscopy. J. Magn. Reson. Ser. A 1995, 117, 295-303.
32. Albert, K., Nieder, M., Bayer, E., Spraul, M. Continuous-flow nuclear magnetic resonance. J. Chromatogr. 1985, 346, 17-24.
33. Griffiths, L. Optimization of NMR and HPLC conditions for LC-NMR. Anal. Chem. 1995, 67, 4091-4095.
34. Griffiths, L. Optimization of liquid chromatography-NMR spectroscopy. Magn. Reson. Chem. 1997, 35, 257-261.
35. Bringmann, G., Rückert, M., Messer, K., Schupp, O., Louis, A.M. Acetogenic isoquinoline alkaloids CXXI. Use of on-line high-performance liquid chromatography-nuclear magnetic resonance spectrometry coupling in phytochemical screening studies: rapid identification of metabolites in Dioncophyllum thollonii. J. Chromatogr. A 1999, 837, 267-272.
36. Bringmann, G., Günthe, C., Schlauer, J., Rückert, M. HPLC-NMR on-line coupling including the ROESY technique: direct characterization of naphthylisoquinoline alkaloids in crude plant extracts. Anal. Chem. 1998, 70, 2805-2811.
37. Garo, E., Wolfender, J.-L., Hostettmann, K. Prenylated flavanones from Monotes engleri: on-line structure elucidation by LC/UV/NMR. Helv. Chim. Acta 1998, 81, 754-763. [An illustrative example of stopped-flow HPLC-NMR.]
38. Cavin, A., Potterat, O., Wolfender, J.-L., Hostettmann, K., Dyatmyko, W. Use of on-flow LC/1H NMR for the study of an antioxidant fraction from Orophea enneandra and isolation of a polyacetylene, lignane, and a tocopherol derivative. J. Nat. Prod. 1998, 61, 1497-1501.
39. Ioset, J.-R., Wolfender, J.-L., Marston, A., Gupta, M.P., Hostettmann, K. Identification of two isomeric meroterpenoid naphthoquinones from Cordia linnaei by liquid chromatography-mass spectrometry and liquid chromatography-nuclear magnetic resonance spectroscopy. Phytochem. Anal. 1999, 10, 137-142.
40. Wolfender, J.-L., Verotta, L., Belvisi, L., Fuzzati, N., Hostettmann, K. Structural investigation of isomeric oxidised forms of hyperforin by HPLC-NMR and HPLCMSn. Phytochem. Anal. 2003, 14, 290-297.
41. Waridel, P., Wolfender, J.-L., Lachavanne, J.-B., Hostettmann, K. Identification of the polar constituents of Potamogeton species by HPLC-UV with post-column derivatization, HPLC-MSn and HPLCNMR, and isolation of a new ent-labdane diglycoside. Phytochemistry 2004, 65, 2401-2410.
42. Gavidia, I., Seitz, H.U., Pérez-Bermúdez, P., Vogler, B. LC-NMR applied to the characterisation of cardiac glycosides from three micropropagated Isoplexis species. Phytochem. Anal. 2002, 13, 266-271.
43. Setzer, W.N., Vogler, B., Bates, R.B., Schmidt, J.M., Dicus, C.W., Nakkiew, P., Haber, W.A. HPLC-NMR/HPLC-MS analysis of the bark extract of Stauranthus perforatus. Phytochem. Anal. 2003, 14, 54-59.
44. Renukappa, T., Roos, G., Klaiber, I., Vogler, B., Kraus, W. Application of highperformance liquid chromatography coupled to nuclear magnetic resonance spectrometry, mass spectrometry, and bioassay for the determination of active saponins from Bacopa monniera Wettst. J. Chromatogr. A 1999, 847, 109-116.
45. Pusecker, K., Albert, K., Bayer, E. Investigation of hop bitter acids by coupling of high-performance liquid chromatography to nuclear magnetic resonance spectroscopy. J. Chrom. A 1999, 836, 245-252.
46. Dachtler, M., Glaser, T., Kohler, K., Albert, K. Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina. Anal. Chem. 2001, 73, 667-674. [An illustrative example of stopped-flow HPLC-NMR.]
47. Waridel, P., Wolfender, J.-L., Lachavanne, J.-B., Hostettmann, K. Ent-labdane glycosides from the aquatic plant Potamogeton lucens and analytical evaluation of the lipophilic extract constituent of various Potamogeton species. Phytochemistry 2004, 65, 945-954.
48. Wolfender, J.-L., Ndjoko, K., Hostettmann, K. The potential of LC-NMR in phytochemical analysis. Phytochem. Anal. 2001, 12, 2-22.
49. Vogler, B., Klaiber, I., Roos, G., Walter, C.U., Holler, W., Sandor, P., Kraus, W. Combination of LC-MS and LC-NMR as a tool for the structure determination of natural products. J. Nat. Prod. 1998, 61, 175-178.
50. * Hansen, S.H., Jensen, A.G., Cornett, C., Bjørnsdottir, I., Taylor, S., Wright, B., Wilson, I.D. High-performance liquid chromatography on-line coupled to highfield NMR and mass spectrometry for structure elucidation of constituents of Hypericum perforatum L. Anal. Chem. 1999, 71, 5235-5241. [An illustrative example of stopped-flow HPLC-NMR.]
51. Wolfender, J.-L., Rodriguez, S., Hostettmann, K., Hiller, W. Liquid chromatography/ ultra violet/mass spectrometric and liquid chromatography/ nuclear magnetic resonance spectroscopic analysis of crude extracts of gentianaceae species. Phytochem. Anal. 1997, 8, 97-104. [An illustrative example of stopped-flow HPLC-NMR.]
52. Bringmann, G., Wohlfarth, M., Rischer, H., Schlauer, J., Brun, R. Extract screening by HPLC coupled to MS-MS, NMR, and CD: a dimeric and three monomeric naphthylisoquinoline alkaloids from Ancistrocladus griffithii. Phytochemistry 2002, 61, 195-204. [An illustrative example of stopped-flow HPLC-NMR.]
53. * Queiroz, E.F., Wolfender, J.-L., Raelison, G., Hostettmann, K. Determination of the absolute configuration of 6-alkylated α-pyrones from Ravensara crassifolia by LC-NMR. Phytochem. Anal. 2003, 14, 34-39. [An illustrative example of stoppedflow HPLC-NMR.]
54. * Guilet, D., Guntern, A., Ioset, J.-R., Queiroz, E.F., Ndjoko, K., Foggin, C.M., Hostettmann, K. Absolute configuration of a tetrahydrophenanthrene from Heliotropium ovalifolium by LC-NMR of its Mosher esters. J. Nat. Prod. 2003, 66, 17-20. [An illustrative example of stopped-flow HPLC-NMR.]
55. * Seco, J.M., Tseng, L.-H., Godejohann, M., Quinoa, E., Riguera, R. Simultaneous enantioresolution and assignment of absolute configuration of secondary alcohols by directly coupled HPLC-NMR of 9-AMA esters. Tetrahedron Asymm. 2002, 13, 2149-2153. [An illustrative example of stopped-flow HPLC-NMR.]
56. * Gil, A.M., Duarte, I.F., Godejohann, M., Braumann, U., Maraschin, M., Spraul, M. Characterization of the aromatic composition of some liquid foods by nuclear magnetic resonance and mass spectrometric detection. Anal. Chim. Acta 2003, 488, 35-51. [An illustrative example of time-sliced HPLC-NMR.]
57. Corcoran, O., Spraul, M. LC-NMR-MS in drug discovery. Drug Discov. Today 2003, 8, 624-631.
58. * Lommen, A., Godejohann, M., Venema, D.P., Hollman, P.C.H., Spraul, M. Application of directly coupled HPLCNMR-MS to the identification and confirmation of quercetin glycosides and phloretin glycosides in apple peel. Anal. Chem. 2000, 72, 1793-1797. [An illustrative example of loop-collection HPLCNMR.]
59. Tseng, L.H., Braumann, U., Godejohann, M., Lee, S.S., Albert, K. Structure identification of aporphine alkaloids by on-line coupling of HPLC-NMR with loop storage. J. Chin. Chem. Soc. 2000, 47, 1231-1236.
60. deKoning, J.A., Hogenboom, A.C., Lacker, T., Albert, K., Brinkmann, U.A.Th. On-line trace enrichment in hyphenated liquid chromatographynuclear magnetic resonance spectroscopy. J. Chromatogr. A 1998, 813, 55-61.
61. Wilcox, C.D., Phelan, R.M. The use of solid-phase extraction columns to effect simple offline LC/MS, LC/NMR, and LC/FTIR. J. Chromatogr. Sci. 1986, 24, 130-133.
62. Nyberg, N.T., Baumann, H., Kenne, L. Application of solid-phase extraction coupled to an NMR flow-probe in the analysis of HPLC fractions. Magn. Reson. Chem. 2001, 39, 236-240.
63. Nyberg, N.T., Baumann, H., Kenne, L. Solid-phase extraction NMR studies of chromatographic fractions of saponins from Quillaja saponaria. Anal. Chem. 2003, 75, 268-274.
64. Griffiths, L., Horton, R. Optimization of LC-NMR III-Increased signal-to-noise ratio through column trapping. Magn. Reson. Chem. 1998, 36, 104-109. [The work presented represents a milestone in development of the HPLC-SPE-NMR method.]
65. * Exarchou, V., Goodejohann, M., van Beek, T.A., Gerothanassis, I.P., Vervoort, J. LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek oregano. Anal. Chem. 2003, 75, 6288-6294. [An illustrative example of application of the HPLC-SPE-NMR method.]
66. * Wang, C.-Y., Lee, S.-S. Analysis and identification of lignans in Phyllanthus urinaria by HPLC-SPE-NMR. Phytochem. Anal. 2005, 16, 120-126. [An illustrative example of application of the HPLC-SPENMR method.]
67. * Seger, C., Godejohann, M., Tseng, L.H., Spraul, M., Girtler, A., Sturm, S., Stuppner, H. LC-DAD-MS/SPE-NMR hyphenation. A tool for the analysis of pharmaceutically used plant extracts: Identification of isobaric iridoid glycoside regiomers from Harpagophytum procumbens. Anal. Chem. 2005, 77, 878-885. [An illustrative example of application of the HPLC-SPE-NMR method.]
68. * Clarkson, C., Stærk, D., Hansen, S.H., Jaroszewski, J.W. Hyphenation of solidphase extraction with liquid chromatography and nuclear magnetic resonance: application of HPLC-DAD-SPE-NMR to identification of constituents of Kanahia laniflora. Anal. Chem. 2005, 77, 3547-3553. [An illustrative example of application of the HPLC-SPE-NMR method.]
69. * Christophoridou, S., Dais, P., Tseng, L.-H., Spraul, M. Separation and identification of phenolic compounds in olive oil by coupling high-performance liquid chromatography with postcolumn solidphase extraction to nuclear magnetic resonance spectroscopy (LC-SPE-NMR). J. Agric. Food Chem. 2005, 53, 4667-4679. [An illustrative example of application of the HPLC-SPE-NMR method.]
70. * Lambert, M., Stærk, D., Hansen, S.H., Sairafianpour, M., Jaroszewski, J.W. Rapid extract dereplication using HPLC-SPE-NMR: Analysis of isoflavanoids from Smirnowia iranica. J. Nat. Prod. 2005, 68, 1500-1509. [An illustrative example of application of the HPLC-SPE-NMR method.]
71. * Miliauskas, G., van Beek, T.A., de Ward, P., Venskutonis, R.P., Sudhölter, E.J.R. Identification of radical scavenging compounds in Rhaponticum carthamoides by means of LC-DAD-SPE-NMR. J. Nat. Prod. 2005, 68, 168-172. [An illustrative example of application of the HPLC-SPENMR method.]
72. * Pukalskas, A., van Beek, T.A., de Ward, P. Development of a triple hyphenated HPLC-radical scavenging detection-DADSPE-NMR system for the rapid identification of antioxidants in complex plant extracts. J. Chrom. A 2005, 1074, 81-88. [An illustrative example of application of the HPLC-SPE-NMR method.]
73. Lambert, M., Stærk, D., Hansen, S.H., Jaroszewski, J.W. HPLC-SPE-NMR hyphenation in natural products research: optimization of analysis of Croton membranaceus extract. Magn. Reson. Chem. 2005, 43, 771-775.
74. * Sandvoss, M., Bardsley, B., Beck, T.L., Lee-Smith, E., North, S.E., Moore, P.J., Edwards, A.J., Smith, R.J. HPLC-SPENMR in pharmaceutical development: capabilities and applications. Magn. Reson. Chem. 2005, 43, 762-770. [An illustrative example of application of the HPLC-SPE-NMR method.]
75. Hoult, D.I., Richards, R.E. Signal-tonoise ratio of nuclear magnetic resonance experiment. J Magn. Reson. 1976, 24, 71-85. [Seminal account of factors affecting sensitivity of NMR detection.]
76. Styles, P., Soffe, N.F., Scott, C.A., Cragg, D.A., Row, F., White, D.J., White, P.C.J. A high-resolution NMR probe in which the coil and preamplifier are cooled with liquid helium. J. Magn. Reson. 1984, 60, 397-404.
77. Kovacs, H., Moskau, D., Spraul, M. Cryogenically cooled probes-a leap in NMR technology. Progr. Nucl. Magn. Reson. Spectr. 2005, 46, 131-155. [A review of theory and applications of cryogenic probe technology.]
78. Peck, T.L., Magin, R.L., Lauterbur, P.C. Design and analysis of microcoils for NMR spectroscopy. J. Magn. Reson. B 1995, 108, 114-124.
79. Schlotterbeck, G., Ross, A., Hochstrasser, R., Senn, H., Kühn, T., Marek, D., Schett, O. High-resolution capillary tube NMR. A miniaturized 5-μL high-sensitivity TXI probe for mass-limited samples, off-line LC NMR, and HT NMR. Anal. Chem. 2002, 74, 4464-4471.
80. 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. Miniaturization of the structure elucidation of novel natural products-two trace antibacterial acylated caprylic alcohol glycosides from Arctostaphylos pumila. Planta Med. 2005, 71, 176-180.
81. Hu, J.-F., Garo, E., Yoo, H.-D., Cremin, P.A., Zeng, L., Goering, M.G., O'Neil-Johnson, M., Eldridge, G.R. Application of capillary-scale NMR for the structure determination of phytochemicals. Phytochem. Anal. 2005, 16, 127-133.
82. 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. Suaveolindole, a new masslimited antibacterial indolosesquiterpene from Greenwayodendron suaveolens obtained via high-throughput natural products chemistry methods. J. Nat. Prod. 2005, 68, 122-124.
83. Hu, J.-F., Garo, E., Yoo, H.-D., Cremin, P.A., Goering, M.G., O'Neil-Johnson, M., Eldridge, G.R. Cyclolignans from Scyphocephalium ochocoa via highthroughput natural product chemistry methods. Phytochemistry 2005, 66, 1077-1082.
84. * Xiao, H.B., Krucker, M., Putzbach, K., Albert, K. Capillary liquid chromatography-microcoil 1H nuclear magnetic resonance spectroscopy and liquid chromatography-ion trap mass spectrometry for on-line structure elucidation of isoflavones in Radix astragali. J. Chromatogr. A 2005, 1067, 135-143. [An illustrative example of application of capillary HPLC-NMR.]
85. * Putzbach, K., Krucker, M., Grynbaum, M.D., Hentschel, P., Webb, A.G., Albert, K. Hyphenation of capillary highperformance liquid chromatography to microcoil magnetic resonance spectroscopy-determination of various carotenoides in a small-sized spinach samples. J. Pharm. Biomed. Anal. 2005, 38, 910-917. [An illustrative example of application of capillary HPLC-NMR.]
86. Olson, D.L., Peck, T.L., Webb, A.G., Magin, R.L., Sweedler, J.V. Highresolution microcoil 1H NMR for masslimited, nanoliter-volume samples. Science 1995, 270, 1967-1970.
87. Olson, D.L., Norcross, J.A., O'Neil-Johnson, M., Molitor, P.F., Detlefsen, D.J., Wilson, A.G., Peck, T.L. Microflow NMR: concepts and capabilities. Anal. Chem. 2004, 76, 2966-2974.
88. Bailey, N.J.C., Marshall, I.R. Development of ultrahigh-throughput NMR spectroscopic analysis utilizing capillary flow NMR technology. Anal. Chem. 2005, 77, 3947-3953.
89. Jansma, A., Chuan, T., Albrecht, R.W., Olson, D.L., Peck, T.L., Geierstanger, B.H. Automated microflow NMR: routine analysis of five-microliter samples. Anal. Chem. 2005, 77, 6509-6515.
90. Webb, A.G. Nuclear magnetic resonance coupled microseparations. Magn. Reson. Chem. 2005, 43, 688-696.
91. Webb, A.G. Microcoil nuclear magnetic resonance spectroscopy. J. Pharm. Biomed. Anal. 2005, 38, 892-903. [A review of capillary probes with solenoidal microcoils.]
92. Wang, H., Ciobanu, L., Webb, A. Reduced data acquisition time in multi-dimensional NMR spectroscopy using multiplecoil probes. J. Magn. Reson. 2005, 173, 134-139.
93. Wang, H., Ciobanu, L., Edison, A.S., Webb, A.G. An eight-coil high-frequency probehead design for high-throughput nuclear magnetic resonance spectroscopy. J. Magn. Reson. 2004, 170, 206-212.
94. Jayawickrama, D.A., Sweedler, J.V. Dual microcoil NMR probe coupled to cyclic ce for continuous separation and analyte isolation. Anal. Chem. 2004, 76, 4894-4900.
95. Kaerner, A., Smittka, T., Unpublished results.