A Importância das Técnicas Acopladas (CL/UV, CL/EM, CL/RMN) para Procura de Princípios Ativos

E. F. Queiroz; K. Hostettmann

doi:10.32712/2446-4775.2006.58

The Importance of Hyphenated Techniques (LV/UV, LC/MS, LC/NMR) in the Search of Active Principles

Vol. 2 No. 03 (2006)
39-53
16/10/2013
01/12/2006
https://doi.org/10.32712/2446-4775.2006.58

E. F. Queiroz
K. Hostettmann

Keywords

Natural Products

Hyphenated Techniques

LC-UV

LC-MS

LCRMN

Abstract

Nature represents an extraordinary reservoir of novel molecules and there is currently a resurgence of interest in natural products as a possible source of new lead compounds for introduction into therapeutical screening programmes. To discover new bioactive natural products, the dereplication of crude extracts performed prior to isolation work is of crucial importance for avoiding the tedious isolation of known constituents. In this respect, chemical screening strategies based on hyphenated techniques such as liquid chromatography-ultraviolet photodiode array detection (LC-UVDAD), liquid chromatography-mass spectrometry (LC-MS), and liquid chromatography-nuclear magnetic resonance (LC-NMR) are more and more extensively used. In the laboratory of Hostettmann’s group, these analytical methods have been fully integrated into the isolation process and are used for the chemical screening of crude plant extracts, in complement with online or at-line bioassays, for rapid localization and identification of new bioactive compounds. In this paper, possibilities and limitations of hyphenated techniques for online natural product identification are discussed. As LC-NMR is playing a key role in this respect, the main part of the paper is dedicated to this technique. In particular, various ways of integrating NMR in the dereplication process are illustrated.

References

AGABABYAN, E.Y.; ARUTYUNYAN, L.S.; MNATSAKANYAN, V.A.; GACH-BAITTS, E.; RADICH, L. Iridoids of Verbascum georgicum. Chemistry of Natural Compounds, v.18, n.4, p.412-417, 1982.
ANDARY, C.; WYLDE, R.; LAFFITE, C.; PRIVAT, G.; WINTERNITZ, F. Structures of verbascoside and orobanchoside, caffeic acid sugar esters from Orobancherapum-genistae. Phytochemistry v. 21, p.1123,-1127, 1982.
ALBERT, K. On-line use of NMR detection in separation chemistry. Journal of Chromatography A, v.703, p.123-147, 1995.
ALBERT, K.; NIEDER, M.; BAYER, E.; SPRAUL, M. Continuous-flow nuclear magnetic resonance. Journal of Chromatography, v. 346, p.17-24, 1985.
ARSLANIAN, R.L.; STERMITZ, F.R. High performace liquid chromatographic analysis of iridoid glycosides from Penstemon rydbergii varieties. Phytochemical Analysis, v.2, p.35, 1991.
ATINDEHOU, K.K.; QUEIROZ, E.F.; TERREAUX, C.; TRAORE, D.; HOSTETTMANN, K. Three new prenylated isoflavonoids from the root bark of Erythrina vogelii. Planta Medica, v. 68, n.2, p.181-182, 2002
BEHNKE, B.; SCHLOTTERBECK, G.; TALLAREK, U.; STROHSCHEIN, S.; TSENG, L.-H.; KELLER, T.; ALBERT, K.; BAYER, E. Capillary HPLC-NMR coupling high-resolution NMR spectroscopy in the nanoliter scale. Analytical Chemistry, v. 68, n.7, p.1110-1115, 1996.
BLAKLEY, C.R.; VESTAL, M.L. Thermospray interface for liquid-chromatography mass-spectrometry. Analytical Chemistry, v.55, p.750-754, 1983.
BRANLEY, P.M. Analysis of carotenoids by high-performance liquid-chromatography and diode-array detection. Phytochemical Analysis, v.3, p.97, 1992.
BRUINS, A.P.; COVEY, T.R.; HENION, J.T. Ion spray interface for combined liquid chromatography/atmospheric pressure ionization mass-spectrometry. Analytical Chemistry, v.59, p.2642-2646, 1987.
COGNE, A.-L.; QUEIROZ, E.F.; WOLFENDER, J.-L.; MARSTON, A.; MAVI, S., HOSTETTMANN, K. On-line identification of unstable catalpol derivatives from
Jamesbrittenia fodina by LC-MS and LC-NMR Phytochemical Analysis, v.14, n.2, 67-73, 2003.
DAHIYA, J.D.; STRANGE, R.N.; BILYARD, K.G.; COOKSEY, C.J.; GARRATT, P.J. 2 Isoprenylated isoflavone phytoalexins from Cajanus cajan. Phytochemistry, v.23, p.871-873, 1984.
DUCREY, B.; WOLFENDER, J.-L.; MARSTON, A.; HOSTETTMANN, K. Analysis of flavonol glycosides of 13 Epilobium species (Onagraceae) by LC-UV and thermospray LC-MS. Phytochemistry, v.38, p.129-137, 1995.
GRÃ„NICHER, F.; CHRISTEN, P.; VUAGNAT, P. Rapid highperformance liquid-chromatographic quantification of valepotriates in hairy root cultures of Valeriana officinalis l var. sambucifolia mikan. Phytochemical Analysis, v.5, p.297, 1994.
HAMBURGER, M.; HOSTETTMANN, K. Bioactivity in plants - the link between phytochemistry and medicine. Phytochemistry, v.30, p.3864-3874, 1991.
HAW, J.F.; GLASS, T.E.; HAUSLER, D.W.; MOTELL, E.; DORN, H.C. Direct coupling of a liquid chromatograph to a continuousflow hydrogen nuclear magnetic-resonance detector for analysis of petroleum and synthetic fuels. Analytical Chemistry, v.52, p.1135, 1980.
HOLT, R.M.; NEWMAN, M.J.; PULLEN, F.S.; RICHARDS, D.S.; SWANSON, A.G. High-Performance liquid chromatographyNMR spectrometry-mass spectrometry: further advances in hyphenated technology. Journal of Mass Spectrometry, v.32, n.1, p.64-70, 1998.
HOSTETTMANN, K.; DOMON, B.; SCHAUFELBERGER, D.; HOSTETTMANN, M. Online high-performance liquidchromatography - ultraviolet visible spectroscopy of phenolic compounds in plant-extracts using post-column derivatization. Journal of Chromatography, v.283, p.137-147, 1984.
HOSTETTMANN, K.; WOLFENDER, J.-L. Application of LC/MS and LC/NMR in the search for new bioactive compounds from plants of the Americas. In Medicinal Plants of the Americas (HOSTETTMANN, K., GUPTA, M.P., MARSTON, M. eds.). Harwood: Amsterdam, 1998.
KINGSTON, D.G.I. High-performance liquid-chromatography of natural-products. Journal of Natural Products, v.42, n.3, p.237-260, 1979.
LINDON, J.C.; NICHOLSON, J.K.; SIDELMANN, U.G.; WILSON, I.D. Directly coupled HPLC-NMR and its application to drug metabolism. Drug Metabolic Review, v.29, n.3, p.705-746, 1997.
MARKHAM, K.R.; MABRY, T.J. Ultraviolet-visible and proton magnetic resonance spectroscopy of flavonoids. In The Flavonoids, Part 1 (Harborne, JB, Mabry, H, eds.), Academic Press, New York, 1975.
MITSCHER, L.A.; DRAKE, S.; GOLLAPUDI, S.R.; OKWUTE, S.K. A modern look at folkloric use of anti-infective agents. Journal of Natural Products, v.50, n.6, p.1025-1040, 1987.
MUELLER-HARVEY, I.; BLACKWELL, P.M.S. An improuved high performace liquid chromatographic post-column derivatization procedure for the UV-VIS spectroscopic characterization of phenolic compounds. Phytochemical Analysis, v.2, p.38, 1991.
MYASE, T.; AKAHORI, C.; KOHSAKA, H.; UENO A. Acylated iridoid glycosides from Buddleja japonica HELMS. Chemical Pharmaceutical Bulletin, v.39, p.2944-2951, 1991.
NYKOLOV, N.; IOSSIFOVA, T.; VASSILEVA, E.; KOSTOVA, I.; STOEV, G. Reverse-phase high-pressure liquidchromatographic analysis of hydroxycoumarins in plant-extracts quantitative determination of hydroxycoumarins in Fraxinus ornus. Phytochemical Analysis, v.4, p.184-185, 1993.
OGG, R.J.; KINGSLEY, P.B.; TAYLOR, J.S. WET, a T-1-insensitive and B-1-insensitive water-suppression method for in-vivo localized H-1 NMR spectroscopy. Journal of Magnetic Resonance B, v.104, p.1-10, 1994.
QUEIROZ, E.F.; WOLFENDER, J.-L.; ATINDEHOU, K.K.; HOSTETTMANN, K. On-line identification of the antifungal constituents of Erythrina vogelii by liquid chromatography with tanden mass spectrometry, ultraviolet absorbance detection and nuclear magnetic resonance spectrometry combined with
liquid chromatographic micro-fractionation. Journal of Chromatography, v.974, p.123-134, 2002a.
QUEIROZ, E.F.; ATINDEHOU, K.; TERREAUX, C.; ANTUS, S.; HOSTETTMANN, K. Prenylated isoflavonoids from the root bark of Erythrina vogelii. Journal of Natural Products, v.65, n.3, p.403-406, 2002b.
SMALLCOMBE, S.H.; PATT, S.L.; KEIFFER, P.A. WET solvent suppression and its application to LC-NMR and high-resolution NMR spectroscopy. Journal of Magnetic Resonance A Series A v.117, p.295-303, 1995.
SPRAUL, M.; HOFMANN, M.; WILSON, D.W.; LENZ, E.; NICHOLSON, J.K.; BALES, J.R. Coupling of hplc with F-19-NMR and H-1-NMR spectroscopy to investigate the human urinary-excretion of flurbiprofen metabolites. Journal of Pharmaceutical Biomedical Analysis, v.11, n.10, p.1009-1115, 1993.
SPRAUL, M.; HOFMANN, M. 750-MHz NMR spectroscopy and food-related LC-NMR applications. Magnetic Resonance in Food Science, v.157, p.77-92, 1995.
STEVENSON, S.; DORN, H.C. C-13 dynamic nuclearpolarization - a detector for continuous-flow, online chromatography. Analytical Chemistry, v.66, n.19, p.2993-2999, 1994.
SWEATMAN, B.C.; FARRANT, R.D.; DANDERSON, P.N.; PHILIPPE, I.; SALMAN, S.R.; NICHLOSON, J. K.; LINDON, J.C. Evaluation of the detection limits of directly coupled 600 MHz, 1 H-13C HPLC-NMR spectroscopy. Journal of Magnetic Resonance Analysis, v.1, p.9-12, 1995
TAHARA, S., IBRAHIM, R. K. Prenylated isoflavonoids - an update. Phytochemistry, v.38, p.1073-1094, 1995.
WALDRON, K.W.; PARR, A.J.; RALPH, J. Cell wall esterified phenolic dimers: Identification and quantification by reverse phase high performance liquid chromatography and diode array detection. Phytochemical Analysis, v.7, n.6, p.305-312, 1996.
WARIDEL, P.; WOLFENDER, J.-L.; NDJOKO, K.; HOBBY, K.R.; MAJOR, H., HOSTETTMANN, K. Evaluation of quadrupole time-of-flight tandem mass spectrometry and iontrap multiple-stage mass spectrometry for the differentiation of C-glycosidic flavonoid isomers. Journal of Chromatography A, v.926, p.29-41, 2001.
WATANABE, N.; NIKI, E.; SHIMIZU, S. An experiment on direct combination of high performance liquid chromatography with FT-NMR (LC-NMR). Jeol News, v.15 A, p.2, 1979.
WHITEHOUSE, R.C.; DREYER, R.N.; YAMASHITA, M.; FENN, J.B. Electrospray interface for liquid chromatographs and mass spectrometers. Analytical Chemistry, v.57, p.675-679, 1985.
WOLFENDER, J.-L.; HOSTETTMANN, K. Liquidchromatographic UV detection and liquid-chromatographic thermospray mass-spectrometric analysis of Chironia
(Gentianaceae) species - a rapid method for the screening of
polyphenols in crude plant-extracts. Journal of Chromatography, v.647, p.191-202, 1993.
WOLFENDER, J.-L.; HOSTETTMANN, K. Applications of liquid chromatography-mass spectrometry to the investigation of medicinal plants. In Phytochemistry of Medicinal Plants. (Arnason, J.T., Mata, R., Romeo, J. eds.). Plenum Press, New York, 1995, pp. 189.
WOLFENDER, J.-L.; RODRIGUEZ, S.; HOSTETTMANN, K.; WAGNER-REDEKER, W. Comparison of liquid chromatography electrospray, atmospheric pressure chemical ionization, thermospray and continuous-flow fast atom bombardment mass spectrometry for the determination of secondary metabolites in crude plant extracts. Journal of Mass Spectrometry and Rapid Communications Mass Spectrometry, v.S35-S46, 1995.
WOLFENDER, J.-L.; RODRIGUEZ, S.; HILLER, W.; HOSTETTMANN, K. LC-UV-MS and LC-NMR analyses of crude extracts of Gentianaceae species. Phytochemical Analysis, v.8, n.3, p.97-104, 1997.
WOLFENDER, J-L.; RODRIGUEZ, S.; HOSTETTMANN, K. LC/MS: a useful tool in the search for new bioactive plant constituents, in Virtual activity, real pharmacology. Differents approaches to the search for bioactive natural compounds, (Verotta, L. ed.). Research Signpost, Trivandrim, India, pp. 137,
YOSHIMURA, I.; YASUHIRO, K.; YOSHIKAZU, Y.; HUNECK, S.; YASUYUKI, Y. Analysis of secondary metabolites from lichen by high-performance liquid-chromatography with a photodiodearray detector. Phytochemical Analysis, v.5, p.197-205, 1994.

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Author(s)

E. F. Queiroz
Aché Laboratórios Farmacêuticos. Departamento de Pesquisa e Desenvolvimento – DMC Rodovia Presidente Dutra Km 222,2, 07034 904 Guarulhos, SP,Brasil
K. Hostettmann
Laboratoire de Pharmacognosie et Phytochimie, École de Pharmacie Genève-Lausanne, Université de Genève, Université de Lausanne CH-1211 Genève 4, Switzerland

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The Importance of Hyphenated Techniques (LV/UV, LC/MS, LC/NMR) in the Search of Active Principles. Rev Fitos [Internet]. 2006 Dec. 1 [cited 2024 Nov. 20];2(03):39-53. Available from: https://revistafitos.far.fiocruz.br/index.php/revista-fitos/article/view/58

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] AGABABYAN, E.Y.; ARUTYUNYAN, L.S.; MNATSAKANYAN, V.A.; GACH-BAITTS, E.; RADICH, L. Iridoids of Verbascum georgicum. Chemistry of Natural Compounds, v.18, n.4, p.412-417, 1982.

[2] ANDARY, C.; WYLDE, R.; LAFFITE, C.; PRIVAT, G.; WINTERNITZ, F. Structures of verbascoside and orobanchoside, caffeic acid sugar esters from Orobancherapum-genistae. Phytochemistry v. 21, p.1123,-1127, 1982.

[3] ALBERT, K. On-line use of NMR detection in separation chemistry. Journal of Chromatography A, v.703, p.123-147, 1995.

[4] ALBERT, K.; NIEDER, M.; BAYER, E.; SPRAUL, M. Continuous-flow nuclear magnetic resonance. Journal of Chromatography, v. 346, p.17-24, 1985.

[5] ARSLANIAN, R.L.; STERMITZ, F.R. High performace liquid chromatographic analysis of iridoid glycosides from Penstemon rydbergii varieties. Phytochemical Analysis, v.2, p.35, 1991.

[6] ATINDEHOU, K.K.; QUEIROZ, E.F.; TERREAUX, C.; TRAORE, D.; HOSTETTMANN, K. Three new prenylated isoflavonoids from the root bark of Erythrina vogelii. Planta Medica, v. 68, n.2, p.181-182, 2002

[7] BEHNKE, B.; SCHLOTTERBECK, G.; TALLAREK, U.; STROHSCHEIN, S.; TSENG, L.-H.; KELLER, T.; ALBERT, K.; BAYER, E. Capillary HPLC-NMR coupling high-resolution NMR spectroscopy in the nanoliter scale. Analytical Chemistry, v. 68, n.7, p.1110-1115, 1996.

[8] BLAKLEY, C.R.; VESTAL, M.L. Thermospray interface for liquid-chromatography mass-spectrometry. Analytical Chemistry, v.55, p.750-754, 1983.

[9] BRANLEY, P.M. Analysis of carotenoids by high-performance liquid-chromatography and diode-array detection. Phytochemical Analysis, v.3, p.97, 1992.

[10] BRUINS, A.P.; COVEY, T.R.; HENION, J.T. Ion spray interface for combined liquid chromatography/atmospheric pressure ionization mass-spectrometry. Analytical Chemistry, v.59, p.2642-2646, 1987.

[11] COGNE, A.-L.; QUEIROZ, E.F.; WOLFENDER, J.-L.; MARSTON, A.; MAVI, S., HOSTETTMANN, K. On-line identification of unstable catalpol derivatives from

[12] Jamesbrittenia fodina by LC-MS and LC-NMR Phytochemical Analysis, v.14, n.2, 67-73, 2003.

[13] DAHIYA, J.D.; STRANGE, R.N.; BILYARD, K.G.; COOKSEY, C.J.; GARRATT, P.J. 2 Isoprenylated isoflavone phytoalexins from Cajanus cajan. Phytochemistry, v.23, p.871-873, 1984.

[14] DUCREY, B.; WOLFENDER, J.-L.; MARSTON, A.; HOSTETTMANN, K. Analysis of flavonol glycosides of 13 Epilobium species (Onagraceae) by LC-UV and thermospray LC-MS. Phytochemistry, v.38, p.129-137, 1995.

[15] GRÃ„NICHER, F.; CHRISTEN, P.; VUAGNAT, P. Rapid highperformance liquid-chromatographic quantification of valepotriates in hairy root cultures of Valeriana officinalis l var. sambucifolia mikan. Phytochemical Analysis, v.5, p.297, 1994.

[16] HAMBURGER, M.; HOSTETTMANN, K. Bioactivity in plants - the link between phytochemistry and medicine. Phytochemistry, v.30, p.3864-3874, 1991.

[17] HAW, J.F.; GLASS, T.E.; HAUSLER, D.W.; MOTELL, E.; DORN, H.C. Direct coupling of a liquid chromatograph to a continuousflow hydrogen nuclear magnetic-resonance detector for analysis of petroleum and synthetic fuels. Analytical Chemistry, v.52, p.1135, 1980.

[18] HOLT, R.M.; NEWMAN, M.J.; PULLEN, F.S.; RICHARDS, D.S.; SWANSON, A.G. High-Performance liquid chromatographyNMR spectrometry-mass spectrometry: further advances in hyphenated technology. Journal of Mass Spectrometry, v.32, n.1, p.64-70, 1998.

[19] HOSTETTMANN, K.; DOMON, B.; SCHAUFELBERGER, D.; HOSTETTMANN, M. Online high-performance liquidchromatography - ultraviolet visible spectroscopy of phenolic compounds in plant-extracts using post-column derivatization. Journal of Chromatography, v.283, p.137-147, 1984.

[20] HOSTETTMANN, K.; WOLFENDER, J.-L. Application of LC/MS and LC/NMR in the search for new bioactive compounds from plants of the Americas. In Medicinal Plants of the Americas (HOSTETTMANN, K., GUPTA, M.P., MARSTON, M. eds.). Harwood: Amsterdam, 1998.

[21] KINGSTON, D.G.I. High-performance liquid-chromatography of natural-products. Journal of Natural Products, v.42, n.3, p.237-260, 1979.

[22] LINDON, J.C.; NICHOLSON, J.K.; SIDELMANN, U.G.; WILSON, I.D. Directly coupled HPLC-NMR and its application to drug metabolism. Drug Metabolic Review, v.29, n.3, p.705-746, 1997.

[23] MARKHAM, K.R.; MABRY, T.J. Ultraviolet-visible and proton magnetic resonance spectroscopy of flavonoids. In The Flavonoids, Part 1 (Harborne, JB, Mabry, H, eds.), Academic Press, New York, 1975.

[24] MITSCHER, L.A.; DRAKE, S.; GOLLAPUDI, S.R.; OKWUTE, S.K. A modern look at folkloric use of anti-infective agents. Journal of Natural Products, v.50, n.6, p.1025-1040, 1987.

[25] MUELLER-HARVEY, I.; BLACKWELL, P.M.S. An improuved high performace liquid chromatographic post-column derivatization procedure for the UV-VIS spectroscopic characterization of phenolic compounds. Phytochemical Analysis, v.2, p.38, 1991.

[26] MYASE, T.; AKAHORI, C.; KOHSAKA, H.; UENO A. Acylated iridoid glycosides from Buddleja japonica HELMS. Chemical Pharmaceutical Bulletin, v.39, p.2944-2951, 1991.

[27] NYKOLOV, N.; IOSSIFOVA, T.; VASSILEVA, E.; KOSTOVA, I.; STOEV, G. Reverse-phase high-pressure liquidchromatographic analysis of hydroxycoumarins in plant-extracts quantitative determination of hydroxycoumarins in Fraxinus ornus. Phytochemical Analysis, v.4, p.184-185, 1993.

[28] OGG, R.J.; KINGSLEY, P.B.; TAYLOR, J.S. WET, a T-1-insensitive and B-1-insensitive water-suppression method for in-vivo localized H-1 NMR spectroscopy. Journal of Magnetic Resonance B, v.104, p.1-10, 1994.

[29] QUEIROZ, E.F.; WOLFENDER, J.-L.; ATINDEHOU, K.K.; HOSTETTMANN, K. On-line identification of the antifungal constituents of Erythrina vogelii by liquid chromatography with tanden mass spectrometry, ultraviolet absorbance detection and nuclear magnetic resonance spectrometry combined with

[30] liquid chromatographic micro-fractionation. Journal of Chromatography, v.974, p.123-134, 2002a.

[31] QUEIROZ, E.F.; ATINDEHOU, K.; TERREAUX, C.; ANTUS, S.; HOSTETTMANN, K. Prenylated isoflavonoids from the root bark of Erythrina vogelii. Journal of Natural Products, v.65, n.3, p.403-406, 2002b.

[32] SMALLCOMBE, S.H.; PATT, S.L.; KEIFFER, P.A. WET solvent suppression and its application to LC-NMR and high-resolution NMR spectroscopy. Journal of Magnetic Resonance A Series A v.117, p.295-303, 1995.

[33] SPRAUL, M.; HOFMANN, M.; WILSON, D.W.; LENZ, E.; NICHOLSON, J.K.; BALES, J.R. Coupling of hplc with F-19-NMR and H-1-NMR spectroscopy to investigate the human urinary-excretion of flurbiprofen metabolites. Journal of Pharmaceutical Biomedical Analysis, v.11, n.10, p.1009-1115, 1993.

[34] SPRAUL, M.; HOFMANN, M. 750-MHz NMR spectroscopy and food-related LC-NMR applications. Magnetic Resonance in Food Science, v.157, p.77-92, 1995.

[35] STEVENSON, S.; DORN, H.C. C-13 dynamic nuclearpolarization - a detector for continuous-flow, online chromatography. Analytical Chemistry, v.66, n.19, p.2993-2999, 1994.

[36] SWEATMAN, B.C.; FARRANT, R.D.; DANDERSON, P.N.; PHILIPPE, I.; SALMAN, S.R.; NICHLOSON, J. K.; LINDON, J.C. Evaluation of the detection limits of directly coupled 600 MHz, 1 H-13C HPLC-NMR spectroscopy. Journal of Magnetic Resonance Analysis, v.1, p.9-12, 1995

[37] TAHARA, S., IBRAHIM, R. K. Prenylated isoflavonoids - an update. Phytochemistry, v.38, p.1073-1094, 1995.

[38] WALDRON, K.W.; PARR, A.J.; RALPH, J. Cell wall esterified phenolic dimers: Identification and quantification by reverse phase high performance liquid chromatography and diode array detection. Phytochemical Analysis, v.7, n.6, p.305-312, 1996.

[39] WARIDEL, P.; WOLFENDER, J.-L.; NDJOKO, K.; HOBBY, K.R.; MAJOR, H., HOSTETTMANN, K. Evaluation of quadrupole time-of-flight tandem mass spectrometry and iontrap multiple-stage mass spectrometry for the differentiation of C-glycosidic flavonoid isomers. Journal of Chromatography A, v.926, p.29-41, 2001.

[40] WATANABE, N.; NIKI, E.; SHIMIZU, S. An experiment on direct combination of high performance liquid chromatography with FT-NMR (LC-NMR). Jeol News, v.15 A, p.2, 1979.

[41] WHITEHOUSE, R.C.; DREYER, R.N.; YAMASHITA, M.; FENN, J.B. Electrospray interface for liquid chromatographs and mass spectrometers. Analytical Chemistry, v.57, p.675-679, 1985.

[42] WOLFENDER, J.-L.; HOSTETTMANN, K. Liquidchromatographic UV detection and liquid-chromatographic thermospray mass-spectrometric analysis of Chironia

[43] (Gentianaceae) species - a rapid method for the screening of

[44] polyphenols in crude plant-extracts. Journal of Chromatography, v.647, p.191-202, 1993.

[45] WOLFENDER, J.-L.; HOSTETTMANN, K. Applications of liquid chromatography-mass spectrometry to the investigation of medicinal plants. In Phytochemistry of Medicinal Plants. (Arnason, J.T., Mata, R., Romeo, J. eds.). Plenum Press, New York, 1995, pp. 189.

[46] WOLFENDER, J.-L.; RODRIGUEZ, S.; HOSTETTMANN, K.; WAGNER-REDEKER, W. Comparison of liquid chromatography electrospray, atmospheric pressure chemical ionization, thermospray and continuous-flow fast atom bombardment mass spectrometry for the determination of secondary metabolites in crude plant extracts. Journal of Mass Spectrometry and Rapid Communications Mass Spectrometry, v.S35-S46, 1995.

[47] WOLFENDER, J.-L.; RODRIGUEZ, S.; HILLER, W.; HOSTETTMANN, K. LC-UV-MS and LC-NMR analyses of crude extracts of Gentianaceae species. Phytochemical Analysis, v.8, n.3, p.97-104, 1997.

[48] WOLFENDER, J-L.; RODRIGUEZ, S.; HOSTETTMANN, K. LC/MS: a useful tool in the search for new bioactive plant constituents, in Virtual activity, real pharmacology. Differents approaches to the search for bioactive natural compounds, (Verotta, L. ed.). Research Signpost, Trivandrim, India, pp. 137,

[50] YOSHIMURA, I.; YASUHIRO, K.; YOSHIKAZU, Y.; HUNECK, S.; YASUYUKI, Y. Analysis of secondary metabolites from lichen by high-performance liquid-chromatography with a photodiodearray detector. Phytochemical Analysis, v.5, p.197-205, 1994.