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Hyphenated Techniques and Their Applications in Natural Products Analysis

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 864))

Abstract

A technique where a separation technique is coupled with an online spectroscopic detection technology is known as hyphenated technique, e.g., GC–MS, LC–PDA, LC–MS, LC–FTIR, LC–NMR, LC–NMR–MS, and CE–MS. Recent advances in hyphenated analytical techniques have remarkably widened their applications to the analysis of complex biomaterials, especially natural products. This chapter focuses on the applications of hyphenated techniques to pre-isolation and isolation of natural products, dereplication, online partial identification of compounds, chemotaxonomic studies, chemical finger-printing, quality control of herbal products, and metabolomic studies, and presents specific examples. However, a particular emphasis has been given on the hyphenated techniques that involve an LC as the separation tool.

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References

  1. Wilson ID, Brinkman UATh (2003) Hyphenation and hypernation: the practice and prospects of multiple hyphenation. J Chromatogr A 1000:325–356

    Article  PubMed  CAS  Google Scholar 

  2. Exarchou V, Fiamegos YC, van Beek TA, Nanos C, Vervoort J (2006) Hyphenated chromatographic techniques for the rapid screening and identification of antioxidants in methanolic extracts of pharmaceutically used plants. J Chromatogr A 1112:293–302

    Article  PubMed  CAS  Google Scholar 

  3. Wolfender JL, Ndjoko K, Hostettmann K (1998) LC/NMR in natural products chemistry. Curr Org Chem 2:575–596

    CAS  Google Scholar 

  4. Wolfender JL, Rodriguez S, Hostettmann K (1998) Liquid chromatography coupled to mass spectrometry and nuclear magnetic resonance spectroscopy for the screening of plant constituents. J Chromatogr A 794:299–316

    Article  CAS  Google Scholar 

  5. Huber L, George SA (1993) Diode array detection in HPLC. Mercel-Dekker, New York

    Google Scholar 

  6. Niessen WMA, Tinke AP (1995) Liquid chromatography-mass spectrometry, general principles and instrumentation. J Chromatogr A 703:37–57

    Article  CAS  Google Scholar 

  7. Niessen WMA (1999) Liquid chromatography–mass spectrometry, 2nd edn. Dekker, New York

    Google Scholar 

  8. Albert K (1995) On-line use of NMR detection in separation chemistry. J Chromatogr A 703:123–147

    Article  CAS  Google Scholar 

  9. Lindon JC, Nicholson JK, Sidelmann UG, Wilson ID (1997) Directly coupled HPLC–NMR and its application to drug metabolism. Drug Metab Rev 29:707–746

    Article  Google Scholar 

  10. Sudmeier JL, Gunther UL, Albert K, Bachovchin WW (1996) Sensitivity onizationn in continuous-flow FTNMR. J Magn Reson A 118:145–156

    Article  CAS  Google Scholar 

  11. Wolfender JL, Abe F, Nagao T, Okabe H, Yamauchi T, Hostettmann K (1995) Liquid chromatography combined with thermospray and continuous-flow fast atom bombardment mass spectrometry of glycosides in crude plant extract. J Chromatogr A 712:155–168

    Article  PubMed  CAS  Google Scholar 

  12. Bringmann G, Messer K, Wohlfarth M, Kraus J, Dumbuya K, Rückert M (1999) 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 71:2678–2686

    Article  CAS  Google Scholar 

  13. Albert K (2002) On-line LC–NMR and related techniques. Wiley, London

    Book  Google Scholar 

  14. Jinno K (2001) Basics and applications of hyphenated-detection system in HPLC: part III – hyphenated techniques in HPLC). Pharm Stage 1:110–131

    Google Scholar 

  15. Jinno K (2001) Basics and applications of hyphenated-detection system in HPLC: part II – detection systems in HPLC. Pharm Stage 1:74–80

    Google Scholar 

  16. Jinno K (2001) Basics and applications of hyphenated-detection system in HPLC: part I – basics and applications in HPLC. Pharm Stage 1:81–94

    Google Scholar 

  17. Elipe MVS (2003) Advantages and disadvantages of nuclear magnetic resonance spectroscopy as a hyphenated technique. Anal Chim Acta 497:1–25

    Article  Google Scholar 

  18. Gohlke R, McLafferty FW (1993) Early gas chromatography/mass spectrometry. J Am Soc Mass Spectrosc 4:367–371

    Article  CAS  Google Scholar 

  19. Jinno K (2001) Infrared detection. In: Cazes J (ed) Encyclopedia of chromatography. Marcel Dekker, New York, NY, USA

    Google Scholar 

  20. Jinno K, Fujimoto C, Hirata Y (1982) An interface for the combination of micro high-performance liquid-chromatography and infrared spectrometry. Appl Spectrosc 36:67–69

    Article  CAS  Google Scholar 

  21. Bourne S, Haefner AM, Norton KL, Grifiths PR (1990) Performance-characteristics of a real-time direct deposition gas-chromatography Fourier-transform infrared spectrometry system. Anal Chem 62:2448–2452

    Article  CAS  Google Scholar 

  22. Kuligowski J, Quintas G, Garrigues S, Lendl B, de la Guardia M (2010) Recent advances in on-line liquid chromatography – infrared spectrometry (LC-IR). Trac-Trends Anal Chem 29:544–552

    Article  CAS  Google Scholar 

  23. Herderich M, Richling E, Roscher R, Schneider C, Schwab W, Humpf HU, Schreier P (1997) Application of atmospheric pressure onization HPLC–MS–MS for the analysis of natural products. Chromatographia 45:127–132

    Article  CAS  Google Scholar 

  24. McMaster MC (2005) LC-MS: a practical user’s guide. John Wiley & Sons, New Jersey

    Book  Google Scholar 

  25. Exarchou V, Krucker M, van Beek TA, Vervoort J, Gerothanassis IP, Albert K (2005) LC–NMR coupling tenchnology: recent advancements and applications in natural products analysis. Magn Reson Chem 43:681–687

    Article  PubMed  CAS  Google Scholar 

  26. Iwasa K, Takahashi T, Nishiyama Y, Moriyasu M, Sugiura M, Takeuchi A, Tode C, Tokuda H, Takeda K (2008) Online structural elucidation of alkaloids and other constituents in crude extracts and cultured cells of Nandina domestica by combination of LC–MS/MS, LC–NMR, and LC–CD analyses. J Nat Prod 71:1376–1385

    Article  PubMed  CAS  Google Scholar 

  27. Logar JK, Malej A, Franko M (2003) Hyphenated high performance liquid chromatography–thermal lens spectrometry technique as a tool for investigations of xanthophyll cycle pigments in different taxonomic groups of marine phytoplankton. Rev Sci Instrum 74:776–778

    Article  CAS  Google Scholar 

  28. Dunayevskiy YM, Vouros P, Winter EA, Shipps GW, Carell T (1996) Application of capillary electrophoresis–electrospray ionisation spectrometry in the determination of molecular diversity. Proc Natl Acad Sci 93:6152–6157

    Article  PubMed  CAS  Google Scholar 

  29. Bernet P, Blaser D, Berger S, Schar M (2004) Development of a robust capillary electrophoresis–mass spectrometer interface with a floating sheath liquid feed. Chimica 58:196–199

    CAS  Google Scholar 

  30. Navas MJ, Jimenez AM (2003) Thermal lens spectroscopy as analytical tool. Crit Rev Anal Chem 33:77–88

    Article  CAS  Google Scholar 

  31. Wilson ID, Brinkman U, Th A (2007) Hype and hypernation: multiple hyphenation of column liquid chromatography and spectroscopy. Trends Anal Chem 26:847–854

    Article  CAS  Google Scholar 

  32. Louden D, Handley A, Taylor S, Lenz E, Miller S, Wilson ID, Sage A, Lafont R (2001) Spectroscopic characterisation and identification of ecdysteroids using high-performance liquid chromatography combined with on-line UV-diode array, FT-infrared and 1  H-nuclear magnetic resonance spectroscopy and time of flight mass spectrometry. J Chromatogr 910:237–246

    Article  CAS  Google Scholar 

  33. Sandvoss M, Weltring A, Preiss A, Levsen K, Wuensch G (2001) Combination of matrix solid-phase dispersion extraction and direct on-line liquid chromatography-nuclear magnetic resonance spectroscopy-tandem mass spectrometry as a new efficient approach for the rapid determination of natural products: application to the total asterosaponin fraction of the starfish Asterias rubens. J Chromatogr A 917:75–86

    Article  PubMed  CAS  Google Scholar 

  34. Sandvoss M, Pham LH, Levsen K, Preiss A, Mugge C, Wuensch G (2000) Isolation and structure elucidation of steroid oligoglycosides from the starfish Asterias rubens by means of direct on-line LC–NMR–MS hyphenation and one- and two-dimensional NMR investigations. Eur J Org Chem 7:1253–1262

    Article  Google Scholar 

  35. Bailey NJC, Stanley PD, Hadfield ST, Lindon JC, Nicholson JK (2000) Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants. Rapid Commun Mass Spectrom 24:679–684

    Article  Google Scholar 

  36. He XE (2000) On-line identification of phytochemical constituents in botanical extracts by combined high performance liquid chromatographic–diode array detection–mass spectrometric techniques. J Chromatogr A 880:203–232

    Article  PubMed  CAS  Google Scholar 

  37. Staerk D, Kesting JR, Sairafianpour M, Witt M, Asili J, Emami SA, Jaroszewski JW (2009) Accelerated dereplication of crude extracts using HPLC–PDA–MS–SPE–NMR: quinoline alkaloids of Halophyllum acutifolium. Phyto-chemistry 70:1055–1061

    Article  PubMed  CAS  Google Scholar 

  38. Kite GC, Veitch NC, Grayer RJ, Simmonds MSJ (2003) The use of hyphenated techniques in comparative phytochemical studies of legumes. Biochem Syst Ecol 31:813–843

    Article  CAS  Google Scholar 

  39. Marin-Loaiza JC, Ernst L, Beuerle T, Theuring C, Cespedes CL, Hartman T (2008) Pyrolizidine alkaloids of the endemic Mexican genus Pittocaulon and assignment of stereoisomeric 1,2-saturated necine bases. Phytochemistry 69:154–167

    Article  PubMed  CAS  Google Scholar 

  40. El-Bazaoui A, Bellimam MA, Soulaymani A (2011) Nine new tropane alkaloids from Datura stramonium, identified by GC/MS. Fitoterapia 82:193–197

    Article  PubMed  CAS  Google Scholar 

  41. Berkov S, Zayed R, Doncheva T (2006) Alkaloid patterns in some varieties of Datura stromonium. Fitoterapia 77:179–182

    Article  PubMed  CAS  Google Scholar 

  42. Iwasa K, Kuribayashi A, Sugiura M, Moriyasu M, Lee D-U, Wiegrebe W (2003) LC–NMR and LC–MS analysis of 2,3,10,11-oxygenated protoberberine metabolites in Corydalis cell cultures. Phytochemistry 64:1229–1238

    Article  PubMed  CAS  Google Scholar 

  43. Zhou J-L, Li P, Li H-J, Jiang Y, Ren M-T, Liu Y (2008) Development and validation of a liquid chromatography/electrospray ionization time-of-flight mass spectrometry method for relative and absolute quantification of steroidal alkaloids in Fritillaria species. J Chromatogr A 1177:126–137

    Article  PubMed  CAS  Google Scholar 

  44. Luterotti S, Franko M, Bicanic D (1999) Ultrasensitive determination of β-carotene in fish oil based supplementary drugs by HPLC–TLS. J Pharm Biomed Anal 21:901–909

    Article  PubMed  CAS  Google Scholar 

  45. Dugo P, Mondello L, Dugo L, Stancanelli R, Dugo G (2000) LC–MS for the identification of oxygen heterocyclic compounds in citrus essential oils. J Pharm Biomed Anal 24:147–154

    Article  PubMed  CAS  Google Scholar 

  46. Su J, Zhang C, Zhang W, Shen Y-H, Li H-L, Liu R-H, Zhang X, Hu X-J, Zhang W-D (2009) Qualitative and quantitative determination of the major coumarins in Zushima by high performance liquid chromatography with diode array detector and mass spectrometry. J Chromatogr A 1216:2111–2117

    Article  PubMed  CAS  Google Scholar 

  47. Louden D, Handley A, Taylor S, Lenz E, Miller S, Wilson ID, Sage A, Lafont R (2001) Spectro­scopic characterisation and identification of ecdysteroids using high-performance liquid chromatography combined with on-line UV-diode array, FT-infrared and 1H-nuclear magnetic resonance spectroscopy and time of flight mass spectrometry. J Chromatogr A 910:237–246

    Article  PubMed  CAS  Google Scholar 

  48. Kumazawa S, Hamasaka T, Nakayama T (2004) Antioxidant activity of propolis of various geographical origins. Food Chem 84:329–339

    Article  CAS  Google Scholar 

  49. Klejdus B, Vitamvásová-Štěrbová D, Kuban V (2001) Identification of isoflavone conjugates in red clover (Trifolium pratense) by liquid chromatography–mass spectrometry after two-dimensional solid-phase extraction. Anal Chim Acta 450:81–97

    Article  CAS  Google Scholar 

  50. Chen Y, Li Z, Xue D, Qi L (1987) Determination of volatile constituents of Chinese medicinal herbs by direct vaporization capillary gas-chromatography mass-spectrometry. Anal Chem 59:744–748

    Article  PubMed  CAS  Google Scholar 

  51. Delazar A, Reid RG, Sarker SD (2004) GC–MS analysis of essential oil of the oleoresin from Pistacia atlantica var mutica. Chem Nat Compd 40:24–27

    Article  CAS  Google Scholar 

  52. Joo E, Dewulf J, Demarcke M, Amelynck C, Schoon N, Muller J-F, Simpraga M, Steppe K, van Langenhove H (2010) Quantification of interferences in PTR–MS measurements of monoterpene emissions from Fagus sylvatica L. using simultaneous TD–GC–MS measurements. Int J Mass Spec 291:90–95

    Article  CAS  Google Scholar 

  53. Ma W-G, Fuzzati N, Wolfender J-L, Hostettmann K, Yang C (1994) Rhodenthoside A, a new type of acylated secoiridoid glycoside from Gentiana rhodentha. Helv Chim Acta 77:1660–1671

    Article  CAS  Google Scholar 

  54. Hostettmann K, Wolfender JL (2001) Application of liquid chromatography/UV/MS and liquid chromatography/NMR for the on-line identification of plant metabolites. In: Tringali C (ed) Bioactive compounds from natural sources: isolation, characterisation and biological properties. Taylor and Francis, London, pp 33–68

    Google Scholar 

  55. Sturm S, Stuppner H (2001) Analysis of iridoid glycosides from Picrorhiza kurroa by capillary electrophoresis and high performance liquid chromatography–mass spectrometry. Chromatographia 53:612–618

    Article  CAS  Google Scholar 

  56. Song Y, Li S-L, Wu M-H, Li H-J, Li P (2006) Qualitative and quantitative analysis of iridoid glycosides in the flower buds of Lonicera species by capillary high performance liquid chromatography coupled with mass spectrometric detector. Anal Chim Acta 564:211–218

    Article  CAS  Google Scholar 

  57. Jia Q (2003) Generating and screening a natural product library for cyclooxygenase and lipoxygenase dual inhibitors. In: Atta-ur-Rahman (ed) Studies in natural products chemistry, vol 29. Elsevier, pp 643–718

    Google Scholar 

  58. Colquhoun JA, Zulu J, Goodfellow M, Horikoshi K, Ward AC, Bull AT (2000) Rapid characterisation of deep-sea actinomycetes for biotechnology screening programmes. Antonie van Leeuwenhoek 77:359–367

    Article  PubMed  CAS  Google Scholar 

  59. Ramakrishna NVS, Nadkarni SR, Bhat RG, Naker SD, Kumar EKSV, Lal B (1999) Screening of natural product extracts for antibacterial activity: early identification and elimination of known compounds by dereplication. Ind J Chem 38B:1384–1387

    CAS  Google Scholar 

  60. Dinan L (2005) Dereplication and partial identification of compounds. In: Sarker SD, Latif Z, Gray AI (eds) Natural products isolation, 2nd edn. Humana Press, New Jersey

    Google Scholar 

  61. Perez-Lopez J-L, Theron R, del Olmo E, Diaz D (2007) NAPROC-13: a database for the dereplication of natural product mixtures in bioassay-guided protocols. Bioinformatics Appl Note 23:3256–3257

    Article  Google Scholar 

  62. Alali FQ, Tawaha K (2009) Dereplication of bioactive constituents of the genus Hypericum using LC–(+,−)-ESI–MS and LC–PDA techniques: Hypericum triquterifolium as a case study. Saudi Pharm J 17:269–274

    Article  Google Scholar 

  63. Xie P-S, Yan Y-Z, Guo B-L, Lam CWK, Yu Q-X (2010) Chemical pattern-aided classification to simplify the intricacy of morphological taxonomy of Epimedium species using chromatographic fingerprinting. J Pharm Biomed Anal 52:452–460

    Article  PubMed  CAS  Google Scholar 

  64. Rossi D, Guerrini A, Maietti S, Bruni R, Paganetto G, Poli F, Scalvenzi L, Radice M, Saro K, Sacchetti G (2011) Chemical fingerprinting and bioactivity of Amazonian Ecuador Croton lechleri Müll. Arg. (Euphorbiaceae) stem bark essential oil: a new functional food ingredient? Food Chem 126:837–848

    Article  CAS  Google Scholar 

  65. Kamenik Z, Hadacek F, Mareckova M, Ulanova D, Kopecky J, Chobot V, Plhackova K, Olsovska J (2010) Ultra-high-performance-liquid chromatography fingerprinting method for chemical screening of metabolites in cultivation broth. J Chromatogr A 1217:8016–8025

    Article  PubMed  CAS  Google Scholar 

  66. Montoro P, Maldini M, Piacente S, Macchia M, Pizza C (2010) Metabolite fingerprinting of Camptotheca acuminata and the HPLC–ESIMS/MS analysis of camptothecin and related alkaloids. J Pharm Biomed Anal 51:405–415

    Article  PubMed  CAS  Google Scholar 

  67. Cai Z, Lee FSC, Wang XR, Yu WJ (2002) A capsule review of recent studies on the application of mass spectrometry in the analysis of Chinese medicinal herbs. J Mass Spectrom 37:1013–1024

    Article  PubMed  CAS  Google Scholar 

  68. Schaneberg BT, Crockett S, Bedir E, Khan IA (2003) The role of chemical fingerprinting: application to Ephedra. Phytochemistry 62:911–918

    Article  PubMed  CAS  Google Scholar 

  69. Ducrey B, Wolfender JL, Marston A, Hostettmann K (1995) Analysis of flavonol glycosides of thirteen Epilobium species (Onagraceae) by LC–UV and thermospray LC–MS. Phytochemistry 38:129–137

    Article  CAS  Google Scholar 

  70. Wolfender J-L, Rodriguez S, Hostettmann K, Hiller W (1997) Liquid chromatography/ultraviolet/mass spectrometric and liquid chromatography/nuclear magnetic resonance spectroscopic analysis of crude extracts of Gentianaceae species. Phytochem Anal 8:97–104

    Article  CAS  Google Scholar 

  71. Grevenstuk T, van der Hooft JJJ, Vervoort J, de Waard P, Romano A (2009) Iridoid and caffeoyl phenylethanoid glycosides of the endangered carnivorous plant Pinguicula lusitanica L. (Lentibulariaceae). Biochem Syst Ecol 37:285–289

    Article  CAS  Google Scholar 

  72. Nishijima M, Araki-Sakai M, Sano H (1997) Identification of isoprenoid quinones by frit-FAB liquid chromatography–mass spectrometry for the chemotaxonomy of microorganisms. J Microbiol Methods 28:113–122

    Article  CAS  Google Scholar 

  73. Ott K-H, Aranibar N, Singh B, Stockton GW (2003) Metabolomics classifies pathways affected by bioactive compounds. Artificial neural network classification of NMR spectra of plant extracts. Phytochemistry 62:971–985

    Article  PubMed  CAS  Google Scholar 

  74. Yamazaki M, Nakajima J-C, Yamanashi M, Sugiyama M, Makita Y, Springob K, Awazuhara M, Saito K (2003) Metabolomics and differential gene expression in anthocyanin chemo-varietal forms of Perilla fructescens. Phytochemistry 62:987–995

    Article  PubMed  CAS  Google Scholar 

  75. Lan K, Zhang Y, Yang J, Xu L (2010) Simple quality assessment approach for herbal extracts using high performance liquid chromatography-UV based metabolomics platform. J Chromatogr A 1217:1414–1418

    Article  PubMed  CAS  Google Scholar 

  76. Shuman JL, Cortes DF, Armenta JM, Pokrzywa RM, Mendes P, Shulaev V (2011) Plant metabolomics by GC–MS and differential analysis. Methods Mol Biol 678:229–246

    Article  PubMed  CAS  Google Scholar 

  77. Huhman DV, Sumner LW (2002) Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer. Phytochemistry 59:347–360

    Article  PubMed  CAS  Google Scholar 

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Sarker, S.D., Nahar, L. (2012). Hyphenated Techniques and Their Applications in Natural Products Analysis. In: Sarker, S., Nahar, L. (eds) Natural Products Isolation. Methods in Molecular Biology, vol 864. Humana Press. https://doi.org/10.1007/978-1-61779-624-1_12

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