Planta Med 2004; 70(8): 764-770
DOI: 10.1055/s-2004-827209
Original Paper
Analytical Methods
© Georg Thieme Verlag KG Stuttgart · New York

Development and Validation of a High Performance Liquid Chromatographic Method for Quantitative Determination of Aporphine Alkaloids from Different Samples of Cassytha filiformis

Caroline Stévigny1 , Marie-Caroline Wautier1 , Jean-Louis Habib Jiwan2 , Patrice Chiap3 , Philippe Hubert3 , Joëlle Quetin-Leclercq1
  • 1Laboratoire de Pharmacognosie, Unité d’Analyse Chimique et Physico-Chimique des Médicaments, Université Catholique de Louvain, UCL 72.30-CHAM, Bruxelles, Belgium
  • 2Laboratoire de Spectrométrie de Masse, Université Catholique de Louvain, Faculté des Sciences, Département de Chimie, Louvain-la-Neuve, Belgium
  • 3Laboratoire d’Analyse des Médicaments, Université de Liège, Département de Pharmacie, CHU, B36, Liège, Belgium
Further Information

Publication History

Received: January 22, 2004

Accepted: May 8, 2004

Publication Date:
24 August 2004 (online)

Abstract

A sensitive and accurate procedure based on an alkaloid extraction coupled to an HPLC-UV-MS determination has been developed for the separation and quantification of the major aporphines in Cassytha filiformis. The extraction step and the liquid chromatography conditions were optimized in order to improve the selectivity of the method. The HPLC mobile phase consisted of a mixture of water containing 10 mM ammonium acetate adjusted to pH 3 with acetic acid-acetonitrile (90 : 10, v/v) (A) and acetonitrile (B) used in a gradient mode (0 to 40 %). The stationary phase was an RP-select B (5 μm) column. The method was completely validated using cassythine, one of the major aporphines in our samples, as reference standard and successfully applied to the determination of these pharmacologically interesting aporphines in seven different batches of C. filiformis. The detection and quantitation limits of cassythine were found to be 13 and 20 μg/mL, respectively. The results showed variations in the total alkaloid content in samples from 0.11 to 0.43 %.

References

  • 1 Cava M P, Rao K V, Douglas B, Weisbach A. The alkaloids of Cassytha americana (C. filiformis L.)  J Org Chem. 1968;  33 2443-6
  • 2 Johns S R, Lamberton J A. Cassytha alkaloids I. New aporphine alkaloids from Cassytha filiformis L.  Aust J Chem. 1966;  19 297-302
  • 3 Chang F R, Chao Y C, Teng C M, Wu Y C. Chemical constituents from Cassytha filiformis II.  J Nat Prod. 1998;  61 863-6
  • 4 Stévigny C, Block S, De Pauw-Gillet M C, de Hoffmann E, Llabres G, Adjakidjé V. et al . Cytotoxic aporphine alkaloids from Cassytha filiformis .  Planta Med. 2002;  68 1042-4
  • 5 Wu Y C, Chang F R, Chao Y C, Teng C M. Antiplatelet and vasorelaxing actions of aporphinoids from Cassytha filiformis .  Phytother Res. 1998;  12 S39-41
  • 6 Neuwinger H D. African traditional medicine: a dictionary of plant use and applications. Stuttgart; Medpharm 2000: pp 99-100
  • 7 Hoet S, Stévigny C, Block S, Opperdoes O, Colson P, Baldeyrou B. et al . Alkaloids from Cassytha filiformis and related aporphines: antitrypanosomal activity, cytotoxicity, and interaction with DNA and topoisomerases.  Planta Med. 2004;  70 407-13
  • 8 Pietta P, Mauri P, Manera E, Ceva P. Determination of isoquinoline alkaloids from Peumus boldus by high performance liquid chromatography.  J Chromatogr. 1988;  457 442-5
  • 9 Betts T J. Chromatographic evaluation of boldine and associated alkaloids in Boldo.  J Chromatogr. 1990;  511 373-8
  • 10 Miraldi E, Ferri S. Quantitative determination of boldine of various percentages from leaves of Peumus boldus Molina.  Rivista Italiana EPPOS. 1996;  7(20) 21-5
  • 11 European Pharmacopeia. 4th Edition Strasbourg; Council of Europe 2002: pp 813-4
  • 12 Sun S W, Lee S S, Huang H M. Determination of lauraceous aporphine alkaloids by high-performance liquid chromatography.  J Pharm Biomed An. 1996;  14 1383-7
  • 13 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 1231-6
  • 14 Stévigny C, Habib Jiwan J L, Rozenberg R, de Hoffmann E, Quetin-Leclercq J. Key fragmentation patterns of aporphine alkaloids by direct inlet ESI multistage mass spectrometry.  Rapid Comm Mass Spectrom. 2004;  18 523-8
  • 15 Kartal M, Altun M L, Kurucu S. HPLC method for the analysis of harmol, harmalol, harmine and harmaline in the seeds of Peganum harmala L.  J Pharm Biomed Anal. 2003;  31 263-9
  • 16 Hubert P, Chiap P, Crommen J, Boulanger B, Chapuzet E, Mercier N. et al . The SFSTP guide on the validation of chromatographic methods for drug bioanalysis: from the Washington Conference to the laboratory.  Anal Chim Acta. 1999;  391 135-48
  • 17 Hubert P, Nguyen-huu J J, Boulanger B, Chapuzet E, Chiap P, Cohen N. et al . Validation of quantitative analytical procedure, Harmonization of approaches.  STP Pharma Pratiques. 2003;  13 101-38
  • 18 Miller J C, Miller J N. Statistics for Analytical Chemistry. 5th edition New York; Ellis Horwood 2000: pp 96-100
  • 19 U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER). Guidance for industry: Bioanalytical Method Validation, May 2001. 

Prof. J. Quetin-Leclercq

Laboratoire de Pharmacognosie

Unité CHAM

UCL 72.30

Avenue E. Mounier 72

1200 Brussels

Belgium

Fax: +32-2-764-72-53

Email: leclercq@cham.ucl.ac.be