[en] The HPLC separation of six alkaloids extracted from Strychnos usambarensis leaves has been
developed and optimized by means of a powerful methodology for modelling chromatographic
responses, based on three steps, i.e. design of experiments (DoE), independent component analysis (ICA) and design space (DS). This study was the first application of a new optimization strategy to a complex natural matrix. The compounds separated are the isomers isostrychnopentamine and strychnopentamine, 10-hydroxyusambarine and 11-hydroxyusambarine, also strychnophylline and strychnofoline. Three LC parameters have been optimized using a multifactorial design comprising 29 experiments that includes 2 center point replicates. The parameters were the percentage of organic modifiers used at the beginning of a gradient profile which consisted in different proportions of methanol (MeOH) and acetonitrile (MeCN), the gradient time to reach 70% of organic modifiers starting from the initial percentage and the percentage of MeCN found in the mobile phase. Subsequent to the experimental design application, predictive multilinear models were developed and used in order to provide optimal analytical conditions. The optimum assay conditions were: methanol/acetonitrile-sodium pentane
sulfonate (pH 2.2; 7.5 mM) (33.4:66.6, v/v) at a mobile phase flow rate of 1mL/min during a 40.6
minutes gradient time. The initial organic phase contained 3.7% MeCN and 96.3% MeOH. The method showed good agreement between the experimental data and predictive value throughout the studied parameters space. Improvement of the analysis time and optimized separation for the compounds of interest was possible due to the original and powerful tools applied. Finally, this study permitted the acquisition of isomers profiles allowing the identification of the optimal collecting period of Strychnos usambarensis.
Angenot L. De l'existence en Afrique centrale d'un poison de flèche curarisant, issu du Strychnos usambarensis. Ann. Pharm. Fr. 1971, 29:353-364. http://hdl.handle.net/2268/28092.
Angenot L., Dubois M., Ginion C., van Dorsser W., Dresse A. Chemical structure and pharmacological (curarizing) properties of various indole alkaloids extracted from an African Strychnos. Arch. Int. Pharmacodyn. Ther. 1975, 215:246-258. http://hdl.handle.net/2268/14380.
Wright C.W., Bray D.H., O'Neill M.J., Warhurst D.C., Phillipson J.D., Quetin-Leclercq J., Angenot L. Antiamoebic and antiplasmodial activities of alkaloids isolated from Strychnos usambarensi. Planta Med. 1991, 57:337-340. http://hdl.handle.net/2268/14427.
Bassleer R., Depauw-Gillet M.C., Massart B., Marnette J.M., Wiliquet P., Caprasse M., Angenot L. Effets de trois alcaloïdes extraits du Strychnos usambarensis sur des cellules cancéreuses en culture. Planta Med. 1982, 45:123-126. http://hdl.handle.net/2268/37269.
Tits M., Desaive C., Marnette J.M., Bassleer R., Angenot L. Antimitotic activity of strychnopentamine, a bisindolic alkaloid. J. Ethnopharmacol. 1984, 12:287-292. http://hdl.handle.net/2268/19636.
Leclercq J., Gillet M.C., Bassleer R., Angenot L. Screening of cytotoxic activities of Strychnos alkaloids (methods and results). J. Ethnopharmacol. 1986, 15:305-316. http://hdl.handle.net/2268/37313.
Bonjean K.A., Gillet M.C., Quentin-Leclercq J., Angenot L., Bassleer R. In vitro cytotoxic activity of two potential anticancer drugs isolated from Strychnos: strychnopentamine and usambarensine. J. Anticancer Res. 1996, 16:1129-1137. http://hdl.handle.net/2268/39255.
Quetin-Leclercq J., Bouzahzah B., Pons A., Greimers R., Angenot L., Bassleer R., Barhason H. Strychnopentamine, a potential anticancer agent. Planta Med. 1993, 59:59-62. http://hdl.handle.net/2268/37332.
Frédérich M., Tits M., Goffin E., Philippe G., Grellier D., De Mol P., Hayette M.P., Angenot L. In vitro and in vivo antimalarial properties of isostrychnopentamine, an indolomonoterpenic alkaloid from Strychnos usambarensis. Planta Med. 2004, 70:520-525. http://hdl.handle.net/2268/14042.
Frédérich M., Bentires-Alj M., Tits M., Angenot L., Greimers R., Gielen J., Bours V., Merville M.P. Isostrychnopentamine, an indolomonoterpenic alkaloid from Strychnos usambarensis, induces cell cycle arrest and apoptosis in human colon cancer cells. J. Pharmacol. Exp. Ther. 2003, 304:1103-1110. http://hdl.handle.net/2268/10834.
Balde E.H.S., Mégalizzi V., Cao M., Angenot L., Kiss R., Van Damme M., Frédérich M. Isostrychnopentamine, an indolomonoterpenic alkaloid from Strychnos usambarensis, with potential anti-tumor activity apoptosis-resistant cancer cells. Int. J. Oncol. 2010, 36:961-965. http://hdl.handle.net/2268/2494.
Gadi Biala R., Tits M., Wauters J.N., Angenot L. A new HPLC method for the assay of alkaloids in Strychnos nux-vomica and Strychnos ignatii. Fitoterapia 1996, 62:163-165. http://hdl.handle.net/2268/29768.
Frédérich M., Tits M., Angenot L. Qualitative and quantitative evaluation of bisindole usambarane alkaloids in Strychnos usambarensis roots by high performance liquid chromatography-diode-array. Phytochem. Anal. 1998, 9:63-66. http://hdl.handle.net/2268/10832.
Dewé W., Marini R.D., Chiap P., Hubert Ph., Crommen J., Boulanger B. Development of response models for optimising HPLC methods. Chemometr. Intell. Lab. Syst. 2004, 74:263-268. http://hdl.handle.net/2268/6036.
Jiju A. Design of Experiments for Engineers and Scientists 2003, Elsevier Science & Technology Books, pp. 6-73.
Box G.E., Hunter W.G., Hunter J.S. Statistics for Experimenters: Design, Innovation, and Discovery 2005, Wiley. 2nd ed.
Lebrun P., Govaerts B., Debrus B., Ceccato A., Caliaro G., Hubert Ph., Boulanger B. Development of a new predictive modelling technique to find with confidence equivalence zone and design space of chromatographic analytical methods. Chemometr. Intell. Lab. Syst. 2008, 91.1:4-16. http://hdl.handle.net/2268/1640.
Debrus B., Lebrun P., Ceccato A., Caliaro G., Rozet E., Nistor I., Oprean R., Rupérez F.J., Barbas C., Boulanger B., Hubert P. Application of new methodologies based on design of experiments, independent component analysis and design space for robust optimization in liquid chromatography. Anal. Chim. Acta 2011, 33-42. http://hdl.handle.net/2268/88084.
Debrus B., Lebrun P., Rozet E., Nistor I., Ceccato A., Caliaro G., Oprean R., Boulanger B., Hubert P. Nouvelle méthodologie pour le développement automatisé de méthodes analytiques en chromatographie liquide pour l'analyse de mélanges de composés inconnus. Spectra Anal. 2009, 268:28-33. http://hdl.handle.net/2268/26406.
Debrus B., Lebrun P., Ceccato A., Caliaro G., Govaerts B., Olsen B.A., Rozet E., Boulanger B., Hubert Ph. A new statistical method for the automated detection of peaks in UV-DAD chromatograms of a sample mixture. Talanta 2009, 79:77-85. http://hdl.handle.net/2268/13236.
LoBrutto R., Jones A., Kazakevich Y.V., McNair H.M. Effect of the eluent pH and acidic modifiers in high-performance liquid chromatography retention of basic analytes. J. Chromatogr. A 2001, 913:173-187.
Kazakevich Y.V., LoBrutto R., Vivilecchia R. Reversed-phase high-performance liquid chromatography behaviour of chaotropic counteranions. J. Chromatogr. A 2005, 1064:9-18.
Flieger J. The effect of chaotropic mobile phase additives on the separation of selected alkaloids in reversed-phase high-performance liquid chromatography. J. Chromatogr. A 2006, 1113:37-44.
Flieger J. Effect of mobile phase composition on the retention of selected alkaloids in reversed-phase liquid chromatography with chaotropic salts. J. Chromatogr. A 2007, 1175:207-216.
Myers R.H., Montgomery D.C., Anderson-Cook C.M. Response Surface Methodology: Process and Product Optimization Using Designed Experiments 2009, Wiley. 3rd ed.
P. Lebrun, B. Boulanger, P. Lambert, Use of Bayesian multivariate prediction models to optimize chromatographic methods, Oral communication, Bayes 2010, Braine l'Alleud, Belgium. http://hdl.handle.net/2268/39052.
P. Lebrun, B. Boulanger, Expected design space: a Bayesian perspective based on modelling, prediction and multi-criteria decision method, Oral communication, NCB 09, Boston, USA. http://hdl.handle.net/2268/39033.
Vanbel P.F., Tilquin B.L., Schoenmakers P.J. Criteria for optimizing the separation of target analytes in complex chromatograms. Chemometr. Intell. Lab. Syst. 1996, 35:67-86.
Hyvärinen A., Oja E. Independent component analysis: algorithms and applications. Neural Netw. 2000, 13:411-430.
Wang G., Ding Q., Hou Z. Independent component analysis and its applications in signal processing for analytical chemistry. Trends Analyt. Chem. 2008, 27:368-376.
R Development Core Team R: A Language and Environment for Statistical Computing 2010.
European Medicines Agency ICH Q8(R2), Guidance on Pharmaceutical Development 2009.