Planta Med 2015; 81(17): 1636-1643
DOI: 10.1055/s-0035-1545912
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Rational and Efficient Preparative Isolation of Natural Products by MPLC-UV-ELSD based on HPLC to MPLC Gradient Transfer

Soura Challal
1   School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, Geneva, Switzerland
,
Emerson Ferreira Queiroz
1   School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, Geneva, Switzerland
,
Benjamin Debrus
1   School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, Geneva, Switzerland
,
Werner Kloeti
1   School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, Geneva, Switzerland
,
Davy Guillarme
1   School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, Geneva, Switzerland
,
Mahabir Prashad Gupta
2   Center for Pharmacognostic Research on Panamanian Flora-CIFLORPAN, University of Panama, Panama, Republic of Panama
,
Jean-Luc Wolfender
1   School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, Geneva, Switzerland
› Author Affiliations
Further Information

Publication History

received 23 October 2014
revised 11 February 2015

accepted 04 March 2015

Publication Date:
29 April 2015 (online)

Abstract

In natural product research, the isolation of biomarkers or bioactive compounds from complex natural extracts represents an essential step for de novo identification and bioactivity assessment. When pure natural products have to be obtained in milligram quantities, the chromatographic steps are generally labourious and time-consuming. In this respect, an efficient method has been developed for the reversed-phase gradient transfer from high-performance liquid chromatography to medium-performance liquid chromatography for the isolation of pure natural products at the level of tens of milligrams from complex crude natural extracts. The proposed method provides a rational way to predict retention behaviour and resolution at the analytical scale prior to medium-performance liquid chromatography, and guarantees similar performances at both analytical and preparative scales. The optimisation of the high-performance liquid chromatography separation and system characterisation allows for the prediction of the gradient at the medium-performance liquid chromatography scale by using identical stationary phase chemistries. The samples were introduced in medium-performance liquid chromatography using a pressure-resistant aluminium dry load cell especially designed for this study to allow high sample loading while maintaining a maximum achievable flow rate for the separation. The method has been validated with a mixture of eight natural product standards. Ultraviolet and evaporative light scattering detections were used in parallel for a comprehensive monitoring. In addition, post-chromatographic mass spectrometry detection was provided by high-throughput ultrahigh-performance liquid chromatography time-of-flight mass spectrometry analyses of all fractions. The processing of all liquid chromatography-mass spectrometry data in the form of an medium-performance liquid chromatography x ultra high-performance liquid chromatography time-of-flight mass spectrometry matrix enabled an efficient localisation of the compounds of interest in the generated fractions. The methodology was successfully applied for the separation of three different plant extracts that contain many diverse secondary metabolites. The advantages and limitations of this approach and the theoretical chromatographic background that rules such as liquid chromatography gradient transfer are presented from a practical viewpoint.

Supporting Information

 
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