Schinus terebinthifolius scale-up countercurrent chromatography (Part I): High performance countercurrent chromatography fractionation of triterpene acids with off-line detection using atmospheric pressure chemical ionization mass spectrometry

• Published in: Journal of Chromatography A 2015-04, Vol.1389, p.39-48
• Main Authors: Vieira, Mariana Neves, Costa, Fernanda das Neves, Leitão, Gilda Guimarães, Garrard, Ian, Hewitson, Peter, Ignatova, Svetlana, Winterhalter, Peter, Jerz, Gerold
• Format: Article
• Language: English
• Subjects: Atmospheric pressure; Barometric pressure; CCC off-line APCI-MS/MS profiling; Chromatography; Consumption; Fractionation; High performance countercurrent chromatography; Ionization; Mathematical analysis; Method optimization; Scale-up; Schinus terebinthifolius; Solvents; Triterpene acids
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2015.02.005

•A methodology for S. terebinthifolius berries extract fractionation was proposed.•Pure masticadienonic and 3β-masticadienolic acids were directly isolated by HPCCC.•Method was optimized to achieve max. throughput with min. solvent/time consumption.•Oleanonic and moronic acids were identified for the first time in the species.•Reconstituted APCI-MS-chromatograms were generated to monitor HPCCC separations. ‘Countercurrent chromatography’ (CCC) is an ideal technique for the recovery, purification and isolation of bioactive natural products, due to the liquid nature of the stationary phase, process predictability and the possibility of scale-up from analytical to preparative scale. In this work, a method developed for the fractionation of Schinus terebinthifolius Raddi berries dichloromethane extract was thoroughly optimized to achieve maximal throughput with minimal solvent and time consumption per gram of processed crude extract, using analytical, semi-preparative and preparative ‘high performance countercurrent chromatography’ (HPCCC) instruments. The method using the biphasic solvent system composed of n-heptane–ethyl acetate–methanol–water (6:1:6:1, v/v/v/v) was volumetrically scaled up to increase sample throughput up to 120 times, while maintaining separation efficiency and time. As a fast and specific detection alternative, the fractions collected from the CCC-separations were injected to an ‘atmospheric pressure chemical ionization mass-spectrometer’ (APCI-MS/MS) and reconstituted molecular weight MS-chromatograms of the APCI-ionizable compounds from S. terebinthifolius were obtained. This procedure led to the direct isolation of tirucallane type triterpenes such as masticadienonic and 3β-masticadienolic acids. Also oleanonic and moronic acids have been identified for the first time in the species. In summary, this approach can be used for other CCC scale-up processes, enabling MS-target-guided isolation procedures.