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.
Key words
natural products - plant extracts - HPLC-UV-ELSD - MPLC-UV-ELSD - UHPLC-TOF-MS - gradient
transfer - isolation
