Application of an integrated LC-UV-MS-NMR platform to the identification of secondary metabolites from cell cultures: benzophenanthridine alkaloids from elicited Eschscholzia californica (california poppy) cell cultures

Abstract

Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for obtaining natural products of medical relevance. Cultures can be optimized for high yields of desired metabolites using rapid profiling assays such as HPLC. We describe an approach to establishing a rapid assay for profiling cell culture expression systems using a novel microscale LC-UV-MS-NMR platform, designed to acquire both MS and NMR each at their optimal sensitivity, by using nanosplitter MS from 4 mm analytical HPLC columns, and offline microdroplet NMR. The approach is demonstrated in the analysis of elicited Eschscholzia californica cell cultures induced with purified yeast extract to produce benzophenanthridine alkaloids. Preliminary HPLC-UV provides an overview of the changes in the production of alkaloids with time after elicitation. At the time point corresponding to the production of the most alkaloids, the integrated LC-MS-microcoil NMR platform is used for structural identification of extracted alkaloids. Eight benzophenanthridine alkaloids were identified at the sub-microgram level. This paper demonstrates the utility of the nanosplitter LC-MS/microdroplet NMR platform when establishing cell culture expression systems.

The integration of LC‐MS and NMR for the analysis of low molecular weight trace analytes in complex matrices

“…The use of long columns, however, leads to long analysis times, and requires a longer time for column equilibration between injections. For LC‐MS‐NMR, the most commonly used columns are 15‐25 cm long . The column internal diameter determines the amount (in mass) of material that can be injected on column, thus larger diameter columns allow for the injection of more material on column.…”

“…Segmented flow sample loading (Figure B), whereby a sample is carried as a droplet in an immiscible fluid (like a droplet of oil in water), facilitates the loading of a concentrated sample into a microcoil probe through large dead volumes. This avoids dispersion and confines all sample into the microcoil probe observe volume, in‐turn increasing sensitivity for NMR analysis …”

“…This avoids dispersion and confines all sample into the microcoil probe observe volume, in-turn increasing sensitivity for NMR analysis. 5,43,89,90 Most applications using microcryoprobes have been conducted An alternative method for restricting samples into the coil region of a standard size NMR probe (5 or 10 mm probe) uses a microscale assembly known as a Shigemi tube. 93 Figures 14B and 14C).…”

Enrichment and analysis of quaternary alkaloids from Zanthoxylum simulans using weak cation exchange solid‐phase extraction coupled with LC–MS

“…At this point, the WCX material, with the advantage of easy to be regenerated, might be applied to enrich and obtain higher content of QAs. WCX chromatography has played an important role in the field of separation and purification of samples, such as drug, urine and plasma samples . With WCX, the effectiveness of separation and purification was markedly improved.…”

“…In addition, with the fast development of separation and analytical technologies, some instruments with high separation efficiency and sensitivity have been applied to the alkaloids analysis from the Zanthoxylum genus, including but not limited to high-speed counter current chromatography (HSCCC), capillary electrophoresis (CE), reversed phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography combined with electrospray ionisation tandem mass spectrometry and nuclear magnetic resonance (LC-ESI/ MS/NMR). [18][19][20][21][22] However, the aforementioned instruments could not improve the analytical results significantly without proper prepurification due to the complexity and high polarity of QAs. Therefore, we aimed to develop a cost-effective and environment-friendly method to enrich and purify QAs from Z. simulans.…”