Procyanidins are polyphenols abundant in dietary fruits, vegetables, nuts, legumes, and grains with a variety of chemopreventive biological effects. Rapid structure determination of these compounds is needed, notably for the more complex polymeric procyanidins. We review the recent developments in the structure elucidation of procyanidins with a focus on mass spectrometric approaches, especially liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization (MALDI) MS/MS.

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Although procyanidins constitute a unique class of polymeric plant secondary metabolites with a variety of biological properties including potent antioxidant activity, structure determination has been challenging and structures of many complex procyanidins remain uncertain. To expedite the characterization of procyanidins, negative ion matrix-assisted laser desorption ionization high-energy collision-induced dissociation tandem time-of-flight (MALDI-ToF/ToF) mass spectra of 20 isolated procyanidins containing catechin and epicatechin subunits with degrees of polymerization up to five were obtained and evaluated. Structurally significant fragmentation pathways of singly charged, deprotonated molecules were identified representing quinone methide, heterocyclic ring fission and retro-Diels-Alder fragmentation. The interpretation of the tandem mass spectra for sequencing A-type, B-type, mixed-type, linear, and branched procyanidins is explained using specific examples of each.

To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts.

The course of alkaloid accumulation and laticifer cell appearance was compared in germinating P. bracteatum seedlings. Seedlings of various ages (0-14 days old) were analyzed for their dopamine, thebaine, morphinan alkaloid immunoreactivity, and benzophenanthridine alkaloid levels. Simultaneous electron microscopic studies revealed that seedlings were devoid of laticifer initials until day 3, where-upon their numbers increased with time. The appearance of appreciable amounts of thebaine only occurred after day 4 of germination. Conversely, dopamine was rapidly formed at the onset of germination and reached millimolar concentrations well before laticifer cells were detected. Benzophenanthridine alkaloid levels remained fairly constant over the period analyzed. These results support the theory that the presence of laticifer cells is necessary for the accumulation of morphinan but neither benzophenanthridine alkaloids nor their mutual precursor, dopamine.

To expedite the identification of active natural products in complex mixtures such as botanical extracts, a Magnetic Microbead Affinity Selection Screening (MagMASS) procedure was developed. This technique utilizes target proteins immobilized on magnetic beads for rapid bioaffinity isolation of ligands from complex mixtures. A MagMASS method was developed and validated for 15-lipoxygenase. As a proof of concept, several North American prairie plants used medicinally by Native Americans were extracted with MeOH and screened. A hit from an extract of Proserpinaca palustris, also known as mermaid weed, was flagged for further characterization using high-resolution tandem mass spectrometry, dereplication, and identification using XCMS online. Through the application of high-resolution product ion tandem mass spectrometry, comparison with natural product databases and confirmation using standards, the hit was identified as quercitrin, which is a known inhibitor of 15-lipoxygenase. The overall workflow of MagMASS is faster and more amendable to automation than alternative methods designed for screening botanical extracts or complex mixtures of combinatorial libraries.

Rationale Electrospray mass spectrometry methods for the analysis of phosphatidylcholines (PCs) routinely include ammonium acetate or ammonium formate in the mobile phase. In an effort to justify and optimize the use of these additives, we investigated possible functions of ammonium compounds in the ionization of PCs. Methods Because PCs contain a quaternary amine, the role of ammonium in neutralizing the negatively charged phosphate group was investigated by using deuterated ammonium acetate, adjusting the pH, varying the organic solvent composition, and by comparing the additives ammonium acetate, ammonium formate and ammonium bicarbonate. Seven PC standards were measured ranging from lyso 1-palmitoyl-sn-glycero-3-phosphocholine to 1,2-dieicosapentaenoyl-sn-glycero-3-phosphocholine as well as a mixture of PCs in a krill oil dietary supplement. Results Under all conditions tested, aqueous acetonitrile provided more abundant formation of protonated PCs than did aqueous methanol. Regardless of the mobile phase composition and electrospray ion source parameters, no [M+NH4]+ ions were detected. Adding deuterated ammonium acetate to the mobile phase failed to form deuterated PCs, indicating that ammonium is not the source of the proton that neutralizes the phosphate negative charge. Instead, water was the source of the proton as deuterated water resulted in the formation of [M+D]+ ions. Addition of organic acids, ammonium formate, ammonium acetate, or ammonium bicarbonate to the mobile phase did not enhance and in most cases suppressed PC ionization. Conclusions Ammonium compounds and organic acids can suppress ionization of PCs when using an aqueous acetonitrile mobile phase during electrospray.

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