- Polyphenolic compounds found in foods such as fruits, vegetables, legumes, nuts and grains, are an integral part of the human diet. Within polyphenols, proanthocyanidins are referred to as condensed tannins and encompass procyanidins, propelargonidins and prodelphinidins. Among these, procyanidins are the largest group. Recent interest in these compounds is related to their potential health benefits, most notably their antioxidant activity. However, there is considerable interest from scientists and the public about their other health benefits.
The complex structure of procyanidins makes their analysis a challenging task. Procyanidins are composed of isomeric catechin and epicatechin monomeric units with A-type and B-type linkages. B-type procyanidins contain C4 → C6 or C4 → C8 interflavan bonds between subunits, whereas A-type compounds are composed of the same C4 → C6 or C4 → C8 interflavan bond with an additional C2 → O7 bond. Procyanidins can assume a linear formation mainly containing C4 → C8 B-type linkages or a branched formation consisting of C4 → C6 B-type, A-type or a mix of A- and B- type linkages. These different linkages give rise to different fragmentation patterns during mass spectrometric and tandem mass spectrometric analyses. The most common fragmentation patterns are due to retro-Diels-Alder (RDA), heterocyclic ring fission (HRF), and quinone methide
(QM) formation. Current mass spectrometry-based methods are time-consuming and lack specificity. Electrospray ionization is more commonly used given the high molecular mass of procyanidins and ease of ionization, however, issues arise with procyanidins mixtures. These electrospray mass spectra are dominated by lower molecular mass components; the signal intensity diminishes as polymer chain length increases and the formation of multiply charged polymers complicates precursor ion selection for tandem mass spectrometric analysis. Therefore, other methods of analysis need to be explored since procyanidins exist in mixed concentrations of varying degrees of polymerization in different plants.
Procyanidin-rich dietary supplements are marketed as botanical products, most commonly as pine bark extract, grape seed extract, green tea extract, and cranberry dietary supplements. Cranberry dietary supplements have been marketed to combat urinary tract infections. Due to the bitter taste of cranberry fruit, cranberry dietary supplements have become popular alternatives for the prevention of urinary tract infections. Cranberries contain mainly A-type procyanidins which have been reported to inhibit the cellular adhesion of P-fimbriated uropathogenic Escherichia coli to uroepithelial cells, which is necessary for the development of urinary tract infections. Due to the popularity of cranberry dietary supplements, there was a surge in the demand of these cranberry products which resulted in increased pricing. This propelled economic adulteration such that some manufacturers added lower cost procyanidins extracted from sources other than cranberries. The most common cranberry adulterants were peanut skins, grape seed, mulberry fruit, plum, maritime pine bark, black bean skin, and black rice. These adulterants pose a risk to consumers resulting in the loss of money, null perceived health benefits, and exposure to potential allergens.
This dissertation presents the development of rapid and efficient mass spectrometry-based analyses of complex procyanidins leading to the authentication of cranberry botanical dietary supplements. Three chapters of original research are presented in this dissertation. The first chapter is a review of procyanidins with a focus on mass spectrometry-based methods of analysis.
The second chapter utilizes ion mobility, a separation technique in which ions are separated by their drift time in an inert gas under a weak electric field. Conventional chromatographic separation of procyanidins requires minutes, whereas ion mobility separations occur in just milliseconds. Using traveling wave ion mobility (TWIMS) on a Waters Synapt Q-ToF mass spectrometer, ions experience roll-overs due to an opposing drift gas. These roll-overs help separate compounds by size and charge. Smaller compounds will travel more quickly; additionally, multi-charged compounds will travel more quickly than singly charged species. Procyanidins are oligomeric and polymeric, linear or branched, making them ideal for ion mobility analysis. Ion mobility has the ability to separate procyanidins by degree of polymerization. In addition to speed, time-of-flight mass analyzers provide a large mass range that is suitable for detection of the larger polymeric forms of procyanidins. The method was developed using procyanidin standards and then applied to unknown cranberry fractions. The optimized method demonstrated the rapid separation of procyanidins by varying degree of polymerization as well as varying linkage types. As expected, lower order procyanidins were easily identified by degree of polymerization. Additionally, procyanidins with different linkage types (A-, B- or mixed) were resolved.
The third chapter utilizes matrix-assisted laser/desorption-time-of-flight tandem mass spectrometry (MALDI-ToF/ToF). MALDI-ToF/ToF also avoids the need for time-consuming chromatographic separation while providing rapid analysis. MALDI is an ionization technique which uses laser energy to create ions from large molecules with little fragmentation. The sample is mixed with a matrix, the laser is pulsed, which causes desorption of the sample and matrix as well as ionization by protonation or deprotonation, and the ions are detected in ToF mass spectrometer. The ToF analyzer is suitable for larger molecules such as polymeric procyanidins, additionally, MALDI produces singly charged species which limits interference from multi-charged species and is therefore ideal for complex procyanidins. MALDI-ToF/ToF mass spectra of procyanidins were characterized by the common fragmentation pathways of RDA, HRF, and QM fission. The tandem mass spectra provided structural information about the number of A- and B-type linkages in each compound as well as the location of the linkages leading to the identification of specific procyanidins.
The fourth chapter utilizes negative electrospray ionization and neutral loss scanning on a triple quadrupole tandem mass spectrometer. The tandem mass spectrometry technique of neutral loss scanning facilitates the identification of precursor ions that fragment to eliminate a stable neutral molecule of structural significance. As previously mentioned, procyanidins have three characteristic fragmentation pathways, which may be utilized for their selective detection. Using neutral loss scanning to follow these characteristic neutral losses, a method was developed to test cranberry botanical dietary supplements for adulteration. To validate this method, cranberry dietary supplements labeled as containing 100% Viccinium macrocarpon Aiton (American Cranberry) were purchased as well as with mixed fruit botanicals used as an adulterated reference. The cranberry supplements were confirmed to contain A-type procyanidins whereas the mixed fruit botanicals showed the presence of A- and B-type procyanidins.
The studies described in this dissertation help advance the analysis of complex procyanidins. Procyanidins are found in a variety of plant products and foods making human exposure widespread. The health promoting properties of procyanidins make them attractive to consumers that embrace a healthful lifestyle. The complexity of these compounds makes them difficult to characterize fully. This research will aid in the structural analyses of procyanidins and indirectly advance the study of the therapeutic effects of these powerful phytonutrients.