HPLC is a universal separation technique that is capable of separating both volatiles and non-volatiles without the need for derivatization. We are developing methods that employ both on-line photodiode array (PDA) detection and mass selective detection, HPLC/PDA/MS. This approach also utilizes an ion-trap mass spectrometer that is capable of normal and tandem mass spectrometry. 47,48 Tandem mass spectrometry allows the isolation of compounds in the gas phase followed by controlled fragmentation to yield structural information.49,50 The combination of these technologies, i.e. HPLC/PDA/MS, yields a powerful tool for profiling AND structural determinations.51,52

Figure 5 Three-dimensional display of the photodiode array absorbance data obtained by HPLC/PDA/MS for a M. truncatula extract. The first dimension is HPLC retention time, second is wavelength and third is absorbance. The data can be rapidly previewed for specific absorbance regions characteristic of functional groups.

On-line photodiode array detection is most useful for the analysis of compounds containing chromophores, such as phenolic compounds including flavonoids, isoflavonoids, coumarins, and pterocarpans. An illustrative three-dimensional photodiode array display for a Medicago truncatula phenolic extract is provided (Fig. 5). The three dimensional data consist of UV absorption spectra from 190 to 500 nm for each point along the chromatogram. The data can be rapidly previewed for unique absorption regions correlating to specific compounds or functional groups. Independent chromatograms can also be constructed for each wavelength to increase the selectivity of the data. The UV data are complemented by the mass selective data. Illustrations of both types of data are provided (Fig. 6). The chromatogram is generated from the ion abundances and mass spectra recorded in the negative-ion, electropsray ionization mode. The mass and UV spectra for the peak eluting at approximately 45 minutes are provided in the inserts and identify the eluting compound as medicarpin, known to have a ?max at 287 nm and a molecular weight of 270. A negative-ion is observed for the deprotonated molecule at m/z 269 with a deprotonated dimer ion observed at m/z 539 confirming the molecular weight as 270.

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Figure 6 Multidimensional data obtained by HPLC/PDA/MS analysis of an alfalfa root extract. The HPLC retention time, the UV absorbance spectrum, and the mass spectrum readily identify the peak eluting at 45 minutes as medicarpin, a known phytoalexin in alfalfa.

   The utility of mass selective detection is greatest when analyzing compounds that do not contain chromophores or when structural information is needed for chemical identification. Triterpene saponins contain very weak chromophores and have long been associated with a variety of biological activities including allelopathy,53 poor digestibility in ruminants,54 deterrence to insect foraging,55 and beneficial antifungal properties. 56 Saponins also possess anti-inflammatory, cholesterol lowering, and anticancer properties.57-59 Saponins isolated from the legume Acacia victoriae have been reported to trigger apoptosis in cancer cells.60

   Saponins consist of triterpenoid or steroidal aglycones that are substituted with a varying number of sugar side chains. Unsubstituted, nonpolar aglycones are classified as sapogenins and two representative structures are included (Fig. 7). Because glycoside conjugates are labile and nonvolatile, they must be ionized using a lower energy technique such as electrospray ionization to retain their integrity during mass analysis. We have been profiling saponins in alfalfa (Medicago sativa) and M. truncatula by using HPLC coupled to an ESI ion-trap mass spectrometer to acquire normal and tandem mass spectra during profiling. 61 The mass spectra have also been used for structural characterization of Medicago saponins. A profile of an alfalfa extract is provided (Fig. 7) that illustrates both the enhanced sensitivity and selectivity of mass selective detection compared to UV detection at 206 nm.

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Figure 7 HPLC/PDA/MS data for a saponin extract from alfalfa root. Comparison of the UV chromatogram and the total ion chromatogram (TIC) from the mass data illustrates the increased sensitivity of mass selective detection for saponins that possess only weak chromophores. Mass spectra and aglycone structures of two common saponins found in alfalfa and M. truncatula are provided for a) soysaponin I and b) 3-glucose-medicagenic acid. The increased selectivity of MS is achieved through molecular weight and fragment information.

 HPLC/PDA/MS has also been used to compare the saponin profiles in multiple cultivars of alfalfa and M. truncatula. Comparative profiles are provided (Fig. 8). It is interesting that these closely related legumes yielded different saponin profiles. The saponin profile of M. truncatula is more complex than alfalfa and may provide a richer source for mining putative pharmaceuticals.

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Figure 8 Comparative saponin profiles for two cultivars of alfalfa and one cultivar of M. truncatula obtained by reverse-phase HPLC/PDA/MS using electrospray ionization and an ion trap mass spectrometer. The profiles illustrate the increased complexity of saponins in M. truncatula and offer a richer source for bio-prospecting of natural products.

References