2012 Annual Report
1a.Objectives (from AD-416):
Research will be conducted to demonstrate a generically-applicable method for the quantitative analysis of pyrrolizidine alkaloids and their N-oxides in plant-derived products. Methods for the extraction, concentration, identification and quantitative analysis of the major Symphytum app. pyrrolizidine alkaloids and their N-oxides will be developed and optimized. This project is a continuation and extension of the previous project funded from March 2009 to March 2011.
1b.Approach (from AD-416):
The entire analytical process will be validated for separate Symphytum sp. based matrices according to the AOAC International Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Sypplements and Botanicals. Standard reference materials will be prepared by isolation and purification of the major pyrrolizidine alkaloids from Symphytum sp. Authentication of standards will include description of characteristics such as physico-chemical properties (e.g. melting point, optical rotation and chromatographic profile), the molecular formula as determined using high resolution mass spectrometry, and a complete structure mapping using nuclear magnetic resonance spectroscopy.
The continual use of the HPLC-esiMS and MS/MS method of analysis for the dehydropyrrolizidine (DHPA) alkaloids has resulted in refinements of the basic procedure, including changes in mobile phase and column characteristics that will potentially facilitate the final development of the method for the comfrey DHPAs. The reproducibility of results is also constantly being monitored (including the range of responses for known amounts of internal standard for example) and these data should help provide a basis for determining the reliability parameters of the comfrey DHPA assay.
However, without ample quantities of the standards, any application of the resultant assay is compromised. Therefore, the research has been directed to utilizing available resources to produce the DHPAs on a larger scale. This has resulted in the evolution of a new stage in the “clean-up” of comfrey extracts that involves a partitioning with dilute acid and n-butanol, the latter removing the large amounts of complicating co-extractives from the DHPAs and their N-oxides.
The crude comfrey root DHPA extract consists of four groups of diastereoisomers. Separation of the components within each group has been the major challenge. Fortuitously, other DHPA-related problems have presented the opportunity to experiment with separation approaches without risking the stocks of comfrey root crude extracts while simultaneously allowing a furthering of DHPA-related knowledge in the literature. These include: a collaborative study of Cryptantha crassipes that determined high levels of lycopsamine-N-oxide in the endangered plant from Texas; a study of Crotalaria juncea (sunn hemp) that unequivocally identified a suite of new DHPAs and confirmed general low levels of DHPAs in the seed commercially available for agricultural use; an investigation of a recent cattle poisoning incident in Arizona that potentially involved Amsinckia menziesii (intermedia). Contrary to all literature reference, analysis showed this Amsinckia chemotype to be a very valuable source of lycopsamine-N-oxide. This is currently being extracted to provide the pure lycopsamine for the comfrey study; two Cryptantha sp. collected in the vicinity of the Arizona poisoning incident were analyzed using the HPLC-esi(+)MS approach. One species produces echimidine and related DHPAs, including a potentially toxic DHPA that was unequivocally identified for the first time, and may provide a source for the standards required for the major comfrey species Symphytum uplandicum. The other Cryptantha produces a previously undescribed, and potentially toxic, alkaloid as the major DHPA. Isolation of this DHPA allowed a successful testing of new silica columns that recently became available for the Biotage flash chromatography system and that may assist in the comfrey project. The structure has been unambiguously elucidated using MS and NMR data.