Method validation of a survey of thevetia cardiac glycosides in serum samples.

A sensitive and specific liquid chromatography tandem mass spectrometry (HPLC-ESI(+)-MS/MS) procedure was developed and validated for the identification and quantification of thevetin B and further cardiac glycosides in human serum. The seeds of Yellow Oleander (Thevetia peruviana) contain cardiac glycosides that can cause serious intoxication. A mixture of six thevetia glycosides was extracted from these seeds and characterized. Thevetin B, isolated and efficiently purified from that mixture, is the main component and can be used as evidence. Solid phase extraction (SPE) proved to be an effective sample preparation method. Digoxin-d3 was used as the internal standard. Although ion suppression occurs, the limit of detection (LOD) is 0.27 ng/ml serum for thevetin B. Recovery is higher than 94%, and accuracy and precision were proficient. Method refinement was carried out with regard to developing a general screening method for cardiac glycosides. The assay is linear over the range of 0.5-8 ng/ml serum. Finally, the method was applied to a case of thevetia seed ingestion.

Cardiac glycosides from Yellow Oleander (Thevetia peruviana) seeds.

Thevetia cardiac glycosides can lead to intoxication, thus they are important indicators for forensic and pharmacologic surveys. Six thevetia cardiac glycosides, including two with unknown structures, were isolated from the seeds of the Yellow Oleander (Thevetia peruviana (Pers.) K. Shum., Apocynaceae). LC-ESI⁺-MS(/MS) analysis under high-resolution conditions used as a qualitative survey of the primary glycosides did not lead to fragmentation of the aglycones. Acid hydrolysis of the polar and non-volatile thevetia glycosides under severe conditions yielded the aglycones of the thevetia glycosides and made them amenable to GC-MS analysis. Comparison of mass spectral fragmentation patterns of the aglycones, as well as high-resolution mass spectrometric and NMR data of four of the primary thevetia glycosides including the two unknowns, revealed the structures of the complete set of six thevetia glycosides. The identified compounds are termed thevetin C and acetylthevetin C and differ by an 18,20-oxido-20,22-dihydro functionality from thevetin B and acetylthevetin B, respectively. The absence of an unsaturated lactone ring renders the glycosides cardio-inactive. The procedures developed in this study and the sets of analytical data obtained will be useful for screening and structure assessment of other, particularly polar, cardiac glycosides.