Analytical Method Development for Ricin Analysis in Environmental and Clinical Samples Based on Lactamyl Agarose Beads Capture and LC-MS/MS(MRM)

Liron feldberg, Analytical chemistry, IIBR, Ness Ziona, ישראל (liron.feldberg@gmail.com)
Ofir Schuster, Analytical Chemistry, Iibr, Ness Ziona, �שר��
Eytan Elhanany, Analytical Chemistry, Iibr, Ness Ziona, �שר��
Orly Levy, Analytical Chemistry, Iibr, Ness Ziona, �שר��
Shmuel Yitzhaki, Analytical Chemistry, Iibr, Ness Ziona, �שר��
Sigalit Gura, Analytical Chemistry, Iibr, Ness Ziona, �שר��

Ricin, a glycosylated proteinous potent toxin which is produced by castor bean plant, Ricinus communis, is considered a likely agent for bioterrorism as a result of its widespread availability, ease of extraction from the plant seeds and high lethality. It belongs to type 2 ribosome-inactivating proteins (RIP-II toxins) which inhibit protein synthesis and cause respiratory failure and death. Hence, rapid, specific, simple and sensitive analytical methods are needed for its unambiguous identification. Most of the current detection methods for ricin are based on immunoaffinity assays. These methods suffer from specific interruptions to ricin analysis, such as the presence of beans, which leads to false positive results. Here we present a new method, antibodies independent, that identify rational selected markers of the ricin protein sequence, that may become applicable both for environmental matrices and clinical fluids. Relying on its lectin properties, the method developed herein is based on carbohydrate binding specificity of the toxin by lactamyl agarose beads, followed by a tryptic digest procedure and LC-MS/MS(MRM) analysis, monitoring the four selected peptides. The use of these beads for chemical adsorption of ricin, enables to decrease the background noise derived from the matrix on one side, and has the potential for multiplex assay of lectins identification (such as RIP-II toxins), on the other. This became possible with the high selectivity and sensitivity enabled by LC-MS/MS(MRM) analysis of specific marker peptides.

In this work we demonstrate the analytical method performance in ricin identification at the presence of various lectin interferences at strict conditions of high concentrations, such as abrin (one of the RIP-II family) or proteins originate from Bean and Peanut. The analytical method was further validated at different environmental soil and serum samples, both spiked with ricin. This rapid, sensitive and specific assay was applied in a real case scenario, for identification of the toxin in a stomach fluid sampled from a women that committed suicide with self- extract injection of castor seeds.