Detection and Quantification of Ricin in Beverages Using Isotope Dilution Tandem Mass Spectrometry

Abstract

The toxic plant protein ricin has gained notoriety due to wide availability and potential use as a bioterrorism agent, with particular concern for food supply contamination. We have developed a sensitive and selective mass spectrometry-based method to detect ricin in tap water, 2% milk, apple juice, and orange juice. Ricin added to beverage matrices was extracted using antibody-bound magnetic beads and digested with trypsin. Absolute quantification was performed using isotope dilution mass spectrometry with a linear ion trap operating in product-ion-monitoring mode. The method allows for identification of ricin A chain and B chain and for distinction of ricin from ricin agglutinin within a single analytical run. Ricin-bound beads were also tested for deadenylase activity by incubation with a synthetic ssDNA oligomer. Depurination of the substrate by ricin was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS). This method was used successfully to extract ricin from each beverage matrix. The activity of recovered ricin was assessed, and quantification was achieved, with a limit of detection of 10 fmol/mL (0.64 ng/mL).

Ricin detection: Tracking active toxin

“…detection (Cho et al, 2009), and LC-MS (Bevilacqua et al, 2010;Kalb and Barr, 2009;McGrath et al, 2011). Notably, by complementing ricin-specific sample enrichment steps with RTA enzyme activity assays, ricin could be selectively identified while confirming A-chain activity in parallel (Becher et al, 2007;Bevilacqua et al, 2010;He et al, 2010;May et al, 1989), allowing distinction from nontoxic monomeric type 1 RIP toxins.…”

“…The optimal assay design would have a rapid and efficient enrichment step, an A-chain activity checkpoint, and a selectivity step that can distinguish ricin from other bioactive toxins. Indeed, streamlined assays have been developed for ricin, where ricin is enriched from samples through the use of ricin specific antibodies (Kalb and Barr, 2009;McGrath et al, 2011). However, this format is unable to confirm B-chain lectin functionality and, therefore cannot guarantee the sequestered ricin is capable of penetrating a host cell membrane and exert toxicity.…”

Identification of RIP-II Toxins by Affinity Enrichment, Enzymatic Digestion and LC-MS

“…43 In a forensic application (next section), the quantification method for ricin was validated with matrix-matched calibration standards in order to mimic the sample composition and reduce systematic errors. The accuracy was determined to 105% with a precision of 4% RSD (n = 3).…”

A Glyco-chip for the Detection of Ricin by an Automated Chemiluminescence Read-out System

“…RCA60 and RCA120 can be identified by mass spectrometry (MS). [8][9][10] For this purpose, the toxin and its agglutinin are isolated from the matrix. For tandem-MS methods, the proteins are digested enzymatically.…”