Rapid, Sensitive and Reliable Ricin Identification in Serum Samples Using LC-MS/MS

Abstract

Ricin, a protein derived from the seeds of the castor bean plant (Ricinus communis), is a highly lethal toxin that inhibits protein synthesis, resulting in cell death. The widespread availability of ricin, its ease of extraction and its extreme toxicity make it an ideal agent for bioterrorism and self-poisoning. Thus, a rapid, sensitive and reliable method for ricin identification in clinical samples is required for applying appropriate and timely medical intervention. However, this goal is challenging due to the low predicted toxin concentrations in bio-fluids, accompanied by significantly high matrix interferences. Here we report the applicability of a sensitive, selective, rapid, simple and antibody-independent assay for the identification of ricin in body fluids using mass spectrometry (MS). The assay involves lectin affinity capturing of ricin by easy-to-use commercial lactose-agarose (LA) beads, following by tryptic digestion and selected marker identification using targeted LC-MS/MS (Multiple Reaction Monitoring) analysis. This enables ricin identification down to 5 ng/mL in serum samples in 2.5 h. To validate the assay, twenty-four diverse naive- or ricin-spiked serum samples were evaluated, and both precision and accuracy were determined. A real-life test of the assay was successfully executed in a challenging clinical scenario, where the toxin was identified in an abdominal fluid sample taken 72 h post self-injection of castor beans extraction in an eventual suicide case. This demonstrates both the high sensitivity of this assay and the extended identification time window, compared to similar events that were previously documented. This method developed for ricin identification in clinical samples has the potential to be applied to the identification of other lectin toxins.

Keywords: LC–MS/MS (MRM); clinical samples; identification; lactamyl-agarose; ricin; serum; suicide.

Figure 1

Assay linearity for the three selected tryptic peptides obtained from ricin spiked to ten pooled individuals’ serum samples, to receive final concentrations of 5–500 ng/mL.

Figure 2

Assay validation in serum samples. LC–MS/MS (MRM) chromatograms of different samples: (A) Typical serum sample spiked with ricin (5 ng/mL). (B) Positive control: PBS spiked with ricin (5 ng/mL). (C) Negative control: naive serum. Each chromatogram represents the higher marker’s intensity of MRM transition.

Figure 3

LC–MS/MS (MRM) chromatograms for the identification of ricin in abdominal fluid. The three selected peptides with their specific two MRM transitions for each peptide are presented. For each peptide (bordered by a rectangle), the upper chromatogram is for the first peptide’s transition, and the lower chromatogram is for the second.