**1. Introduction**

Ricin is a highly toxic protein which can be extracted from the castor bean seeds (*Ricinus communis* L.). This plant is present in all Brazilian regions and explored commercially for its oil, which is mainly used for the production of lubricants, fuel and drugs. Currently, Brazil is the fourth world producer of castor bean oil, just behind India, China and Mozambique [1,2]. The production of 1.0 ton of oil generates around 1.2 ton of residue, known as castor cake [3]. The literature reports different values for the ricin content in the castor cake, varying between 0.04% and 0.08% (w/w), depending on the

cultivars, the extraction method, and the analysis [4–6]. Castor cake is an excellent source of nutrients for cattle; however, its content of ricin can intoxicate the animals. In addition, the disposal of this residue in the environment represents a risk for the population. The detoxification methods proposed so far for the castor cake are expensive, time and energy demanding, and do not guarantee the total destruction of ricin without formation of other toxic products. The analyses are usually based on oral toxicity and other experiments with animals that can be influenced by several factors like species, age, and feeding time. Spectrometric techniques for identification and quantification of the products formed have rarely been used for these studies [7,8].

Ricin has toxicity similar to the neurotoxic agen<sup>t</sup> sarin and can be easily extracted from the castor bean (*R. communis* L.) seeds as a fine white powder, water soluble, and stable at a large range of pH. For this reason, it is considered a chemical/biological warfare agen<sup>t</sup> scheduled by both the chemical weapons convention (CWC) [9], and the biological weapons convention (BWC) [10]. It can be disseminated in the air as fine particles with a diameter smaller than 5 microns or used to contaminate water supplies or agricultural products. This turns ricin into a perfect agen<sup>t</sup> for terrorist attacks and a matter of big concern for national authorities worldwide [11–14].

The structure of the ricin molecule is made up of two di fferent chains, named RTA and RTB, connected by a disulfide bond. RTA is an N-glycosidase containing 267 amino acids arranged in eight α-helices and eight β-strands, distributed in three structural domains, forming a "U" shaped cleft containing the protein active site. RTB is a lecithin composed of 262 amino acids, containing neither α-helices nor β-strands [12,15,16]. Due to its mechanism of action in the organism, and for being a heterodimer, ricin is classified as a ribosome inactivating protein (RIP) of type II [16–18]. RTB is responsible for the binding of ricin to the terminal galactose residues of the glycolipids and glycoproteins present on the surface of eukaryotic cells [12]. This enables the formation of a vesicle surrounding the toxin, which guides it into the inner part of the cell through endocytosis. Once inside the endosome, many ricin molecules are transported back to the outside of the cell or for the lysosomes, where they are degraded. However, some of them manage to reach the Golgi complex, following in retrograde movement, until the endoplasmic reticulum, where their disulfide bonds are cleaved, splitting RTA and RTB. After, RTA is transferred to the cytosol where it reacts specifically with the ribosomal RNA (rRNA) 28S of the ribosomal subunit 60S, provoking the hydrolysis of the N-glycoside bond of the adenine residue at position 4324 (A4324) [13,19–21].

The current decontamination process of people exposed to ricin consists only on the removal of clothes, followed by washing the skin with running water [11]. In cases of ingestion, the patient should be immediately submitted to gastrointestinal lavage [22]. There is no specific antidote for poisoning with ricin yet, neither a commercial vaccine [22,23]. Many works have been performed in the last decade towards the development of a vaccine, including tests with humans. Results have been promising, however, no final product has been approved ye<sup>t</sup> [22–26].

The most common methods used for detection of ricin are based on enzyme-linked immunosorbent assay, like the ELISA method, or in bioassays where the inactivation of an RNA substrate is measured. Other techniques also used are sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), real-time quantitative-polymerase chain reaction (RTQ-PCR), and toxicological analyses in cell culture and in guinea pigs [7,27–33]. In this work, we report for the first time the accelerated solvent extraction (ASE) as an e fficient method for ricin extraction from the seeds of castor bean (*R. communis* L.) followed by the combined use of SDS-PAGE, matrix-assisted laser desorption/ionization/time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS/MS for a fast and unambiguous identification of ricin for forensic purposes. Additionally, this method was further successfully used to detect the presence of ricin in gamma-irradiated samples.
