**3. Conclusions**

Our results showed that the ASE method was e fficient and rapid for the extraction of ricin samples from castor bean seeds. For the best of our knowledge, it is the first time that this method is employed to ricin extraction. This method can be improved for future works, including subsequent steps of protein purification, and comparison with other forms of sample preparation reported in the literature [4,14,32,35]. In addition, the use of ASE combined with SDS-PAGE, MALDI-TOF MS and MALDI-TOF MS/MS, has provided a fast and unambiguous identification method for ricin that can be used in real cases of forensic investigation of suspected samples.

The irradiation of samples provoked a strong and gradual reduction in the intensity of the molecular mass signal of ricin measured by MALDI-TOF MS. The signal related to the molecules that remained intact after irradiation at 30 kGy, was so small that it was not possible to distinguish it from the noise. The loss of molecular mass, however, did not imply in the complete destruction of the protein or elimination of the toxicity. Despite the initial results, ricin showed quite resistant to gamma-ray irradiation. This is illustrated by the fact that even after exposure to a dosage of 30 kGy the sample still presented toxic activity, being able to remove the adenine residue from the nucleotide sequence of the DNA substrate. These results can be attributed to two main factors: The first is related to the very low toxicity of ricin already reported by Olsnes [15] who relates that a single unit of RTA is capable of inactivating thousands of ribosomes per minute. This makes any residual remnants of ricin potentially active. The second reason is that probably the mass loss provoked by the irradiation did not alter considerably the active site of the toxin, located in a specific region of RTA. In fact, the principal trypsin peptides of the ricin chains, including the ones related to the toxic activity, were identified in all samples, including the sample irradiated at 30 kGy.

The SDS-PAGE separation technique used followed by trypsin digestion and analyzed by MALDI-TOF MS, showed an important tool of identification, in this case, making it possible to di fferentiate ricin from other proteins with similar structures, like the RCA120, for example. Besides, the confirmation by a second method, where the amino acids sequence of the peptides was verified by MALDI-TOF MS/MS, provided higher credibility to the identification.
