GUANIDINE COMPOUNDS OF A BRAZILIAN PUFFER FISH Sphoeroides spengleri

MORALES, R.A.V.(1,2), PIRES JR, O.R.(1), OLIVEIRA, J.S.(3), FREITAS, J.C.(3), SCHWARTZ, C.A.(1), BLOCH JR, C.(2)

(1)Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, UnB, (2)Embrapa - Recursos Genéticos e Biotecnologia - DF, (3)Departamento de Fisiologia, Instituto de Biociências e Biologia Marinha - USP.

Puffer fish toxicity derives from the presence of guanidine compounds such as TTX and analogs probably acquired from food chain or from symbiotic bacterial strains present in their skin or digestive tract. In this work we analysed the guanidine compounds present in Sphoeroides spengleri, a brazilian puffer fish from São Sebastião Channel using LC-FLD and LC-MS/MS. 85 specimens of S. spengleri were submitted to toxin extraction with methanol 70%-acid acetic1%, evaporated and ressuspended in deionized water. The semi purified extract was submitted to an HPLC system using a post-column fluorescent detection system (LC-FLD) and to an LC-MS/MS system, carried out under a selection ion mode (SIM). The m/z values of 320Da, 290Da, 302Da, 304Da and 336Da were chosen  as precursor ions, fragmented by collision-induced dissociation. The retention time data from the LC-FLD system showed the presence of TTX,        4-epiTTX, 6-epi-TTX, 4,9-anidroTTX and TDA while the SIM data confirmed the presence of these compounds and revealed 4 other analogs 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX in puffer extracts. The identity of the 320Da, 302Da and 304Da ions was achieved by their fragmentation pattern and the formation of characteristic daughter ions such as TTX(302Da, 284Da, 256Da,178Da and 262Da), 4,9-anhydroTTX(256Da, 210Da, 195Da, 162Da and 135Da) and 5-deoxyTTX(286Da, 176Da, 162Da, and 146Da), according to Shoji, Y. et al., 2001(Analytical Biochem.290,10-17). The identification of puffer toxins is somewhat limited in an LC-FLD detection system, due to C9-base analog different fluorescent intensities and conversion efficiencies. LC-MS/MS is thus a powerful tool, capable of determining guanidine compounds previously gone undetected by current used methods.

CORRESPONDENCE TO:

Pires Jr, O.R., Laboratório de Toxinologia, Departamento de Ciências Fisiológicas – Instituto de Ciências Biológicas – Universidade de Brasília, Brasília, 70910-900, DF, Brasil, Email: osmindo@unb.br