Journal of Pharmaceutical and Biomedical Analysis 43 (2007) 1499–1506 Short communication Analysis of guazatine mixture by LC and LC–MS and antimycotic activity determination of principal components Elena Dreassi a,∗ , Alessandra Tania Zizzari a , Silvia D’Arezzo b , Paolo Visca b,c , Maurizio Botta a a Dipartimento Farmaco Chimico Tecnologico, Universit` a degli Studi di Siena, via A. Moro 2, Siena I-53100, Italy b Istituto Nazionale di Malattie Infettive “Lazzaro Spallanzani” I.R.C.C.S., via Portuense 292, Roma I-00149, Italy c Dipartimento di Biologia, Universit` a “Roma Tre”, viale G. Marconi 446, Roma I-00146, Italy Received 1 September 2006; received in revised form 9 October 2006; accepted 17 October 2006 Available online 1 December 2006 Abstract Guazatine is a non-systemic contact fungicide, a mixture of reaction products from polyamines, comprising mainly octa-methylenediamine, iminodi(octamethylene)diamine, octamethylenebis(imino-octamethylene) diamine and carbamonitrile. In this work, the analysis of guazatine mixture by LC and LC–MS has been treated for the first time. In the guazatine mixture diamine derivatives account for 40% of the constituents of guazatine, triamines for 46%, tetramines for 11% and other amine derivatives for 3%. The most abundant individual components are the fully guanidated triamine (GGG, 30.6%) and the fully guanidated diamine (GG, 29.5%) followed by the monoguanidated diamine (GN, 9.8%) and a diguanidated triamine (GGN, 8.1%). The identification and separation of main components of commercial guazatine was performed through a new LC–MS method. The separation was performed on an Alltima C 18 column using linear gradient elution (formic acid in water and acetonitrile) with UV-detection at 200 nm and the identification was performed by ESI + -mass spectrometry analysis. The main components (GN, GG, GNG, GGN, GGG and GGGG) were then purified and separated from the mixture. Antimycotic activity of guazatine derivatives was determined on different species and strains belonging to genus Candida. The results obtained suggest that GNG and GGGG components can further be developed in new antifungal compounds with high potential for the treatment of Candida infections. © 2006 Elsevier B.V. All rights reserved. Keywords: HPLC/UV/MS; Guazatine; Candida; Antimycotic activity; MICs 1. Introduction Guazatine is a non-systemic contact fungicide which disturbs the membrane function of fungi [1]. WHO has classified guaza- tine as moderately hazardous with an oral LD 50 value in rats of 280 mg/kg bw [1]. It is widely used in agriculture to control a wide range of seed-borne diseases of cereals. On citrus fruit, guazatine is used as a bulk dip after harvest and in the packing line as a spray [1]. Guazatine acetate, the salt that is used in practice, is a mixture of reaction products from polyamines, comprising mainly octa-methylenediamine, iminodi(octamethylene)diam- ine, octamethylenebis(imino-octamethylene) diamine, and ∗ Corresponding author. Tel.: +39 0577234321; fax: +39 0577234333. E-mail address: dreassi@unisi.it (E. Dreassi). carbamonitrile. A coding system is used for the compounds that make up guazatine. In this system ‘N’ represents any amino group. Thus, NN stands for H 2 N–(CH 2 ) 8 –NH 2 , NNN stands for H 2 N–(CH 2 ) 8 –NH–(CH 2 ) 8 –NH 2 and so on. ‘G’ stands for any amino group (NH or NH 2 ) of the above which is guanidated. For example GG stands for H 2 N–C(NH)NH–(CH 2 ) 8 –NH–C(NH)–NH 2 [1]. The guanidated diamines and triamines are the most abun- dant components of guazatine [2]. A typical composition of free guazatine is described in Table 1. It can be seen that diamine derivatives account for ca. 40% of the constituents of guaza- tine, triamines for ca. 46%, tetramines for ca. 11% and other amine derivatives for ca. 3%. The most abundant individual components are the fully guanidated triamine (GGG, 30.6%) and the fully guanidated diamine (GG, 29.5%), followed by the monoguanidated diamine (GN, 9.8%) and by a diguanidated triamine (GGN, 8.1%). 0731-7085/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2006.10.029