Identification and quantitative determination of glutathione-related urinary metabolites of fotemustine, a new anti-cancer agent

1. Potential sulphur-containing metabolites of the anticancer agent, fotemustine, were synthesized, namely thiodiacetic acid (TDA), S-2-hydroxyethyl N-acetyl-L-cysteine (2-HE-NAC), N-acetyl-L-cysteine (NAC), S-methyl N-acetyl-L-cysteine (M-NAC), S-carboxymethyl-L-cysteine (CM-Cys), S-carboxymethyl N-acetyl-L-cysteine (CM-NAC), their corresponding sulphoxides and sulphones. Their chemical structures and stabilities were confirmed and derivatization methods were developed for their analysis by sulphur-selective g.l.c. (g.l.c.-FPD) and g.l.c.-mass spectrometry. 2. Four methods for isolation of potential metabolites of fotemustine were developed. Quantification of metabolites, derived in various ways was carried out by g.l.c.-atomic emission detection (AED) or g.l.c.-mass spectrometry. 3. Male Wistar rats (n=4) were given a single i.p. dose of 40mg/kg fotemustine. Urine excretion of TDA (18.4+1.9% in 24h) and TDA sulphoxide (12.0+1.6% in 24h) was significant; 32.7 + 4.6% of the fotemustine dose was excreted as TDA, and TDA sulphoxide in 48 h. NAC was excreted in rat urine at 1% of the dose. No other potential glutathione-derived metabolites of fotemustine were excreted. 4. Male Wistar rats (n = 4) were also treated i.p. with fotemustine at 5,20 and 40 mg/kg, to investigate dose dependency and the time course of excretion of TDA. Excretion of TDA in 48 h urine decreased from 32 + 2 to 17 π 2% dose (mean π SD) with increasing dose of fotemustine