Analysis of mutagenic activity in human urine after concentration on different resins and high-performance liquid...

Smokers' urine was tested for mutagenic activity on Salmonella typhimurium strain TA1538 with metabolic activation after adsorption on different resins and desorption with organic solvents. The amounts of XAD-2 were 1.25 and 5 g/100 ml urine, the amounts of alumina, cyanopropyl and C18 were all 5 g/100 ml and extrelut 80 g/100 ml. Adsorbed organic chemicals were eluted with acetone from XAD-2, with dichloromethane from extrelut and with a series of solvents from the other resins (hexane, toluene, dichloromethane and methanol). All columns gave similar results, with the exception of extrelut, which had poor recovery of mutagenic activity. Higher resin/urine ratios and sequences of columns gave better results. The organic eluates from XAD-2 columns loaded with the urine of patients treated with cyclophosphamide and melphalan were mutagenic on strain TA1535 with S9, and some mutagenic activity was also detectable in the aqueous eluate. Cisplatin was adsorbed on XAD-2, C18 and extrelut, but was eluted only from extrelut using dimethylformamide as a solvent. Smokers' urine was separated into several fractions with high-performance liquid chromatography, using C-18 columns with a series of solutions of 2.5 mM phosphoric acid and acetone or with a gradient of methanol. Several fractions containing dissolved organic compounds and no histidine were mutagenic with metabolic activation, but the overall mutagenic activity was still lower than the one detected with one-step chromatography on XAD-2. Using XAD-2 resins with a high ratio of resin to urine still seems to be the method of choice for studying urinary mutagenicity.