Chromatographic separation and quantitative determination of the metabolites of di-(2-ethylhexyl) phthalate from urine of laboratory animals.

Free, glycine-conjugated, and glucuronide-conjugated metabolites of di-(2-ethylhexyl) phthalate may be stripped from urine with XAD-2 resin, derivatized, and quantitatively analyzed by liquid chromatography on a nitrile column with UV and/or radioactivity monitors. One class of metabolites requires reversed-phase chromatography or gas-liquid chromatography for its resolution. Relative molar responses of the hydrogen flame-ionization detector to these metabolites have been determined. Packed gas chromatography columns (OV-3, OV-210, cyclohexanedimethanol succinate) and fused-silica capillary columns (SP2100 and FFAP) are useful for quantitative analysis under appropriate conditions. The simplest gas chromatographic procedure permitting complete quantitative analysis requires hydrolysis of conjugates, formation of methyl esters of carboxyl groups, butyration of hydroxyl groups and chromatography on OV-3. Typical distributions of di-(2-ethylhexyl) phthalate metabolites in urine from mice, hamsters, and guinea pigs are presented.