On-line clean-up by multidimensional liquid chromatography-electrospray ionization tandem mass spectrometry for high throughput quantification of primary and secondary phthalate metabolites in human urine.
We developed a new and fast multidimensional on-line HPLC-method for the quantitative determination of the secondary, chain oxidized monoester metabolites of diethylhexylphthalate (DEHP), 5-hydroxy-mono-(2-ethylhexyl)-phthalate (5OH-MEHP) and 5-oxo-mono-(2-ethylhexyl)-phthalate (5oxo-MEHP) in urine samples from the general population. Also included in the method were the simple monoester metabolites of DEHP, dioctylphthalate (DOP), dibutylphthalate (DBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Except for enzymatic hydrolysis for deconjugation of the metabolites no further sample pre-treatment step is necessary. The phthalate metabolites are stripped from urinary matrix by on-line extraction on a restricted access material (LiChrospher((R)) ADS-8) precolumn, transferred in backflush-mode and chromatographically resolved by reversed-phase HPLC. Eluting metabolites are detected by ESI-tandem mass spectrometry in negative ionization mode and quantified by isotope dilution. Within a total run time of 25 min we can selectively and sensitively quantify seven urinary metabolites of six commonly occurring phthalate diesters including the controversial di(2-ethylhexyl)phthalate (DEHP). The detection limits for all analytes are in the low ppb range (0.5-2.0 microgram/l urine). First results on a small non-exposed group (n=8) ranged for 5OH-MEHP from 0.59 to 124 microgram/l, for 5oxo-MEHP from <LOQ to 73.0 microgram/l, and for MEHP from <LOQ to 41.1 microgram/l. The other short chain monoester metabolites were detectable in every sample with mean concentrations for MnBuP of 36.5 microgram/l, for MBzP of 7.19 microgram/l and MEP of 1.0 mg/l. With this rapid and economic method we can determine the internal exposure of the general population to DEHP and other phthalates as well as the body burden of occupationally and medically exposed subjects. The results can help to rank the risks of phthalates in the areas of carcinogenesis, peroxisome proliferation and endocrine disruption. Since secondary, functionalized metabolites of DEHP are included in the method an enduring problem of the past is excluded: sample contamination in the pre-analytical and analytical phase by both di- and monoesters.
Koch
Holger M
HM
Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine, University of Erlangen-Nuremberg, Schillerstr. 25-29, D-91054, Erlangen, Germany.
Gonzalez-Reche
Luis M
LM
Angerer
Jürgen
J
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D010795:Phthalic Acids / Q000652:urine*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010795:Phthalic Acids; C032279:phthalic acid