Simultaneous determination of some phthalate metabolites, parabens and benzophenone-3 in urine by ultra high pressure liquid chromatography tandem mass spectrometry

Phthalates, parabens and 2-hydroxy-4-methoxybenzophenone or benzophone-3 are thought to
act as endocrine disrupting chemicals, being able to disrupt the endocrine balance and
therefore able to lead to some hormonal diseases. Numerous large-scale biomonitoring studies
have detected the biomarkers of these compounds in more than 75% of the general
population. To assess the exposure to these chemicals, we developed an analytical method
based on a Solid Phase Extraction (SPE) prior to ultra high pressure liquid chromatographytandem
mass spectrometry (UPLC-MS/MS) for the simultaneous measurement of seven
phthalate metabolites (monobenzyl phthalate, mono-n-butyl phthalate, mono-iso-butyl
phthalate, mono-2-ethylhexyl phthalate, mono-2-ethyl-5-hydroxyhexyl phthalate, mono-2-
ethyl-5-oxohexyl phthalate, monoethyl phthalate), four parabens (methyl paraben, ethyl
paraben, n-propyl paraben, n-butyl parabens) and benzophenone-3 in human urine. The
distinction between unconjugated, glucuro- and sulfoconjugated forms was achieved using
different enzymatic hydrolyses. The whole procedure was validated according to the total
error approach, and was demonstrated to be linear (regression coefficient ranging from 0.987
to 0.998) and accurate (inter and intra assay precision <17.71%, relative bias <5.87%) in the
dosing range of concentrations. The limits of quantification (LOQs) obtained ranged between
0.30 and 1.23 ng/ml depending on the analyte. The reliability of the method was proven in
passing successfully the German External Quality Assessment Scheme (G-EQUAS).
Moreover, the urine from 25 volunteers were analyzed for the determination of glucuro-,
sulfo- and free species separately. Phthalate metabolites, parabens and benzophenone-3 were
positively detected in almost all urine samples, with detection rates ranging from 40 to 100%.
Levels measured ranged from <LOQ to 2207 ng/ml varying widely depending on the
compound and the individual. In our small participating population, most of the phthalate
metabolites were excreted predominately as glucuroconjugated forms while parabens and
benzophenone-3 were detected as glucuro- and sulfoconjugated species in variable
proportions according to the target compound.