Determination of 12 urinary phthalate metabolites in Norwegian pregnant women by core-shell high performance liquid chromatography with on-line solid-phase extraction, column switching and tandem mass spectrometry.

Abstract

Phthalates (dialkyl or alkyl phenyl esters of phthalic acid, benzene-1.2-dicarboxylic acid) are a group of industrial chemicals that have been used for more than 50 years. Phthalates are ubiquitous and can potentially have adverse effects on humans. The present study presents an accurate, sensitive and automated analytical method for measuring 12 phthalate metabolites (free and conjugated) in human urine using on-line solid phase extraction coupled to high performance liquid chromatography - electrospray ionization - tandem mass spectrometry. A small volume of urine sample (300μL) is required. Glucoronidated phthalate metabolites are deconjugated by incubation with glucoronidase enzyme (Escherihia coli-K 12) and the reaction is stopped by adding formic acid. This is the only sample preparation needed prior to injection into the column switching system. Thus, the method involves minimal sample handling and minimizes possible contaminations from the surroundings. The method was validated by spiking synthetic urine at 5-8 levels in the range of 0.1-500ng phthalate metabolites/mL synthetic urine. The method is sensitive with limits of detection in the low nanogram range, and rapid with a total run time about 25min. The accuracy was between 90 and 120 % and the intermediate precision was given as relative standard deviation was below 20% for most of the compounds. The high sensitivity, high throughput and minimal manual handling make the method suitable for large-scale biomonitoring studies. The present method was applied for the determination of phthalate metabolites in urine samples from 116 pregnant women, a subproject within the Norwegian Mother and Child Cohort Study. Concentrations of all the twelve phthalate metabolites was >LOQ in 100% of the samples analysed. Mean urinary concentrations for different phthalate metabolites ranged from 1 to 100ng/mL, the highest concentrations were observed for di-2-ethylhexyl phthalate (DEHP) metabolites and lowest for di-iso-nonyl phthalate (DiNP) metabolites. The urinary concentrations for most of the phthalate metabolites in the present study were found to be in the same range as found in other studies of pregnant women.