Quantifying Phthalate Metabolites in Human Meconium and Semen Using Automated Off-Line Solid-Phase Extraction Coupled with On-Line SPE and Isotope-Dilution High-Performance Liquid Chromatography−Tandem Mass Spectrometry

Abstract

We developed an analytical method using off-line solid-phase extraction (SPE) coupled with on-line SPE and isotope-dilution high-performance liquid chromatography-tandem mass spectrometry (HPLC−MS/MS) to determine the concentrations of phthalate metabolites in human meconium and in semen. First, we used off-line SPE to remove interfering proteins and other biomolecules from the samples. Then, we preconcentrated the phthalate metabolites in the extract using on-line SPE before measuring them by HPLC−MS/MS. For most of the analytes, the limits of detection ranged between 0.2 and 0.7 ng/g for meconium and between 0.3 and 0.7 ng/mL for semen. The recovery after off-line SPE varied for most analytes between 65 and 99% at concentrations ranging from 3.0 to 30.0 ng/mL in semen and between 67 and 103% at concentrations ranging from 2.0 to 10.0 ng/mL in meconium. Precision measured by the relative standard deviation ranged from 3.2 to 19.1% for intraday and from 3.9 to 18.6% for interday. We validated this novel approachwhich is applicable to other biological matrixes, including serum and breast milkon spiked samples and on five meconium samples and one pooled semen sample from people with no known occupational exposure to phthalates.