Improved quantitative detection of 11 urinary phthalate metabolites in humans using liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry

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Abstract

Phthalates are widely used as industrial solvents and plasticizers, with global use exceeding four million tons per year. We improved our previously developed high-performance liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometric (HPLC–APCI-MS/MS) method to measure urinary phthalate metabolites by increasing the selectivity and the sensitivity by better resolving them from the solvent front, adding three more phthalate metabolites, monomethyl phthalate (mMP), mono-(2-ethyl-5-oxohexyl)phthalate (mEOHP) and mono-(2-ethyl-5-hydroxyhexyl)phthalate (mEHHP); increasing the sample throughput; and reducing the solvent usage. Furthermore, this improved method enabled us to analyze free un-conjugated mono-2-ethylhexyl phthalate (mEHP) by eliminating interferences derived from coelution of the glucuronide-bound, or conjugated form, of the mEHP on measurements of the free mEHP. This method for measuring phthalate metabolites in urine involves solid-phase extraction followed by reversed-phase HPLC–APCI-MS/MS using isotope dilution with 13C4 internal standards. We further evaluated the ruggedness and the reliability of the method by comparing measurements made by multiple analysts at different extraction settings on multiple instruments. We observed mMP, monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mEHP, mEHHP and mEOHP in the majority of urine specimens analyzed with DEHP-metabolites mEHHP and mEOHP present in significantly higher amounts than mEHP.

Introduction

Many people are routinely exposed to phthalates (diesters of phthalic acid) because of their wide use as industrial solvents and plasticizers. After human exposure and absorption, phthalate diesters are metabolized to their respective monoesters and their oxidative products that are partially glucuronidated and excreted through urine and feces [1], [2], [3], [4], [5]. The metabolism is reported to be rapid, with a large portion being excreted within a short time [2], [6]. The proportion of the diester that is converted in vivo to its specific monoester or other oxidative metabolite is phthalate-dependent [6]. Exposure to dibutyl phthalate (DBP) and diethyl phthalate (DEP) resulted in excretion of their respective monoesters as the primary metabolites, whereas for di(2-ethylhexyl) phthalate (DEHP), the oxidative metabolites predominate. Some phthalates and their metabolic products are responsible for reproductive [7], [8] and developmental toxicities in animals [9], [10]. However, little information is known about the effects of phthalate exposure on humans. To understand any adverse health outcomes associated with phthalate exposure, reliable information about the exposures must be obtained. In exposure assessment of suspected toxic chemicals, measurement of internal dose produces valuable information [11], [12]. Hence, urine and serum are widely used as matrices for measuring the internal dose of toxic chemicals. Both phthalate diesters [4], [13], [14] and their respective monoesters (Fig. 1) [1], [15], [16] have been used as urinary or serum biomarkers of phthalate exposure. We recently published the urinary levels of metabolites of selected phthalates in non-representative [1], [15] and representative [17] US populations. Measurable levels of the monoesters of DEP and DBP [1], [15], [17], which are widely used in many consumer products such as perfumes, cologne, soap, shampoo, nail polish and cosmetics, were reported. Low levels of monoesters of more hydrophobic diesters such as dicyclohexyl phthalate (DCHP), DEHP, DiNP, and DOP were also reported [1], [17], indicating either low exposure, bioaccumulation or different path of metabolism or excretion compared with more hydrophilic diesters.

We previously developed a sensitive high-performance liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometric (HPLC–APCI-MS/MS) method to assess exposure to phthalates using monoesters as the biomarkers for exposure [18]. We modified this method to include three important additional analytes, to greatly improve the chromatography of low-molecular mass hydrophilic analytes, to better resolve them from the solvent front, to analyze free un-conjugated mEHP by eliminating the interferences derived from coelution of the glucuronide-bound form (or conjugated form) of the mEHP on measurements of the free mEHP, to increase the sample throughput of the method and to make it cost effective. We expanded the method to measure 11 phthalate metabolites, monomethyl phthalate (mMP), monoethyl (mEP), mono-n-butyl (mBP), monocyclohexyl (mCHP), monobenzyl (mBzP), mEHP, mono-n-octyl (mOP), mono-3-methyl-5-dimethylhexyl (iso-nonyl, mNP), and mono-3-methyl-7-methyloctyl phthalate (iso-decyl, mDP), mono-(2-ethyl-5-oxohexyl)phthalate (mEOHP) and mono-(2-ethyl-5-hydroxyhexyl)phthalate (mEHHP) in human urine with the detection limits in the low ng/ml (Table 1) range using 13C4-labeled analytes as the internal standards (Fig. 1) for nine of the above analytes while 13C4 mBP is using as the internal standard for DEHP-metabolites mEOHP and mEHHP. In the analysis of total phthalate monoesters, the completion of the deglucuronidation was monitored as a quality assurance step by monitoring the deglucuronidation of 4-methyl-umbelliferryl-glucuronide.

Section snippets

Reagents

Analytes mMP, mEP, mBP, mCHP, mBzP, mEHP, mOP, mNP, mDP, mEOHP and mEHHP (>99.9%), 13C4-stable isotope-labeled internal standards of mMP, mEP, mBP, mCHP, mBzP, mEHP, mOP, mNP, mDP (>99.9%, Fig. 1) and 13C4 4-methyl-umbelliferone internal standard (Fig. 1) were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Acetonitrile and water (HPLC grade), phosphoric acid (85%), ethyl acetate (99.8%), monosodium phosphate monohydrate (ultrapure bioreagent), ammonium hydroxide (30%),

Results and discussion

We modified our method for measuring urinary phthalate metabolites (Fig. 1) in humans to greatly improve the overall performance of the method. We obtained linear calibration curves for all analytes over three orders of magnitude with correlation coefficient exceeding 0.99 (Fig. 2). The use of an appropriate chromatographic condition prior to mass spectrometric analysis was necessary in order to determine all phthalate monoester analytes in one chromatographic run. Our previous method [18] with

Acknowledgements

The authors acknowledge Antonia Calafat for her input in this project. The use of trade names is for identification only and does not constitute endorsement by the US Department of Health and Human Services or the Centers for Disease Control and Prevention.

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