Automated solid phase extraction and quantitative analysis of human milk for 13 phthalate metabolites

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Abstract

While the demonstrated benefits associated with breastfeeding are well recognized, breast milk is one possible route of exposure to environmental chemicals, including phthalates, by breastfeeding infants. Because of the potential health impact of phthalates to nursing children, determining whether phthalates are present in breast milk is important. We developed a sensitive method for measuring 13 phthalate metabolites in breast milk using automated solid phase extraction (SPE) coupled to isotope dilution–high-performance liquid chromatography (HPLC)–negative ion electrospray ionization–tandem mass spectrometry. We used D4-phthalate diesters to unequivocally establish the presence in human breast milk of enzymes capable of hydrolyzing the ubiquitous phthalate diesters to their respective monoesters. The analytical method involves acid-denaturation of the enzymes after collection of the milk to avoid hydrolysis of contaminant phthalate diesters introduced during sampling, storage, and analysis. The method shows good reproducibility (average coefficient of variations range between 4 and 27%) and accuracy (spiked recoveries are ∼100%). The detection limits are in the low ng/ml range in 1 ml of breast milk. We detected several phthalate metabolites in pooled human breast milk samples, suggesting that phthalates can be incorporated into breast milk and transferred to the nursing child.

Introduction

Diesters of phthalic acid, commonly known as phthalates, are a group of industrial chemicals with many commercial uses such as solvents, additives, and plasticizers. The potential for nonoccupational exposure to phthalates is high given their use in a vast range of consumables such as personal-care products (e.g., perfumes, lotions, cosmetics), paints, industrial plastics, and certain medical devices and pharmaceuticals [1], [2], [3], [4], [5], [6]. Human exposure to phthalates can occur via ingestion, inhalation, and dermal routes, and intravenous and parenteral absorption in patients undergoing medical procedures that involve the use of medical devices containing phthalates. Upon exposure, phthalates are rapidly hydrolyzed to their monoesters. These monoesters may be further metabolized by oxidation and/or glucuronidation and excreted in urine and feces [1], [2], [3], [4].

Several phthalates are carcinogenic in animal models [4], [7], [8]. In addition, some phthalates and their metabolic products act functionally as antiandrogens during the prenatal period [9], [10], [11] and cause reproductive and developmental toxicities in animals [12], [13], [14], [15]. Exposure to high doses of dibutyl phthalate (DBP) results in spontaneous abortion of rat pups, demasculinization of fetal male rats, and testicular atrophy in rats, mice, ferrets, and guinea pigs [3], [10]. DBP reduces the production of testosterone by the fetal testis through an antiandrogenic mechanism [16]. Studies in male rodents exposed to high doses of di-2-ethylhexyl phthalate (DEHP) indicate that the testes are a primary target [4]; mono-2-ethylhexyl phthalate (MEHP), a metabolite of DEHP, may be the ultimately active testicular toxicant [17], [18]. Long-term exposures of adult female rats to DEHP also appear to have deleterious effects, including hypoestrogenic anovulatory cycles and polycystic ovaries [19]. DEHP appears to suppress estradiol production in the ovary, leading to anovulation [20].

Little information is known about the effects of phthalate exposure on humans. To evaluate the potential adverse effects of exposure to phthalates, accurate methods for measuring the amount of phthalates absorbed by the body must be developed. The measurement of monoester phthalate metabolites in urine and serum has been used for assessment of exposure to phthalates [21], [22], [23], [24], [25], [26], [27], [28], [29], [30]. These studies suggest that exposure to phthalates is widespread.

Interest is increasing in monitoring breast milk for environmental contaminants because breast milk is the major route of exposure to these contaminants by the breastfeeding infant. Data on the levels of phthalates in milk are necessary for assessing the potential impact of phthalates to nursing mothers and their children. Trace levels of the phthalate diesters DBP (14 ng/ml) and DEHP (19 ng/ml) were detected in the milk of one person [31]. Because of the ubiquitous presence of phthalate diesters in the environment and hence the likelihood of contamination during the collection, storage, and measurement processes, measuring phthalate metabolites would be preferable [32]. However, measurements of phthalate metabolites in human milk are susceptible to contamination from the ubiquitous phthalate diesters that can be hydrolyzed to their respective monoesters by milk esterases [33], [34]. Therefore, if the enzyme activity is not eliminated, the milk phthalate monoester measurements will be artificially high.

We report here a method for the quantitative detection of 13 phthalate metabolites in human milk. The method involves the initial acid denaturation of the milk enzymes to eliminate the esterase activity. The phthalate metabolites are extracted from breast milk using an automated solid phase extraction (SPE) procedure, separated from other extracted components in the eluate by reverse phase high-performance liquid chromatography (HPLC), and detected by isotope dilution–negative ion electrospray ionization–tandem mass spectrometry. We also report the use of D4-phthalate diesters to unequivocally establish the presence in human breast milk of esterases.

Section snippets

Analytical Standards and Reagents

Monomethyl phthalate (mMP), monoethyl phthalate (mEP), monocyclohexyl phthalate (mCHP), monobenzyl phthalate (mBzP), mono-n-butyl phthalate (mBP), mono-2-ethylhexyl phthalate (mEHP), mono-2-ethyl-5-oxohexyl phthalate (mEOHP), mono-2-ethyl-5-hydroxyhexyl phthalate (mEHHP), mono-n-octyl phthalate (mOP), and mono-3-methyl-5-dimethylhexyl phthalate (isononyl, mNP) (>99.9%), their 13C4-labeled internal standards (>99.9%), 13C4-4-methyl-umbelliferone, and ring-deuterium-labeled phthalate diesters,

Results and discussion

Breast milk is possibly the major route of exposure to certain xenobiotics by the breastfeeding infant. Because of the potential impact of these chemicals to lactating mothers and their children, interest is increasing in monitoring breast milk for environmental contaminants, including phthalates. We have developed a sensitive method to assess human exposure to phthalates through this pathway by measuring phthalate metabolites in breast milk.

We used D4-phthalate diesters to unequivocally

Acknowledgements

This research was supported in part by an appointment (A.R. Slakman) to the Research Participation Program at the Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and CDC.

References (41)

  • H.G. Wahl et al.

    J. Chromatogr. A

    (1999)
  • E. Mylchreest et al.

    Toxicol. Sci.

    (1998)
  • M. Ema et al.

    Reprod. Toxicol.

    (2001)
  • E. Mylchreest et al.

    Reprod. Toxicol.

    (2002)
  • T.J.B. Gray et al.

    Food Chem. Toxicol.

    (1984)
  • B.J. Davis et al.

    Toxicol. Appl. Pharmacol.

    (1994)
  • H.M. Koch et al.

    J. Chromatogr. B

    (2003)
  • H.M. Koch et al.

    Environ. Res.

    (2003)
  • H.M. Koch et al.

    Int. J. Hyg. Environ. Health

    (2004)
  • D.T. Rossi et al.

    J. Pharm. Biomed. Anal.

    (1997)
  • C. Polson et al.

    J. Chromatogr. B

    (2003)
  • P.M. Emmett et al.

    Early Hum. Dev.

    (1997)
  • M.J. Silva et al.

    J. Chromatogr. B

    (2003)
  • ATSDR, Toxicological Profile for Diethyl phthalate (DEP), http://www.atsdr.cdc.gov/toxprofiles (accessed 11 August...
  • ATSDR, Toxicological Profile for Di-n-octyl phthalate (DNOP), http://www.atsdr.cdc.gov/toxprofiles/tp95.html (accessed...
  • ATSDR, Toxicological Profile for Di-n-butyl phthalate (DBP), http://www.atsdr.cdc.gov/toxprofiles (accessed 11 August...
  • ATSDR, Toxicological Profile for Di(2-ethylhexyl)phthalate (DEHP), http://www.atsdr.cdc.gov/toxprofiles (accessed 11...
  • R.M. David, R.H. McKee, J.H. Butala, R.A. Barter, M. Kayser, in: E. Bingham, B. Cohrssen, C.H. Powell (Eds.), Patty’s...
  • W.W. Huber et al.

    Crit. Rev. Toxicol.

    (1996)
  • W.M. Kluwe et al.

    Environ. Health Perspect.

    (1982)
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