Phthalate separation success can be secured by GC-MS

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  • Published: Feb 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: Phthalate separation success can be secured by GC-MS

Phthalates linked to many health conditions

Phthalate plasticisers are ubiquitous in the environment, and have long been a source of health concern, particularly in their role as endocrine disrupters.

Phthalates are common plasticizers, found in vast range of items, such as flooring, furnishings, cosmetics and some foodstuffs. The EU has restricted the use of phthalates in toys and childcare products in an attempt to reduce children’s exposure.

Phthalates are classed as anti-androgen endocrine disruptors (affecting the action of male hormones). Maternal and/or childhood exposure is considered likely to raise the risk of genital abnormalities, low sperm count, amongst others. Anti-androgenic effects have been shown in animal studies and are also suspected to occur in humans.

In addition to anti-androgenic effects, phthalates have been tentatively linked to many other conditions, such as diabetes, cardiovascular disease, obesity, breast cancer and children’s mental development. However, a 2014 review looking at the possible effects of phthalates as ‘obesogens’ (promoting obesity, diabetes and cardiovascular disease) concluded that the various studies examined tended to lack reproducibility, and no firm conclusions could be drawn from them.

Currently, exposure to the various phthalates is usually estimated by the measurement of their metabolites in urine. Phthalates are broken down in the body, so normally the levels of metabolites are higher than those of the phthalates themselves. LC-MS/MS is the usual technique used, but GC-MS methods are less expensive, allowing more extensive monitoring. Since phthalate metabolites, such as aryl acids, are usually polar, derivatisation methods are needed. Some of these methods, such as methyl derivatisation with diazomethane, involve the use of toxic agents. Others, such as the formation of trimethyl silyl derivatives, give rise to compounds susceptible to hydrolysis. Yoshida’s work involved looking at better derivatisation methods, particularly for di-n-pentyl phthalate (DnPP), di-n-hexyl phthalate (DnHP) and dicyclohexyl phthalate (DcHP), for which good GC methods are currently lacking.

Derivatisation and GC-MS for phthalate metabolites

Yoshida derivatised phthalate metabolites to give the stable t-butyldimethylsilyl derivatives, using the derivatisation agent N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). Stock solutions of the t-butyldimethylsilyl derivatives of nine phthalate metabolites (each specific for a particular phthalate) were prepared or purchased. Urine samples were taken from seven non-smokers without any particular occupational exposure to phthalates. The samples were first treated with sulfatase enzyme to remove any conjugation of the phthalate metabolites via sulfate linkages, since such conjugation often occurs, and then derivatised. GC-MS was carried out using a gradient of 70 °C to 280 °C. The MS used an electron ionisation (EI) source with selected ion monitoring (SIM). Runs were carried out on the urine samples, along with samples spiked with known quantities of the various metabolites. The peak separation (including DnPP, DnHP and DcHP metabolites), accuracy and precision of the new method were found to be good. The limits of detection (LOD) for the nine metabolites ranged from 0.1 to 0.4 μg/l, while the limits of quantification (LOQ) ranged from 0.3 to 1.3 μg/l. The results detected metabolites from eight of the nine phthalates examined in the seven volunteers. Only mono-n-pentyl phthalate (from di-n-pentyl phthalate) was absent. The highest levels were found for mono-n-butyl phthalate MnBP (derived from di-n-butyl phthalate, DBP, a very common phthalate), which ranged from 10 to 22 μg/l in the seven subjects. Even dicyclohexyl phthalate (DcHP), which is not always looked for in such studies, was found in six out of seven subjects, although at low levels (maximum 0.5 μg/l of metabolite).

Better methods promise better phthalate studies

The new GC-MS method is cheaper than LC-MS/MS, while giving good detection of a variety of phthalate metabolites. It should be possible to use this method at a greater number of locations, thus allowing studies on the effects of phthalates to enrol more subjects, giving a clearer picture of health effects. While phthalates probably have some adverse effects on humans, this area of research has not always been helped by numerous small-scale studies, which may sometimes just be interpreting statistical ‘noise’ as worrying results.

Related Links

Biomedical Chromatography, 2017, Early View paper. Toshiaki Yoshida. Analytical method for urinary metabolites as biomarkers for monitoring exposure to phthalates by gas chromatography/mass spectrometry.

Critical Reviews in Toxicology, 2014, 44, 151-175. Goodman et al. Do phthalates act as obesogens in humans? A systematic review of the epidemiological literature.

Wikipedia, Phthalate

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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