Gender Bender Steroids in the Alabama Mud Detected by UHPLC

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  • Published: Feb 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Gender Bender Steroids in the Alabama Mud Detected by UHPLC

Steroids in estuaries pose a threat to wildlife

Steroid pollutants tend to pass through sewage works and accumulate in estuaries, where they act as endocrine disruptors and may harm wildlife. Scientists from Auburn University, Alabama have developed a new UHPLC-MS/MS method for the detection of these steroids from estuarine sediments.

A number of steroids find their way into waterways, some from plants and animals, while others are derived from humans. Human urine contains some natural steroids, along with others from various pharmaceuticals, such as oral contraceptives, hormone replacement therapy agents, anti-inflammatory steroids and anabolic steroids. The steroidal compounds pass through sewage works and can accumulate in estuaries, where they can act as endocrine disruptors, interfering with the reproduction and development of a variety of creatures, both vertebrates and invertebrates. In the past, much attention has been focused on the effect of pesticides in the environment, while that of pharmaceuticals has tended to be ignored. Nowadays, environmental scientists are beginning to redress the balance, looking at compounds such as steroids in the environment.

Quick methods for the analysis of steroids in sediments are required, although current techniques often involve lengthy extraction techniques, followed by HPLC methods with long run times. The Auburn University group looked at developing improved extraction techniques, followed by fast detection using UHPLC (ultra-high performance liquid chromatography).

New methods of extraction and UHPLC in environmental steroid analysis

The Auburn scientists took sediment core samples from Perdido Bay in Alabama, an estuary on the Gulf of Mexico, near to the coastal town of Orange Beach. The extraction conditions were optimised. For best results, magnesium sulphate, Hydromatrix™ diatomaceous earth sorbent and an extraction liquid composed of 70:30 methanol : water were added to the samples; the mixture was vortexed and then centrifuged to remove the solids. The supernatant liquid was then vortexed and centrifuged again in the presence of Agilent ‘Bond Elut SPE’, before a final filtration. The SPE material eliminated non-steroidal compounds that otherwise interfered with the analysis.

UHPLC was carried out with an Agilent C-18 Poroshell 120 UHPLC column with gradient elution. The mobile phases were aqueous 1 mM ammonium fluoride and methanol:acetonitrile (65:35), with the organic mobile phase increasing from 30% to 100% during the run, which was only 15 minutes long. Mass spectrometry was carried out with an Agilent 6460 triple quadrupole mass spectrometer with a Jet Stream /ESI (electrospray ionisation) source. The ammonium fluoride in the aqueous phase gave good ionisation of the steroids. The overall method was validated, showing good linearity and clearly separating the 17 steroid standards examined. These steroids covered four different groups: oestrogens, glucocorticoids, androgens and progestins. Very low limits of detection (LOD) were seen, ranging from 0.001 to 0.391 pg/injection, with most compounds being less than 0.050 pg/injection. The limits of quantification (LOQ) ranged from 0.006 to 3.125 pg/injection.

During the analysis of samples, four deuterated steroids were added as internal standards to the filtered solution prior to injection. These would be diminished by the system at the same rate as their non-deuterated analogues, so allowing for any losses. Of the 17 steroids separated by the method, only ten were detected in the sediment samples. The most abundant proved to be dihydrotestosterone (DHT) and androsterone (both probably of natural origin, from human and animal urine, etc.), with concentrations of 57–137 pg/g for the former and 39–129 pg/g for the latter. Pharmaceutical steroids were also present at low levels, such as levonorgestrel from oral contraceptives, found at levels of 1–3 pg/g, and the anti-inflammatory agent prednisolone, found at levels of 6–15 pg/g.

Fast method for steroid analysis from environmental samples

The new method allows the fast and accurate determination of steroid levels from environmental samples, covering four classes of steroids in a single run. It should aid the construction of maps of steroid concentration in the environment, so that areas with the highest levels can be identified, and action taken to reduce steroidal accumulation.

Related Links

Rapid Communications in Mass Spectrometry, 2017, Early View paper. Mulabagal et al. An UHPLC-MS/MS quantitative method for trace analysis of potential endocrine disrupting steroid hormones in estuarine sediments.

Environmental Science and Pollution Research, 2011, 18, 226-236. Cailleaud et al. Uptake and elimination, and effect of estrogen-like contaminants in estuarine copepods: an experimental study.

Environmental Science & Technology, 2010, 44, 2661-2666. Fick et al. Therapeutic levels of levonorgestrel detected in blood plasma of fish: Results from screening rainbow trout exposed to treated sewage effluents.

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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