Steroids in transsexual eels sorted by LC-MS/MS and DMS

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  • Published: May 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Steroids in transsexual eels sorted by LC-MS/MS and DMS

Female hormone levels in farmed eels require monitoring

Currently, commercial eel farming involves taking young eels, so-called ‘glass eels’, from rivers and then growing them on in fish farms. The glass eels can develop into either males or females, but under farmed conditions crowding leads to their transformation almost exclusively into males, rather than the heavier and more commercially desirable females. Female hormones, such as 17β-estradiol, can be added to eel feed to ensure feminisation. Accurate monitoring of hormone levels is necessary, both for fish management and regulatory purposes. There should be no excess hormone present when fish enter the human food chain.

Steroid hormones are typically detected by immunoassays, LC-MS or GC-MS (in the latter case derivatisation is usually needed). The Canadian scientists decided to use LC-MS/MS in combination with differential ion mobility spectrometry (DMS). The latter technique takes place between the HPLC and MS system. It produces ions using an ESI source, then separates them based on their different mobility in an electric field in the gas phase.

LC-MS/MS in combination with DMS used for steroid hormone detection

The researchers examined three steroids hormones in American eel samples: the 17β-estradiol added to feed, and the related compounds estrone and estriol. All three compounds occur naturally in fish to some extent. Fish were taken for muscle tissue sampling at different time points, and wild female eels were also caught for sampling. Muscle samples were homogenised and then extracted with acetonitrile under sonication.

HPLC was carried out using a ThermoScientific UltiMate 3000 RSLCnano instrument, fitted with a Phenomenex Luna 3μm C18 column, using gradient elution with acetonitrile and water, both containing 0.05% ammonium hydroxide. A SciEx SelexION source was used for the differential ion mobility (DMS), with isopropanol as modifier (i.e. a volatile substance introduced into the ion mobility source in order to improve selectivity). In this case, both the use of the modifier and careful adjustment of the DMS parameters were required in order to separate the steroid ions by DMS. Finally, MS/MS was carried out using a Sciex QTrap 5500 triple quadrupole linear ion trap mass spectrometer. Selective reaction monitoring was optimised to give specific transitions for the three steroids of interest.

Overall, the combined set-up gave good separation, but it should be noted that the three main compounds could be separated by the HPLC alone under optimum conditions. Switching on the DMS helped to give cleaner spectra. The system was used to measure the decline in 17β-estradiol in eel muscle after the withdrawal of this supplement from their diet. It was found that the steroid in question reached background levels by day five after withdrawal, the same as those in untreated farmed eels. The final levels were somewhat lower than those found in wild female fish. Estrone was only noted at low levels just after the last feeding with 17β-estradiol, while no estriol was detected.

DMS enhances LC-MS/MS, but is it worth it?

The combination of LC-MS/MS with DMS was successfully used in the monitoring of eel hormones. The results clearly showed that feminisation with added 17β-estradiol did not give any long-term accumulation of female hormones. However, although DMS helped to give cleaner spectra, adequate separation was still achieved without its use. It is not clear whether many laboratories will consider the extra expense of a DMS system to be worthwhile for steroid analysis, although there are undoubtedly some complex analyses where it can significantly improve performance.

Related Links

Rapid Communications in Mass Spectrometry, 2017, Early View paper. Cohen et al. Analysis of 17β-estradiol, estriol and estrone in American eel (Anguilla rostrata) tissue samples using liquid chromatography coupled to electrospray-differential ion mobility tandem mass spectrometry.

Bioanalysis, 2015, 7, 853-856. Campbell et al. Differential mobility spectrometry: a valuable technology for analyzing challenging biological samples.

Bird, Yale Environment 360, 10th October 2013: “In Japan, captive breeding may help save the wild eel.”

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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