Analysing aldehydes: UHPLC reveals the chemical weapons that crush crustacean reproduction

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  • Published: Dec 1, 2016
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Analysing aldehydes: UHPLC reveals the chemical weapons that crush crustacean reproduction

Damage response chemicals target crustaceans

Microscopic diatoms may be small, but these silica-clad plankton have their own chemical weapons, producing compounds, such as polyunsaturated aldehydes, that inhibit some of their predators. Image credit: Nicola Angeli/MUSE via Wikimedia Commons

Diatoms are some of the most successful organisms on earth. These microscopic phytoplankton occur in vast numbers in the oceans, but can also be found in your local river or garden pond. It has been estimated that diatoms account for a massive 45% of the primary production of organic material from carbon dioxide in the oceans.

When attacked, damage to their structure induces some species of diatom to make a variety of defensive chemicals, such as polyunsaturated aldehydes (or PUAs), which are believed to inhibit attackers. Other chemicals, such as hydroxylated fatty acids, are also released. All these chemicals are ‘oxylipins’, that is, compounds arising from the enzymatic oxidation of fatty acids.

‘Grazers’ on diatoms include copepods, which are tiny shrimp-like crustaceans, numerous in plankton. A number of studies have found that polyunsaturated aldehydes, along with related oxylipins, can inhibit both the hatching of copepod eggs and the development of the larvae. However, not all studies have found significant effects on copepod reproduction and further research is needed in this area.

Polyunsaturated aldehydes are also believed to act as messengers between diatoms, and to play a role in their interactions with bacteria. Scientists have only just begun to unravel the complex behaviour and chemistry of the different types of plankton.

Solving the problem of oxylipin analysis

Progress in teasing out the effects of polyunsaturated aldehydes and related compounds has been slowed by the cumbersome analytical methods used to detect and quantify these oxylipins. Most previous studies have employed time-consuming derivatisation methods, in which the polyunsaturated aldehydes are first converted to suitable derivative compounds, which are then analysed by gas chromatography (GC) or high-performance liquid chromatography (HPLC). Great care must be taken to ensure complete derivatisation, otherwise inaccurate measurements will result. Furthermore, most derivatisation methods suitable for polyunsaturated aldehydes fail to detect all the other oxidation compounds of interest, meaning more than one method needs to be employed. A fast, direct method was needed to enable experiments to be conducted more rapidly and with greater accuracy.

The Jena researchers, in a paper in Limnology and Oceanography: Methods, tried directly analysing the polyunsaturated aldehydes, without any derivatisation, using ultra-high performance liquid chromatography (UHPLC). This technique uses smaller column particles and higher pressures than traditional HPLC, allowing better resolution and shorter run times.

Samples of diatoms cultured in the laboratory were treated with ultrasound, the resulting cell damage causing the production of polyunsaturated aldehydes. The broken cells were extracted with methanol and UHPLC was run on the extracts. The individual compounds were identified by mass spectrometry, using an atmospheric pressure chemical ionisation (APCI) source.

The amounts of different polyunsaturated aldehydes produced varied between the three different species of diatom examined. One complicating factor was the existence of different geometric isomers of some of the aldehydes, which led to some peak splitting. Complete baseline separation of the isomers was not always achieved under the conditions used. The authors suggested that their direct method of analysis indicated that the different isomers were produced by the diatoms, rather than occurring as an artefact of the analytical process. In addition to polyunsaturated aldehydes, various oxygenated fatty acids were also detected.

Fast, single analytical runs now possible

The new method allows the detection of polyunsaturated aldehydes and other relevant oxylipins in a single short run, without any of the awkward derivatisation methods used in the past. The limits of detection (LODs) are low at <1.0 to 5.0 × 10-8 mol L-1 and the authors consider that the method would be suitable for field samples, but caution that large particles and herbivorous plankton may need to be removed. Researchers now have an improved tool to investigate the details of the chemical warfare and signalling agents used by diatoms.

Related Links

Limnology and Oceanography: Methods, 2016, Early View paper. Kuhlisch et al. A fast and direct liquid chromatography-mass spectrometry method to detect and quantify polyunsaturated aldehydes and polar oxylipins in diatoms.

Journal of Experimental Marine Biology and Ecology, 2011, 401, 13-19. Barriero et al. Diatom induction of reproductive failure in copepods: The effect of PUAs versus non-volatile oxylipins.

Wikipedia, Diatoms

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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