LC on a nano-scale measures steroids in the whale

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  • Published: Jun 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: LC on a nano-scale measures steroids in the whale

Steroid measurements in living whales are a challenge

A number of species of whale are critically endangered and require active monitoring of their status and fitness. Analysis of steroid hormones in blubber can give crucial information regarding stress levels and breeding capacity. However, the amount of blubber that can be safely taken from live whales is extremely limited, giving very small sample sizes. Traditionally, enzyme-linked immunosorbent assays (ELISA) have been used to detect steroids in blubber, but these require 75–150 mg of sample, which consumes most of the material available. In addition, ELISA assays are typically limited to one steroid at a time, are subject to interference by antibodies and tend to give different results depending on the ELISA kit suppliers.

The researchers from Texas and Alaska developed a novel nano-LC-MS/MS to detect steroids in small blubber samples from the Eastern Grey Whale. Nano-HPLC gives better separation than conventional HPLC and also allows the use of very small sample sizes. The researchers determined the levels of progesterone, testosterone and hydrocortisone using cholic acid as a surrogate internal standard. The latter was chosen as a suitable steroid not found in whale blubber.

Nano-LC-MS/MS used to quantify steroids in whale blubber

For each determination, 50-mg samples of whale blubber were taken and sliced. They were placed in lysing matrix A with ethanol and homogenised. The homogenate was extracted in turn with ethanol, acetone and finally diethyl ether, the solids being separated by centrifugation prior to the next extraction. Evaporation of the various solvent extracts was carried out giving a combined residue, which was partitioned between hexane and acetonitrile, cooling to -20 °C to ensure separation of the two solvents. The hexane layer was discarded and the partition was repeated with fresh hexane, which was again discarded. The acetonitrile layer was filtered and evaporated. The residue was then taken up in aqueous 5 nM cholic acid solution, containing acetonitrile (40%) and formic acid (0.1%), for injection into the nano-HPLC system.

HPLC was carried out using a Dionex UltiMate 3000 RSLCnano system with a Thermo Scientific Acclaim PepMap RSLC column and a 1-μl injection volume. Gradient elution was used with acetonitrile and water, both containing 0.1% formic acid, as the two solvents. The flow rate was only 300 nL/min, thus solvent use was low. A Thermo LTQ XL linear ion trap mass spectrometer was employed, with ESI (electron spray ionisation) in positive mode. Selected reaction monitoring (SRM) was carried out, giving specific product ion transitions for each steroid.

Clear separation of the three steroids and the internal standard was obtained. The limits of detection (LOD) were low at 7.2 fg/μl for testosterone, 7.8 fg/μl for progesterone and 9 fg/μl for hydrocortisone, while the limits of quantification (LOQ) were 14, 16 and 18 fg/μl, respectively. These values are much lower than literature values using conventional HPLC. The method also gave good linearity and precision.

Spiking experiments with progesterone, using blubber samples from a male whale that initially contained none of this steroid, showed good recovery levels. Additionally, the method was tested on three whale blubber samples, showing high levels of testosterone in an adult male, high levels of progesterone in an adult female and lower steroid levels in a juvenile male, as would be expected.

Steroid analysis in small samples improved using nano-HPLC

The nano-LC-MS/MS method allowed simultaneous detection of traces of different steroids in small samples of whale blubber. The nano-HPLC gave a clear advantage in sensitivity, while also using much less solvent. Such techniques can be extended to many other situations where sample quantity is limited.

Related Links

Rapid Commun. Mass Spectrom. 2017, 31, 1088-1094. Hayden et al. Nano-LC-MS/MS analysis of steroids from gray whale blubber.

LCGC North America, 2011, 29, 926-934. Rieux et al. Nano LC: Principles, evolution, and state-of-the-art of the technique.

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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