Arthropod molting hormones (ecdysteroids) are present in the blood of insectivorous bats
In this study, blood samples of eight insectivorous bat species were analyzed for the presence of ecdysteroids with highly sensitive targeted ultra-high-performance liquid chromatography coupled to high-resolution quadrupole-orbitrap mass spectrometry method (SIM). Nine ecdysteroids were detected in bat blood.
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All chemicals (water, methanol, acetonitrile, formic acid) for sample preparation and analysis with LC-MS grade were purchased from VWR (Radnor, PA, USA). Poststerone (20E)-oxime as internal standard was synthesized from poststerone (Bogdán et al. 2018). The mass spectrometer was operated in positive mode using single ion monitoring (SIM) for quantitative analysis of ecdysteroids and HRMS/MS parallel reaction monitoring (PRM) for their identification. The heated ESI source was used with the following conditions: capillary temperature 262.5 °C, S-Lens RF level 50, spray voltage 4.0 kV, sheath gas flow 50, spare gas flow 2.5, and auxiliary gas flow 12.50 in arbitrary units. For SIM mode the maximum injection time (IT) was 50 ms with a resolution of 35,000 (FWHM) and the automatic gain control (AGC) setting was defined as 1×106 charges. In PRM mode with resolution of 17,500 (FWHM), the AGC setting was defined as 1×106 charges, the maximum IT was set to 50 ms and 20 eV collision energy was used. The precursor ion window was set to 1 Da in both scan modes. The UHPLC system was controlled with MassLynx V4.1 SCN 901 (Waters, Milford, MA, USA). The control of mass spectrometer, data acquisition and processing were performed by Xcalibur 4.3 software (Thermo Fisher Scientific, Waltham, MA, USA). In whole blood samples, the ecdysteroids were reliably confirmed by UHPLC-HRMS/MS analysis which were based on literature data and the fragmentation pattern of the reference standards (Hornok et al. 2019). The mass tolerance for the processing of UHPLC-HRMS spectra was set to 5 ppm. Three replicate quantitative analyses of samples were performed by external calibration using an internal standard and the linear calibration curves of ecdysteroids were based on analyte/IS peak area ratios against concentrations.