Abstract
Rationale: Wildlife scientists are quantifying steroid hormones in a
growing number of tissue types and employing novel methods which must
undergo validation before application. This study tested the accuracy
and precision of liquid chromatography-tandem mass spectrometry
(LC-MS/MS) methods for use on blubber samples from short-finned pilot
whales (Globicephala macrorhynchus ). We expanded upon a method
for corticosteroid quantification by increasing the number of analytes
and optimizing internal standards application.
Methods: We optimized a method for the quantification of seven steroid
hormones using LC-MS/MS with a C18 column. We assessed the accuracy and
precision of this updated C18 method and an existing Biphenyl method for
use with short-finned pilot whale blubber tissue by conducting a
spike-recovery experiment and calculating percent recovery and relative
standard deviation (RSD) for each analyte. To explore the potential for
running this method more cost-effectively with fewer matched internal
standards (IS), we compared the performance of multiple internal
standards for each analyte.
Results: The C18 method produced reliable quantitation for the seven
target adrenal steroids. The measurement of all 11 adrenal and gonadal
analytes was both accurate and precise, with percent recoveries between
82 % to 110 % and RSDs below 10 %. IS comparisons showed 10 of 11
analytes could be calculated accurately and precisely with at least one
of the IS substitutes. Though many internal standard substitutions met
percent recovery and RSD requirements, some of these substitutions
significantly altered the analyte concentrations calculated.
Discussion: The methods developed and tested in this study provide
reliable detection and quantification of 11 steroid hormones, including
DHEA, which has not been previously quantified in blubber. These methods
can be used for more comprehensive assessments of adrenal and gonadal
steroid hormones from whales. Laboratories can reduce costs through IS
substitution but should consider how these substitutions might affect
results.