Quantification of 2-hydrazinopyridine derivatized steroid hormones in fathead minnow (Pimephales promelas) blood plasma using LC-ESI+/MS/MS.
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abstract
Fathead minnows (Pimephales promelas) comprise a species-of-choice for the hazard assessments of various environmental contaminants, including compounds capable of disrupting endocrine function. Towards this end, the use of liquid chromatography coupled with mass spectrometry (LC-MS) and/or tandem mass spectrometry (MS/MS) is gaining common use for the quantification of steroid hormones as biomarkers of endocrine stress in small-fish toxicological studies. In this work, 2-hydrazinopyridine (2-HP) was used to derivatize and quantify the physiologically relevant steroid hormones of: 17-hydroxypregnenolone, progesterone, 11-ketotestosterone, 11-deoxycortisol and 17,20-dihydroxypregnenone, in the blood plasma of male and female fathead minnows. Liquid chromatographic separation was achieved using a Waters Sunfire C(18) column (2.1 mm50 mm with a 3.5 m particle size) and Milli-Q water:methanol (both with 0.1% formic acid) mobile phase over a gradient of 15 min. All mass analyses were conducted using electrospray ionization in the positive mode with tandem mass spectrometry (ESI+/MS/MS). This is the first such application of 2-HP derivatization for the quantifications of the structurally and functionally diverse C19 androgen of 11-ketotestosterone; C21 progestogens of 17-hydroxypregnenolone, progesterone and17,20-dihydroxypregnenone; and C21 corticosteroid of 11-deoxycortisol, in fathead minnow blood plasma. The limits of detection (LOD) were set to the lowest calibration standard that gave a signal-to-background response of 3, and were: 0.16 ng/ml for progesterone, 0.63 ng/ml for 17-hydroxypregnenolone, 11-deoxycortisol and 17,20-dihydroxypregnenone, and 1.25 ng/ml for 11-ketotestosterone. This study demonstrates the application of 2-HP derivatization for the analysis of a variety of steroid hormones representative of endocrine function in a species of fish commonly used in toxicological studies.
