title: Quantification of 2-hydrazinopyridine derivatized steroid hormones in fathead minnow (Pimephales promelas) blood plasma using LC-ESI+/MS/MS.
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: 17α-hydroxypregnenolone, progesterone, 11-ketotestosterone, 11-deoxycortisol and 17α,20β-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 mm×50 mm with a 3.5 μm 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 17α-hydroxypregnenolone, progesterone and17α,20β-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 17α-hydroxypregnenolone, 11-deoxycortisol and 17α,20β-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.
authors.affiliation: Department of Biology, Institute of Applied Sciences, University of North Texas, 1704 West Mulberry, Denton, TX 76203, USA.
authors.forename: D
authors.initials: D
authors.lastname: Hala
authors.forename: M D
authors.initials: MD
authors.lastname: Overturf
authors.forename: L H
authors.initials: LH
authors.lastname: Petersen
authors.forename: D B
authors.initials: DB
authors.lastname: Huggett
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D003530:Cyprinidae / Q000097:blood*; D005260:Female; D008297:Male; D011282:Pregnenediones / Q000097:blood*; D011728:Pyridones / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D013739:Testosterone / Q000031:analogs & derivatives* / Q000097:blood
chemical_list: D011282:Pregnenediones; D011728:Pyridones; D013739:Testosterone; C062607:2-hydrazinopyridine; C003600:11-ketotestosterone