Analysis of androgenic steroid Girard P hydrazones using multistage tandem mass spectrometry

Abstract

Seven androgenic steroids have been converted into steroid hydrazones using Girard P hydrazine and analysed by electrospray ionisation multistage tandem mass spectrometry. The cationic derivatives 17alpha-testosterone hydrazone, 17beta-nortestosterone hydrazone, 17beta-bolasterone hydrazone, 17alpha-boldenone hydrazone, 17beta-fluoxymesterone hydrazone, 17alpha-trenbolone hydrazone and 4-chloroandrosten-3,17-dione hydrazone show good response in positive ion mode with enhancements for the method of up to 33 times relative to the native species. Detailed characterisation of fragmentation pathways reveals structurally specific ions formed by fragmentation of the hydrazine moiety. Comparison of structural similarities among the androgenic steroids allows recognition of common ions/fragmentation processes as well as analyte-specific transitions. The suitability of the derivatisation approach in the screening of heifer urine for the presence of administered hormones has been demonstrated using partially purified urine spiked with a mixture of androgenic steroids.

Unique potentiometric detection systems for HPLC determination of some steroids in human urine

“…Derivatization temperature and reaction time of 17-KS were chosen to maximize the quantitative yield (A99.9%, as determined by performed UV monitoring of reaction progress at 280 nm) of final acetohydrazone product, and to simplify and optimize maintaining period of this derivatization step as much as possible, when the ethanol/glacial acetic acid (9:1 v/v) was employed as the solvent with pH range of 4 -5, and at least 15-fold excess of the reagent was used. Similar to other reports [24,27,39] we did not observe the degradation of 17-KS in such reaction conditions. In our results there is no evidence of degradation because of the high level of reproducibility of the reaction, and the linearity of the calibration lines as shown in Fig.…”

“…In addition, no contamination of HPLC system with the introduction of excessive quantities of GP was observed. However, in performed additional experiments, when this un-reacted GP reagent was separated from the GP derivatized ketosteroid(s) in calibration solutions or in spiked human urine samples by the offline SPE procedure -as recommended earlier in the case of the LC-MALDI-MS [27][28][29][30] and LC-ESI-MS procedures [24][25][26] -and using a Bakerbond-spe C18 cartridge (500 mg/6 mL, J.T. Baker) with 10% aqueous methanol i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim (6 mL) in the washing step and methanol (1 mL) for final elution, no substantial improvement in the response of developed potentiometric detector was observed in our study (data not shown).…”

LC‐MS/MS profiling for detection of endogenous steroids and prostaglandins in tissue samples

“…In the latter case, the type of matrix and all steps of sample preparation must be considered. Notably, LODs are likely to be lower after derivatization of hormones to their oximes 4, 11, Girard P hydrazones 28, 29 or dansyl derivatives 14, 17, 30, since ESI ionization efficiency is greater, and/or more selective for these derivatives than for the underivatized hormones. However, it should be emphasized that derivatization is usually a group‐characteristic process and hence it can be difficult (if not impossible) to find a generic derivatization method for simultaneous analysis of different classes of compounds.…”

Cerebrospinal Fluid Steroidomics: Are Bioactive Bile Acids Present in Brain?

“…group to enhance their mass spectrometric detection (10,(32)(33)(34)(35)(36)(37)(38). We have selected the Girard P derivative that effectively tags a positively charged quaternary nitrogen group to the steroid skeleton.…”