Development and application of a UHPLC‐MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum

Abstract

A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone, androsterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisol, corticosterone, aldosterone, 11-deoxycortisol, 11-deoxycorticosterone, dihydrotestosterone, estrone and estradiol) has been developed in equine serum using the ultra-high-performance liquid chromatography-tandem mass spectrometry technique. A total of 400 µl of sample was deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in ultra-high-performance liquid chromatography-tandem mass spectrometry triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60% and with the percentage of coefficient of variation lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922-0.9986, and the limits of detection and limits of quantification were in the range of 0.002-2 and 0.0055-5.5 ng ml , respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml ) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of the steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25-51.26 ng ml ) and cortisol (range 32.57-52.24 ng ml ), followed by 17-OH-pregnenolone, dihydrotestosterone and pregnenolone. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous analysis by LC–MS/MS of 22 ketosteroids with hydroxylamine derivatization and underivatized estradiol from human plasma, serum and prostate tissue

“…However, the majority of the published LC-MS methods have been developed for the analysis of steroids from serum samples. These include also very sensitive methods; some of these tend to have focused only on a few steroids [6,7], while others have examined up to dozens of steroids, but with somewhat greater lower limits of quantification (LLOQ) [8][9][10][11]. There are much fewer methods intended to analyze steroid profiles from different tissues, such as the prostate [12][13][14][15][16].…”

Compensate for or Minimize Matrix Effects? Strategies for Overcoming Matrix Effects in Liquid Chromatography-Mass Spectrometry Technique: A Tutorial Review

“…According to the FDA definition, when no blank matrix is available, it may be impossible to find a matrix similar to the sample without the target analytes, because they are ubiquitous substances, such as hormones [55] in plasma or serum. But in practice, a blank matrix may be unavailable simply because quantities are limited, for example in the case of cerebrospinal fluid (CSF) [56] or previous samples.…”

“…The use of surrogate matrix could be a useful approach, especially for such biological matrices as urine [71][72][73], serum [55,70], and cerebrospinal fluid [74]. Surrogate matrices reported in the literature are neat solvent, stripped and artificial matrices.…”

Liquid chromatography–mass spectrometry applications for quantification of endogenous sex hormones

“…The investigators at NIST have revealed the photosensitivity of progesterone, which must be considered during sample handling, which in their case includes a pH adjustment (pH 9.8) followed by hexane LLE (Tai et al, 2006). The m / z transition used for +ESI multiple reaction monitoring (MRM) (314.8 ➔ 97.1) is the same as or similar to what is used in many other progesterone assays moving forward (Desai, Harwood, & Handelsman, 2019; Farke et al, 2011; Gaikwad, 2013; Galligan et al, 2018; Gao et al, 2015; Genangeli et al, 2017; Gibson, Bucknall, Golebiowski, & Stapleton, 2019; Gomez‐Gomez et al, 2020; Häkkinen et al, 2018; Higashi et al, 2011; Lee, Lee, Hong, Chung, & Choi, 2016; McCulloch & Robb, 2017; Olisov et al, 2019; Schiffer, Adaway, Baranowski, Arlt, & Keevil, 2018; Surowiec, Koc, Antti, Wikström, & Moritz, 2011; Tai et al, 2006; Voegel et al, 2018; Zhou & Cai, 2020).…”

“…The relative “cleanliness” of the plasma and serum matrices allows some investigators to rely on protein precipitation alone to prepare samples for LC–MS/MS. Not surprisingly, the most frequently used solvents include methanol (Broccardo et al, 2013; McCulloch & Robb, 2017; Xu et al, 2017) and acetonitrile (Subhash Chandra Bose et al, 2013; Genangeli et al, 2017). The relevance of this approach is emphasized by the fact that most testosterone in circulation is protein bound (Tai et al, 2007).…”