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2016-01-28 - Colloque/Présentation - communication orale - Allemand - 1 page(s)

Hambye Stéphanie , Tonoli David, Jeanneret Fabienne, Rudaz Serge, "A new UHPLC-MS/MS method for the determination of testosterone, androstenedione, and 17a-hydroxyprogesterone in human serum using isotopic internal calibration. A new way for endogenous steroids quantification?" in HTC-14 ; 14th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology, Ghent, Belgique, 2016

  • Codes CREF : Chimie analytique (DI1314), Pharmacocinétique (DI3431), Sciences pharmaceutiques (DI3400), Techniques séparatives (DI2729)
  • Unités de recherche UMONS : Analyse pharmaceutique (M130)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé)

Abstract(s) :

(Anglais) Quantification of steroids in biological matrices is a challenging area of research. Identification and quantification of this particular family of compounds can be problematic especially because of their structural similarities and their relatively low concentrations. Strategies of quantification in bioanalysis are mainly performed using external calibrations, which can be time and money consuming. Moreover, the inherent presence of endogenous steroids in biological fluids at unknown concentrations makes the use of surrogate steroid-free matrices necessary to build calibration curves. To overcome these drawbacks, an UHPLC-MS/MS quantification method based on isotopic internal calibration was evaluated to quantify steroids. For this purpose, three representative endogenous steroids present in human serum, namely androstenedione (AD), 17-hydroxyprogesterone (17-OHP) and testosterone (T), were selected. Instead of quantifying by interpolation from external calibration curves, levels of targeted steroids (CA) were determined from the Analyte (A)/internal standard (IS) area ratio, the concentration of the isotopic internal standard (CIS) and a predetermined response factor (RF) (see relation 1). The latter was established as the signal ratio between equimolar solutions of the analyte and its corresponding isotopic labelled standard. Deuterated (D) compounds were selected as IS (T-d3, AD-d7 and 17-OHP-d8). C_A= (Area A)/(Area IS) ×C_IS×RF (1) The UHPLC-MS/MS method was developed and optimized on a triple quadrupole (QqQ) instrument equipped with an ESI source operating in positive mode. The Selected Reaction Monitoring (SRM) transitions monitored were m/z 289.2>109.0 (T), 292.2>109.0 (T-d3), 287.2>109.1 (AD), 294.1>113.0 (AD-d7), 331.2>109.0 (17-OHP), 339.2>100.0 (17-OHP-d8). Chromatographic separation was performed on a core-shell stationary phase (Kinetex C18 column, 1.7 µm, 150 x 2.1 mm) using a linear gradient of water and acetonitrile, supplemented with 0.1 % formic acid [1]. The method includes an extraction step, based on supported liquid extraction (SLE) on a 96-well plate format, which is quick and convenient when dealing with numerous samples. The method was validated according to the SPSTF guidelines, using the accuracy profiles strategy [2]. Accuracy profiles were built, with two independent samples (n=2) at four levels of concentration (k=4), repeated on 6 different series (j=6). Acceptability limits were set to the conventional level of ± 30 % and β=80%, as recommended by the FDA for bioanalyses. The validated concentration ranges were confirmed from 0.1 to 10 ng/mL, 0.2 to 4 ng/mL and 0.4 to 4 ng/mL, for testosterone, 17-OHP and androstenedione, respectively. The developed method represents an original and rapid strategy of quantification that perfectly fits the needs for the clinical determinations of the tested analytes. Furthermore, this concept could easily be extended to other steroids of interest. 1. Tonoli, D., et al., Steroidomic Footprinting Based on Ultra-High Performance Liquid Chromatography Coupled with Qualitative and Quantitative High-Resolution Mass Spectrometry for the Evaluation of Endocrine Disrupting Chemicals in H295R Cells. Chemical Research in Toxicology, 2015. 28(5): p. 955-966. 2. Hubert, P., et al., Harmonization of strategies for the validation of quantitative analytical procedures: A SFSTP proposal—part I. Journal of Pharmaceutical and Biomedical Analysis, 2004. 36(3): p. 579-586.