Analysis of glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry

Abstract

Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids, which is expected to have properties including accuracy, specificity, sensitivity, and, if possible, rapidity. In this context, the ability of ultra-high-performance supercritical-fluid chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS-MS) to provide reliable and accurate phase II analysis of steroids was assessed. Four stationary phases with sub-2 μm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH fluorophenyl) were screened for their capacity to separate several conjugated steroid isomers. Analytical conditions including stationary phase, modifier composition and percentage, back pressure, column temperature, and composition and flow rate of make-up solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated, and UHPSFC-MS-MS analysis revealed its ability to provide sensitive (limits of quantification: 0.1 ng mL(-1) and 0.5 ng mL(-1) for sulfate and glucuronide steroids, respectively) and reliable quantitative performance (R(2) > 0.995, RSD < 20%, and bias < 30%) through the use of suitable labeled internal standards. Comparison with UHPLC-MS-MS was performed, and UHPSFC-MS-MS obtained better performance in terms of sensitivity. Finally, as a proof of concept, this so-called green technology was used in a chemical-food-safety context to profile steroid conjugates in urine samples from bovines treated with estradiol. Graphical Abstract Glucuronide and sulfate steroids analysis in urine by ultra-high performance supercritical fluid chromatography hyphenated tandem mass spectrometry.

Identification and characterization of novel long‐term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC‐CI‐MS/MS

“…The ‘steroid profile’, a set of endogenous steroids used as markers for testosterone abuse, is analysed concurrently and the use of E. coli is a regulatory requirement for these steroids. Recent publications however have illustrated the potential advantage of detecting sulfates for long‐term detection of steroid abuse, which resulted in a further increased research focus . In this light, the excretion form of the two new long‐term metabolites, oxyM9 and mestM22, was investigated.…”

“…Recent publications however have illustrated the potential advantage of detecting sulfates for long-term detection of steroid abuse, which resulted in a further increased research focus. [38][39][40][41][42][43][44][45] In this light, the excretion form of the two new long-term metabolites, oxyM9 and mestM22, was investigated. Excretion urine samples were prepared with β-glucuronidase (from E. coli), β-glucuronidase/aryl sulfatase (from H. pomatia) and without enzyme, resulting in the free + glucuronide fraction, free + glucuronide + sulfate fraction and free steroid fraction, respectively.…”

A novel, fast and sensitive supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method for analysis of arachidonic acid metabolites

“…7,18,19 The most important parameter influencing the retention of the analytes was the chemistry of the stationary phase. 13,16 In spite of the fair availability of different UPC 2 columns, with known chemistry of the stationary phases, selecting the best column for the method development still remains challenging. 11 Mass spectrometric sensitivity and overall chromatographic resolution of the target analytes including geometrical isomers such as PGD 2 and PGE 2 provided a basis for the selection of column.…”

“…9,11 Over the years, many studies have demonstrated successful application of SFC for the separation of diverse species of lipids including phospholipids, lysophospholipids, sphingolipids, ceramides, glycerolipids, 10,12 vitamins, 13 fatty acids, 14 oxylipins, 15 and steroids. 16 More importantly, the use of SFC has enabled the comprehensive separation of isomers in very short run time. 11,13,14 Under such context, the aim of this study was to develop a fully validated supercritical fluid chromatography-tandem mass spectrometry method (SFC-MS/ MS) for the rapid identification and quantification of a number of endogenous eicosanoids.…”

Specific characterization of non‐steroidal selective androgen peceptor modulators using supercritical fluid chromatography coupled to ion‐mobility mass spectrometry: application to the detection of enobosarm in bovine urine

“…Supercritical fluid chromatography (SFC) [16] represents an interesting separation alternative that has already shown its potential in enhancing detection performances of residues of growth promoters. [17] Using mainly liquid carbon dioxide as a mobile phase, this separation technique is based on similar retention mechanisms of both gas and liquid chromatography, depending on the mobile phase density, the flow rate, the column temperature and the stationary phase's properties [18] with a larger range of solvent selectivity choice. De facto, it enables an increased separation of relatively similar and numerous chemical compounds as described in for peptides,, [19] drugs, chiral or not, [20] or even lipids separation.…”