用高分辨率精准质量质谱检测运动员兴奋剂康立龙葡糖苷酸

Detection of Stanozolol Glucuronides in Human Sports Drug Testing by Means of High-Resolution, Accurate-Mass Mass Spectrometry

Key Words

Sports doping, antidoping testing, Q Exactive Focus, long-term metabolite, anabolic agents, 16-oxo-stanozolol, stanozolol glucuronide, epistanozolol

Goal

To demonstrate the utility of direct dilute-and-shoot analysis of glucuronic acid conjugates of stanozolol by means of liquid chromatography and high-resolution, accurate-mass mass spectrometry in sports antidoping testing. To characterize and validate, by means of commercially available 3’-OH-stanozolol glucuronide, “dilute and inject” and confirmation methods, which will allow for the unambiguous identification of stanozolol misuse in routine doping-control samples.

Introduction

The analysis of the anabolic steroid stanozolol (Figure 1a) has proved to be challenging for gas chromatography mass spectrometry (GC-MS) methods due to stanozolol’s peculiar physicochemical properties. The uncovering of stanozolol abuse by means of its major urinary metabolite 3’-OH-stanozolol (Figure 1b) as accomplished by Schänzer and Donike1 initiated investigations into the metabolic fate of this anabolic agent. The molecular features of stanozolol and its metabolites demand sophisticated derivatization and separation steps for GC/MS-based methodologies. Methods based on liquid chromatography with electrospray-ionization tandem mass spectrometry (LC-MS/MS), on the other hand, provide benefits such as lower limits of detection (LODs) and detection windows with expanded metabolite identification. 3’-OH-stanozolol glucuronide (Figure 1c) is the latest metabolite analyzed at 25–50 pg/mL in human urine. In the present study, the use of high-resolution, accurate-mass mass spectrometry for the detection of 3’-OH-stanozolol glucuronide is outlined. Complementary information on N-conjugated glucuronide metabolites of stanozolol and 17-epistanozolol and the use of these in routine doping controls is provided.

Conclusion

In this study, the utility of direct dilute-and-shoot analysis of glucuronic acid conjugates of stanozolol by means of liquid chromatography and high-resolution, accurate-mass mass spectrometry in sports antidoping testing was assessed and demonstrated. Characterized and validated by means of commercially available 3’-OH-stanozolol glucuronide, dilute-and-inject and confirmation methods were established, which allowed for the unambiguous identification of stanozolol misuse in routine doping control samples. Additionally, new long-term metabolites for the detection of stanozolol abuse were observed in administration study urine samples. These new target analytes, assigned as stanozolol-N-glucuronide and 17-epistanozolol-N-glucuronide, were characterized by mass spectrometry, and hydrolysis experiments. Both proved particularly useful as target compounds, enabling the determination of drug abuse for up to 28 days post-administration of 5 mg of stanozolol. Since high-resolution, accurate-mass mass spectrometry has been found to be essential for the successful identification of lowest amounts of stanozolol metabolites in human urine, expansion of its use in doping control is encouraged.