Development of the Comprehensive Method for Steroid Analysis by GCxGC-HR-TOFMS

Importance of the Topic

Comprehensive two-dimensional gas chromatography coupled to high-resolution time-of-flight mass spectrometry (GCxGC-HR-TOFMS) addresses the challenge of separating closely related steroid isomers, ensuring reliable detection and quantitation in biological and complex samples.

Study Objectives and Overview

The study aimed to develop a workflow for analyzing 33 steroid standards spanning progestogens, androgens, estrogens, glucocorticoids, and mineralocorticoids in a single run. A custom accurate mass library was created, and a multidimensional separation method was optimized to achieve baseline resolution of all analytes.

Methodology

Steroid standards were derivatized by reacting carbonyl groups with methoxyamine HCl followed by silylation using MSTFA+1% TMCS or TMSI. Sample preparation was automated via a GERSTEL Double Rail Autosampler, performing drying, reagent addition, heating, and agitation. Data acquisition and processing utilized ChromaTOF software along with Simply GCxGC for chromatographic method development and accurate mass library construction.

Used Instrumentation

• Agilent 7890 Gas Chromatograph with cryogenic thermal modulator
• LECO Pegasus HRT+ 4D high-resolution TOFMS (25 000 resolving power at m/z 219)
• GERSTEL Double Rail Autosampler
• ChromaTOF and Simply GCxGC software platforms
• HP-1MS first-dimension column and BPX-50 second-dimension column

Key Results and Discussion

All 33 steroids were successfully separated and identified within complex mixtures. The accurate mass library facilitated definitive compound assignment based on molecular ions, major fragments, and retention indices. Contour plots illustrated clear baseline resolution of isomeric pairs, demonstrating the method's robustness for both targeted and non-targeted steroid screening.

Benefits and Practical Applications

The developed GCxGC-HR-TOFMS workflow offers high sensitivity, enhanced separation, and accurate mass confirmation, making it suitable for clinical diagnostics, anti-doping control, pharmaceutical research, and quality assurance in complex matrices.

Future Trends and Potential Applications

Further work will focus on optimizing derivatization protocols for biological fluids, validating the method with urine samples spiked with steroids, and integrating high-throughput automation. Expansion of the accurate mass library and coupling with complementary techniques will broaden the scope of steroidomics studies.

Conclusion

GCxGC-HR-TOFMS combined with a custom accurate mass library provides a powerful platform for comprehensive steroid analysis, delivering reliable separation and confident identification of isomeric metabolites in complex samples.

References

1. Groessl M, et al. Comprehensive Steroid Analysis by GCxGC-TOFMS. Proceedings of the 65th ASMS Conference on Mass Spectrometry and Allied Topics; 2017.
2. LECO Corporation. Simply GCxGC Software; Accessed 2023.