Development of Comprehensive Steroid Analysis Methods by GCxGC-HR-TOFMS

Importance of the Topic

Identification and quantification of steroid metabolites in biological matrices is critical for diagnosing hormonal disorders and conducting biomedical research. Many steroids exist as structurally similar isomers, complicating their separation and identification by conventional one-dimensional gas chromatography or liquid chromatography. Two-dimensional GC coupled with high-resolution time-of-flight MS (GCxGC-HR-TOFMS) addresses these challenges by enhancing chromatographic resolution, sensitivity, and detection confidence.

Objectives and Study Overview

This study aimed to develop a comprehensive analytical workflow for 33 steroids spanning progestogens, androgens, estrogens, glucocorticoids, and mineralocorticoids. Key objectives included creating an accurate mass library for confident compound identification, optimizing multidimensional separation, and demonstrating method applicability to a mixed standard sample.

Methodology and Instrumentation

Individual steroid standards were derivatized using methoxyamine HCl in pyridine followed by silylation with MSTFA+1% TMCS or TMSI. Derivatized analytes were first analyzed separately to generate high-resolution spectra and build a custom accurate mass library (AML). Comprehensive two-dimensional separations were achieved with an Agilent 7890 GC equipped with a LECO cryogenic thermal modulator, using an HP-1MS first dimension column and a BPX-50 second dimension column. Modulation occurred every 3 seconds. Detection employed a LECO Pegasus HRT+ 4D HR-TOFMS with a mass range of m/z 40–1000 at 200 spectra/s. Data acquisition, AML creation, peak finding, and compound identification were performed using ChromaTOF and Simply GCxGC software.

Used Instrumentation

• Gas chromatograph: Agilent 7890 GC with split injection (1 µL, split 100:1, 250 °C).
• Modulator: LECO cryogenic thermal modulator (modulation period 3 s; hot pulse 0.9 s).
• Columns: 1D HP-1MS (15 m×0.25 mm×0.25 µm), 2D BPX-50 (2 m×0.25 mm×0.25 µm); guard columns installed.
• Mass spectrometer: LECO Pegasus HRT+ 4D HR-TOFMS (R=25 000 at m/z 219).
• Software: ChromaTOF and Simply GCxGC for instrument control and data analysis.

Main Results and Discussion

The custom AML enabled unambiguous identification of all 33 derivatized steroids in the mixed sample. GCxGC contour plots demonstrated baseline separation of closely related isomers. Accurate mass confirmation of molecular ions and key fragment ions, along with retention indices and library matches, provided high confidence in compound assignments. The method also proved suitable for non-targeted steroid screening in complex matrices.

Benefits and Practical Applications

• Enhanced resolution of isomeric steroids, reducing coelution and false positives.
• Improved sensitivity and detection limits through focused peak capacity.
• Accurate mass measurements and library matching enable reliable identification.
• Applicable to clinical diagnostics, doping control, environmental monitoring, and forensic analysis.

Future Trends and Applications

Ongoing work includes optimizing derivatization protocols for biological matrices (e.g., urine) and validating the workflow with spiked and authentic samples. Future developments may integrate automated sample preparation, quantitative calibration, and machine-learning algorithms for pattern recognition in steroidomics.

Conclusion

GCxGC-HR-TOFMS combined with a custom accurate mass library offers a robust, high-resolution platform for comprehensive steroid analysis. The method reliably separates and identifies a wide range of steroid isomers, supporting advanced research and routine applications in biochemical and clinical laboratories.

References

• [1] Grössl M. et al. Comprehensive steroid analysis by GCxGC-TOFMS. Proceedings of the 65th ASMS Conference, Indianapolis, IN, 2017.
• [2] LECO Corporation. Simply GCxGC software overview.