MSACL 2025 Abstract

Flexible Steroid Hormone Quantification: Analytical Equivalence of Two C8 Columns for LC-MS/MS

John Rohloff (1), Ayham Al Ahmad (1), Marcel Willi Debong (1), Juliane Kramer (2)
(1) Tecan, IBL International, Hamburg, Germany, (2) KNAUER Wissenschaftliche Geräte GmbH, Berlin, Germany

John Rohloff, M.Sc. (Presenter) Tecan, IBL International >> POSTER (PDF)

Relevant Financial Disclosures (within past 24 months, reported on Jul 14, 2025) Other Potential Conflicts Tecan, IBL-International, Hamburg, Germany / Employee Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Ensuring the continuous availability of consumables is a key requirement in LC-MS analysis. Relying on a single chromatographic column can create supply chain vulnerabilities and limit laboratory flexibility. This study investigates whether the Tecan Steroid Panel LC-MS, originally developed for a specific C8 column, can also be reliably applied using the KNAUER Eurospher II C8 HPLC column, thereby providing an alternative for robust and flexible steroid hormone quantification.

METHODS:
The Steroid Panel LC-MS method, based on solid phase extraction (SPE) and subsequent LC-MS analysis, was applied to human serum samples from both male and female subjects. Fourteen steroid hormones were measured in parallel using both the original kit-provided C8 column and the KNAUER Eurospher II 100-3 C8 column. For the KNAUER column, only the flow rate was adjusted (from 0.35 to 0.50 mL/min); all other chromatographic and mass spectrometric parameters, as well as sample preparation, remained unchanged. Method comparison was performed on at least 40 samples per analyte, using Passing-Bablok regression and calculation of correlation coefficients (r). Chromatographic separation was evaluated, particularly for analytes with similar m/z transitions.

RESULTS:
The method comparison demonstrated excellent agreement between the two columns for all 14 steroid hormones, with correlation coefficients (r) ranging from 0.909 to 0.998. Passing-Bablok regression analysis confirmed the comparability of quantitative results. Baseline separation was achieved for critical analytes such as 17-OH-progesterone, 11-deoxycorticosterone, cortisone, and cortisol, which are essential for reliable quantification due to overlapping mass transitions. The workflow remained efficient, and no significant changes in sample preparation or instrument handling were required.

CONCLUSION:
The Tecan Steroid Panel LC-MS can be used interchangeably with both the original kit-provided C8 column and the KNAUER Eurospher II C8 column, without compromising analytical performance or reliability. This flexibility enhances supply chain security and supports robust implementation in laboratories. The results provide a foundation for future validations and broader adoption of alternative columns in routine steroid hormone analysis.