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Abstract INTRODUCTION
The Tecan Steroid Panel LC-MS Kit is widely used for the quantitative determination of a broad spectrum of steroids in human serum and plasma. The standard protocol, as described in the Instructions for Use (IFU), includes a validated solid phase extraction (SPE) and a drying step using an external evaporator at elevated temperature. However, the need for additional equipment and the desire for streamlined, automated workflows have prompted the development of alternative methods. Having a completely automated workflow is needed by some laboratories, whereas other investigators prefer a semi-automated solution, offering more flexibility and options. This study aimed to optimize the sample preparation process by introducing a new drying method directly on the Tecan Resolvex A200 system using a nitrogen stream using positive pressure, and to evaluate its comparability to the established protocol. Nevertheless, this would be the first step to set up a complete automated workflow.
METHODS
The new drying method involved evaporating eluates at ambient temperature (18–21°C) under a nitrogen stream at 6 bar, eliminating the need for a separate evaporator. Feasibility was assessed through six independent runs, each including calibration standards, controls, and spiked samples. In order to compare the streamlined drying method with the external evaporation, spiked serum and plasma samples were analyzed. Measurements were conducted several times, over a timespan of days and with different lots to eliminate possible variabilities. For method comparison, a parallel study was conducted: one set of samples was processed according to the IFU (drying at 40°C on an external evaporator), while the other set was dried on the Resolvex A200 using the streamlined method under ambient temperatures using an evaporation plate. Quantification was performed by LC-MS/MS according to IFU, and results were evaluated using Passing-Bablok regression and correlation analysis to assess agreement between methods according to CLSI guideline EP09-A3.
RESULTS
The new drying method achieved complete evaporation of eluates within 58–90 minutes, depending on matrix composition, at ambient temperature. The deviations in drying times are caused by room temperature variances and the sample matrix. Therefore, for setting the drying time in an automation protocol, worst-case conditions were tested and led to a drying time of 90 min in the streamlined protocol. Overall, feasibility studies demonstrated high reproducibility across six independent runs, whereby all controls met their specific ranges mentioned in the QC certificate.
In the method comparison study, results from the new and standard protocols showed excellent agreement for all 18 steroid analytes. Passing-Bablok regression coefficients (R) ranged from 0.947 to 1.00, with most analytes exceeding 0.99, indicating strong linear correlation. All regression intercepts fell within the 95% bootstrap confidence intervals. For key analytes such as androstenedione, aldosterone, and progesterone, the regression lines were nearly identical to the line of identity, confirming method equivalence. Slight discrepancies observed for DHEA and DHEAS were traced to chromatographic column aging rather than the extraction or drying method. Overall, the alternative protocol provided results fully comparable to the IFU, supporting its use as a valid alternative in research workflows.
CONCLUSION
This study demonstrates that this streamlined protocol on the Tecan Resolvex A200 is fully comparable to the standard IFU protocol for the Tecan Steroid Panel LC-MS Kit. The alternative approach eliminates the need for additional drying equipment, streamlines the workflow, and supports further automation in sample preparation. The agreement between methods across a wide range of steroid analytes confirms that users can confidently adopt these alternative procedures without compromising data quality. Any deviation from the Instruction for Use will need to be validated by the customer. These findings expand the flexibility of sample preparation and pave the way for further integration of automated solutions in steroid quantification by LC-MS. |
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
