Extraction of a Comprehensive Steroid Panel from Human Urine Using EVOLUTE® EXPRESS ABN Prior to LC/MS-MS Analysis

Significance of the Topic

In clinical and research settings, accurate quantification of steroid hormones in urine is essential for endocrine diagnostics, therapeutic monitoring, doping control and metabolic studies. Comprehensive profiling of multiple steroids demands robust sample preparation to achieve sensitivity, selectivity and reproducibility.

Study Objectives and Overview

This study aimed to develop and validate an efficient solid-phase extraction (SPE) protocol using EVOLUTE EXPRESS ABN plates for the simultaneous extraction of 19 steroid analytes from human urine, followed by LC–MS/MS analysis. Emphasis was placed on high recovery, low variability and compatibility with both manual and automated workflows.

Methodology

An internal standard mix (DHT-D3 and Aldosterone-D3) was spiked into 200 µL urine, diluted 1:1 with water and loaded onto EVOLUTE EXPRESS ABN SPE wells. After optional conditioning and equilibration, the protocol comprised:

• Wash 1 with water (500 µL) and Wash 2 with 60:40 water:methanol (500 µL) to remove interferences
• Elution of steroids with methanol (200 µL)
• Evaporation under nitrogen at 40 °C and reconstitution in 50:50 aqueous/mobile phase B prior to injection

Chromatographic separation employed a C18 UHPLC column (40 °C, 0.4 mL/min gradient of 0.2 mM NH4F and methanol) with a 5 µL injection. MS/MS detection used MRM transitions in both positive and negative modes on a Shimadzu triple quadrupole instrument.

Used Instrumentation

• SPE: EVOLUTE EXPRESS ABN 10 mg 96-well plates, Biotage PRESSURE+ 96 Positive Pressure Manifold; Biotage SPE Dry evaporator
• UHPLC: Shimadzu Nexera X2 with ACE C18 (100 × 2.1 mm, 1.7 µm) and guard cartridge
• MS/MS: Shimadzu 8060 triple quadrupole with electrospray interface

Main Results and Discussion

The optimized SPE method yielded analyte recoveries above 75% (n=7) with RSDs below 5% for most steroids, including polar metabolites like DHEAS when eluted with methanol. Chromatography demonstrated baseline resolution of isobaric steroids at 5 ng/mL spiking. Calibration curves in the 1–1000 pg/mL range exhibited excellent linearity (r2 > 0.999). Limits of quantification ranged from sub-pg/mL to hundreds of pg/mL depending on the analyte.

Benefits and Practical Applications

• Streamlined sample preparation compatible with manual and automated platforms
• High throughput and reproducibility suitable for routine clinical and research laboratories
• Broad dynamic range and low detection limits support diverse applications in endocrinology, doping analysis and metabolic profiling

Future Trends and Opportunities

Advances may include integration with high-resolution mass spectrometry for steroidomics, miniaturized SPE formats to reduce solvent use, and expanded panels incorporating conjugated metabolites. Automated workflows and AI-driven data processing will further enhance throughput and data quality in clinical and environmental monitoring.

Conclusion

The presented SPE–LC–MS/MS workflow enables reliable, high-sensitivity quantification of a comprehensive steroid panel from human urine with strong reproducibility and adaptability to automated platforms, meeting the demands of modern bioanalytical laboratories.