High speed UHPLC-MS/MS determination of multiple steroids in human serum using the Nexera MX system for multiplex analysis

Significance of the Topic

Steroid hormones are pivotal regulators of metabolism, inflammation and immune responses. Accurate profiling of multiple steroids supports diagnosis and monitoring of endocrine disorders such as congenital adrenal hyperplasia, Cushing’s syndrome and polycystic ovarian syndrome. Conventional immunoassays often lack specificity and multiplex capability. High-speed UHPLC-MS/MS addresses these limitations by enabling simultaneous quantification of numerous steroid analytes with high sensitivity and selectivity, representing a valuable advancement for clinical laboratories and research settings.

Objectives and Study Overview

This study aimed to adapt a validated UHPLC-MS/MS method for simultaneous determination of a panel of 16 steroids in human serum onto the Shimadzu Nexera MX dual-stream multiplex LC-MS/MS platform. The objectives were to compare chromatographic performance, calibration linearity, limits of quantification and method precision under overlapping injection conditions versus a conventional single-stream approach, and to quantify improvement in sample throughput.

Methodology and Sample Preparation

Synthetic surrogate serum was spiked with target steroids and stable-isotope internal standards. After dilution and loading onto an SLE+ cartridge, steroids were extracted with dichloromethane, evaporated and reconstituted in water/methanol (1:1, v/v). Samples were maintained at 5 °C prior to injection. Calibration and QC samples were prepared to match extracted matrix concentrations.

Instrumentation Used

• UHPLC System: Shimadzu Nexera MX Dual Stream with two parallel LC channels.
• Mass Spectrometer: Shimadzu LCMS-8050/8060 triple-quadrupole with HESI source.
• Column: Restek Raptor Biphenyl, 2.7 µm, 50 × 3 mm (two columns).
• Mobile Phases: Water and methanol with additives; flow rate 0.8 mL/min; injection 30 µL; column 30 °C.
• Gas Settings: Nebulizing 3.0 L/min N₂, drying 10 L/min N₂, heating 10 L/min air; interface 400 °C, DL 150 °C, block 500 °C.
• Dual-Stream Gradient: Offset gradients on each channel to maximize continuous operation.

Key Results and Discussion

Calibration curves exhibited excellent linearity from low-pg/mL to mid-ng/mL levels with LOQs between 0.5 and 100 pg/mL. Comparison of single-stream and dual-stream modes showed negligible differences in accuracy (<10% bias) and precision (RSD ≤5%). QC samples at two concentration levels achieved accuracy within ±10% and RSD below 5% for key analytes including 17β-estradiol and androstenedione. Chromatographic overlays confirmed consistent retention times and peak shapes under overlapping injection conditions, demonstrating the robustness of the multiplexed workflow.

Benefits and Practical Applications

• Up to two-fold increase in sample throughput without expanding instrument footprint.
• Maintained analytical performance in sensitivity, precision and accuracy.
• Optimized MS duty cycle by reducing idle times between injections.
• Ideal for large-scale clinical surveys, routine hormone panels and specialized endocrinology diagnostics.

Future Trends and Opportunities

Emerging directions include expanded steroidomics panels, fully automated sample preparation, integration with advanced data processing for real-time QC, and extension to other small-molecule biomarkers. Advancements in column technologies and high-resolution MS promise further gains in selectivity, throughput and analytical depth for complex biological matrices.

Conclusion

The transfer of a high-speed UHPLC-MS/MS steroid panel to the Nexera MX dual-stream platform successfully doubled sample throughput while preserving stringent analytical standards, offering a scalable solution for high-capacity, reliable hormone quantification workflows.

References

• Loftus N, Moreau S, Levi M, Grüning A. High speed UHPLC-MS/MS determination of multiple steroids in human serum using the Nexera MX system. MSACL 2016 EU.