Ultra-fast Multiplexed LC/MS/MS Using Newly Designed Online SPE Column for the Simultaneous Analysis of Steroid Hormones in Human Serum

Significance of the Topic

Multiplexed online SPE-LC/MS/MS enables rapid, high-throughput and sensitive measurement of steroid hormones in human serum. Steroid profiling is critical in clinical diagnostics and pharmaceutical research but challenged by low analyte concentrations and complex matrices. Integrating online solid phase extraction (SPE) with a four-channel LC/MS/MS platform addresses sample preparation bottlenecks, reduces matrix effects and enhances reproducibility.

Objectives and Study Overview

The study aimed to develop and validate an ultra-fast, multiplexed LC/MS/MS method for simultaneous quantitation of 17 steroid hormones in human serum. Key goals included achieving baseline separation of isobaric steroids, establishing linear calibration over a wide dynamic range, confirming accuracy and precision at multiple QC levels, and demonstrating improved sample throughput.

Methodology and Instrumentation

• Sample Preparation: Protein precipitation of serum with acetonitrile, centrifugation, nitrogen drying and reconstitution in 50% methanol.
• System Configuration: Nexera QX equipped with a four-channel online SPE module paired with the LCMS-8060NX triple quadrupole mass spectrometer.
• Columns: Prototype trap column (1.0 × 50 mm) for SPE; biphenyl analytical column (2.1 × 50 mm, 1.7 μm).
• Mobile Phases: A – 0.2 mM ammonium fluoride in water; B – 0.2 mM ammonium fluoride in methanol; C – 0.1% formic acid in a MeOH:ACN:IPA:water (1:1:1:1) mixture.
• Mass Spectrometry: Heated electrospray ionization at 400°C interface temperature, 150°C DL temperature, 500°C heat block, optimized gas flows for nebulizing, heating and drying.
• Workflow Steps: Alternating injection streams trap analytes on SPE columns, elute to the analytical column, wash the SPE columns and perform sequential analysis across four channels.
• Carryover Control: Automated needle rinse and column washing effectively eliminated residual analyte peaks in blank injections.

Main Results and Discussion

• Chromatographic Resolution: Achieved complete baseline separation of isobaric pairs such as 17-hydroxyprogesterone vs 11-deoxycorticosterone and 21-deoxycortisol vs 11-deoxycortisol vs corticosterone.
• Calibration and Accuracy: Linear dynamic range from 1 to 10,000 pg/mL for 18 steroids, correlation coefficients (R²) greater than 0.993, and accuracy within 85–115% at low, mid and high QC levels.
• Carryover Reduction: Needle inner rinse reduced carryover to below detectable limits after 10,000 pg/mL injections.
• Throughput Enhancement: Single-stream analysis required 10.3 min per injection; four-channel multiplexing halved cycle time to 5.0 min, effectively doubling sample throughput.

Benefits and Practical Applications

• High Sensitivity: Low detection limits facilitate clinical measurement of trace-level steroids.
• Wide Dynamic Range: Accommodates diverse concentration levels across hormone panels.
• Automated Workflow: Online SPE streamlines sample preparation, reducing hands-on time and potential errors.
• Enhanced Throughput: Multiplexed channels support large-scale studies and high-volume testing in clinical and pharmaceutical laboratories.

Future Trends and Applications

Multiplexed online SPE-LC/MS/MS is poised for integration with automated sample handling and advanced data processing. Expanding SPE column chemistries, adopting high-resolution mass analyzers and leveraging machine learning for spectral deconvolution could extend analyte coverage and refine quantitation. Cloud-based data management and real-time quality control may further enhance clinical and research workflows.

Conclusion

The developed four-channel online SPE-LC/MS/MS method delivers rapid, robust quantitation of steroid hormones with excellent chromatographic separation, sensitivity and throughput. This platform offers a powerful solution for high-volume clinical assays and research studies by minimizing sample pretreatment and maximizing analytical performance.

References

1. Imoto E., Johnson V., Miller L., Fujimura D., Matsubara T. Ultra-fast Multiplexed LC/MS/MS Using Newly Designed Online SPE Column for the Simultaneous Analysis of Steroid Hormones in Human Serum. Shimadzu Scientific Instruments R&D Center (US); Application Development Center (US); Shimadzu Corp. Lab Consumables Business Unit (Japan).