Integration of steroids analysis in serum using LC-MS/MS with full-automated sample preparation

Importance of the Topic

Rapid and reliable measurement of steroid hormones in serum is essential for clinical diagnostics, therapeutic monitoring and research in endocrinology. Traditional manual extraction methods are time-consuming, subject to operator error and limit laboratory throughput. Integrating a fully automated sample preparation with high-sensitivity LC-MS/MS analysis addresses these challenges and enhances reproducibility and efficiency.

Objectives and Overview of the Study

This study aimed to develop and validate a streamlined workflow for simultaneous quantification of ten key steroid hormones (cortisol, aldosterone, 11-deoxycortisol, corticosterone, 17-OHP, androstenedione, DHEA, DHEAS, progesterone and testosterone) in human serum. The focus was on coupling an automated sample preparation platform to a triple quadrupole LC-MS/MS system to boost throughput while maintaining analytical performance.

Methodology

The automated protocol employs protein precipitation with acetonitrile containing internal standards, followed by filtration. The filtrate is transferred directly from the CLAM-2000 system to the LC-MS/MS auto-sampler. Chromatographic separation uses a trap-and-elute setup with a MAYI-ODS trap column and a Kinetex Biphenyl analytical column (50×2 mm, 2.6 µm) operated at 40 °C. A binary gradient of 1 mM ammonium fluoride in water (Mobile Phase A) and methanol (Mobile Phase B), supplemented by 10 mM ammonium formate (Mobile Phase C), is applied at 0.3 mL/min over 12 minutes.

Used Instrumentation

• Shimadzu CLAM-2000 automated sample preparation system
• Shimadzu Nexera series HPLC with MAYI-ODS trap and Kinetex Biphenyl column
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer with heated ESI interface

Main Results and Discussion

Calibration curves for all steroids showed excellent linearity (r2>0.997) across clinically relevant concentration ranges. Intra-assay precision (n=3) at seven concentration levels, including the lower limit of quantitation (LLOQ), yielded coefficients of variation below 10%. Automated preparation reduced sample handling time from approximately 60 minutes to 6 minutes. Parallel processing of sample prep and LC-MS/MS runs enabled continuous throughput enhancement.

Benefits and Practical Applications

• Significant reduction in manual labor and associated variability
• High analytical precision and accuracy for steroid profiling
• Enhanced laboratory throughput by enabling parallel workflows
• Applicability to clinical research, endocrinology diagnostics and quality control settings

Future Trends and Applications

Future developments may include multiplexed analysis of broader steroid panels, integration of microfluidic sample handling, and implementation of data-driven QC algorithms. Further miniaturization and coupling with high-resolution mass spectrometry could expand applications to point-of-care testing and personalized medicine.

Conclusion

The combination of fully automated sample preparation with sensitive LC-MS/MS quantification provides a robust, high-throughput solution for steroid analysis in serum. This workflow minimizes human error, enhances reproducibility and supports large-scale studies in clinical and research laboratories.