Quantitative Determination of a Panel of Endogenous Steroids in Human Serum by LC/MS/MS - Using an Agilent Supported Liquid Extraction (SLE) Chem Elut S Plate

Importance of the Topic

In clinical research, accurate quantification of endogenous steroids in human serum is essential for hormonal profiling, disease diagnosis, and therapeutic monitoring. Liquid chromatography tandem mass spectrometry offers high specificity but requires robust sample preparation to remove matrix interferences and phospholipids while maintaining analyte recovery and throughput.

Objectives and Study Overview

This study aimed to develop and validate a high throughput supported liquid extraction workflow using Agilent Chem Elut S 2 mL 96 well plates for simultaneous determination of 15 endogenous steroids in human serum by LC MS MS. Method performance was compared with traditional liquid liquid extraction and diatomaceous earth based SLE in terms of recovery, reproducibility, matrix effect and phospholipid depletion.

Methodology and Instrumentation

Serum samples were spiked with stable isotope labelled internal standards then loaded onto Chem Elut S plates. Analytes were eluted by gravity using three sequential 400 microliter aliquots of methyl t butyl ether ethyl acetate 1 1 mixture. Extracts were dried under nitrogen at 40 degrees C and reconstituted in 1 1 methanol water. LC separation employed an Agilent ZORBAX RRHD Eclipse Plus C18 column with a gradient of 0.2 mM ammonium fluoride in water and methanol. Detection was performed on an Agilent G6490 triple quadrupole mass spectrometer with JetStream electrospray ionization in dynamic MRM mode.

Used Instrumentation

• Agilent 1290 Infinity II UHPLC system
• Agilent 6490 Triple Quadrupole LC MS MS
• Agilent Chem Elut S 2 mL 96 well plates
• 96 well positive pressure manifold and nitrogen evaporator

Main Results and Discussion

Calibration curves covered 5 to 10000 pg per mL for most steroids with R squared greater than 0.99 using 1 over x2 weighting. Limits of quantitation were 5 pg per mL for most analytes with slightly higher values for estradiol, testosterone and selected corticosteroids due to matrix interferences. Three day accuracy ranged from 80 to 120 and inter day precision RSD was below 15. Compared to liquid liquid extraction, the Chem Elut S workflow increased recoveries by 10 to 20 and reduced labor and risk of emulsion formation. Relative to diatomaceous earth based SLE, the synthetic sorbent delivered more consistent water holding capacity well to well, improved reproducibility and equivalent or better recoveries. The supported liquid extraction also achieved greater phospholipid depletion than traditional LLE and competitor SLE, enhancing method cleanliness and sensitivity.

Benefits and Practical Applications

• Significant labor and time savings over traditional LLE
• High throughput 96 well plate compatibility and automation readiness
• Robust recoveries and reproducibility across 15 steroids
• Enhanced matrix cleanup and phospholipid removal
• Wide calibration range suitable for clinical research and QA QC

Future Trends and Applications

Integration of supported liquid extraction into automated platforms will further increase throughput and reproducibility. Extending this approach to additional small molecules or protein bound analytes could broaden clinical and industrial applications. Coupling with high resolution or ion mobility mass spectrometry may enable even lower limits of detection and improved structural elucidation. Ongoing advances in sorbent materials and micro extraction formats are anticipated to drive future innovations in sample cleanup workflows.

Conclusion

The Agilent Chem Elut S supported liquid extraction method offers a streamlined, reliable and sensitive workflow for quantifying a panel of endogenous steroids in human serum by LC MS MS. Its compatibility with 96 well automation, superior reproducibility and matrix cleanup make it highly suited for clinical research and high throughput analytical laboratories.

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