Absolute Analytical Sensitivity Utilizing the new Xevo™ TQ Absolute IVD for the UPLC™- MS/MS Analysis of a Panel of Steroid Hormones for Clinical Research

Significance of the Topic

The accurate quantification of steroid hormones is critical in clinical research due to their role in metabolism, immune function, salt and water balance, and sexual development. Traditional immunoassays suffer from cross-reactivity and limited specificity. UPLC-MS/MS overcomes these limitations by providing high sensitivity, selectivity, and precision, enabling reliable profiling of structurally related steroids in complex biological matrices.

Aims and Study Overview

This study evaluates the analytical performance of the Xevo TQ Absolute IVD mass spectrometer coupled to an ACQUITY UPLC I-Class FL system for quantifying a panel of six steroid hormones in only 100 µL of serum. Key objectives included demonstrating sensitivity, linearity, precision, and robustness of the method for clinical research applications.

Methodology and Instrumentation

The workflow combines automated SPE sample preparation and fast UPLC-MS/MS analysis:

• Sample Preparation: 100 µL serum processed on a Hamilton STAR robot using protein precipitation followed by Oasis MAX µElution SPE to remove interferences.
• Chromatography: Waters CORTECS UPLC C18 column with a water/methanol/ammonium fluoride gradient, total cycle ~7 minutes.
• Mass Spectrometry: Xevo TQ Absolute IVD with StepWave XS optics and Xtended Dynamic Range for MRM detection of androstenedione, 17-OHP, DHEA, progesterone, DHT, and testosterone.

Main Results and Discussion

Baseline separation of all isobaric analytes was achieved with consistent retention times. Calibration curves for each hormone exhibited linearity (r2>0.99) across clinical ranges with biases within ±15%. Precision studies showed within-run and total CVs ≤6.8% at low, mid, and high QC levels. Low-level detection limits (CV<20%, S/N>10) were 20.8 pg/mL for androstenedione, 17.9 pg/mL for 17-OHP, 11.9 pg/mL for progesterone, and 3.3 pg/mL for testosterone.

Benefits and Practical Applications

• High sensitivity and selectivity allow accurate quantification of multiple steroids from minimal sample volumes.
• Robust method suitable for high-throughput clinical research and biomarker studies.
• Automated workflow reduces hands-on time and variability.

Future Trends and Possibilities

The integration of advanced MS instrumentation with automated sample preparation is expected to expand multiplexed panels, support emerging biomarkers, and advance personalized medicine. Trends include further miniaturization, increased throughput, and incorporation into regulated IVD workflows for diagnostic applications.

Conclusion

The Xevo TQ Absolute IVD coupled with UPLC offers an efficient and sensitive solution for comprehensive steroid hormone analysis, delivering excellent linearity, precision, and low detection limits from small serum volumes, with potential to enhance clinical research studies.

Reference

Waters Corporation. Absolute Analytical Sensitivity Utilizing the new Xevo TQ Absolute IVD for the UPLC-MS/MS Analysis of a Panel of Steroid Hormones for Clinical Research. Application Note, March 2023.