Exemplary performance data for steroids

Importance of the Topic

Accurate quantification of steroid hormones in biological specimens underpins clinical diagnostics, therapeutic monitoring, and research into endocrine disorders. Advances in liquid chromatography–tandem mass spectrometry (LC-MS/MS) have improved specificity and sensitivity for low-level analytes, supporting reliable in vitro diagnostics.

Objectives and Study Overview

This application note demonstrates the analytical performance of the Thermo Scientific Vanquish MD HPLC coupled with the TSQ Quantis MD triple-stage quadrupole mass spectrometer for measuring estrone and testosterone in human matrices. The goal is to illustrate method precision, accuracy, linearity, and suitability for routine clinical laboratory use.

Methodology and Instrumentation

Sample Preparation

• Methyl tert-butyl ether extraction
• Transfer of extract into HPLC vials

Calibration and Quality Controls

• Seven-point calibrators and three QC levels prepared by UTAK Laboratories
• Concentration ranges: 0.14 to 7.6 ng/mL for estrone, 0.27 to 6.2 ng/mL for testosterone

Chromatographic Conditions

• Vanquish MD HPLC
• Mobile phase A: 0.5 mM ammonium fluoride in water
• Mobile phase B: methanol
• Run time: 6 minutes

Mass Spectrometry

• TSQ Quantis MD Series with both heated electrospray ionization and APCI
• Polarity modes: negative for estrone, positive for testosterone
• Selected reaction monitoring transitions for quantifier and qualifier ions

Software Tools

• TSQ Quantis MD software for system calibration and diagnostics
• TraceFinder LDT Software for quantitation

Results and Discussion

Linearity and Accuracy

• Calibration curves exhibited R2 ≥ 0.9978 across both ion sources
• %Difference between assigned and measured calibrator values < ±15%

Precision

• Intra-run CV ≤ 7% at low, medium, and high QC levels
• Consistent performance in both HESI and APCI modes

Chromatographic and Spectral Confirmation

• Clear separation of estrone and testosterone peaks within 6 minutes
• Distinct MS2 spectra with reliable quantifier and qualifier ion ratios

Benefits and Practical Applications

Implementation of this LC-MS/MS workflow offers clinical laboratories a robust solution for steroid profiling, enabling high throughput and reproducible results. The modular compatibility of the Vanquish MD HPLC and TSQ Quantis MD MS ensures streamlined validation and maintenance under in vitro diagnostic regulations.

Future Trends and Possibilities

Emerging developments in high-resolution MS and multiplexed assays may further expand analyte panels, while automation of sample prep and data processing could reduce turnaround times. Integration with laboratory information systems will support real-time QC monitoring and compliance.

Conclusion

The Thermo Scientific Vanquish MD HPLC coupled with the TSQ Quantis MD Series mass spectrometer demonstrates reliable steroid quantification with excellent accuracy, precision, and speed. This platform meets rigorous in vitro diagnostic performance criteria and is well suited for clinical steroid hormone analysis.

Reference

Clinical and Laboratory Standards Institute. Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline CLSI document C62-A. Wayne, PA; 2014.