Sensitive LC-MS/MS Assay for Total Testosterone Quantification on Unit Resolution and High-Resolution Instruments

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Testosterone is a vital androgenic hormone with broad physiological roles.
• Existing immunoassays struggle with accuracy at low total testosterone concentrations (<100 ng/dL), impacting female and pediatric testing.
• Accurate measurement of total testosterone is crucial for various clinical diagnostics.

Purpose of the Study:

• To develop and validate a precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for total testosterone.
• To enhance accuracy in quantifying low testosterone levels.
• To ensure assay harmonization across different mass spectrometry platforms.

Main Methods:

• Serum samples underwent dilution and conditioning.
• Analyte extraction utilized supported liquid extraction cartridges.
• Chromatographic separation was performed on a C18 column with a 5-minute runtime.
• Assay validation was conducted on Triple Quad 6500, API 4500, and Orbitrap instruments per CLSI guidelines.

Main Results:

• Achieved a limit of quantitation of 0.5 ng/dL and a measurement range of 2-1200 ng/dL.
• Demonstrated excellent method validation with within-day CV <10% and between-day CV <15%.
• Correlated well with a reference method, showing consistency across TQ 6500, API 4500, and Orbitrap platforms.
• Confirmed suitability of both serum-separator and serum-activator tubes.

Conclusions:

• A robust and validated total testosterone LC-MS/MS assay is established for routine clinical use.
• The assay provides accurate quantification, especially at low concentrations.
• Successful harmonization across triple quadrupole and high-resolution mass spectrometers ensures broad applicability.