Direct total and free testosterone measurement by liquid chromatography tandem mass spectrometry across two different platforms

作者：Rhea Jeanne M; French Deborah; Molinaro Ross J^*

来源：Clinical Biochemistry, 2013, 46(7-8): 656-664.

DOI：10.1016/j.clinbiochem.2013.01.005

• Mass spectrometry
• Total testosterone
• Free testosterone
• Serum
• Method comparison
• Internal standard

Objectives: To develop and validate liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the direct measurement of total and free testosterone in patient samples on two different analytical systems.
Design and methods: An API 4000 and 5000 triple quadropoles were used and compared; the former is reported to be 3-5 times less sensitive, as was used to set the quantitation limits. Free testosterone was separated from the protein-bound fraction by equilibrium dialysis followed by derivatization. Either free or total testosterone, and a deuterated internal standard (d(3)-testosterone) were extracted by liquid-liquid extraction. The validation results were compared to two different clinical laboratories.
Results: The use of d(2)-testosterone was found to be unacceptable for our method. The total testosterone LC-MS/MS methods on both systems were linear over a wide concentration range of 1.5-2000 ng/dL. Free testosterone was measured directly using equilibrium dialysis coupled LC-MS/MS and linear over the concentration range of 2.5-2500 pg/mL. Good correlation (total testosterone, R-2=0.96; free testosterone, R-2=0.98) was observed between our LC-MS/MS systems and comparator laboratory. However, differences in absolute values for both free and total testosterone measurements were observed while a comparison to a second published LC-MS/MS method showed excellent correlation. Free and total testosterone measurements correlated well with clinical observations.
Conclusions: To our knowledge, this is the first published validation of free and total testosterone methods across two analytical systems of different analytical sensitivities. A less sensitive system does not sacrifice analytical or clinical sensitivity to directly measure free and total testosterone in patient samples.

• 出版日期2013-5