47. A Rapid Analysis of Testosterone in Serum using Two Dimensional HPLC System
Poster: Tue 2:00-3:00PM
M P George
MP George*, Andre Szczesniewski(A-USA,ex1)

Agilent Technologies, Inc., Santa Clara, CA 95051. (*All the correspondence to All the Authors are employees of Agilent Technologies, Inc.)
Tandem Mass Spectrometry has gained momentum for the analysis of small molecules in biological compounds such as testosterone and 25-OH Vitamin D in both the research and clinical applications). These applications employ High Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (LC/MS/MS). We have developed a simple method using two dimensional liquid chromatography coupled with tandem mass spectrometry for the analysis of testosterone in serum and plasma. Our study demonstrates that two dimensional Liquid Chromatography is rapid and simple for the everyday routine analysis of testosterone and this technique also eliminated extensive solid-phase or liquid-liquid sample preparation procedure. We performed correlation study with real patient specimens.

Samples are prepared for tandem mass spectrometry analysis by protein precipitation using acetonirile, and centrifugation. The supernatant is evaporated to dryness and the extracts are re-dissolved with methanol: water 50:50 Ratio.

Two HPLC pumps are connected serially through a six port two position valve. The first HPLC Pump and the column are used as sample clean up to reduce ion suppression and interferences. The chromatographic separation is run under acidic condition with 0.1% formic acid in both aqueous and organic solvents.

The testosterone is eluted to the second column using the ‘Heart-Cut Technique” by setting the six-port valve timing in the Mass Hunter Acquisition Software. The compound is further separated chromatographically on the Rapid Resolution Column. The first LC column is C-8 Cartage 2.1X30 mm 3.5u particles and the second column is C-18 Eclipse plus 2.1X50 mm 1.8u particles. Agilent 6430 Tandem Mass Spectrometry with APCI source in MRM mode is used. Quantiation is achieved using Mass Hunter Quantitative Software with isotopic internal standard method and nine point calibrators. All the instrument components are operated as one system by the software.

Experimental Data:
For testosterone the limit of detection is 2ng/dL and the limit of quantitation is 5ng/dL. The linearity of the assay is from 5ng/dL to 2000 ng/dL. Fifty serum samples which were previously analyzed by a reference lab by LC-MS/MS are used for the correlation study. Ion suppression is less than 5% and no interference was observed. The correlation results are presented in this study. The run time for this assay is 4.8 minute.