Automated Method for High-Throughput LC-MS/MS Quantitation of Testosterone from Serum: An Improved Validated Method

99.5% Recovery of Testosterone

Analysis of testosterone is helpful when investigating endocrine disorders such as hypogonadism, polycystic ovarian syndrome in women, and early or late onset of puberty in boys. The naturally occurring low levels of endogenous testosterone, found in females and children, has resulted in quantitation by LC-MS/MS being the preferred analytical method for achieving relevant sensitivity and specificity (1). Automated sample preparation of testosterone is desirable to accommodate the high sample throughput, robustness, and efficiency demands of the analysis.

An improved automated method for the extraction of testosterone in serum was developed using ZnSO4 and Low Porosity Filtration Tips- Ultra Pure (LPFT-UP) on a MICROLAB® NIMBUS96 system (Hamilton Company, Reno, NV). The fully automated method incorporates a ZnSO4 solution with acetonitrile on a Hamilton Heater Shaker (HHS) for improved recovery and precision during protein precipitation. LPFT-UP containing high purity filtration media are used to remove matrix interferences and achieve very low limits of detection. Using this quick (< 2 minutes) and effective, DPX patent-pending, Tip-on-Tip (ToT) technology, the LPFT-UP provide an automated filtration alternative to traditional centrifugation and filtration. This method allows for up to 96 samples to be protein precipitated, filtered, and ready for injection in under 5 minutes.

Sample Preparation Workflow

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Pre-scripted Method on Hamilton Robotics

DPX ProductSample VolumeOffline ProcessingOnline Processing
Catalog #:DPX170068U
Ultra-Pure Grade Low Porosity Filtration tips for low detection level methods
75 µLSolution Prep< 5 minutes

This workflow can be adapted for varying sample volumes. DPX can support automation on other Hamilton systems (STAR, STARlet or VANTAGE).

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Learn more about pre-scripted methods for Hamilton Robotics


1. Run Zhang Shi, Huub H. van Rossum, Raffick A.R. Bowen, “Serum testosterone quantitation by liquid chromatography-tandem mass spectrometry: Interference from blood collection tubes” Clinical Biochemistry 2012, 45, 1706-1709.