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Quantitation of endogenous steroids or similar molecules in body fluids is challenging due to their low levels, the interfering matrix, the small volumes of sample and the presence of many similar compounds in the sample. SRM analysis using a triple quadrupole LC-MS/MS system has been routinely employed for quantitative analysis of these analytes. Although, in many cases, SRM offers the desired sensitivity and specificity, some key assays still suffer from interferences and a lack of robustness. High-resolution, accurate mass spectrometry based on Orbitrap™ technology offers for the first time an attractive alternative with the unique benefits of specificity, sensitivity and, more importantly, ease of use. Ultra-high resolution of up to 140K FWHM and better than 3 ppm mass accuracy on both precursor and fragment ions help minimize interferences due to isobaric precursor ions and fragment ions obtained in a collision cell (isobaric for triple quadrupole MS/MS or any unit resolution instrument).
Here we describe the quantitative workflow solutions for vitamin D metabolites, testosterone and other steroids, estrogens and cholesterol.

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Workflow Overview for Testosterone

Quantitative targeted analysis of testosterone in plasma, urine and oral fluid is important for research into a variety of endocrine disorders. A fast SRM-based analysis using LC-MS/MS is the standard method. Depending on body fluid and the source of the sample, testosterone levels can vary from a few pg/mL to hundreds of pg/mL. The water losses during ionization, extensive fragmentation in the collision cell and isobaric matrix interferences make it difficult to achieve the desired LLOQ, dynamic range, and specificity at the needed speed. The ultra-high resolution Thermo Scientific Q Exactive MS offers an easier-to-use alternative to the SRM analysis performed using high-end triple quadrupole LC-MS/MS systems.
The workflow described here uses a simple targeted MS/MS approach at 70,000 FWHM resolution. The data is acquired in Full Scan MS/MS mode and the quantitation is based on extracted ion chromatograms on the two or more fragment ions using a 3 ppm window around the m/z of interest. This application demonstrates the capability of the Q Exactive™ MS coupled to a uHPLC system for quantitative analysis. An LLOQ of 10 pg/mL using ion ratio confirmation and a dynamic range up to 500 pg/mL can be easily obtained in a 5 minute run.




Quantitative analysis of Testosterone in plasma using Q Exactive

He K, Kozak M.
Slide Presentation

Evaluation of quantitative performance for testosterone analysis in plasma on a novel quadrupole Orbitrap mass spectrometer

He K, Kozak M.
ASMS 2011 Poster