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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.
For additional resources, search the Orbitrap Science Library
Overview
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.
Resources
Quantitative analysis of Testosterone in plasma using Q Exactive
He K, Kozak M.
Slide Presentation
He K, Kozak M.
ASMS 2011 Poster
Sample Preparation
Sample Preparation Workflow for Testosterone
A simple protein precipitation of plasma using organic solvents followed by vortexing, freezing and centrifugation is needed before the supernatant is injected. While this offers a robust quantitative analysis, for much longer uninterrupted operation without source or MS cleaning, a TurboFlow™ online sample cleanup is recommended. The cleanup of the TurboFlow columns minimizes ion suppression, removes interferences and extends the life of the analytical columns.
Resources
Quantitative analysis of Testosterone in plasma using Q Exactive
He K, Kozak M.
Slide Presentation
He K, Kozak M.
ASMS 2011 Poster
Mass Spectrometry
Mass Spectrometry Workflow for Testosterone
A Thermo Scientific Q Exactive mass spectrometer with an APCI source was coupled to a Thermo Scientific Accela 1250 uHPLC or Transcend system. The MS was operated in Full Scan targeted MS/MS mode at a resolution of 70,000 FWHM during the entire LC run. The column eluent was diverted to waste for a specified period of time after the sample injection before being sent to the MS. The Q Exactive™ MS was calibrated at the beginning of the day and used for the entire analysis. No lock mass or internal calibration was used.
Resources
Quantitative analysis of Testosterone in plasma using Q Exactive
He K, Kozak M.
Slide Presentation
He K, Kozak M.
ASMS 2011 Poster
Data Analysis
Data Analysis Workflow for Testosterone
Thermo Scientific TraceFinder software, which supports quantitation using both low and high resolution MS systems, was used for automated processing and reporting. The quantitative processing method included the exact m/z of fragments monitored and their retention times. The extracted ion chromatograms for fragments were obtained for calibrators, QCs and samples. The area under the peak for the quantifier ion was used for creating calibration curves and calculating values of QCs and samples; the area for the other fragment was used as a qualifier. The ion ratios were measured to rule out interferences. Automatic reports were created as selected in the method.
Resources
Quantitative analysis of Testosterone in plasma using Q Exactive
He K, Kozak M.
Slide Presentation
He K, Kozak M.
ASMS 2011 Poster
Thermo Fisher Scientific
United States
