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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 25-OH Vitamin D
Quantitative analysis of 25-OH Vitamin D3 and 25-OH Vitamin D2 in plasma is performed in analytical labs for a variety of research purposes. A fast SRM-based analysis has become the research method of choice. Vitamin D metabolites, even though present at reasonably high levels in human plasma, present certain challenges for quantitative analysis. The water losses during ionization and extensive fragmentation in the collision cell make it difficult to achieve the desired LLOQ, dynamic range, and specificity at the needed speed. The Thermo Scientific Exactive Plus ultra-high resolution MS offers an easier-to-use alternative to SRM analysis.
The workflow described here uses simple full-scan MS at 70,000 FWHM resolution. The data is acquired in Full Scan mode, and the quantitation is based on extracted ion chromatograms using a 3 ppm window around the m/z of interest. This application demonstrates the capability of the Exactive Plus™ MS coupled to a uHPLC system for quantitative analysis of the above two metabolites. A LLOQ of 1 ng/mL and dynamic range up to 150 ng/mL can be easily obtained in a 3 minute run.
Resources
Quantitative Analysis of Vit D Metabolites in Human Plasma using Exactive System
Kozak M.
Slide Presentation
Quantitation of 25OH Vit D using online TurboFlow Extraction and Exactive Plus System
Berube M.
Slide Presentation
Berube M, Dibussolo J, Lafontaine C, Shi Y, Espourteille F.
Poster Note

Rochat B.
ASMS 2012 Presentation
Sample Preparation
Sample Preparation Workflow for 25-OH Vitamin D
A simple protein precipitation of plasma using organic solvents followed by vortexing 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 Vit D Metabolites in Human Plasma using Exactive System
Kozak M.
Slide Presentation
Quantitation of 25OH Vit D using online TurboFlow Extraction and Exactive Plus System
Berube M.
Slide Presentation
Berube M, Dibussolo J, Lafontaine C, Shi Y, Espourteille F.
Poster Note

Rochat B.
ASMS 2012 Presentation
Mass Spectrometry
Mass Spectrometry Workflow for 25-OH Vitamin D
The Thermo Scientific Exactive Plus 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 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 Exactive Plus™ 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 Vit D Metabolites in Human Plasma using Exactive System
Kozak M.
Slide Presentation
Quantitation of 25OH Vit D using online TurboFlow Extraction and Exactive Plus System
Berube M.
Slide Presentation
Berube M, Dibussolo J, Lafontaine C, Shi Y, Espourteille F.
Poster Note

Rochat B.
ASMS 2012 Presentation
Data Analysis
Data Analysis Workflow for 25-OH Vitamin D
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 molecules monitored and their retention times. The extracted ion chromatograms were obtained for calibrators, QCs and samples. The area under the peak was used for creating calibration curves and calculating values of QCs and samples. Automatic reports were created as specified in the method.
Resources
Quantitative Analysis of Vit D Metabolites in Human Plasma using Exactive System
Kozak M.
Slide Presentation
Quantitation of 25OH Vit D using online TurboFlow Extraction and Exactive Plus System
Berube M.
Slide Presentation
Berube M, Dibussolo J, Lafontaine C, Shi Y, Espourteille F.
Poster Note

Rochat B.
ASMS 2012 Presentation
Thermo Fisher Scientific
United States
