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Orbitrap_SciLib
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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 Cholesterol & Metabolites

 
Quantitative targeted analysis of cholesterol and its metabolites in various body fluids is performed in analytical labs for numerous research purposes. Depending on the body fluid and the source of the sample, the levels of cholesterol and metabolites 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 coupled to a uHPLC system enables a powerful yet easy-to-use workflow for researchers. It enables both quantitative and qualitative analysis in a single run.
 
The workflow described here uses selected ion monitoring (SIM) at 70,000 FWHM resolution. The data is acquired for multiple precursor ions at any give time 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 Q Exactive™ MS coupled to a uHPLC system for quantitative analysis of approximately 15 cholesterol molecules including metabolites. An LLOQ of 1 ng/mL and a dynamic range up to 10,000 ng/mL can be easily obtained in a 5 minute run. The analysis demonstrates the ability to quantify these analytes in organic solvent extracted mouse brain.
 
 



Workflows_Clin_Endo_CholMeta(1).jpg


 

Resources

 

Quantitation of Cholesterol Using Q Exactive System

Dreyer M, Second T.
Slide Presentation

Sample Preparation

Sample Preparation Workflow for Cholesterol & Metabolites

 
Micro-dissected mouse brain was extracted using petroleum ether. The extract was dried and reconstituted in an appropriate solvent for injection.

Literature Highlights

Sample Preparation Workflow for Cholesterol & Metabolites

 
Micro-dissected mouse brain was extracted using petroleum ether. The extract was dried and reconstituted in an appropriate solvent for injection.

Resources

 

Quantitation of Cholesterol Using Q Exactive System

Dreyer M, Second T.
Slide Presentation

Mass Spectrometry

Mass Spectrometry Workflow for Cholesterol & Metabolites

 
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 SIM 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.
 
QExactive_Schematic.jpg

Resources

 

Quantitation of Cholesterol Using Q Exactive System

Dreyer M, Second T.
Slide Presentation

Data Analysis

Data Analysis Workflow for Cholesterol & Metabolites

 
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 specified in the method.
  MWorkflows_Endocrine1_DA_.jpg

Resources

 

Quantitation of Cholesterol Using Q Exactive System

Dreyer M, Second T.
Slide Presentation

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Last update:
‎10-15-2021 11:05 AM
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