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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 Estrogenic Compounds

 
Quantitative targeted analysis of estrogenic compounds and their metabolites in various body fluids is performed in analytical labs for a variety of research purposes. Depending on body fluid and the source of the sample, the levels of these compounds can vary from a few pg/mL to hundreds of pg/mL. 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 simple selected ion monitoring (SIM) at 70,000 FWHM resolution for the quantitative analysis of Estradiol, Estrone, Estriol and their metabolites in solvent. The data is acquired for multiple precursor ions at any give time and the quantitation is performed by extracting 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 the estrogenic compounds. An LLOQ of 10 pg/mL and a dynamic range up to 500 ng/mL can be easily obtained in solvents in a 25 minute run.
 



 Workflows_Clin_Endo_Estrogenic(1).jpg


 

Resources

 

Quantitative and Qualitative Analysis of Estrogenic Compounds Using Q Exactive system

Dreyer M, Kalbron J.
Slide Presentation

Sample Preparation

Sample Preparation Workflow for Estrogenic Compounds

 
Standards were dissolved in mobile phase to the required concentration.


Resources

 

Quantitative and Qualitative Analysis of Estrogenic Compounds Using Q Exactive system

Dreyer M, Kalbron J.
Slide Presentation

Mass Spectrometry

Mass Spectrometry Workflow for Estrogenic Compounds

 
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.
 

Scheme_Q-Exactive-color-RGB.jpg

Resources

 

Quantitative and Qualitative Analysis of Estrogenic Compounds Using Q Exactive system

Dreyer M, Kalbron J.
Slide Presentation

Data Analysis

Data Analysis Workflow for Estrogenic Compounds

 
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 the analytes and their retention times. 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. Automatic reports were created as specified in the method.
 
MWorkflows_Endocrine1_DA_.jpg

Resources

 

Quantitative and Qualitative Analysis of Estrogenic Compounds Using Q Exactive system

Dreyer M, Kalbron J.
Slide Presentation

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Last update:
‎10-15-2021 11:59 AM
Updated by:
AnalyteGuru
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