Orbitrap Illustration

25-OH Vitamin D

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 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.





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


Analysis of 25-Hydroxyvitamin D In Serum Using Automated On Line Sample Preparation Technique With High Resolution Orbitrap Mass Spectrometer

Berube M, Dibussolo J, Lafontaine C, Shi Y, Espourteille F.
Poster Note

Quan / Qual Analyses in Clinical Research using a Second Generation Exactive High Resolution Mass Spectrometer

Rochat B.
ASMS 2012 Presentation