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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Quantitative analysis of wide ranging steroids in human serum is challenging due to low endogenous levels, the presence of many similar molecules and matrix interferences. An SRM-based analysis needs significant sample preparation. The challenge to achieve the desired sensitivity and dynamic range comfortably is further aggravated by the water losses during ionization with both electrospray ionization and APCI sources and the extensive fragmentation in the collision cell.
The workflow described here uses simple full-scan MS at 100,000 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 Thermo Scientific Exactive Plus MS coupled to a uHPLC system for quantitative analysis of ten steroids.
He K, Kozak M.
He K, Kozak M.
Thomas A, Geyer H, Schänzer W, Crone C, Kellmann M, Moehring T, Thevis M.
Anal Bioanal Chem. 2012 May;403(5):1279-89.
He K, Kozak M.
ASMS 2011 Poster
The quantitative analysis of steroids described here was performed using the Perkin Elmer CHS™ MSMS Steroids Kit available commercially in Europe. Protein precipitation using the daily precipitation solution was followed by drying and reconstitution. The calibrators and QC were processed using the protocol provided with the kit.
The Thermo Scientific Exactive Plus mass spectrometer with an APCI source was coupled to a Thermo Scientific Accela 1250 uHPLC system. The MS was operated in Full Scan MS mode at a resolution of 100,000 FWHM during the entire LC run. The column eluent was diverted to waste after the sample injection for a specified period of time before being sent to the MS. The Exactive Plus™ MS was calibrated at the beginning of the day and used for the entire analysis. No lock mass or internal calibration was used.
Thermo Scientific TraceFinder software, which supports quantitation using both low and high resolution MS systems, was used for automated data 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.