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 the Ten Steroid Panel
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
Sample Preparation Workflow for the Ten Steroid Panel
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.
Mass Spectrometry Workflow for the Ten Steroid Panel
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.
Data Analysis Workflow for the Ten Steroid Panel
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.
Orbitrap of Choice for the Ten Steroid Panel
The Thermo Scientific Exactive Plus system, operated in Full Scan MS mode at a resolution of 100,000 FWHM and mass accuracy better than 3 ppm, has demonstrated the needed specificity, sensitivity and dynamic range for the analysis of steroids. The simplicity of operation and lower cost makes it a very affordable and robust alternative to SRM analysis for assays requiring mid to high sensitivity, for example, quantitation of steroids in adult male serum or plasma samples.
For ultra-high sensitivity analysis using high-resolution MS, the Thermo Scientific Q Exactive system is an ideal solution that provides the LLOQ in the range of 10 pg/mL in serum. The Thermo Scientific Transcend system equipped with TurboFlow™ technology is well suited for online sample preparation for long uninterrupted operation and multiplexing for high throughput.