B-177 Selective and sensitive quantitation of 18 steroids in human serum using Thermo Scientific™ Stellar™ mass spectrometer

Background Successful biomarker quantitation in complex matrices requires sensitivity and specificity to ensure the measured signal is attributed to the targeted analytes. To reduce background interferences and provide greater specificity, alternative fragmentation mechanisms and/or MS3 fragmentation are an efficient solution. The Thermo Scientific™ Stellar™ mass spectrometer (MS) is a hybrid quadrupole, dual-pressure linear ion trap MS that offers two types of orthogonal, yet complementary, collision-induced dissocation (CID) with rapid MS2 and MS3 scans. This poster is the first to demonstrate the advantages of utilizing multiple collision activations and MS3 scans provided by Stellar MS to the selective and sensitive quantification of 18 steroids in serum. Methods Commercially available 18 steroids standards, including 4 progestagens, 5 androgens, 2 estrogens, 3 mineralocorticoids, and 4 glucocorticoids, and their corresponding internal standards were serially diluted in 0.05% bovine serum albumin to generate calibrator samples over 4-orders of magnitude concentration range. Quality control human serum samples were purchased from Chromsystem. Steroids were extracted with protein precipitation, dried, and reconstituted in 50% methanol for the LCMS analysis. Multiple fragmentation schemes were evaluated and optimized on the Stellar MS, and the data was analyzed in Thermo Scientific™ TraceFinder™ software for the quantitative performance, including detection limit, quantitation limit and linearity. Results The detection of 18 steroids were optimized in Stellar MS under multiple fragmentation mechanisms (beam-type CID, resonance-type CID, and MS3 in different combinations) with polarity switching. Breakdown curves were generated to facilitate characteristic fragment ion selection to ensure high sensitivity and selectivity. Adaptive RT function was utilized to adjust the scheduled retention time window of the analyte in real-time to minimize any chromatographic changes such as aging column. Analytical performance of the calibration curves with R2 > 0.98 were achieved using a weighting factor of 1/x, and the limit of quantification (LOQ) values are established with % RSD < 20, ¦% Diff¦ < 20, with relative ion ratio meeting the values specified by EU Council Directive 96/23/EC. Comparing to the MS2 beam-type CID commonly used in contemporary triple quadruple MS, steroids such as cortisone, estradiol, and dihydrotestosterone benefited from the alternative, resonance-type CID and MS3 acquisition modes, resulting in a 2-10 fold increase of the LOQ values. Conclusion Stellar MS provides unique selectivity allied to fast acquisition for high-throughput and sensitive biomarker quantitation optimal for routine analysis