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Abstract Introduction:
Hormonal steroids regulate most body functions and the dysregulation of these molecules can play a role in the pathophysiology of human disease. Early techniques to measure endogenous steroids include immunoassay and gas chromatography-mass spectrometry (GC-MS). Immunoassays are problematic because they lack specificity for low-level steroids due to interference from endogenous steroids present at higher levels. In contrast, GC-MS offers higher specificity and is sensitive to low-level steroids, however, it requires extensive sample preparation via derivatization. More recently, steroid analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a sensitive and specific technique with a simplified sample preparation.
Here, the analysis of hormonal steroids by LC-MS/MS is explored using the ZenoTOF 7600 system. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were calculated for steroids and steroid levels were measured in plasma samples. EAD-derived fragment ions were used to structurally characterize the steroids of interest. EAD generated structure-specific fragment ions that were sufficient to distinguish steroid isomers and isobars during analysis without requiring extensive chromatographic development.
Methods:
Stock solutions of analytes and internal standards were diluted in PBS to generate standard curves using technical replicates to determine the LOD and LLOQ of steroids. PBS was used as a matrix surrogate because plasma contains significant amounts of endogenous steroids. Deuterated internal standards were added to NIST 1950 plasma samples at a final concentration of 1 ng/mL to quantify endogenous steroids. For plasma samples and quality control (QC) samples, 5 µL of the internal standard mixture was added to 200 µL of sample with and 300 µL DI water. Samples were shaken @ 700 rpm for 5 minutes, and the protein was precipitated by adding 250 µL of 0.1M zinc sulfate and shaking @ 600 rpm for 5 minutes after which 500 µL cold methanol was added. Samples were again shaken at 500 rpm for 5 minutes, centrifuged at 2637 rcf for 10 minutes at room temperature, and the supernatant was collected and loaded onto a preconditioned HLB SPE 30mg (30 µm) plate. The plate was washed with DI water and steroids were eluted with acetonitrile. The eluant was dried with N¬2, and samples were reconstituted in 50 µL 50:50 methanol:water.
Prepared samples were separated by high-performance liquid chromatography (HPLC) using a Kinetex biphenyl column from Phenomenex (2.6 µm particle size, 100 x 2.1 mm). Analytes were eluted from the column using a biphasic gradient. Mobile phase A was water with 0.2mM ammonium fluoride and mobile phase B was methanol. The flow rate was 400 µL/min. The column temperature was held constant at 50°C and the injection volume was 15 µL.
Data were acquired with a ZenoTOF 7600 system using 2 methods. The LLOQ and LOD of various steroids were determined using the scheduled high-resolution multiple reaction monitoring scan mode (sMRMHR). For DHEA-sulfate and estrone, pseudo MRM transitions were used (precursor ion to precursor ion) due to inefficient fragmentation. Experiments to structurally characterize steroids via EAD-based fragmentation were performed using a data-dependent acquisition (DDA) scan mode.
Results:
Standard curves analyzed using the sMRMHR scan mode run with CID-based fragmentation yielded LLOQ values ranging from 0.01 to 25 ng/ml and LODs ranging from 0.01 to 25 ng/mL to values for all compounds tested. However, distinguishing structural isomers is a significant challenge in bioanalysis. Traditional LC-MS/MS methodologies use CID to generate fragments for quantification. If isomers and/or isobars interfere with the analysis of CID-based fragments, then time-consuming chromatographic methods must be used to resolve these compounds. EAD-generated fragments have been shown to enable high compound specificity without the need for extensive chromatographic method development. CID product ion spectra for the isomers 11-deoxycorticosterone and 17-hydroxyprogesterone, both have masses of m/z 331.227. CID based MRM transitions are not sufficient to distinguish these molecules. In contrast, EAD-based fragmentation generated rich MS/MS data for the same pair of isomers. Three unique EAD-based fragments derived from 11-deoxycorticosterone that were not present in the spectrum for 17-hydroxyprogesterone. Similarly, 2 unique fragments were found for 17-hydroxyprogesterone. These product ions can be used in sMRMHR experiments using EAD-based fragmentation to increase the specificity of the assay by distinguishing between these 2 isomers.
Conclusions:
In this study, the ZenoTOF 7600 system enabled rapid and sensitive analysis of hormonal steroids in human plasma. The use of EAD enhanced structural details, surpassing the limitations of CID. Simultaneous CID- and EAD-based fragmentation supported high-throughput analysis while improving analyte specificity, marking a significant advancement in hormonal steroid analysis.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
