|
Abstract INTRODUCTION:
Steroid analysis is essential in clinical diagnostics and research due to the significant role steroids play in regulating metabolism, immune responses, and reproductive functions. Further, accurate quantification of steroid levels is vital for diagnosing and managing various health conditions.
Historically, steroids have been measured using immunoassays and GC-MS, but these methods have limitations. For example, immunoassays often suffer from cross-reactivity, leading to inaccurate results, while GC-MS requires extensive sample preparation. Therefore, LC-MS/MS has become the preferred method, offering high sensitivity and specificity with minimal sample preparation. LC-MS/MS can simultaneously measure multiple steroids in a single sample, making it ideal for clinical research and diagnostics.
High sensitivity is crucial in steroid analysis because many steroids exist in very low concentrations within biological samples. Enhanced sensitivity can also enable reduced injection volumes, which is beneficial when sample volumes are limited, particularly in clinical settings or research involving rare specimens.
OBJECTIVES:
The objective of this work was to develop a method for the analysis of 18 steroids in solvent using a next generation mass spectrometer, the SCIEX 7500+ system. The advantages of this high sensitivity were explored by comparing the results with the previous generation of triple quadrupole mass spectrometer, the QTRAP 6500+ system.
METHODS:
Analytical grade standards for the target analytes were purchased from commercial sources, and a mixed stock solution was prepared in methanol. Calibration standards were prepared in 30:70 (v/v) methanol/water covering greater than 5 orders of magnitude.
The calibration standards were injected on a SCIEX ExionAE system using a Phenomenex Kinetex C18 column for chromatographic separation. The gradient run was 16 min with mobile phases of 0.05mM ammonium fluoride in water (“A”) and 0.05mM ammonium fluoride in methanol (“B”). The injection volume was 10 µL. The samples were analyzed using the SCIEX 7500+ system with an OptiFlow Pro source operated in electrospray ionization mode. Samples were also run on a QTRAP 6500+ system for sensitivity comparison.
RESULTS:
The SCIEX 7500+ system showed high levels of sensitivity, allowing for the low-level quantitation of almost all steroids at sub-pg levels, reaching as low as 2.5 fg on column for some compounds. Linearity (1/x weighting, r2 > 0.999) was achieved for all steroids across the calibration range. Average accuracy and precision were assessed for 3 replicates and was within acceptable validation requirements with average accuracy of 85-115% and precision of < 15% for three replicate injections at all concentrations.
The sensitivity differences between the 7500+ and 6500+ systems were investigated by calculating the relative gains in peak area and S/N (n=3) for each MRM transition at the LLOQ concentration determined on the QTRAP 6500+ system. Across all transitions, the average area gain was 14-fold and the average increase in S/N was 4.7-fold demonstrating the significant sensitivity increase of the 7500+ system. In addition to sensitivity, the assay reproducibility was assessed by comparing the RSD% at the same concentrations for 3 replicate injections. Overall, the 7500+ system showed better precision (i.e., lower RSD% values). Specifically, the CV% was 2.1% for the 7500+ system as compared to 5. 9% for the QTRAP 6500+ system.
DISCUSSION:
This technical note demonstrates a method for the analysis of 18 steroids in solvent, achieving lower limits of quantitation (LLOQ) as low as 2.5 fg on column on the SCIEX 7500+ system. The advantage of this high sensitivity was explored by comparing the results with the previous generation of MS, a QTRAP 6500+ instrument. The results showed an increase in sensitivity for the SCIEX 7500+ system, shown in significant gains for both peak area, and signal-to-noise (S/N), utilizing optimization of the Q0 dissociation (Q0D) parameter, a feature unique to the improved front-end technology of the SCIEX 7500+ system. This increase in sensitivity would potentially allow for simplified sample preparation, reduced injection volume or a decrease in the detection levels previously achieved in similar workflows. |
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
