Quantification of steroid hormones in plasma using a surrogate calibrant approach and UHPLC-ESI-QTOF-MS/MS with SWATH-acquisition combined with untargeted profiling

Abstract

In spite of demonstrated lack of accuracy and consistency, quantification of steroid hormones is still most commonly executed via immunoassays. Mass spectrometric methods with triple quadrupole instruments are well established and, because of their proven robustness and sensitivity, best suited for targeted analysis. However, recent studies have shown that high-resolution mass spectrometers, like quadrupole time-of-flight instruments (QTOF), show comparable performance in terms of quantification and can generate additional sample information via untargeted profiling workflows. We demonstrate that adequate accuracy and selectivity for estradiol and testosterone can be achieved with a QTOF by data-independent acquisition with sequential window acquisition of all theoretical fragment-ion mass spectra (SWATH). Besides potential combination of targeted quantification and untargeted profiling, SWATH offers advantages with respect to sensitivity because the reduced total number of MS/MS experiments could be used to increase accumulation time without increasing cycle time. By applying a surrogate calibrant method leading to successful validation, a reliable method for absolute steroid quantification and high potential for steroid profiling has been developed. Linear calibration was achieved in the range from 10 to 1,000 pg mL for C-estradiol and from 20 to 15,000 pg mL for C-testosterone. Results for inter-day precision (C-estradiol: 4.5-10.2%; C-testosterone: 5.1-7.8%) and inter-day accuracy (C-estradiol: 94.6-112.8%; C-testosterone: 98.2-107.7%) were found to be well acceptable. Eventually, the method has been utilized to measure clinical samples of a study in which male volunteers obtained transdermal estradiol patches and sex hormone levels were quantified in plasma.

Pulsatilla Decoction Can Treat the Dampness-Heat Diarrhea Rat Model by Regulating Glycerinphospholipid Metabolism Based Lipidomics Approach

“…Recent lipidomics analysis has commonly used the liquid chromatography-mass spectrometry (LC-MS) technology, and is mainly divided into untargeted or non-targeted and targeted analysis (Lee and Yokomizo, 2018). Among them, the untargeted analysis model can detect various types of lipids in a sample without bias, and perform systematic analysis (Drotleff et al, 2018). Targeted analysis is mainly used to elucidate specific lipid molecules with great selectivity and specificity achieved in the quantitative analysis (Xu et al, 2018).…”

A combined targeted/untargeted LC-MS/MS-based screening approach for mammalian cell lines treated with ionic liquids: Toxicity correlates with metabolic profile

“…S1) has to be applied. Response factors of uniformly-13 C, 15 N-labelled amino acids were determined in comparison to authentic amino acids by their respective slope ratios (see SI Table S5) and applied to obtain corrected calibration functions (eq. 2) (SI Figure S5).…”

“…A sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) method with variable Q1 window widths and narrow Q1 target windows of 5 Da for most of the amino acids was selected as the optimal acquisition mode. Due to the absence of a true blank matrix, 13 C, 15 N-isotopically labelled amino acids were utilized as surrogate calibrants, instead of proteinogenic amino acids. Partial least squares (PLS) analysis of the median effective concentrations (EC 50 ) of 9 selected ionic liquids showed a correlation with their metabolic profile measured by the untargeted screening.…”

“…However, the new generation of high-resolution mass spectrometers (QTOF and quadrupole-orbitraps) show improved performance for quantitative analysis [11,12]. Hence, there is an increasing interest in performing qualitative and quantitative analysis in a single run [11,[13][14][15][16]. In general, HRMS-based untargeted metabolomics is typically performed by data-dependent acquisition (DDA) (also called information-dependent acquisition, IDA).…”

Recent developments and emerging trends of mass spectrometric methods in plant hormone analysis: a review

“…High resolution enhances the sensitivity of quantitative analysis [105]. One of the mainstream industries where LC-MS is constantly implemented is the doping control analysis, which employs different mass analyzers such as triple quadrupole (QqQ) [106], time of flight (TOF) [107] and quadrupole time of flight (QTOF) [108]. These qualitative and quantitative approaches are also applied in the detection of plant hormones.…”