Analyst version 1

Analyst ^® version 1.7 (AB Sciex) was used for instrument control and data acquisition. Data analysis was completed using MultiQuant version 3.0 (AB Sciex). Confidence intervals and reference ranges were calculated using R programming language, with the bias-corrected percentile confidence interval with bootstrapping (n = 999) using the boot package. Reference intervals were created for each sample for the whole cohort, for men and women separately and for those with and without AD biomarkers. Comparisons of median concentrations for each steroid between men and women and healthy volunteers and those with and without AD biomarkers were carried out using the Wilcoxon test.

The same system as with LC-MS analysis was used. The mobile phases in this case were ultrapure water with 0.1% (v/v) acetic acid (A) and MeOH with 0.1% (v/v) acetic acid (B). The flow rate was 350 μL min⁻¹ and the column heater temperature was 55 °C. An elution gradient was applied, starting with 30% B, held for 3 min. Afterwards B was ramped to 95% within 11 min and held constant for the next 4 min, decreased back to 30% B within 0.5 min and held for the next 7.5 min to re-equilibrate the column. The injection volume was 10 μL, too. Parameters used to produce fragment ions in selected reaction monitoring mode (SRM) and collision energies (CE) are given in Table 1. The electrospray ionization source (ESI) was operated in the positive ionization mode.
The parameters used for ionization were a temperature of 400 °C, 4500 V ion spray voltage, an entrance potential (EP) of 10 V, a declustering potential (DP) of 90 V, a collision cell exit potentials (CXP) of 15 V, a curtain gas with 35 psi (1 psi = 0.0689 bar = 6890 kg m⁻¹ s⁻² (SI unit)), a nebulizer gas (GS1) with 62 psi, a turbo gas (GS2) with 62 psi and a collision gas with 8 psi. Analyst® version 1.6.2 software (SCIEX) was used to control the instrument, acquire data and evaluate the results.

Quinoline and its derivative contents were analyzed using gas chromatography–mass spectrometry (GC/MS) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). A detailed description of the sample preparation and chromatographic methods is included in Felczak et al. [24 (link)]. Additionally, qualitative analysis involving the loss of the neutral glucose molecule (162 Da) was carried out using an Eksigent microLC 200 System (Sciex Framingham, MA, USA) connected with an AB Sciex 4500 QTRAP mass spectrometer (Sciex Framingham, MA, USA). A C18 column (50 mm × 0.5 mm, particle size: 2.7 μm, (Sciex Framingham, MA, USA)) maintained at 45 °C was used for the chromatographic separation of quinoline metabolites. The eluents with a constant flow of 15 μL/min were water (A) and acetonitrile (B). Both eluents were supplemented with 0.1% formic acid. The injection volume was 5 µL. The mass spectra of quinoline metabolites were collected at collision energy settings of 30 ± 15 and 50 ± 10 V. The gradient of eluents and the microESI ion source parameters are presented in Table 3. The negative mode was retained for quinoline metabolite analysis. Analyst™ version 1.6.2 software (Sciex Framingham, MA, USA) was used in the qualitative analysis of quinolone metabolites.

Samples were analyzed using an Agilent^® Series 3200 liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled to an ABSCIEX^® API 4000 mass spectrometer (SCIEX, Framingham, MA, USA) and a Sunfire^® C18 (Waters Corp., Milford, MA, USA) chromatographic column of 3.5 µm and 2.1 × 150 mm.
The analytes were chromatographically separated using a mobile phase gradient of 0.1% formic acid in water (Phase A) and 0.1% formic acid in methanol (Phase B). The flow rate for this mobile phase was set at 0.2 mL min⁻¹, the injection volume was set at 25 µL, and the column temperature was set at 30 °C.
The criteria to identify CTC and 4-epi-CTC was the detection of precursor ions with masses of 479.0 Da. Similarly, precursor ions of mass 451 Da were regarded as indicative of TC-d₆. Meanwhile, fragmented ions had masses of 444.0, 154.0, and 416.0 Da for CTC, 4-epi-CTC, and TC-d₆, respectively.
As for the chromatographic integration of the samples, we used the Analyst^® version 1.6.2 software (SCIEX, Framingham, MA, USA).

The sample preparation, extract analysis, metabolite identification, and quantification were performed at Wuhan Metware Biotechnology Co., Ltd., WuHan, China following¹ the standard procedures, which were previously described by Yuan et al. (2018) (link), Cao et al. (2019) (link), and Zhang et al. (2019) (link). Metabolite data analysis was carried out using the Analyst version 1.6.1 software (AB SCIEX, ON, Canada).