6495 triple quadrupole

The Agilent 6495 Triple Quadrupole (Santa Clara, USA) served as a mass analyzer. The heated ESI ionization source (Agilent JetStream Technology) in the positive mode was used. Nitrogen served as a nebulizing, sheath, drying, and collision gas. Sheath gas temperature was set to 350 °C and drying gas temperature to 250 °C. The data were collected in the MRM mode; the transitions monitored and the respective collision energies are listed in Table 1. The LC-MS system was controlled with the Agilent MassHunter Workstation software version B.07.00. For peak integration and quantitative calculations, the Agilent MassHunter Quantitative Analysis software version B.07.00 was used.Table 1

MRM transitions used for analysis with collision energies (CE)

Compound	Precursor ion	Product ion
		Quantifier		Qualifier
	m/z	m/z	CE, V	m/z	CE, V
Spironolactone	341	107	38	341	2
Canrenone	341	107	38	341	2
7-Alpha-methylthiospironolactone	389	341	14	323	14
Spironolactone-D3	344	107	32	341	2

The solutions were analyzed via high-performance liquid chromatography (Agilent 1260 Infinity) coupled with diode array and mass spectrometry detection (6495 triple quadrupole, Agilent technology, Cheadle, UK). The stationary phase was a Phenomenex C18 column (250 × 4.6 mm; 5 µm). The mobile phase was a gradient combining 0.1% (v/v) formic acid added in both solvents: pure water and 0.1% formic acid (solvent A) and acetonitrile and 0.1% acid formic (solvent B). The gradient program was set as follows: 0 to 35 min, 95% A → 0%; 35–37 min, 95 → 5% B; 37–40 min, 95% A/5% B with a flow rate of 0.4 mL/min⁻¹. The injection volume was 5 μL, and column temperature was set to 30 °C with a UV wavelength of 254 nm.
The UV spectra of ONC201 were recorded with a spectrophotometer UV 7 (Mettler Toledo; Viroflay, France); the pH of the studied solutions was fixed by the use of sodium hydroxide and chlorohydric acid.

A Shimadzu Nexis 2030 gas chromatograph equipped with a programmed split/splitless injector and an AOC 6000 multifunction autosampler (Shimadzu, Kyoto, Japan), and a Shimadzu 8040 138 NX tandem mass spectrometry (Shimadzu, Kyoto, Japan) were used to perform gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) confirmation. An SH-Rxi-5Sil MS (30 m 140 × 0.25 mm id × 0.25 μm film) capillary column was used. A 1290 Infinity UHPLC system was linked to a 6495 Triple Quadrupole LC-MS/MS device added with a jet stream EI source (Agilent, Santa Clara, CA, USA). Data were acquired and analyzed on an Agilent MassHunter Workstation B.07.00. Chromatographic isolation was finished on an Agilent ZORBAX Eclipse Plus C₁₈ column (50 mm × 2.1 mm, 1.8 μm) with gradient elution.
Samples were prepared using a GENIUS 3 vortex agitator (IKA, Stauffen, Germany), a CL31R multispeed refrigerated centrifuge (Thermo Scientific, Waltham, MA, USA), a WD12 water bath nitrogen blowing instrument (Aosheng Instrument, Hangzhou, China), and a CK2000 high-throughput tissue grinder (Thmorgan Biotechnology, Beijing, China).