Jetstream source

An Agilent 1260 LC system operating in normal flow mode at 400 μl min⁻¹ was coupled to an Agilent 6460QQQ Mass Spectrometer equipped with an Agilent Jet Stream source and running MassHunter version B.05.00; 10 μg of peptides was separated on an Ascentis Express Peptide C18 column [2.7 μm particle size, 160 Å pore size, 5 cm length × 2.1 mm i.d., coupled to a 5 mm × 2.1 mm i.d. guard column with similar particle and pore size, operating (CG1) at 60°C; Sigma‐Aldrich]. Peptides were ionized by using an Agilent Jet Stream source operating in positive‐ion mode with the following parameter settings: sheath gas flow = 11 l min⁻¹; sheath gas temperature = 400°C; nozzle voltage = 1000 V; nebulizing pressure = 45 psi; chamber voltage = 5000 V. A 25‐min method with the following gradient was used: 95% Buffer A (2% acetonitrile, 0.1% formic acid), 5% Buffer B (98% acetonitrile, 0.1% formic acid). Buffer B is increased to 40% over 17 min, followed by an increase to 80% B in 30 sec, where it is held for 1 min. Buffer B is ramped back down to 5% in 30 sec and equilibrated for 6 min prior to the next injection. Peptide quantification was achieved by summing the integrated peak areas of two validated SRMs. Summed peaks were averaged for all peptides associated with subcellular compartments.

Quantitative analysis was performed using Agilent 1290 Infinity II high-performance liquid chromatograph was equipped with Agilent 6470 triple-quadrupole mass spectrometer coupled with an Agilent Jet Stream source in electrospray ionization (ESI) mode (Agilent Technologies, CA, USA). The water extract of Paeoniae Radix Alba (1 mg) was dissolved in 1 mL of 100% ACN. 100 μL of the sample was mixed with an equal volume of internal standard (1 ng/mL Gliclazide in acetonitrile). Paeoniflorin separation solution (5 μL) was injected onto an Agilent Zorbax Eclipse Plus C18 column (2.1 mm × 50 mm id, 1.8 μm) and separated. The column and autosampler tray was kept at 40°C and 4°C, respectively. The mobile phase consisted of HPLC-grade water with 10 mM ammonium formate in distilled water as solvent A and acetonitrile as solvent B (A : B = 20 : 80 isocratic) with isocratic elution at a flow rate of 0.2 mL/min. The total run time was 2.5 min per sample. Quantification was performed with MS/MS detection set in the positive in multiple reaction monitoring modes for the analyte and IS. The amount of paeoniflorin (including 100 ng/mL) is analyzed to be 4.21 ± 0.40 ng/mL (Figure 1).

Untargeted polar metabolomics chromatographic separation was conducted by following the indications reported by Jian Li et al. [53 (link)]. For the immobile phase, a Waters XBridge BEH Amide (HILIC) column (150 mm, 2.1 mm, and 2.5 µm) at 25 °C with a flow rate of 0.35 mL/min was used. The mobile phase consisted of a mixture of water (A) and ACN (B), both with 0.2% formic acid. The gradient used was as follows: From 0 to 3 min, an isocratic gradient was maintained, with A at 10% and B at 90%. From 3 to 13 min, a linear gradient was used, with A at 52% and B at 48%. From 13 to 15 min, an isocratic gradient was maintained, with A at 52% and B at 48%. From 15 to 16 min, a linear gradient was used, with A at 10% and B at 90%. From 16 to 20 min, an isocratic gradient was maintained, with A at 10% and B at 90%. At 20 min, the run was stopped. Spectrometric data were acquired in the range of 40–1700 m/z in both negative and positive polarity. The Agilent JetStream source was used with the following settings: gas temperature (N₂) at 200 °C, drying gas flow at 10 L/min, nebulizer pressure at 50 psi, and sheath gas temperature at 300 °C with a flow of 12 L/min.

Testosterone and corticosterone quantification was determined using Agilent’s Infinity II UHPLC in line with a 6495 triple quadrupole mass spectrometer and MassHunter workstation software (8.0.8.23.5). Briefly, DSRCT xenograft and PDX samples were homogenized using water containing internal standard (Cerilliant, T070) extracted with tert-butyl methyl ether (Sigma 34875), dried under nitrogen, and derivatized using hydroxylamine hydrochloride (Sigma 431362). The recovered ketoxime steroids were reconstituted in methanol/water (1:1 v/v) and injected into the Infinity II UHPLC. Ketoxime steroids were separated using a Chromolith reverse-phase column (RP-18 endcapped 100–2 mm, Sigma 152006) and introduced into a JetStream source (Agilent) for triple quadrupole analysis. Data were analyzed and quantified using MassHunter software (Agilent)^{39 (link),40 (link)}.

LC-Q/TOF MS analysis was carried out with the use of a 1290 infinity II high-performance liquid chromatograph and a 6530 Accurate-Mass Q-TOF mass spectrometer equipped with a JetStream source (both Agilent Technologies, Santa Clara, CA) for the identification of compounds. Chromatographic separation of the samples was performed under the same lipid profiling conditions to those above. The source was operated in the positive ionization mode as follows: ion spray 4000 V; gas temperature and sheath gas temperature 300 and 350°C, respectively; nebulizer (N2) 40 psi; and sheath gas flow 12 L/min. Data-dependent acquisition was used in the mass range of 50−1700 m/z for both MS and MS/MS with a collision energy of 25 V. The calibration of the spectral range was performed using Tuning & Performance Standards for the LC/MS solution (Agilent Technologies) and a fifth-order non-linear calibration curve was adopted. To perform the real-time lock mass correction, a lock mass solution including purine (m/z 121.0509) and HP-921 [hexakis (1H,1H,3H-tetrafluoro-pentoxy) phosphazene] (m/z 922.0098) was delivered by a built-in pump. The accurate mass Q-TOF MS and MS/MS data were processed with the use of MassHunter Workstation Software (Agilent Technologies).