Triple quad 4500

The detection of Compounds 1a, 1b, 1c, and 1d for degradation of 1 (See Scheme 2), were identified by an ABSciex Triple Quad 4500 (UHPLC-MS/MS) mass spectrometer equipped with a Turbo Ion Spray (AB Sciex) ion source. A microsyringe pump delivered the mixed reaction of 1 with piperidine in DMSO at infinite time dissolved in 10% (vol/vol) acetonitrile into the ESI source at a flow rate of 10 μL/min. ESI and the QQ (linear trap) mass spectrometer were operated in the negative-ion mode for detecting 1a and 1c and the positive mode for 1b and 1d by using the multiple reaction monitoring (MRM) scan types. Main conditions: curtain gas nitrogen flow = 10 mL min-1; ion spray voltage = −4,500 eV; declustering potential = −60 eV; entrance potential = −10 eV; collision cell exit potential = −12 eV; source temperature was set at 300°C and source gas GS1 and GS2 were set to 12 and 0, respectively. All data were acquired using Analyst 1.6.2 (AB Sciex).

Plasma and urine concentrations of dotinurad and its major metabolites (glucuronide and sulfate conjugate) were measured using liquid chromatography (LC)–tandem mass spectrometry (MS/MS) at Fuji Yakuhin Co., Ltd.
For dotinurad concentration measurement: Agilent 1100 series (Agilent Technologies, USA) for LC, API3000 (AB SCIEX, USA) for MS, Inertsil ODS-3 (150 × 2.1 mm, 3 μm; GL Sciences, Inc., Japan) for column analyses, and 5 mmol/L ammonium acetate solution/methanol for the mobile phase. The limit of quantification was 1 ng/mL for the dotinurad concentration measurement in plasma and urine.
For dotinurad metabolite concentration measurement: Nexera X2, Prominence (Shimadzu, Japan) for LC, and Triple Quad 4500 (AB SCIEX) for MS. The limit of quantification was 1 ng/mL for dotinurad metabolite concentrations in plasma and urine, for both glucuronide conjugate and sulfate conjugate. Uric acid concentration in serum and urine was measured using the enzyme method, at the study site.

Melting points were determined on a Kofler Thermogerate apparatus and were uncorrected. Infrared spectra were recorded on a JASCO FT/IR-400 spectrophotometer. Nuclear magnetic resonance spectra were recorded, unless otherwise specified, on a Bruker AM-400 instrument using deuterated chloroform or dimethylsulfoxide solutions containing tetramethylsilane as an internal standard. ESI/MS experiment was carried out on an UHPLC Eksigent1 coupled with MS detector ABSciex1, Triple Quad 4500 model equipment. HRMS-ESI-MS experiments were performed using a Thermo Scientific Exactive Plus Orbitrap spectrometer with a constant nebuliser temperature of 250 °C. The experiments were carried out in positive or negative ion mode, with a scan range of m/z 300.00–1510.40 and a resolution of 140,000. The samples were infused directly into the ESI source via a syringe pump at flow rates of 5 µL·min⁻¹ through the instrument’s injection valve. Thin layer chromatography (TLC) was performed using Merck GF-254 type 60 silica gel. Column chromatography was carried out using Merck type 9385 silica gel. The purity of the compounds was determined by TLC and high-resolution mass spectrometry (HRMS) and for 4s by HLPC (Figure S8).

The concentrations of TRP and KYN in the sera and CSF of subjects were measured by the combination of high-performance liquid chromatography (LC-30AD, Shimadzu, Kyoto, Japan) and triple quadruple mass spectrometry (Triple Quad 4500, AB Sciex, Boston, MA, USA). The parameters of the mass spectrometer and the mobile phase were set as previously described (Wang et al., 2018 (link)). IDO1 activity was determined according to the KYN/TRP ratio.

First, 200 ng Col-0 mRNA was digested into nucleosides by Nuclease P1 (NEB and shrimp alkaline phosphatase (NEB) in 50 μl RNase-free water, and incubated at 37 °C overnight. The mixture was diluted to 100 μl, and 10 μl from each sample was injected into an LC-MS/MS system consisting of a high-performance liquid chromatographer (Shimadzu) equipped with a C18-T column (Weltech) and a Triple Quad 4500 (AB SCIEX) mass spectrometer in positive ion mode by multiple-reaction monitoring. Mass transitions of m/z 268.0–136.0 (A), m/z 245.0–113.1 (U), m/z 244.0–112.1 (C), m/z 284.0–152.0 (G), and m/z 282.0–150.1 (m⁶A) were monitored and recorded. The concentration of nucleosides was quantified according to the standard curves generated against pure commercial nucleosides (MCE).

Flavonoids were identified using an ABSciex triple Quad 4500 mass spectrometer equipped with an electrospray (TurboV) interface coupled to an Eksigent Ekspert Ultra LC100 with an Ekspert Ultra LC100-XL autosampler system (AB/Sciex Concord, ON, Canada). Chromatographic separation occurred using a gradient elution with (A) 0.1% formic acid and (B) methanol as the mobile phase as follows: 0–1 min, 15% B; 1–17 min, 15–100% B; 17–21 min 100–100% B; 21–22 min, 100–15% B; and 22–25 min, 15–15% B. The instrument was operated using an injection volume of 50 μL, a flow rate of 0.5 mL/min, and an end-capped column (LiChrospher 100 RP-18; 125 mm × 4 mm i.d., 5 μm; Merck, Darmstadt, Germany) maintained at 50 °C. Calibration curves for quantification were constructed using commercially available standards. Table 1 shows the parameters used for compound identification.

Analysis of 54 pesticides was performed on Triple Quad 4500 (AB SCIEX, Framingham, MA, USA). Separation of target compounds was achieved on Waters ACQUITY UPLC^®BEH C18 column (100 mm × 2.1 mm, 1.7 μm) at a temperature of 40 °C. The mobile phase consisted of 0.1% formic acid (FA) aqueous solution containing 4 mM ammonium acetate (A) and methanol (B). Gradient elution procedure was: 5–20% B at 0–1 min, 20–40% B for 0.10 min, 40–60% B for 1.9 min, 60–80% B for 0.1 min, 80–95% B for 1.9 min and holding for 2 min, 95–5% B for 2 min then holding for 1 min. The flow rate of the mobile phase was 0.30 mL/min and the injection volume was 2 μL.
Mass spectrometry analyses of pesticides were conducted in both positive mode (ESI⁺) and negative mode (ESI⁻). Mass spectrometry parameters were set as follows: curtain gas 35 psi, collision gas 9, ion source gas 1 (GS1) 55 psi, ion source gas 2 (GS2) 55 psi, ionspray voltage 5500 V (ESI⁺) and 4500 V (ESI⁻), temperature 550 °C. MRM parameters and retention time of 54 pesticides are shown in Table 1.