6470 triple quad

We used an Agilent Technologies 1290 Infinity II LC system, including an autosampler, binary pumps and a thermostatted column compartment with 6470 Triple Quad (Agilent Technologies, Santa Clara, CA, USA). Other necessary equipment were MS 40+ Vacuum Products (Agilent Technologies, Santa Clara, CA, USA) and a nitrogen generator NM32LA (Peak Scientific, Inchinnan, UK). The sample separation was carried out on a reversed phase column SB-C8, 1.8 µm, 2.1 × 100 mm (Agilent Technologies, Santa Clara, CA, USA).

LC–MS experiments were performed on an Agilent 1290 Infinity II equipped with a Phenomenex Synergi 2.5 µm Fusion-RP 100 Å (100 × 2 mm) coupled to an Agilent 6470 Triple Quad equipped with electron spray ionization. Of each sample, 18 µL were injected without prior filtering. Chromatographic separation was carried out at 35 °C with a flow rate of 0.35 ml/min using a linear gradient of two solvents: 5 mM ammonium acetate pH 5.3 as solvent A and acetonitrile as solvent B (gradient: 0–1 min hold at 0% B, 1–5 min increase to 10% B, 5–7 min increase to 40% B, 7–8 min hold at 40% B, 8–8.5 min decrease to 0% B, 8.5–11 min hold at 0% B). Optimized MS parameters for each compound can be found in Supplementary Data 6. Quantification was done using calibration curves of synthetic standards and stable isotope labeled internal standards (20 ng in 1 µL was automatically added by the instrument per sample) for each nucleoside⁷³ using Agilent’s MassHunter software (version 9.0.647.0). To obtain the calibration curves, a solution containing synthetic standards of all nucleosides was serially diluted by factor 1:2 (twelve calibration levels). The highest injected amounts were 100 pmol (C, U, G, A), 20 pmol (Ψ), or 5 pmol (all other nucleosides).

A UHPLC Agilent 1290 Infinity II LC coupled with a G7167B autosampler, a G7120A pump, and a GT116B MCT oven were connected to an Agilent 6470 Triple Quad LC/MS/MS mass detector with a MassHunter ChemStation. The column was a ZORBAX Eclipse Plus C18 (2.1 mm × 150 mm, 1.8 μm). The UHPLC analysis was conducted using a binary gradient: solvent A (H₂O 5 mM in ammonium formate + 0.1% formic acid) and solvent B (methanol 5 mM ammonium formate + 0.1% formic acid).
The elution gradient was T = 0, A 95%; T = 3.50 min, A 60%; T = 17 min, A 2%; and 10 min of post-run A 95%. The run’s total duration was 20 min, and retention times were 13.69 and 14.24 for fipronil and fipronil sulfone, respectively. The flow was 0.3 mL/min.
The sample (2 μL) was injected in negative and positive modes. Mass detector conditions were gas and sheath-gas temperature 350 °C, gas flow 10 L/min, sheath-gas flow 12 L/min, nebulizer 30 psi, and capillary negative 3000 V. MRM transitions were m/z 435–330 and 435–250 for fipronil, 451–415 and 451–282 for fipronil sulfone.