Accela uhplc

The extraction and the quantification of 3SH precursors in grape berries and musts was performed as described [38 ]. In brief, the purification was carried on a mix of 1 mL of grape juice obtained by defrosting of 2 g of frozen berry powder in the presence of sulfur dioxide (200 mg L⁻¹) or directly from grape must, 1 mL of water and a final concentration of 50 μg L⁻¹ of the internal standard solution containing a deuterated form of the glutathionylated S-conjugate of 3SH ((3-S-hexan-1-ol)-glutathione-d₃) percolated through a SPE columns (LC-18 500 mg 6 mL, Supelco France, Saint Germain-Laye, France). Precursors were eluted with 3 mL of methanol/water (30/70; v/v) in hemolysis tubes. The flow-through was subsequently evaporated and residues were dissolved in 700 μL of aqueous formic acid solution (0.1 %). Quantification was done using an Accela UHPLC (Thermo Fisher Scientific) connected in series to an Exactive (Thermo Fisher Scientific, Bremen, Germany) mass spectrometer equipped with a heated ESI ion source (LC-ESI-MS). The separation was performed on a Synchronis aQ column (100 × 2.1 mm i.d., 1.7 μm, Synchronis aQ, Thermo Scientific, Bremen, Germany) with a flow rate of 300 μL min⁻¹ of solvent A (0.1 % aqueous formic acid) and solvent B (0.1 % formic acid in acetonitrile). The ion source was operated in the positive ion mode at 3.5 kV.

The UPLC separation was conducted on a Thermo TSQ Quantum Access Max equipped with Accela U-HPLC and autosampler (Thermo Fisher Scientific, Waltham, MA). A reversed-phase Hypersil GOLD column (100×2.1 mm, 1.9 μm, Thermo) was used, and 95% water (containing 0.1% formic acid) and 5.0% methanol was used as mobile phase at a flow rate of 0.2 mL/min. The eluate from the HPLC column was directly introduced into an ESI-triple quadrupole mass spectrometer (TSQ Quantum Access MAX). The mass spectrometer was operated in the positive ion mode. For Selective Reaction Monitoring (SRM) analysis, collision energy was performed at 15 eV. The fragmentor voltage was 90 V, and nitrogen was used as nebulizer gas. The desolvation gas (nitrogen) was heated to 300 °C and delivered at a flow rate of 9.0 L/min. The capillary voltage was set at 3,500 V. The injection volume is 5.0-15.0 μL for the digested DNA. Then, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in cellular DNA was detected in the form of the mononucleotide 8-oxodG by monitoring the transitions of m/z 284.1→168.1. The amount of 8-oxodG was calibrated by the standard curve.

A quality control sample (QC) taken from all samples was prepared in order to periodically assess the reproducibility of the measurements. The separation of the analytes contained in the saffron samples was achieved with a Fortis UPLC C₁₈ column (2.1 mm × 100 mm, 1.7 µm, Fortis Technologies Ltd., Cheshire, UK). The hyphenated LC-HRMS system comprised of an Accela UHPLC equipped with an autosampler, a vacuum degasser, a binary pump and a temperature-controlled column (Thermo Scientific, Germany) coupled to an Orbitrap Discovery XL, which was equipped with an IonMAX ion source (Thermo Scientific, Bremen, Germany). The mobile phase consisted of 0.1% aq. formic acid (v/v) (solvent A) and 0.1% formic acid in LC–MS grade ACN (v/v) (solvent B). The gradient program was for solvent B: 5% at 0 min, 5% at 3 min, 95% at 24 min, 95% at 26 min, 5% at 28 min, 5% at 30 min. The overall analysis time spanned for 30 min, whereas the injection volume was 5 µl keeping a flow rate of 400 µl min⁻¹. The positive ionization ESI mode was used using a mass range of 100–1000 amu. The “big three” approach, employing parallel scans, was used. The samples were centrifuged using a Mikro 200R centrifuge (Hettich Lab Technology, Tuttlingen, Germany), and for the solvent evaporation was performed on a GeneVac HT-4X EZ-2 series evaporator Lyospeed ENABLED (Genevac Ltd., Ipswich, UK).

High-resolution LC-MS analyses were carried out to quantify sclareol and manool in the different extracts, using an LTQ-orbitrap spectrometer coupled with an Accela UHPLC (Thermo Fisher Scientific). Pure compounds were used to set up and validate the method. Mass spectra were acquired in positive high-resolution single ion monitoring (hrSIM) mode, to maximize selectivity and sensitivity. Ions 291.2685 [sclareol-H₂O + H]⁺ and 273.2582 [manool-H₂O + H]⁺ were monitored for the two compounds. Chromatography was performed on a Kinetex C8 column (100 × 2.1 mm, 1.7 µm; Phenomenex) using a mixture of 0.1% formic acid in water (Eluent A) and methanol (Eluent B) as mobile phase. Compound elution was achieved through a gradient from 55 % to 85 % of B over 7 min. Using this method, a Lower Limit of Detection (LLOD) of 0.1 µg/mL was measured for both compounds, whereas the Lower Limit of Quantization (LLOQ) was 0.2 µg/mL, and the response was linear over a 0.3–6 µg/mL concentration range (Figure S12, Supplementary material). Different samples were analyzed in triplicate, injecting 10 µL of each 1 mg/mL extract.

Lipids were extracted by a previously published methyl-tert-butyl ether protocol^{53 (link)} and an aliquot of the lipid extract was resuspended in 100 µl isopropanol:chloroform:methanol (90:5:5 v/v/v). Fourfold experiments of MUG-Mell 1, C8 and D5 were carried out. Data were acquired in data dependent acquisition mode by an LTQ Orbitrap Velos Pro instrument (Thermo Scientific) coupled to an Accela UHPLC (Thermo Scientific) according to previously published protocols^{54 (link),55 (link)} with 100.000 mass resolution at m/z 400. Data were analyzed by Lipid Data Analyzer (LDA), a custom developed lipidomics software package⁵⁶ .

RP HPLC was performed using a Shimadzu Prominence HPLC system and a Phenomenex Luna C₆-Phenyl column (5μm, 250 x 10 mm). The mass spectrometry data for 2 and 3 were acquired with a Bruker MaXis Quadrupole Time-of-Flight MS coupled to a Waters Acquity UPLC system operated by Bruker Hystar software, and for 1 and 4 with an Accela UHPLC (Thermo Scientific, USA) apparatus with an 80 Hz photodiode array detector (PDA) coupled to a Q-Exactive Plus Orbitrap mass analyzer (Thermo Scientific, USA). NMR spectra of 1–4 were obtained in CDCl₃ with a Varian Unity-Inova 500 MHz spectrometer. The LC-HRESIMS and MS/MS spectra of organic extracts of Atta sexdens workers were acquired with a UPLC (Shimadzu) coupled to a micrOTOF II mass spectrometer (Bruker Daltonics).