Q exactive hybrid quadruple orbitrap mass spectrometer

An aliquot (1 μL) of each reconstituted extract (0.2 μg/μL) was injected and analyzed on an UltiMate 3000 UHPLC coupled to a Q Exactive Hybrid Quadruple-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Samples were chromatographically resolved at a flow rate of 0.45 mL/min on a C₁₈-reverse-phase column (HSS T3, 2.1 mm i.d. ×100 mm, 1.8-μm particle size; Waters, Milford, MA) by mixing mobile-phase solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) to generate the following gradient: 0 to 1 min, 2% B; 1 to 10 min, 2 to 30% B; 10 to 12 min, 30 to 50% B; 12 to 20 min, 50 to 75% B; 20 to 22 min, 75 to 98% B; 22 to 23 min, 98 to 2% B; 23 to 27 min, 2% B. The mass spectrometer was operated in the positive/negative switching ionization mode over a scan range of 150–2000 m/z. The presence of monomers, dimers, trimers, and tetramers was monitored using the [M + H]⁺m/z values of 229, 445, 681, and 907, respectively. Ions yielded in the [M–H]⁻ mode were not used for mQTL mapping due to ionization issues. UV/visible absorbance data were simultaneously collected using the UltiMate 3000 UHPLC diode array detector throughout each separation; spectra with λ_max values of 280 nm and 306 nm were consistent with eluting stilbenoids.

A Thermo Scientific™ Q Exactive™ hybrid quadruple Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a HESI-II probe was employed. The positive and negative HESI-II spray voltages were 3.7 kV and 3.5 kV, respectively, the heated capillary temperature was 320 °C, the sheath gas pressure was 30 psi, the auxiliary gas setting was 10 psi, and the heated vaporizer temperature was 300 °C. Both the sheath gas and the auxiliary gas were nitrogen. The collision gas was also nitrogen at a pressure of 1.5 mTorr. The parameters of the full mass scan were as follows: a resolution of 70,000, an auto gain control target under 1 × 10⁶, a maximum isolation time of 50 ms, and an m/z range of 150–1500. The parameters of the dd-MS2 scan were as follows: a resolution of 17,500, an auto gain control target under 1 × 10⁵, a maximum isolation time of 50 ms, a loop count of the top 10 peaks, an isolation window of m/z 2, a normalized collision energy of 30v and an intensity threshold under 1 × 10⁵. The LC-MS system was controlled using Xcalibur 2.2 SP1.48 software (Thermo Fisher Scientific), and the data were collected and processed with the same software.