Triple quadrupole mass spectrometer

For the hormonal analysis, fresh material was frozen in liquid N, ground, and freeze-dried. Fresh tissue (0.5 g) was immediately homogenized in 2.5 mL of ultrapure water, and 100 ng mL⁻¹ of a mixture of internal standards ((2H6-ABA (to quantify ABA), 2H4-SA (to quantify SA and propylparaben (to quantify phenolic compounds like ferulic acid (FA) and chlorogenic acid (CGA)), (Sigma–Aldrich, St. Louis, MO, USA)) were added prior to extraction. The samples were centrifuged at 5000 rpm for 45 min at 4 °C. The supernatant was partitioned against diethylether, dried in a speed vacuum and resuspended in 90:10 H₂O:MeOH. [78 (link)]. After extraction, a 20 µL aliquot was injected directly into an ultra-high performance liquid chromatography (UPLC) system with an ACQUITY UPLC BEH C18 column (1.7 μm 2.1 × 50 mm) (Waters, Mildford, MA, USA), which was interfaced with a triple quadrupole mass spectrometer (TQD, Waters, Manchester, United Kingdom). Version 4.1 of the MASSLYNX NT software (Micromass) was used to process the quantitative data from the calibration standards and plant samples. The concentrations of hormones and phenolic compounds were determined in each sample by normalizing the chromatographic area for each compound with the fresh weight of the corresponding sample.

Harmaline and harmine concentrations were simultaneous quantitative determined on a Waters-ACQUITY™ UPLC system (Waters Corp., Milford, MA, USA) using an ACQUITY UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm particle size). Mass spectrometric detection was performed using a triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with electrospray ionization in positive ionization mode, and all other instrumental parameters were set according to our previous study (Li et al., 2016 (link)). The UPLC-ESI-MS/MS method was well-validated (data not shown) and the analytical method was successfully applied to determine the concentration of harmine and harmaline in the HBSS buffers. Figure S2 presents the representative of typical MRM chromatograms of blank HBSS, blank HBSS spiked with harmine, harmaline and IS, and IS-spiked HBSS sample collected at 30 min after administration of harmine and harmaline.

For analysis of SCFA/MCFA, a liquid–liquid extraction procedure was used [25 (link)]. For quantification a Trace GC Ultra gas chromatograph (Thermo Fisher Scientific, San Jose, CA, USA) was used, equipped with an autosampler PAL combi-xt autosampler (CTC Diagnostics AG, Zwingen, Switzerland) coupled to a TSQ Quantum XLS tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The GC–MS data processing was performed using a qualitative and quantitative software package, XCALIBUR™ 2.2 (Thermo Fisher Scientific, San Jose, CA, USA).
For analysis of polyphenol metabolites, the extraction procedure was as described previously [26 (link)]. The MS system used was a Waters Xevo TQ (Milford, MA, USA) triple quadrupole mass spectrometer, coupled with an electrospray interface and polarity switching option during acquisition. The LC-MS data processing was performed using qualitative and quantitative packages in Waters MassLynx 4.1 and TargetLynx software.