Tracefinder

The characterization chemical ingredients of P. longum Linn were carried out as described previously study [23 (link),24 (link)]. Dionex UltiMate 3000 system (LabX, Midland, ON, Canada) with a Thermo Q-Exactive mass spectrometer (UHPLC-MS/MS, Thermo Fisher Scientific) were used for analysis. All tested compounds were separated using an Acquity BEH C18 column (100 × 2.1 mm, 1.7 μm) with water and acetonitrile containing 0.1% formic acid. The mass spectra of P. longum Linn analyzed using a heated electrospray ionization source. The full scan mass spectra were acquired in positive ion mode at a scan range of 100–1500 m/z in data-dependent MS2 scan mode. Data acquisition and analysis were based on Xcalibur and TraceFinder software (Thermo Fisher Scientific). The reference standards, magnoflorine and piperine, were purchased from Targetmol (Wellesley Hills, MA, USA).

Human plasma samples were thawed on ice, and 100 ul were extracted with 900 ul of cold extraction buffer containing 40:40:20 methanol:acetonitrile:water [v:v:v]. After 30 min in an orbital mixer at 4°C, samples were sonicated for 10 min in an ice-cooled bath-type sonicator and centrifuged for 10 min at 16000xg at 4°C. Supernatants were collected and dried in a SpeedVac until complete dryness. Standard curves of Nam were prepared with concentrations ranging from .005 to 50 ug/ml (expected limit of detection .1–.5 ng/ml). Standards were processed in the same way as samples. Dried down samples and standards were derivatized using methoxyamine and MSTFA/FAMEs solution (N-methyl-N-trimethylsilyl-trifluoracetamid/Fatty acid methyl esters) following standard procedures (Lisec et al., 2006 (link); Caldana et al., 2013 (link)). After that, samples were analyzed in a GC-EI-MS (Q Exactive GC Orbitrap system, ThermoFisher) using a 30-m DB-35M capillary column. Representative fragments from the GC-EI-MS analysis of Nam were extracted using TraceFinder (Version 4.1, ThermoFischer) and quantified using the linear range of the obtained standard curve. All analysis were performed using peak areas, transformed into Z-scores, for easier comparison among experiments.

Initially, 3 × 10⁶ cells were pelleted (300g for 2 min at room temperature) and the medium aspired followed by immediate addition of 1.5 mL ice-cold 80% MeOH and snap-frozen in liquid nitrogen. The samples were subsequently thawed on ice, vortexed for 30 s before being snap-frozen again and the procedure repeated for a total of three cycles. Finally, any undissolved fractions were pelleted (12,000g for 10 min at 0 °C) and the supernatant dried under a light flow of N₂ before the samples were resuspended in 30 μL 50% acetonitrile (pH 9) and diluted two times in 10 mM ammonium acetate in 90% acetonitrile (pH 9). Analysis of the intracellular metabolites was performed by MS-Omics. Overall, the LC–MS method was modified from Hsiao et al.^{42 (link)} using a Thermo Scientific Vanquish LC coupled to a Thermo Q Exactive HF MS with a heated electrospray ionization interface operated in negative and positive ionization mode. For the untargeted analysis peak areas were extracted using Compound Discoverer (vers: 3.0.0.294, Thermo Fisher Scientific Inc.), while the targeted analysis was conducted using TraceFinder (vers 4.1, Thermo Fisher Scientific Inc.).

The chromatographic data
was processed by peak detection, retention time alignment, and peak
integration followed by isotope ratio filtering. This resulted in
a processed dataset with peak areas from extracted ions at specific
retention times corresponding to different compounds. Data processing
was done in Tracefinder (version 4.1, Thermo Scientific, MA, U.S.)
using the analysis mode for unknown screening, which enables nontargeted
screening of data. Peak picking was done with the deconvolution plugin
(version 1.3, Thermo Scientific, MA, U.S.). The retention time alignment
window was set to 10 s, the accurate mass tolerance to 10 ppm, the
signal-to-noise (s/n) threshold to 5, the total ion-chromatogram intensity
threshold to 500,000, the ion overlap window to 90–99%, and
the response threshold to 10,000.
The extracted peaks were automatically
time-aligned and integrated in the unknown screening view using the
Avalon detection algorithm and the nearest RT detection method with
seven smoothing points. Data representing compounds present in blank
samples were manually removed from each crude HD dataset. The datasets
for spiked soil and textile samples were processed and manually merged
after removing peaks found in blank samples. The variation in the
spiked matrix data was higher than in the crude HD data, so the m/z deviation threshold was increased to
0.01 to permit merging.