Masshunter workstation qualitative analysis software

The metabolite profiles of GAL were determined using an LC-QTOF-MS instrument (Agilent 1290 Infinity LC instrument coupled to an Agilent 6540 series QTOF-MS equipped with an ESI source and a diode-array detector (DAD)) in positive ion mode. Chromatographic separation was conducted on an Agilent Poroshell 120 EC-C18 column (150 mm length x 4.6 mm inner diameter, particle size 2.7 μm). The gradient elution was performed using 0.1% formic acid water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 200 μL/min. The injection volume was 1.0 μL, and the column temperature was maintained at 35°C. Data acquisition was controlled using the Mass Hunter Workstation Software Qualitative Analysis (version B.08.00, Agilent Technologies, California, United States of America). The phytochemical compounds present in the extract samples were identified by comparing retention times, mass data, and fragmentation patterns with a compound database in the library search of the Agilent MassHunter Personal Compound Database and Library (Agilent Technologies). Peaks with similarity scores of 80% compared to the database were selected to confirm peak identification (Sun et al., 2015 (link); Zhu et al., 2022 (link)).

Before annotating the features of interest described above, their calculated retention time and m/z value were compared with the experimental values of the main peaks found on the spectra extracted from the extracted ion chromatograms (EIC), which were obtained from the total ion chromatograms (TIC) of the samples. Only the features of interests that corresponded to peaks in terms of retention time and m/z value were kept for the annotation. This was performed using the molecular feature extraction algorithm of the MassHunter Workstation software – Qualitative analysis (Agilent, USA). The features were annotated using the online databases METLIN (metabolite database), KNApSAcK (plant species-metabolite relationship database [71 (link)]), HMDB (Human Metabolome Database) and PubChem (chemical database) and using published data [15 (link), 20 , 72 (link)–75 (link)]. A maximum error of 10 ppm was allowed between the neutral measured mass and the monoisotopic exact m/z values.

Molecular low weigh metabolites were extracted from algal cells using MeOH, then derivatized with bis(trimethylsilyl)trifluoroacetamide to gain trimethylsilyl derivatives and analyzed using a gas chromatograph (7890B GC System) with mass selective detector MSD 5977A (Agilent Technologies, USA) equipped with HP-5MS fused silica column (30 m × 0.25 mm × 0.25 μm)³ . Metabolites were identified using an automatic system of processing data (Agilent MassHunter Workstation Software Qualitative Analysis) supplied by the National Institute of Standards and Technology (NIST) database.

The samples were analyzed using a Zorbax Eclipse XDB-C18 column (5 µm, 4.6 × 150 mm), run on the Agilent 1200 Series HPLC system (Santa Clara, CA, USA), connected to a diode array detector, followed by the 500TR Series Flow Scintillation Analyzer (Packard Bioscience, Meriden, CT, USA) or the Agilent 6540 UHD Accurate Quadrupole Time-of-Flight MS/MS with an Agilent Jet Stream™ ESI source in negative mode(Santa Clara, CA, USA). The elution was performed with a solvent system of ACN/water/formic acid (98.9%/1.0%/0.1% v/v/v) (A) and water/formic acid (99.9%/0.1% v/v) (B), 0–8 min isocratic (35%A:65%B), 9–17 min gradient to 100% A, 18–30 min 100% A at a flow rate of 1 mL min⁻¹ [26 (link)]. The data were analyzed by Agilent MassHunter Workstation Software Qualitative Analysis, Version B.05.00 Build 5.0.519.0. Eicosanoids were identified by comparing their retention times and mass spectra with those of authentic standards.

Analyses of the butanol residues of each plant were performed with UHPLC Agilent 1290 infinity system with a DAD G4212A and MS Agilent G6540A Q-TOF with Agilent Jet Stream technology electrospray ionization. Separation was performed on a Phenomenex Luna Omega column (C18, 1.9 u, 90 A°, 75 × 2.0 mm) using gradient mixtures of 0.1% formic acid (solvent A) and MeCN supplemented with 0.1% formic acid (solvent B). Gradient: 0.0−8.0 min, 0→30% B; 8.0−8.1 min, 30→98% B; 8.1−9.1 min, 98% B; 9.1−9.2 min, 98→5% B; 9.2−10.0 min, 5% B; flow rate, 0.6 mL/min; injection volume, 1 μL; oven temperature, 40 °C. Data analysis was performed by MassHunter Workstation Software Qualitative Analysis (B.07.00, B.10.00, Agilent) using automatic mass spectrum integration. LC-Q-TOF-MS/MS analyses were performed in positive and negative ionization modes to obtain maximum information on its composition. The metabolites were characterized based on their mass spectra, using the precursor ion and comparison of the fragmentation patterns with molecules described in the literature [15 (link)]. The putative identification of these compounds is summarized in Table 1, where the compounds are listed according to their retention times in the total ion chromatogram (TIC).

The raw data obtained by UHPLC-Q-TOF-MS/MS were converted into mzData through Agilent MassHunter Workstation Software Qualitative Analysis, version B.07.00, build 7.7.7024.29 SP2 (Agilent Technologies, Santa Clara, CA, USA) to be analyzed with XCMS Online version 3.7.1 (www.xcmsonline.scripps.edu (accessed on 10 June 2020)) [49 (link)]. Information was analyzed by pairwise analysis: MCF-7 untreated cells versus MCF-7 treated with VPA. The parameters selected for the jobs were those available for a UPLC/UHD Q-TOF, which included: m/z tolerated deviation of 15 ppm, peak width from 5 to 20 s, signal/noise threshold of 6, mzdiff of 0.01, mzwid of 0.015, bw of 5, minfrac and minsamp of 0.5 and 1, respectively, a fold change of 1.5, p-value threshold of 0.05 for significant features and 0.01 for highly significant features. An unpaired parametric t-test (Welch t-test) was used to evaluate the results. Putative metabolite annotation and identification were performed through the METLIN database available through XCMS, allowing a 5 ppm error and 0.015 m/z absolute error for the annotation and a 10 ppm tolerance for the database search using human as the selected biosource for the identification [50 (link)]. Only those features that were identified through this method were further considered as putative metabolites.

Reaction products were generated by preparing unlabeled (12C14N) and isotope-labeled (13C15N) sodium cyanide solutions at 0.135 M, pH = 7.4. Next a stock solution of 20 mM glyoxylate was prepared in water. Glyoxylate was added to the cyanide solutions generating a final concentration of 20 µM glyoxylate and incubated (1 µL of 20 mM glyoxylate, pH = 7.4, was added to 1 mL of cyanide, pH = 7.4) for 2 h. The resulting mixture was diluted tenfold with HPLC-grade methanol and then infused directly into an Agilent 6550 iFunnel Q-TOF Mass Spectrometer equipped with a dual AJS-ESI source. The MS-only full scan and/or targeted MS/MS scan in positive mode was acquired through Agilent MassHunter Workstation LC/MS Data Acquisition software (version B.05.01). MS1 and MS2 spectra were obtained and compared using Agilent MassHunter Workstation Qualitative Analysis software (version B.06.00) for peak identification.