Q exactive high resolution mass spectrometer

An Acquity UPLC system (Waters Corporation, Milford, MA, USA) combined with a Q-Exactive high-resolution mass spectrometer (Thermo Fisher, Waltham, MA, USA) with an Orbitrap mass analyzer was used. Samples (5 μL) were injected onto an ACQUITY UPLC BEH Shield RP18 column (150 × 2.1 mm, particle size 1.7 μm) (Waters, Manchester, MA, USA), with a flow rate of 0.35 μL min⁻¹ at 50 °C. Mobile phases contained 0.1% (v/v) formic acid in water (A) (LC-MS grade, Merck, Darmstadt, Germany) and acetonitrile (B) (LC–MS grade, Merck). A multi-step linear gradient was as follows: 5% B—1.5 min, 80% B—10.5 min, 98% B—11.5 min, 5% B—13 min.
Mass spectrometry analysis was performed using heated electrospray ionization (H-ESI) in positive and negative modes. A 3.5 kV and 2.5 kV ion spray voltage was applied for positive and negative ionization, respectively. Ion source temperature was 320 °C. Data were acquired in Full MS/data-dependent MS2 mode in the 100–1500 m/z range. The resolution of Full MS was 70,000 and of ddMS2 17,500. Normalized collision energy in the ddMS2 experiment was set to 30%. Xcalibur software (ThermoFisher Scientific, Waltham, MA, USA) was used for system operation, data acquisition, and data analysis [58 (link)].

Ripe Areca seed was thinly sliced into chips and dried under the sunshine for three days. The dry Areca chips were grinded and sieved with a 200-mesh filter. After that, Areca powder was extracted by the maceration method using a methanol analyzer solvent. The composition of Areca extract was tested using LC-MS-MS metabolomic. The sequence of extraction mechanisms up to the Areca nut composition test is shown in Figure 1.
Areca nut extract was dissolved in 0.1% formic acid in acetonitrile LC-MS gade, flowing in an analytical column Hypersil GOLD aQ, 50 × 1 mm, particle size 1.9 μm. Analytical flow rate was 40 μL/min with run time of 70 minutes and oven temperature of 30°C. Samples were analyzed on Thermo Scientific Q Exactive High-Resolution Mass Spectrometer, full scan at 70,000 resolution, MS2 dependent data at 17,500 resolution, and run time of 70 minutes. Data was processed using software: Compound Discoverer with mzCloud MS/MS Library.
The LC-MS test result shows that Areca extract contains guvacoline, arecoline, choline, epicatechin, trigonelline, and other molecules in small amounts. Epicatechin is among these molecules that can bind to vegetable oils, as shown in Figure 1. Epicatechin has positively charged hydroxyl, which attracts negatively charged carbonyl in vegetable oils. This attractive force makes epicatechin evenly scattered in vegetable oils.

The assessments of untargeted metabolomics were conducted by BGI Tech (Guangzhou, China). The experimental procedure included metabolite extraction and LC–MS/MS detection. Briefly, 25 mg tissues were weighed and extracted by directly adding 800 µL of precooled extraction reagent (methanol: acetonitrile: water (2:2:1, v/v/v)), internal standards mix was added for quality control of sample preparation. The samples were homogenized for 5 min using TissueLyser (JXFSTPRP, China), followed by 10 min of sonication and an hour of incubation at – 20 °C. The supernatant from the centrifugation of the samples was then transferred for vacuum freeze drying after 15 min at 25,000 rpm and 4 °C. The metabolites were resuspended in 600 µL of 10% methanol and sonicated for 10 min at 4 °C. After centrifuging for 15 min at 25,000 rpm, the supernatants were transferred to autosampler vials for LC–MS analysis. To evaluate the consistency of the entire LC–MS analysis, a quality control (QC) sample was created by pooling the same volume of each sample. We employed a tandem Q Exactive high-resolution mass spectrometer from Thermo Fisher Scientific (USA) and Waters 2D UPLC (USA) for metabolite separation and identification.

Sample collection, sample preparation, and metabolomics analysis were performed as previously described [11 (link)]. In short, untargeted metabolomics was performed by direct-infusion high-resolution mass spectrometry, using a TriVersa NanoMate system (Advion, Ithaca, NY, USA) controlled by Chipsoft software (version 8.3.3, Advion, Ithaca, NY, USA), mounted onto the interface of a Q-Exactive high-resolution mass spectrometer (Thermo Scientific™, Bremen, Germany). For each sample, technical triplicates were analyzed, infusing each sample three times into the mass spectrometer. Scan range was 70 to 600 m/z.Mass peak annotation was performed by matching the m/z value of the mass peak with a range of two parts per million to monoisotopic metabolite masses present in the HMDB, version 3.6 [27 (link)]. Annotations of metabolites that can occur endogenously were selected: >1800 unique m/z per batch, corresponding to >3800 metabolite identifications, in line with previous analyses [11 (link)]. For each mass peak per patient sample, the deviation from the intensities in the control samples was indicated by a Z-score, calculated by Z-score = (intensity patient sample − mean intensity control samples)/standard deviation intensity control samples. Z-scores were calculated for both patient and control samples (Figure 6).

A fecal sample (100 mg) was added to 100 μL of pure water. The solution was centrifuged at 16,000 g for 15 min at 4°C and 800–850 μL of the supernatant was transferred into a new tube. Concentrated samples were dried in a vacuum and then added to 40 μL of 15 mg/mL methoxyamine pyridine, which was then vortexed for 30 s, and incubated at 37°C for 90 min. Next, 40 μL of BSTFA reagent (containing 1% TMCS) was added to the mixture and incubated at 70°C for 60 min. The mixture was centrifuged at 12,000 rpm for 5 min and the supernatant was transferred to an injection bottle (Dunn et al., 2011 (link)). Twenty microliters of each sample was used for quality control (QC).
To improve metabolite coverage, non-targeted metabolomics analysis was performed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and a Q Exactive high-resolution mass spectrometer (Thermo Fisher Scientific, USA) was used to acquire data in positive and negative ion modes. LC-MS/MS data was processed by Compound Discoverer 2.1 and included intelligent peak extraction, metabolite identification, and peak alignment.

The detection was performed using a tandem Q Exactive high-resolution mass spectrometer (Thermo Fisher Scientific, USA). The chromatographic column used was a CSH C18 (1.7 μm, 2.1 × 100 mm, Waters, USA). In positive ionization mode, the mobile phases consisted of an aqueous solution containing 10 mM ammonia formate, 0.1% formic acid, and 60% acetonitrile (liquid A) and a solution containing 10 mM ammonia formate, 0.1% formic acid, 90% isopropanol, and 10% ACN (liquid B). Additionally, in negative ionization mode, an aqueous solution containing 10 mM ammonia formate and 60% ACN (liquid A) and a solution containing 10 mM ammonia formate, 90% isopropanol, and 10% acetonitrile were used.