Triple toftm 5600

A total of 50 mg of each intestinal content sample was accurately weighed, and the metabolites were extracted using a 400 µL methanol: water (4:1, v/v) solution. The mixture was allowed to settle at − 20 °C and treated by high throughput tissue crusher Wonbio-96c (Shanghai Wanbo Biotechnology Co., Ltd) at 50 Hz for 6 min, followed by vortexing for 30 s and ultrasound at 40 kHz for 30 min at 5 °C. The samples were placed at − 20 °C for 30 min to allow the proteins to precipitate. After centrifugation at 13,000g at 4 °C for 15 min, the supernatant was carefully transferred to sample vials with 20 μL of 2-chloro-l-phenylalanine (0.3 mg/mL) for LC–MS/MS analysis. The Ultra Performance Liquid Chromatography (UPLC) system was coupled to a quadrupole-time-of-flight mass spectrometer (Triple TOFTM 5600+, AB Sciex, USA) equipped with an electrospray ionization (ESI) source operating in positive and negative mode. To monitor the stability of the analysis, quality control was prepared by two different methods. One is to extract and mix the same volume of each sample, the other is to set internal standard (2-chloro-l-phenylalanine). In the process of instrument analysis, a mixed sample was inserted every 8–10 samples. A relative standard deviation (RSD) of Internal standard < 30%, which represents the stability and repeatability of the system.

Metabolites of mature Euryale seeds were detected by LC-MS as previously described (Wu et al., 2013 (link); Wang et al., 2015 (link)). Briefly, 4 g power of the sample was extracted in 40 ml extraction buffer (methanol: double distilled water = 4:1, v/v) by ultrasonication. Then, the samples were centrifuged at 10,000 rpm for 5 min; the upper phase was dried under a stream of nitrogen gas and redissolved with 1 ml chromatographic methanol. Each sample solution was injected into the reverse-phase C₁₈ column (250 mm × 4.6 mm, 5 μm, Agilent, United States) for LC-MS analysis (Ultra-Fast Liquid Chromatography, Shimadzu, Japan; Triple TOFTM 5600, AB Sciex, United States). The qualitative analysis and the resolution of the chromatogram of the target components were carried out using the MS-DIAL software 3.98. The differences in metabolites were calculated using one-way ANOVA and post hoc statistical analysis.

Chromatographic separation of the metabolites was performed on ExionLC AD system (AB Sciex, USA) equipped with an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters, Milford, USA). The UPLC system was coupled to a quadrupole-time-of-flight mass spectrometer (Triple TOFTM5600+, AB Sciex, USA). The optimal conditions were set as listed (Supplementary Tables S2, S3). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile: isopropanol (1:1, v/v). During the period of analysis, the rest samples were stored at 4 °C.

¹H NMR spectra were recorded on a Bruker Avance III 400 MHz NMR spectrometer (Bruker BioSpin GmbH, Ettlingen, Germany), using CDCl₃ as the solvent. FTIR spectra of the sample were recorded with a FTIR spectrometer (Affinity-1, Shimadzu, Kyoto, Japan), and the number of scans was 16 at a resolution of 1 cm⁻¹. ESI+ mass spectrometry data were obtained by a high-resolution mass spectrometer (Triple TOFTM 5600+, AB SCIEX, Sun Valley, CA, USA). UV-vis spectra were recorded with a UV-vis spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan).

Fecal sample (50 mg) was weighted to an EP tube, and 400 µL extract solution (methanol: water = 4: 1) was added. Then they were homogenized by a Wonbio-96c high-throughput tissue crusher (Shanghai Wanbo Biotechnology Co., Ltd.) for 6 min at 50 Hz. Then, they were vortexed for half a minute and ultrasonicated for 30 min at 40 kHz (incubated in ice water). The mixture was incubated for 30 minutes at -20°C to precipitate proteins and then centrifuged at 13,000 g for 15 min at 4°C. The supernatant of the samples was transferred to fresh EP tubes and analyzed by ultra-performance liquid chromatography (UPLC) system coupled to a quadrupole-time-of-flight mass spectrometer (Triple TOFTM5600+, AB Sciex, USA) equipped with a electrospray ionization (ESI) source operating in both positive mode and negative ion mode. Additionally, 10 μL of supernatant from each sample was combined and mixed to serve as the quality control (QC) sample, which was injected randomly (every 8 samples) to screen the consistency of the analysis.

High pressure liquid chromatography of nectar and muscle was performed on a Shimadzu Nexera system (Shimadzu, Columbia, MD, USA) coupled to a hybrid quadrupole-time of flight mass spectrometer (TripleTOF^TM 5600, AB SCIEX). Raw LC-MS/MS data files were imported into MarkerView software (AB SCIEX) for initial data processing including feature detection, peak alignment, peak integration, and principal component analysis. A portion of metabolites was identified using the Mass Spectrometry Metabolite Library of Standards library (IROA Technologies, LLC), which contains >600 metabolites. Details of LC-MS/MS analysis can be found in the supplementary files (S1 Appendix).