A total of eight flavonoids ((+)-catechin, taxifolin, myricetin, eriodictyol, luteolin, morin, apigenin and naringenin) in TSE were quantified according to the method of our previous study [25 ] using the UPLC-MS/MS (Thermo Fisher Scientific, Waltham, MA, USA). Shortly, TSE was solved in water and then filtered through a 0.45 μm nylon 6–6 filter and analyzed. The analysis was carried out by UPLC (Ultimate 3000 DGLC, Thermos Fisher Scientific, USA) equipped with a quadrupole orbitrap mass spectrometry (Q-Exactive) with an ESI source. HSS T3 C18 (100 × 2.1 mm, Waters Acquity) was employed for chromatographic separation. The mobile phase consisted of acetonitrile (Solvent A) and 0.1% aqueous formic acid (Solvent B). The analysis was performed under a gradient program of 0–1 min—5% B, 1–1.5 min—10% B, 1.5–4.5 min—13% B, 4.5–15 min—17% B, 15–20 min—27% B, 20–20.5 min—95% B, 20.5–23 min—95% B, 23–23.2 min—5% B, 23.2–28 min—5% B. The flow rate was 0.3 mL/min, the injection volume was 2 μL and the column temperature was maintained at 40 °C. Mass spectra were obtained in both positive and negative ionization modes. The capillary voltage was 3.0 kV with a source temperature of 320 °C. The collision energy was 20, 30, 40 eV. Quantification of the analytes were calculated by standard calibration curves. All the data were obtained as average values in triplicate experiments.
Uplc ms ms
Manufactured by Thermo Fisher Scientific
Sourced in United States
The UPLC-MS/MS is an analytical instrument that combines Ultra-Performance Liquid Chromatography (UPLC) with tandem mass spectrometry (MS/MS) technology. It is used for the separation, identification, and quantification of complex chemical compounds with high sensitivity and resolution.
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Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000 dglc, by Thermo Fisher Scientific (1 mentions)
Syncronis c18 column, by Thermo Fisher Scientific (1 mentions)
Dextran sulfate sodium salt, by MP Biomedicals (1 mentions)
Interleukin 6 (il 6), by Keygen Biotech (1 mentions)
Il 1β, by Keygen Biotech (1 mentions)
Gapdh, by Keygen Biotech (1 mentions)
Q exactive orbitrap, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Merck Group (1 mentions)
Wz 50c6 microinfusion pump, by Smith & Nephew (1 mentions)
Polypropylene centrifuge tube, by Corning (1 mentions)
Sodium chloride (nacl), by Sinopharm (1 mentions)
Compound discoverer 2, by Thermo Fisher Scientific (1 mentions)
Acquity uplc tqd, by Waters Corporation (1 mentions)
Uplc ms, by Thermo Fisher Scientific (1 mentions)
Microlab star system, by Hamilton Company (1 mentions)
11 protocols using uplc ms ms
1
Quantitative Analysis of Flavonoids in TSE
2
Quantification of PTX and IR780 in Samples
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The amount of PTX in the samples were determined through ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS, ThermoFisher Scientific). Thermo Syncronis C18 column (50 × 2 mm, 1.7 μm) was used at a column temperature of 40 °C. Acetonitrile/0.1% formic acid (68:32, v/v) was used as the eluent at a flow rate of 0.3 mL/min. An electrospray ionization source was used for positive-ion detection. Settings for the remaining parameters are presented in Additional file 1 : Table S4. The endogenous substances in biological samples did not interfere with the detection of PTX (Additional file 1 : Figure S8), and specificity was deemed good. To assay the biological samples, a linear relationship was established between the peak area and the concentration of PTX with a range of 2–5000 ng/mL (r = 0.9999), with an extraction recovery of more than 90%.
IR780 was detected using a UV-Vis Cary 8454 instrument (Agilent Technologies Inc., Santa Clara, CA, USA) at a wavelength of 780 nm. The IR780 concentration in the range of 0.25–5 µg/mL showed a linear relationship (r = 0.999) with the absorbance, and IR780 was recovered at a rate of > 90%.
IR780 was detected using a UV-Vis Cary 8454 instrument (Agilent Technologies Inc., Santa Clara, CA, USA) at a wavelength of 780 nm. The IR780 concentration in the range of 0.25–5 µg/mL showed a linear relationship (r = 0.999) with the absorbance, and IR780 was recovered at a rate of > 90%.
3
Bachu Mushroom Bioactive Compounds
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Dried fruit bodies of the Bachu mushroom were obtained from Bachu County in Xinjiang Province, China. All chemical reagents used in high-performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis were of mass spectrometry grade and purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Dextran sulfate sodium salt (36–50 kDa) was obtained from MP Biomedicals (Santa Ana, CA, USA). Antibodies against TNF-α, IL-6, IL-1β, IL-10, iNOS, COX-2, and GAPDH were purchased from KeyGEN Biotech (Nanjing, China).
4
Quantification of Neonatal Metabolites
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In neonatal blood samples, 82 metabolites, including 12 kinds of amino acids and 70 kinds of carnitines, were identified and quantified with nonderivative methods using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC−MS/MS) (Thermo Scientific, Germany). In detail, 3.5-4.0 mm punches were punched from dried blood spots and extracted with 100 μL of 80% aqueous methanol and 0.1% formic acid (Sigma Aldrich, USA) in a thermomixer at 45°C and 700 rpm for 45 min. Extracted liquid was transferred to HPLC vials for direct detection. An ALC system consisting of a Dionex Ultimate 3000 UHPLC quaternary system pump, a column department, and an autosampler (Thermo Scientific, Germany) and a Q Exactive Orbitrap (Thermo Scientific, Germany) were used to analyze the metabolites one by one to obtain molecular weight, structure and other information. Blood samples were tested in both positive and negative modes. The test for the quality control sample was repeatedly conducted every 20 samples to ensure the stability of the whole measurement process. The concentrations of neonatal metabolites were obtained by calculating the signal strength ratio between each analyte and an internal standard.
5
Skin Microdialysis for TMP-ME Absorption
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Male SD rats were randomly assigned to either TMP-ME or MN-assisted TMP-ME groups (n = 5 for each). The skin microdialysis experiments for the TMP-ME group were conducted following methods described previously [34 ]. For the MN-assisted TMP-ME group, the protocol was identical, except that the rat skin was pretreated with MNs by manual insertion for 2 min. For both groups, rats were intraperitoneally anesthetized with urethane (1.3 g/Kg), and their abdominal hair was clipped. Microdialysis tubing (20 mm long, 200 µm inner diameter, 13,000 Da cut-off molecular weight) was inserted into the skin and perfused with 30% ethanol in phosphate-buffered saline (PBS) at 0.22 mL/h using a WZ-50C6 Micro Infusion pump (Smiths Medical, Norwell, MA, USA). an hour and a half after perfusion, 2 mL of TMP-ME was applied to the rat skin, and dialysate samples were collected from the tubes every 0.5 h for 8 h. TMP levels in the dialysate samples were determined using UPLC-MS/MS (Thermo Fisher Scientific Inc., Waltham, MA, USA).
6
Rapid Filtration for Metabolite Extraction
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Because of the massive leaking of metabolites into the quenching reagent in our previous experiments and other studies [15 (link)], we adapted a rapid filtration protocol for sampling instead of quenching. 1 mL broth was filtered through a vacuum filtration device, then the filter cake was washed by precooled 2.3% (m/m) sodium chloride solution. This step was performed as soon as possible. Subsequently, the filter cake with filter paper was transferred rapidly to 10 mL 75% (v/v) ethanol solution and the extraction continued for 4 min at 95 °C. To accurately determine metabolite’s concentration, 30 μL of 13C-labeled cell extract was added to every sample as internal standard for isotope dilution mass spectrometry (IDMS) before extraction. The supernatant was evaporated through a rotary evaporator at −101 kPa (relative pressure) and then stored at −80 °C before determination by UPLC-MS/MS (Thermo Fisher Scientific Corporation, Waltham, USA).
7
Multiresidue Extraction and UPLC-MS/MS Analysis
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The samples, stored at −20 °C, were allowed to acclimate to room temperature. Once at room temperature, 5 g aliquots were weighed and mixed with 10 mL water and 15 mL acetonitrile in 50 mL polypropylene centrifuge tubes (Corning, Shanghai, China). The sample was vortexed for 2 min and then subjected to ultrasound in an ultrasonic generator (Kun Shan Ultrasonic Instruments Co., Ltd., Kunshan, China) for 3 min before the addition of 1 g NaCl (Sinopharm Inc., Shanghai, China). and 4 g anhydrous MgSO4 (Sinopharm Inc., Shanghai, China). After this, the mixture was vortexed for 1 min before centrifugation at 4000 rpm for 8 min. Then 8 mL of supernatant was transferred into a 10 mL centrifuge tube with 0.15 g N-Propylethane-1,2-diamine (Sinopharm Inc., Shanghai, China) and 0.9 g anhydrous MgSO4. Then, after vortexing for 1 min and centrifugation at 4000 rpm for 8 min, 5 mL of the supernatant was transferred into a 20 mL thread screw neck vial (ANPEL Instrument Inc., Shanghai, China). Finally, the liquid was dried by nitrogen gas and reconstituted with 0.5 mL of 50% methanol (ANPEL Instrument Inc., Shanghai, China) with 0.05% formic acid (Thermo Scientific, Massachusetts, USA), through a 0.22 µm nylon filter (ANPEL Instrument Inc., Shanghai, China) before analysis with UPLC–MS/MS (Thermo Scientific, Massachusetts, USA).
8
Untargeted Serum Metabolome Assessment
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The serum metabolome was assessed using an untargeted approach via UPLC-MS/MS (Thermo Fisher Scientific, Waltham, MA, USA). The sample pretreatment methods and UPLC-MS/MS analysis method were carried out as described previously [6 (link),25 (link)], with minor modification. In brief, we removed the protein from the serum sample and then dried the supernatant for testing. Raw data were preprocessed by compound discoverer 2.1 software (Thermo Fisher Scientific). Pathway enrichment analyses were processed in MetaboAnalyst 5.0 (https://www.metaboanalyst.ca (accessed on 10 September 2022)). The visualization results of the models were obtained with MetaboAnalyst 5.0.
9
Metabolomic Profiling of IDH3α-modified Neural Cells
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Cells (7.5 × 106; NHAs with CRISPR-mediated IDH3α KO or NHAs overexpressing IDH3α and their corresponding control cell lines) were washed twice with DPBS and then snap frozen in liquid nitrogen. At Metabolon, samples were then processed by the automated Microlab STAR System (Hamilton). Recovery standards were added to all samples for quality control measures. Methanol was added to all samples. Each sample was shaken for 2 min followed by centrifugation to release metabolites. All samples had aliquots that underwent UPLC-MS/MS (Thermo Fisher Scientific) with reverse phase (RP) plus positive ion mode electrospray ionization (ESI), with RP plus negative ion mode ESI, or following hydrophilic interaction liquid chromatography column (Waters UPLC BEH Amide 2.1 mm × 150 mm, 1.7 μm) elution with negative ion mode ESI. Samples were reconstituted with solvents appropriate for each method and run alongside standards. Data were analyzed using Metabolon hardware and software. Compounds were identified on the basis of a narrow retention index that was then compared to a library of purified standards and MS/MS scores comparing the experimental spectrum to the library spectrum.
10
Quantification of Folate Production
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All folate produced by LLC test samples, 1 mL of each, were suspended into 10 mL of a bicarbonate buffer 0.5 mmol•L -1 with 0.5% of dieritrotietol (DTT) and 1% of ascorbic acid, at pH 7.2 (with internal standard of 100 ng•mL -1 of 13 C folic acid and 13 C 5-MeTHF). After that, all samples were submitted to boiling for 10 min and cooled down in ice at the end. After that the samples were centrifuged in an Amicon filter (5 kDa), at 13,000g, during 50 min at 4 °C.
To quantify the amount of folate produced, different standards were used, such as folic acid, THF, 5-MeTHF, 5-FoTHF, 10-MeTHF and 10-FoTHF. To perform this experiment an Ultra performance liquid chromatography -tandem mass spectrometer (UPLC -MS/MS, Thermo Fisher, Waltham, USA), an AcQuity UPLC-TQD with an AcQuity HSS T3 1.8 μm 2.1 × 150 mm column at 45 °C was used (Waters, Milford, USA). The gradient mobile phase was 0.1% of formic acid (A) and 99% of acetonitrile (B) at 0.4 mL•min -1 (Motta, 2015) .
To quantify the amount of folate produced, different standards were used, such as folic acid, THF, 5-MeTHF, 5-FoTHF, 10-MeTHF and 10-FoTHF. To perform this experiment an Ultra performance liquid chromatography -tandem mass spectrometer (UPLC -MS/MS, Thermo Fisher, Waltham, USA), an AcQuity UPLC-TQD with an AcQuity HSS T3 1.8 μm 2.1 × 150 mm column at 45 °C was used (Waters, Milford, USA). The gradient mobile phase was 0.1% of formic acid (A) and 99% of acetonitrile (B) at 0.4 mL•min -1 (Motta, 2015) .
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