Trp, 5-hydroxytryptamine (5-HT), kynurenine (Kyn) and quinolinic acid (Quin), and LPS (Escherichia coli, serotype 0111:4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Benzoyl chloride and ammonium formate were obtained from Aladdin (Shanghai, China). LC-MS grade methanol, acetonitrile, and formic acid were purchased from Fisher Scientific. Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). NMDAR antibody was purchased from Abcam (Cambridge, UK). The real-time PCR system used in this study was the Thermo Fisher ABI StepOne Plus system. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis was performed on an Agilent 1290 Infinity II UHPLC system coupled to a 6470A triple quadrupole mass spectrometer (Santa Clara, CA, United States).
6470a triple quadrupole mass spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6470A triple quadrupole mass spectrometer is a high-performance analytical instrument designed for accurate and sensitive quantification of target compounds in complex matrices. It utilizes three quadrupole mass filters to provide enhanced selectivity and sensitivity for a wide range of applications.
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Lab products found in correlation
Agilent 1290 infinity 2 uhplc system, by Agilent Technologies (2 mentions)
Abi steponeplus system, by Thermo Fisher Scientific (1 mentions)
Lc ms grade methanol, by Thermo Fisher Scientific (1 mentions)
1290 infinity 2 uhplc system, by Agilent Technologies (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
5 hydroxytryptamine 5 ht, by Merck Group (1 mentions)
Kynurenine kyn, by Merck Group (1 mentions)
Quinolinic acid quin, by Merck Group (1 mentions)
Spermidine, by Merck Group (1 mentions)
L arginine, by Merck Group (1 mentions)
Spermine, by Merck Group (1 mentions)
Putrescine, by Merck Group (1 mentions)
L ornithine, by Merck Group (1 mentions)
4 protocols using 6470a triple quadrupole mass spectrometer
1
Quantification of Neuroactive Metabolites
2
Simultaneous Quantification of Lignans and Neurotransmitters
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Microdialysis samples were analyzed using a 1290Ⅱ ultra-high performance liquid chromatograph (Agilent) and a 6470A triple quadrupole mass spectrometer (Agilent). The liquid phase determination conditions for Schisandra lignans were consistent with those described in Section 2.4 . For the neurotransmitter substances, UPLC separation was carried out on a Venusil ASB C18 (5 μm, 4.6 × 250 mm) column maintained at 35 °C and with a flow rate of 0.5 mL min−1. Mobile phases consisted of 0.12% formic acid-water (A) and 0.06% formic acid-(80% methanol-water) (B). The elution gradient of B was as follows: 0–2 min, 20%–22% B; 2–8 min, 22%–24% B; 8–9 min, 24%–100% B; 9–14 min, 100% B; 14–15 min, 100%–20% B; 15–18 min, 20% B.
Mass spectrometry analysis utilized an AJS ESI jet point spray ionization source. In the multiple reaction monitoring mode (MRM), 15 lignan components and four neurotransmitters were quantified in the positive mode. The parameters were optimized as follows: sheath temperature: 350°C; sheath flow rate: 11 L min−1; dry gas temperature: 300°C; dry gas flow rate: 10 L min−1; capillary voltage: 4,000 V; nozzle voltage: 2000 V; nebulizer pressure: 40 Psi; EMV: 200 V. The specific mass spectrometry parameters can be found inSupplementary Table S1 .
Mass spectrometry analysis utilized an AJS ESI jet point spray ionization source. In the multiple reaction monitoring mode (MRM), 15 lignan components and four neurotransmitters were quantified in the positive mode. The parameters were optimized as follows: sheath temperature: 350°C; sheath flow rate: 11 L min−1; dry gas temperature: 300°C; dry gas flow rate: 10 L min−1; capillary voltage: 4,000 V; nozzle voltage: 2000 V; nebulizer pressure: 40 Psi; EMV: 200 V. The specific mass spectrometry parameters can be found in
3
Quantitative Polyamine Analysis in ESCC Cells
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The ESCC cells with/without Fn infection for 24 h were collected in 80% aqueous methanol. After ultrasonic treatment, the lysates were centrifuged, and the supernatant was dried and dissolved in 50 μL of 100 mM sodium carbonate. The chemical derivatization was initiated after adding 50 μL of 2% benzoyl chloride in acetonitrile. After derivatization, the sample was isometrically mixed with benzoyl-13C6 chloride-derivatized standards (as internal standards) prior to ultra-high-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) analysis. The UHPLC-MS/MS analysis was performed on an Agilent 1290 Infinity II UHPLC system coupled to a 6470A Triple Quadrupole mass spectrometer (Agilent, Santa Clara, United States).
The reagents used to prepare for standard solution, such asl- arginine, l- ornithine, putrescine, spermine, and spermidine, were purchased from Sigma-Aldrich. N1-acetylspermidine and N1-acetylspermine were purchased from Shanghai Yuanye Bio-Technology Co (Shanghai, China).
The reagents used to prepare for standard solution, such as
4
Quantitative Muscle Metabolite Analysis
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The frozen muscle tissue samples (25 ± 2 mg), spiked with 200 µL of cold methanol, were homogenized in a frozen state by a TissueLyser (JX-24; Jingxin, Shanghai, China) with beads at 40 Hz for 4 min. After centrifugation (15,000× g and 4 • C) for 15 min, the whole supernatant was evaporated to dryness under nitrogen gas, and the dried residues were derivatized at room temperature for 10 min after additions of 50 µL sodium carbonate and 50 µL benzoyl chloride (2%). The derivatized samples were isometrically mixed with a stable isotope labeled according to internal standards. The quality control (QC) sample was obtained by isometrically pooling all the prepared samples. A solution of multiple standards with 5 µM was prepared by mixing a single standard stock solution. The calibration curve solutions were obtained by serially diluting the mixed standard working solution and isometrically mixing with internal standards, respectively. The samples of the calibration curve covered a range of 0.01-5 µM. The samples were detected and analyzed by using liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, which was performed on an Agilent 1290 Infinity II UHPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to a 6470A Triple Quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).
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