Valinomycin was extracted with three-fold volumes of ethyl acetate from cell-free reactions. After centrifugation at 16,000 g for 5 min, the organic fraction was transferred to a fresh 1.5 mL microcentrifuge tube, air dried, and resuspended in methanol for Valinomycin analysis. Valinomycin quantification was performed with an Agilent 6470 Triple Quadrupole LC/MS System equipped with an Eclipse Plus C18 column (2.1×50 mm, 1.8 μm). For Valinomycin detection, 2 μL of each sample was injected and separated at a flow rate of 0.4 mL/min with elution buffers A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid) through a linear gradient elution from 80 to 100% B over 2.5 min, a 100% B wash for 7.5 min, and a post time wash for 10 min with 80% B. Valinomycin concentrations were calculated according to a calibration curve prepared with commercial Valinomycin (Sigma) as a standard. All measurements were performed in triplicate.
6470 triple quadrupole lc ms system
Manufactured by Agilent Technologies
Sourced in United States
The 6470 Triple Quadrupole LC/MS System is a mass spectrometry instrument designed for quantitative and qualitative analysis. It combines liquid chromatography (LC) and triple quadrupole mass spectrometry (MS) technology to provide precise and sensitive detection of target analytes.
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Lab products found in correlation
Valinomycin, by Merck Group (1 mentions)
Eclipse plus c18 column, by Agilent Technologies (1 mentions)
Lcms 8050, by Shimadzu (1 mentions)
1290 infinity 2 system, by Agilent Technologies (1 mentions)
It tof, by Shimadzu (1 mentions)
Agilent 1290 infinity 2 multisampler, by Agilent Technologies (1 mentions)
Masshunter workstation version 10, by Agilent Technologies (1 mentions)
Eclipse plus c18 rrhd column, by Agilent Technologies (1 mentions)
4 protocols using 6470 triple quadrupole lc ms system
1
Quantification of Valinomycin by LC-MS
2
Aminorex Detection in Urine and Plasma
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An Agilent Technologies 6150 B Quadrupole LC/MS System and an Agilent 64 70 Triple Quadrupole LC/MS System in positive ionization modes were used for screening and confirmatory analysis respectively. Each instrument used 1290 Infinity Auto Injectors and thermostated column compartments. The 6150B used an Agilent Eclipse AAA, 3 × 100 mm liquid chromatography column. The LC gradient consisted of 100% A (100%/0.1%formic acid) for 5 min and then a linear gradient to 100% B (90% methanol/10% 0.1 formic acid) at 20 min at a flow of 0.4 mL/minute. Column compartment was 40 °C. The 6470 Triple Quadrupole used an Agilent Poroshell EC-120, 3.0 × 100 LC column. An LC gradient of acetonitrile/formic acid in the following composition was used: (90% formic acid/10% acetonitrile) for 1 m, (5% formic acid/95% acetonitrile) for 4.5 min and (90% formic acid/10% acetonitrile) at 5 min. Flow rate was 0.4 ml/minute and column temperature was 50 °C. Limit of Detection was 1 ng/mL and urinary concentrations of Aminorex were estimated to be approximately in the range of 10 ng/ml. No Aminorex was detected in the post-administration plasma samples, and no Barbarin was detected in either the plasma or urine samples.
3
Quantitative LC-MS Analysis of Peptides
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The LC-MS analysis of the peptides was performed on a Shimadzu LCMS-8050 equipped with a triple quadrupole mass spectrometer (using a UHPLC Nexera X2 system) and on an Agilent 6470 Triple Quadrupole LC/MS System equipped with a standard Jet Stream ESI source and Agilent Technologies 1290 Infinity II system. The LC-MS analysis on both instruments were carried out in SIM (selected ion monitoring) mode and with a Q1Q3 scan. The LC system was operated with a mobile phase consisting of solvent A: 0.1% formic acid in H2O and solvent B: 0.1% formic acid in MeCN. The gradient conditions (B %) were from 5 to 80% B, within 15 min. The flow rate was 0.2 mL/min, and the injection volume 5 μL. The separation was performed on an Aeris Peptide XB-C18 column (50 mm × 2.1 mm) with a 3.6 μm bead diameter. The peptide samples were dissolved in 400 μl of a water: acetonitrile mixture (80: 20). Most analysis were carried out on a Shimadzu IT-TOF, which is a hybrid system consisting of an ion trap and a time-of-flight mass analyzer; it also includes an electrospray (ESI) ion source. In our experiments, we set the potential between the spray needle and the orifice to 4.5 kV. The LC separation on this instrument was performed in the same condition as described above.
4
Quantifying Metalaxyl-M and Azoxystrobin
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Metalaxyl-M and azoxystrobin were analyzed by high-performance liquid chromatography (Agilent 1290 Infinity II Multisampler, Agilent Technologies, City of Santa Clara, CA, USA) and a tandem triple quadrupole mass spectrometer (6470 Triple Quadrupole LC/MS System, Agilent Technologies, City of Santa Clara, CA, USA) with an electrospray ionization (ESI) source operated in positive ion mode (ESI+). An Agilent EclipsePlusC18 RRHD column (50 mm × 2.1 mm, 1.8 μm) was used for chromatographic separation at a temperature of 40 °C. The mobile phases were acetonitrile (A) and 0.1% (v/v) formic acid aqueous solution (B). The gradient elution procedure is shown in Table S1 . The flow was 0.30 mL/min, and the sample injection volume was 1.5 μL. The gas temperature was set at 280 °C and the gas flow rate at 7 L/min for MS detection working conditions. The sheath gas temperature was set at 320 °C and the sheath gas flow was 11 L/min, and the capillary voltages were controlled at 4000 V under positive ion detection mode. Analytes were determined in multiple reaction monitoring (MRM) mode. For instrument control, data acquisition, and processing, MassHunter Workstation version 10.1 (Agilent Technologies, City of Santa Clara, CA, USA) was used. Under the operating conditions given above, the two compounds were quantified based on the acquisition parameters as listed in Table S2 .
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