The concentrations of 13C4SA in plasma and tissues were determined using a 1290 Infinity II series UHPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled with an AB Sciex Qtrap 6500+ mass spectrometer (Concord, Canada). A Waters Atlantis Premier BEH C18 AX column (2.1 × 100 mm, 1.7 μm particle size) was used, and the column temperature was maintained at 30 °C. The gradient mode consisted of mobile phases A (0.9% formic acid in water) and B (0.9% formic acid in ACN) as follows: 0–1.5 min (0%–0% B), 1.5–4.0 min (5%–30% B), 4.0–4.5 min (30%–30% B), 4.5–5.0 min (30%–0% B), and 5.0–5.5 min (0%–0% B). The flow rate was 0.3 mL/min. Meanwhile, 5-μL prepared samples were injected for analysis. The multiple reaction monitoring mode with negative electrospray ionization was used to quantify 13C4SA and IS. The source temperature and ion spray voltage were 350 °C and −4500 kV, respectively. The ion source gas 1 flow pressure was 50 psig, and the gas 2 flow setting was 50 psig. The curtain gas pressure was 40 psig, and collision gas is medium. The optimized mass parameters for 13C4SA and CAD4 are listed in Table 1 .
Atlantis premier beh c18 ax column
Manufactured by Waters Corporation
Sourced in United States
The Atlantis Premier BEH C18 AX column is a high-performance reversed-phase liquid chromatography column. It features a hybrid organic-inorganic stationary phase with both alkyl and ion-exchange functionality, designed to provide enhanced selectivity and separation capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Targetlynx, by Waters Corporation (2 mentions)
Acquity uplc 1 class system, by Waters Corporation (2 mentions)
Xevo tq s triple quadrupole mass spectrometer, by Waters Corporation (2 mentions)
1290 infinity 2 series uhplc system, by Agilent Technologies (1 mentions)
Xevo tq xs mass spectrometer, by Waters Corporation (1 mentions)
Uplc 1 class ftn system, by Waters Corporation (1 mentions)
Vanquish uplc system, by Thermo Fisher Scientific (1 mentions)
Xcalibur, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap elite, by Thermo Fisher Scientific (1 mentions)
5 protocols using atlantis premier beh c18 ax column
1
Quantification of 13C4SA in Plasma and Tissues
2
Quantitative Nucleotide Analysis via UPLC-MS
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LC separation of nucleotides (cAMP, AMP, ATP, cGMP, GMP, GDP, and GTP) was performed with a UPLC I-Class FTN system (Waters) equipped with an Atlantis Premier BEH C18 AX column (2.1 mm x 50 mm, 1.7μm, Waters). Separation was performed at a flow rate of 400 μl/min with the following LC gradient: 0–1 min: 3% B, 1–4 min: 3–20% B, 4–5 min: 20–85% B, and 5–5.5 min: 85% B; solvent A, 0.2% (v/v) formic acid in water; solvent B, 0.2% (v/v) formic acid in acetonitrile. The UPLC system was directly coupled to a Xevo TQ-XS mass spectrometer (Waters) with an electrospray ionization (ESI) source. Multiple-reaction monitoring (MRM) was performed using specific transitions for the nucleotides cAMP, AMP, ATP, cGMP, GMP, GDP, and GTP.
3
Quantitative Nucleotide Analysis via LC-MS
4
SCFA Quantification via UPLC-MS/MS
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All derivatised SCFAs were separated by an Atlantis PREMIER BEH C18 AX column (2.1 × 150 mm, 1.7 µm, Waters) on a Vanquish UPLC system (Thermo Scientific, Waltham, MA, USA) with sample tray and column compartments that are correspondingly maintained at 15 °C and 55 °C. Mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), and gradient elution was conducted by increasing mobile phase B from 5 to 50% in 20 min and then 100% in the next 3 min. Flow rate and injection volume were 0.2 mL/min and 5 µL, respectively.
Derivatised SCFAs were detected by an LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific, Waltham, MA, USA) with a heated electrospray ionisation source. Capillary temperature and probe heater temperature were 300 °C, and the sheath, auxiliary and sweep gases were at 30, 10 and 0 units, respectively. The mass spectrometer was operated in positive (4.2 kV) ionisation mode with a full scan (120–1200 m/z) at 60,000 resolution (FTMS mode). SCFAs were identified using Xcalibur (Thermo Fisher Scientific) based on retention time and accurate mass matching. The quantification of SCFA was performed using external calibration curves.
Derivatised SCFAs were detected by an LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific, Waltham, MA, USA) with a heated electrospray ionisation source. Capillary temperature and probe heater temperature were 300 °C, and the sheath, auxiliary and sweep gases were at 30, 10 and 0 units, respectively. The mass spectrometer was operated in positive (4.2 kV) ionisation mode with a full scan (120–1200 m/z) at 60,000 resolution (FTMS mode). SCFAs were identified using Xcalibur (Thermo Fisher Scientific) based on retention time and accurate mass matching. The quantification of SCFA was performed using external calibration curves.
5
UPLC-MS/MS Quantification of Metabolites
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Quantification of metabolites in cell lysates or in the in vitro reactions was carried out using an Acquity I-class UPLC system coupled to Xevo TQ-S triple quadrupole mass spectrometer (both Waters, US). The UPLC was performed using an Atlantis Premier BEH C18 AX column with the dimension of 2.1 × 100 mm and particle size of 1.7 μm (Waters). Mobile phase A was 20 mM ammonium formate at pH 3 and acetonitrile was mobile phase B. The flow rate was kept at 300 μl min−1 consisting of a 2 min hold at 2% B, followed by linear gradient increase to 100% B during 5 min. The column temperature was set at 25°C and an injection volume of 1 μl. An electrospray ionization interface was used as ionization source. Analysis was performed in positive ionization mode. Metabolites were analyzed using multiple-reaction monitoring with argon as the collision gas, and detected based on retention times and MS/MS parameters of chemical standards (S1 Fig ). Quantification was made using standard curve in 0–1 mM concentration range. 15N5-adenosine 5′-monophosphate (Sigma) was added to standards and samples as internal standard (0.5 μM). TargetLynx (Waters) was used for data analysis.
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