Other phosphorylated metabolites (e.g., sugar phosphate, 2PG, and PEP) and nucleotide sugars (ADPG and UDPG) were analyzed using an anion-exchange LC-MS/MS method described in Alonso et al. (2010) (link) with slight modifications. Metabolites were reconstituted in 100 μL of water from lyophilized extract, and 10 μL of extracts was injected into an ACQUITY UPLC pump system (Waters, Milford, MA, USA) coupled with a Xevo ACQUITY TQ Triple Quadrupole Detector (Waters, Milford, MA, USA). Metabolites were separated by an IonPac AS11 analytical column (2 × 250 mm, Dionex) equipped with an IonPac guard column AG11 (2 × 50 mm, Dionex) at a flow rate of 0.35 mL min−1. A multi-step gradient was applied with mobile phase A (0.5 mM KOH) and mobile phase B (75 mM KOH): 0–2 min, 100% A; 2–4 min, 100%–93% A; 4–13 min, 93%–60% A; 13–15 min, 0% A; 15–17 min, 100% A. The KOH concentration was suppressed by a post-column anion self-regenerating suppressor (Dionex ADRS 600, Thermo Scientific), with a current of 50 mA and flow rate of 3.5 mL min−1. An IonPac ATC-3 Anion Trap Column (4 × 35 mm), conditioned with 2M KOH, was used to remove contaminant ions from KOH solvents. Mass spectra were acquired using MRM in negative ESI mode. Parent-product ion transitions for metabolites were described in Supplemental Table S2 .
Dionex adrs 600
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dionex ADRS 600 is an anion self-regenerating suppressor used in ion chromatography systems. It is designed to reduce background conductivity and enhance analyte detection.
Automatically generated - may contain errors
Lab products found in correlation
Xevo acquity tq triple quadrupole detector, by Waters Corporation (2 mentions)
Ionpac guard column ag11, by Thermo Fisher Scientific (2 mentions)
Acquity uplc pump system, by Waters Corporation (2 mentions)
Ionpac as11, by Thermo Fisher Scientific (1 mentions)
Dionex egc 3 koh rfic, by Thermo Fisher Scientific (1 mentions)
Ics 5000, by Thermo Fisher Scientific (1 mentions)
Ionpac as11 analytical column, by Thermo Fisher Scientific (1 mentions)
3 protocols using dionex adrs 600
1
Quantification of Phosphorylated Metabolites
2
Nitrite Analysis by Ion Chromatography
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Analyses of nitrite were carried out by ion chromatography (ICS-5000, Thermo Fisher Scientific, Waltham, MA, USA) with suppressed conductivity detection (Thermo Scientific Dionex ADRS 600 (4 mm) RFIC Suppressor, recycle mode, 112 mA current). The chromatographic separation was achieved on a Dionex IonPac AS18 column (250 × 4 mm) operated at 30 °C, coupled to the Dionex IonPac AG18 guard column (50 × 4 mm) (Thermo Fisher Scientific, Waltham, MA, USA). Gradient at a constant flow rate of 1.0 mL/min was performed using eluent generator cartridge (Thermo Scientific Dionex EGC III KOH RFIC). 4 mM potassium hydroxide (KOH) was kept constant for 15.0 min, increased to 30 mM KOH in 1.0 min, increased to 45 mM KOH within 4.0 min and kept constant for 5.0 min. The injection volume was 50 µL. The precision (repeatability) of the method was checked in 6 replicates and was 0.4% RSD for nitrite in CP and 12.8% RSD for nitrite in Afatinib FCTs. The accuracy of the method was assessed by the recovery which was 104% and 95% for nitrite in CP and in Afatinib FCTs, respectively. The linearity of the method in the range LOQ -100 × LOQ was evaluated as R 2 (0.999).
3
Quantitative Analysis of Phosphorylated Metabolites
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Nucleotide sugars and additional phosphorylated intermediates (i.e. 2PG and phosphoenolpyruvate) were analyzed by anion exchange chromatography–tandem mass spectrometry (AEC-MS/MS) by an ACQUITY UPLC pump system (Waters, Milford, MA, USA) coupled with a Xevo ACQUITY TQ Triple Quadrupole Detector (Waters, Milford, MA, USA). Metabolites were separated by an IonPac AS11 analytical column (2 × 250 mm, Dionex) equipped with an IonPac Guard Column AG11 (2 × 50 mm, Dionex) at a flow rate of 0.35 mL min−1. A multistep gradient was applied with mobile phase A (0.5 mM KOH) and mobile phase B (75 mM KOH): 0 to 2 min, 100% A; 2 to 4 min, 100% to 93% A; 4 to 13 min, 93% to 60% A; 13 to 15 min, 0% A; and 15 to 17 min, 100% A. The KOH concentration was suppressed by a postcolumn anion self-regenerating suppressor (Dionex ADRS 600, Thermo Scientific, Waltham, MA, USA), with a current of 50 mA and flow rate of 3.5 mL min−1. An IonPac ATC-3 Anion Trap Column (4 × 35 mm), conditioned with 2 M KOH, was used to remove contaminant ions from KOH solvents.
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