For the reverse-phase liquid chromatography (LC), an Agilent 1290 series HPLC system (Agilent, Santa Clara, CA, USA) was employed. The chromatographic separation was performed on Acquity UPLC BEH shield RP18 columns (2.1 × 100 mm; 1.7 m; Waters, MA, USA) [43 (link),44 (link)]. Samples were injected with a volume of 10 µL. The LC was coupled to a Agilent 6495 triple quadrupole (QQQ) mass spectrometer (Agilent, Santa Clara, CA, USA). (see Supplementary Materials for detail Information).
6495 qqq triple quadrupole mass spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6495 QQQ triple quadrupole mass spectrometer is a high-performance analytical instrument designed for sensitive and precise quantitative analysis. It features a triple quadrupole configuration, which enables efficient precursor ion selection, fragmentation, and detection of target analytes. The 6495 QQQ offers outstanding sensitivity, selectivity, and reproducibility, making it a versatile tool for a wide range of applications.
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Lab products found in correlation
Rrhd sb cn column, by Agilent Technologies (2 mentions)
Agilent 1290 series hplc system, by Agilent Technologies (1 mentions)
Eclipse plus c18 column, by Agilent Technologies (1 mentions)
Agilent masshunter quantitative analysis software, by Agilent Technologies (1 mentions)
Xbridge amide column, by Waters Corporation (1 mentions)
1290 infinity hplc system, by Agilent Technologies (1 mentions)
8 protocols using 6495 qqq triple quadrupole mass spectrometer
1
Quantitative Reverse-Phase LC-MS/MS Analysis
2
N-Linked Glycopeptide Analysis by LC-MS/MS
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For N-linked glycopeptide analysis, samples were digested with trypsin to obtain glycopeptides. Before trypsin digestion, the samples were first reduced with 2 µL of 550 mM dithiothreitol (DTT) at 65 °C for 50 min in 50 mM ammonium bicarbonate (NH4HCO3) solution. Then 4 µL of 450 mM iodine acetamide (IAA) was used to alkylate samples for 25 min in the dark to prevent the re-formation of disulfide bonds between cysteines. One µg of trypsin was added to digest samples in a 37 °C water bath for 18 h overnight. To stop the digestion, samples were stored at −20 °C for 1 h. The instrument used was an Agilent 1290 infinity ultra-high-pressure liquid chromatography (UHPLC) system coupled to an Agilent 6495 triple quadrupole (QQQ) mass spectrometer. Solvents of the 10 min LC gradient include solvent A of 3% acetonitrile and solvent B of 90% acetonitrile in nanopore water. Samples were first separated with an Agilent Eclipse plus C18 column (RRHD 1.8 µm, 2.1 × 150 mm) connected to an Agilent Eclipse plus C18 trap column (RRHD 1.8 µm, 2.1 × 5 mm). The tandem mass spectrometry mode operated on the instrument was dynamic multiple reaction monitoring (MRM). Data collected from the instrument were analyzed with the Agilent MassHunter Quantitative Analysis software (B.05.02, Agilent, Santa Clara, CA, USA).
3
Targeted Metabolite Identification via HPLC-MS
4
Quantification of Nucleotides by LC-MS/MS
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For measurement of nucleotides and deoxy-nucleotides prior to mass spectrometry analysis, the dried extract was suspended in 50 µL of methanol: water (50:50) containing 0.1% formic acid. Samples were delivered to the MS via reverse phase chromatography using a RRHD SB-CN column (1.8 µm, 3.0 × 100 mm, Agilent Technologies) at 300 µl/min. Gradient spanning 2% B to 98% B over a 15-minute period followed by 98% B to 2% B for a 1-minute period. Then gradient is continued for a 4-minute time period to re-equilibrate the column. Buffers A and B were comprised of 0.1% formic acid in water and acetonitrile, respectively.
Ten microliters were injected and analyzed using a 6495 QQQ triple quadrupole mass spectrometer (Agilent Technologies) coupled to a 1290 series HPLC system via Selected Reaction Monitoring (SRM). Metabolites were measured using positive ionization mode with an ESI voltage of +4000 ev, respectively. Approximately 9–12 data points were acquired per detected metabolite.
Ten microliters were injected and analyzed using a 6495 QQQ triple quadrupole mass spectrometer (Agilent Technologies) coupled to a 1290 series HPLC system via Selected Reaction Monitoring (SRM). Metabolites were measured using positive ionization mode with an ESI voltage of +4000 ev, respectively. Approximately 9–12 data points were acquired per detected metabolite.
5
Quantification of Nucleotides by LC-MS/MS
6
Targeted Metabolite Identification via HPLC-MS
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The normal phase chromatographic separation was also used for targeted identification of metabolites. This analysis employed solvents containing water (solvent A), with solvent A modified by the addition of 5mM Ammonium acetate (pH 9.9), and 100% acetonitrile (ACN) solvent B). The binary pump flow rate was 0.2 ml/min with a gradient spanning 80 % B to 2 % B over a 20-minute period followed by 2% B to 80% B for a 5-min period and followed by 80% B for 13-minute time period. The flow rate was gradually increased during the separation from 0.2 mL/min (0–20 mins), 0.3 mL/min (20.1–25 min), 0.35 mL/min (25–30 min), 0.4 mL/min (30–37.99 min) and finally set at 0.2 mL/min (5 min). Metabolites were separated on a Luna Amino (NH2) column (4µm, 100A 2.1×150mm, Phenominex) that was maintained in a temperature controlled chamber (37°C). All the columns used in this study were washed and reconditioned after every 50 injections. Ten microliters were injected and analyzed using a 6495 QQQ triple quadrupole mass spectrometer (Agilent Technologies) coupled to a 1290 series HPLC system via Selected Reaction Monitoring (SRM). Metabolites were measured using negative ionization mode with an ESI voltage of −3500 ev, respectively. Approximately 9–12 data points were acquired per detected metabolite.
7
Quantitative Metabolite Analysis by Triple Quadrupole Mass Spectrometry
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Targeted metabolites were analyzed using a 6495 QQQ triple quadrupole mass spectrometer (Agilent Technologies) coupled to a 1290 series HPLC system via Selected Reaction Monitoring (SRM). Metabolites were measured using positive and negative ionization modes with an ESI voltage of + 3000 and −3500 ev, respectively. Approximately 9–12 data points were acquired per detected metabolite28 (link),35 (link).
8
Quantification of Plasma Glibenclamide
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