Jan 11, 2026
Data Acquisition and Analysis of Derivatized Hexose/Glucose by LC-QQQ-MS
- Uche Medoh1,2,3,4,5,
- Ali Ghoochani1,2,3,4,
- Monther Abu-Remaileh1,2,3,4
- 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
- 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
- 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
- 5Arc Institute, Palo Alto, CA 94304, USA
Protocol Citation: Uche Medoh, Ali Ghoochani, Monther Abu-Remaileh 2026. Data Acquisition and Analysis of Derivatized Hexose/Glucose by LC-QQQ-MS. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbw1xygpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 07, 2025
Last Modified: January 11, 2026
Protocol Integer ID: 221909
Keywords: glucose by lc, analysis of derivatized hexose, glucose, derivatized hexose, triple quadrupole mass spectrometer, throughput metabolite quantification, ultivo triple quadrupole mass spectrometer, using lc, chromatographic condition
Abstract
This protocol outlines the steps for the separation, detection, and quantification of derivatized hexose/glucose using LC-QQQ-MS. Samples are analyzed with a C18 column coupled to a 1290 II LC system and an Ultivo triple quadrupole mass spectrometer. The procedure includes chromatographic conditions, MS settings, and data analysis workflows for high-throughput metabolite quantification.
Materials
Materials:
- Derivatized hexose/glucose samples
- Eclipse Plus C18 column (4.6 x 100 mm, 3.5 µm, Agilent)
- Agilent 1290 Infinity II LC system
- Agilent Ultivo triple quadrupole (QQQ) MS with ESI source
- LC/MS-grade water, acetonitrile, isopropanol
- Ammonium formate (10 mM)
- Formic acid (0.1%)
- MassHunter Qualitative Analysis Software
- Quant-My-Way QQQ Quantitative Analysis Software
- Microsoft Excel
Troubleshooting
Materials:
Materials:
- Derivatized hexose/glucose samples
- Eclipse Plus C18 column (4.6 x 100 mm, 3.5 µm, Agilent)
- Agilent 1290 Infinity II LC system
- Agilent Ultivo triple quadrupole (QQQ) MS with ESI source
- LC/MS-grade water, acetonitrile, isopropanol
- Ammonium formate (10 mM)
- Formic acid (0.1%)
- MassHunter Qualitative Analysis Software
- Quant-My-Way QQQ Quantitative Analysis Software
Reagents and Mobile Phases:
- Mobile Phase A: 10 mM ammonium formate + 0.1% formic acid in 60:40 (v/v) water:acetonitrile
- Mobile Phase B: 10 mM ammonium formate + 0.1% formic acid in 90:10 (v/v) isopropanol:acetonitrile
Procedure:
Connect Eclipse Plus C18 column to the LC system.
Ensure column oven is set to 30°C.
Set autosampler temperature to 4°C.
Perform external mass calibration with standard mix every 7 days.
Sample Injection:
- Inject 3 µl of each sample with fast polarity switching enabled.
LC Gradient:
| Time (min) | %B | |
| 0.0 | 50 | |
| 3.0 | 100 | |
| 6.0 | 100 | |
| 6.1 | 50 | |
| 8.0 | 50 |
Flow rate: 0.400 ml/min
Mass Spectrometer Settings
· Spray Voltage: 3.6 kV (positive mode), 2.5 kV (negative mode)
· Gas Temperature: 250°C
· Sheath Gas Temperature: 400°C
· Gas Flow: 11 l/min
· Sheath Gas Flow: 12 l/min
· Nebulizer: 28 psi
MRM Transitions
| Compound | Precursor Ion (m/z) | Product Ion (m/z) | Fragmentor (V) | Collision Energy (eV) | |
| Benzoyl-Hexose/Glucose | 718.0 | 231.0 | 100 | 11 | |
| Benzoyl-Hexose/Glucose | 718.0 | 105.0 | 100 | 37 | |
| Benzoyl-Hexose/Glucose | 718.0 | 579.1 | 100 | 5 |
Data Analysis
Use MassHunter Qualitative Analysis software to validate peak alignment and retention times. Confirm identification by comparing MS/MS spectra to standards and evaluating both transitions for consistent relative intensity. Use Quant-My-Way for QQQ quantification with MRM data. Export raw peak areas to Microsoft Excel for further processing and normalization.
Consistent peak profiles for derivatized hexose/glucose with reproducible retention times and MRM transition ratios. Accurate relative quantification should be observed when using validated transitions and verified against standard fragmentation patterns.