Application description
This method focuses on the separation of compounds integral to the glutamate metabolomic cycle utilizing High-Performance Liquid Chromatography (HPLC) coupled with Mass Spectrometry (MS). The separation is conducted on an Amaze TH Tri-Modal Hydrophilic Interaction Liquid Chromatography (HILIC) Column, tailored to handle polar and ionizable compounds effectively.
Compound Properties:
Glutamate: As a neurotransmitter and key player in amino acid metabolism, glutamate exhibits water solubility and contains both acidic and basic functional groups, allowing its interaction under HILIC conditions.
Alpha-Ketoglutarate (α-Ketoglutaric Acid): An intermediate in the TCA cycle, alpha-ketoglutarate is vital for cellular energy metabolism. It is water-soluble and possesses a keto acid functional group, contributing to its behavior in HILIC chromatography.
Glutamine: A conditionally essential amino acid involved in protein synthesis and nitrogen transport, glutamine is water-soluble and contains both acidic and basic functional groups, suitable for separation under HILIC conditions.
Glutamic Acid: Another neurotransmitter, glutamic acid is involved in various physiological functions. It is water-soluble and contains an acidic functional group, allowing for interaction in HILIC chromatography.
Gamma-Aminobutyric Acid (GABA): Serving as a neurotransmitter that inhibits neuronal activity, GABA is water-soluble and possesses both acidic and basic functional groups, making it compatible with HILIC chromatography.
LC-MS Conditions:
Mobile Phase: Comprising a mixture of water and organic solvent, typically acetonitrile, supplemented with additives like ammonium formate or ammonium acetate to enhance ionization efficiency for MS detection.
Gradient Elution: Employing a gradient of organic solvent to achieve optimal separation of compounds throughout the chromatographic run.
Mass Spectrometry: Coupled with HPLC for sensitive and selective detection and quantification of metabolites based on their mass-to-charge ratio (m/z).
By leveraging this method, efficient separation and detection of glutamate metabolomic cycle compounds can be achieved, facilitating comprehensive analysis of metabolic pathways and related biological processes.
Conditions of Experiment
| Column: |
Amaze MH |
| Separation Modes: |
HILIC, cation-exchange, Anion-exchange |
| Column Dimenstions: |
3x100 mm, 3 um, 100 A |
| Mobile Phase: |
ACN/water/ammonium formate/Formic acid |
| Detection: |
Mass Spectrometry (MS) |
| Sample: |
various-0.1 mg/ml |
| Injection: |
3 uL |
| Flow rate: |
0.6 ml/min |
Analytes
| Class of compounds: |
Amino acid, Metabolite, Neurotransmitter, Organic acid, Supplement |
| Nature of compounds: |
Acidic, Basic, Hydrophilic, Polar, Zwitterionic |
| Compounds: |
Glutamic acid, Glutamine, Ketoglutaric acid, 4-Aminobuyric acid (GABA) |
