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Protocol for liquid chromatography/mass spectrometry of glutathione conjugates using postcolumn solvent modification
M F Bean1, S L Pallante-Morell, D M Dulik
1Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Analytical Chemistry
|January 15, 1990
Summary
A new liquid chromatography/mass spectrometry (LC/MS) method improves the analysis of glutathione conjugates. Post-column addition of organic modifiers enhances both separation and detection sensitivity for these compounds.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Mass Spectrometry
Background:
- Glutathione conjugates are crucial biomarkers in toxicology and drug metabolism.
- Analyzing these polar compounds using liquid chromatography/mass spectrometry (LC/MS) presents challenges due to incompatible mobile phase requirements for chromatography and ionization.
- Thermospray ionization (TSI) often requires specific solvent conditions that may hinder chromatographic separation.
Purpose of the Study:
- To develop a novel LC/MS protocol for the sensitive and effective analysis of glutathione conjugates.
- To overcome the limitations of traditional LC/MS methods in analyzing labile and polar glutathione conjugates.
- To investigate the impact of mobile phase composition on both chromatographic separation and TSI efficiency.
Main Methods:
- Development of a thermospray liquid chromatography/mass spectrometry (LC/MS) protocol.
- Optimization of solvent conditions for both high-performance liquid chromatography (HPLC) and TSI.
- Utilizing post-column addition of organic modifiers to the mobile phase.
- Analysis of various glutathione conjugates, including those of menadione, styrene oxide, and haloalkanes.
Main Results:
- Direct injection TSI with 30-60% acetonitrile in aqueous ammonium acetate yielded protonated molecular ions for several glutathione conjugates.
- High organic modifier percentages, while enhancing ionization, precluded chromatographic separation.
- Post-column addition of organic modifiers enabled excellent chromatographic separations and sensitive mass spectra.
- Class-characteristic fragment ions were observed due to cleavage of the gamma-glutamyl-cysteine amide bond.
Conclusions:
- The developed post-column addition methodology significantly improves LC/MS analysis of glutathione conjugates.
- This approach balances the need for chromatographic separation of polar compounds with sensitive detection via TSI.
- Understanding the role of desolvation in fragmentation pathways enhances molecular ion sensitivity in thermospray ionization.