Improved LC/MS Methods for the Analysis of Anionic Analytes

Significance of the Topic

The accurate detection of anionic compounds is critical in metabolomics, environmental analysis and pharmaceutical research. Metal contamination in LC/MS systems can degrade peak shapes and sensitivity for negatively charged analytes. Conventional chelators such as EDTA improve chromatography but often cause ion suppression, limiting detection limits.

Study Objectives and Overview

This work evaluates medronic acid as a mobile phase additive to enhance chromatographic performance of metal‐sensitive analytes with minimal ion suppression. Key aims include

• Comparing medronic acid against EDTA for HILIC LC/MS of phosphorylated metabolites, organic acids and polar pesticides
• Profiling nutrient consumption and waste secretion in mammalian cell culture
• Assessing intracellular metabolomic shifts in K562 leukemia cells treated with methotrexate
• Improving phosphopeptide detection via reversed‐phase LC/MS

Methodology and Instrumentation

Chromatographic separations employed Agilent InfinityLab Poroshell 120 HILIC-Z columns and AdvanceBio Peptide Mapping columns on a 1290 Infinity Binary LC. Mass analysis used 6545 Q-TOF, 6490 Triple Quad with iFunnel, and 6550 iFunnel Q-TOF systems. Mobile phases consisted of 100 mM ammonium acetate at pH 9 with 5 µM medronic acid additive. K562 cells were cultured in RPMI medium, treated with 5 µM methotrexate or vehicle for 16 hours, and metabolites were extracted for LC/MS profiling.

Main Results and Discussion

Key findings include

• Medronic acid restores sharp peak shapes for nucleotides, organic acids and polar pesticides with minimal reduction in signal intensity
• Underivatized amino acids were reliably separated and quantified by HILIC LC/MS
• Fast profiling of cell culture media enabled quantification of glucose uptake, amino acid consumption and secreted metabolites over multiple days
• Methotrexate treatment in K562 cells increased fructose 1,6-bisphosphate and extracellular adenosine, reflecting drug action on purine metabolism
• Reversed-phase LC/MS of phosphopeptides showed enhanced detection across mono- to tetra-phosphorylated sequences without the need for enrichment

Applications and Practical Benefits

This medronic acid method offers

• Improved sensitivity and peak fidelity for metal‐sensitive analytes
• High-throughput monitoring of cell culture performance in bioprocessing and drug screening
• Streamlined workflows for metabolomics and phosphoproteomics without extensive sample preparation
• Broader applicability to environmental and food safety testing of polar contaminants

Future Trends and Potential Applications

Ongoing developments will explore integration of medronic acid additives in ion‐exchange and ion-pairing chromatography, automation for large sample sets, and pairing with high-resolution MS for deep proteome and metabolome coverage. Emerging applications may include real-time bioreactor monitoring and clinical biomarker discovery.

Conclusion

The introduction of low-concentration medronic acid into mobile phases significantly enhances LC/MS analysis of anionic and phosphorylated analytes, overcoming limitations of traditional chelators. This approach broadens the scope of quantitative workflows in metabolomics, cell culture analytics and phosphoproteomics.

References

1. J J Pesek, M T Matyska, S M Fischer Improvement of peak shape in aqueous normal phase analysis of anionic metabolites J Sep Sci 34 24 3509-16 2011
2. F P Veras et al Fructose 1 6-bisphosphate a high-energy intermediate of glycolysis attenuates experimental arthritis by activating anti-inflammatory adenosinergic pathway Sci Rep 5 15171 2015