Analysis of Underivatized Bisphosphonate Drugs Using HILIC-MS

Significance of the Topic

Bisphosphonate drugs are critical in managing bone-related disorders such as osteoporosis and Paget’s disease. Their highly polar nature and multiple phosphonic acid groups make direct analysis challenging. Developing a robust HILIC-MS approach for underivatized bisphosphonates simplifies workflows and improves throughput in pharmaceutical analysis.

Objectives and Study Overview

This work aimed to establish a HILIC-MS method for five bisphosphonate drugs without chemical derivatization. Key objectives included comparing two HILIC stationary phases, optimizing mobile phase buffer strength, and evaluating column-to-column reproducibility to ensure reliable quantitation.

Methodology

Stock solutions (1 mg/mL) of etidronic, alendronic, ibandronic, risedronic, and zoledronic acids were prepared in water and stored at 4 °C. Initial screening employed a generic 5–50% aqueous gradient (10 mM ammonium formate, pH 3) on ACQUITY Premier BEH Amide and Atlantis Premier BEH Z-HILIC columns. A refined gradient (20–50% aqueous) and buffer strengths of 10, 20, and 40 mM were tested. Final evaluation measured retention, peak shape, and symmetry across three Z-HILIC columns from different production batches.

Instrumentation Used

• Liquid chromatography: ACQUITY™ Premier Binary Solvent Manager with PDA detector
• Columns: Atlantis Premier BEH Z-HILIC (2.1 × 50 mm, 1.7 μm) and ACQUITY Premier BEH Amide (2.1 × 50 mm, 1.7 μm)
• Mass spectrometry: Xevo™ TQ-S micro triple quadrupole with negative-mode ESI
• Software: MassLynx™ V4.1 with TargetLynx™ application

Key Results and Discussion

Both columns retained underivatized bisphosphonates, but the Z-HILIC phase provided stronger anion-exchange interactions, notably enhancing etidronic acid retention. Increasing buffer concentration to 40 mM improved peak sharpness and reduced tailing for highly acidic analytes. Optimized conditions yielded consistent retention times, symmetry factors between 1.14 and 1.44, and relative standard deviations below 5% for key metrics.

Benefits and Practical Applications

• Eliminates time-consuming derivatization, reducing sample preparation steps
• Enhances sensitivity and specificity through direct HILIC-MS detection of polar analytes
• Delivers high reproducibility suitable for routine pharmaceutical QC and bioanalysis

Future Trends and Opportunities

Emerging HILIC phases with advanced zwitterionic and hybrid surface chemistries are expected to further improve selectivity for multiphosphorylated compounds. Coupling these developments with high-resolution MS and automated sample handling could extend this approach to metabolomics and therapeutic monitoring of other highly polar drugs.

Conclusion

The optimized HILIC-MS method on an Atlantis Premier BEH Z-HILIC column enables direct, reproducible analysis of underivatized bisphosphonate drugs, removing the need for derivatization and streamlining workflow efficiency.

Reference

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