LC-MS Analysis of Underivatized Bisphosphonate Drugs Using Mixed-Mode Reversed-Phase/Anion-Exchange Chromatography

Importance of the Topic

Bisphosphonates are widely prescribed for bone density disorders such as osteoporosis and Paget’s disease. Their strong polarity and acidic nature pose analytical challenges, traditionally addressed by derivatization to improve retention and detection. Developing a rapid LC-MS approach that bypasses derivatization simplifies workflows and enhances sensitivity in pharmaceutical and clinical laboratories.

Objectives and Study Overview

This work aimed to establish a 6-minute mixed-mode reversed-phase/anion-exchange LC-MS method for four common underivatized bisphosphonate drugs. The study evaluated retention, separation efficiency, and method reproducibility using a novel column with high-performance surface technology.

Methodology and Used Instrumentation

The approach combined reversed-phase and anion-exchange interactions to retain highly polar analytes without chemical modification.

• Sample preparation: Four bisphosphonate standards (1 mg/mL in 90:10 water:acetonitrile) combined into a mixed standard and stored at 4 °C in polypropylene vials.
• Chromatography: ACQUITY UPLC H-Class Plus System with HPS tubing; Atlantis Premier BEH C18 AX column (1.7 µm, 2.1 × 50 mm) at 40 °C; mobile phases: water (A), methanol (C), 200 mM ammonium formate pH 3 (D); flow rate 0.4 mL/min; injection volume 2 µL; gradient elution with 90:10 water:acetonitrile washes.
• Mass spectrometry: ACQUITY QDa Detector in negative ESI-SIR mode; capillary voltage 0.8 kV; cone voltage 15 V.
• Data analysis: Empower Chromatography Data System.

Main Results and Discussion

The mixed-mode column delivered clear retention and baseline separation of all four bisphosphonates within 6 minutes. High-performance surface (HPS) hardware significantly reduced nonspecific adsorption compared to stainless steel, yielding between 20 % and 100 % larger peak areas and improved precision (RSD < 3 % vs. up to 8 %). This enhancement eliminated lengthy column conditioning and minimized analyte–metal interactions, supporting reliable quantitation without derivatization or ion-pair reagents.

Benefits and Practical Applications

• Rapid analysis: Complete separation in under 6 minutes.
• No derivatization or ion-pair reagents needed, simplifying sample preparation and reducing cost.
• High sensitivity and reproducibility enabled by reduced adsorption.
• Compatibility with standard LC-MS platforms for pharmaceutical QA/QC and generic drug development.

Future Trends and Potential Applications

Advances may include tailored mixed-mode phases for designer bisphosphonates, integration with high-resolution mass spectrometry for complex biological matrices, and high-throughput screening in drug discovery. Further improvements in surface technologies could expand applications to other polar acidic compounds.

Conclusion

This study demonstrates a robust, high-throughput LC-MS method for underivatized bisphosphonates using mixed-mode RP/AX chromatography with high-performance surfaces. The method offers fast analysis, strong retention, and enhanced sensitivity, streamlining workflows in analytical and clinical laboratories.

Reference

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