A new IC-MS/MS approach for the analysis of bisphosphonates in horse plasma

Significance of the Topic

Bisphosphonates are widely used to treat bone disorders in horses but are banned in racing under international regulations. Their high polarity and strong chelating properties make routine detection in complex biological matrices technically challenging. A robust, sensitive and rapid analytical approach is essential for compliance in equine doping control and broader pharmaceutical and clinical applications.

Objectives and Overview of the Study

This work aimed to develop a simplified ion chromatography tandem mass spectrometry protocol for simultaneous detection and confirmation of both nitrogen containing and non­n itrogen containing bisphosphonates in horse plasma. The focus was on eliminating derivatization steps, reducing sample preparation time, and ensuring high sensitivity and selectivity.

Methodology and Instrumentation Used

Sample Preparation

• Aliquot of 200 µL horse plasma spiked with a trifluoromethylphenyl thiomethylene biphosphonic acid internal standard
• Acidic protein precipitation followed by addition of water
• Sequential solid phase extraction using HLB polymeric sorbent for cleanup and weak anion exchange sorbent for bisphosphonate retention
• Elution with water, methanol and ammonium hydroxide, evaporation and reconstitution in water, injection volume 50 µL

Chromatographic Conditions

• IonPac AS18-Fast 4 µm column, 150 × 2 mm
• Potassium hydroxide gradient generated electrochemically from 35 to 100 mM over 7 minutes and returned to starting conditions by 14 minutes
• Flow rate 0.25 mL/min, suppressed conductivity detection, total run time 14 minutes

Mass Spectrometry

• TSQ Altis triple quadrupole with H-ESI source, spray voltage 3500 V, transfer tube and vaporizer at 325 °C
• Sheath gas 50, auxiliary gas 10, sweep gas 1
• SRM acquisition with optimized precursor to product ion transitions and collision energies for seven bisphosphonates and internal standard
• Chromeleon CDS 7.2 for data acquisition and processing

Main Results and Discussion

The method achieved baseline separation of seven bisphosphonates within 14 minutes. Conductivity detection alone showed co-elution of several analytes, whereas SRM traces provided individual compound resolution. Limits of detection were below 50 fg/µL, and calibration was linear from 0.05 to 10 pg/µL. In horse plasma spiked with 5 ng/mL tiludronic acid, product ion chromatograms delivered a signal-to-noise ratio of 150. Matrix interference at retention times of target transitions was minimal, demonstrating the method’s robustness.

Benefits and Practical Applications of the Method

• Elimination of derivatization steps and reduction of sample preparation complexity
• Comprehensive quantification of nitrogen and non-nitrogen bisphosphonates in a single run
• High selectivity and sensitivity suitable for doping control laboratories
• Rapid screening and confirmatory analysis enabling high throughput
• Compatibility with existing IC and MS infrastructure

Future Trends and Potential Applications

The IC-MS/MS approach could expand to other highly polar or chelating compounds requiring low detection limits. Advances in suppressor technology and SRM multiplexing may further increase throughput. Integration with autosamplers and online SPE could create fully automated workflows for clinical diagnostics, environmental monitoring, and pharmaceutical quality control.

Conclusion

This IC-MS/MS protocol offers a powerful, derivatization-free solution for the analysis of bisphosphonates in horse plasma. It delivers rapid, sensitive and selective detection and confirmation, streamlining analytical workflows in doping control and potentially across diverse fields requiring analysis of polar analytes.

References

1. International Federation of Horseracing Authorities Article 6 of the International Agreement on Breeding, Racing and Wagering IFHA 2018
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