Ion Chromatography Assay for Ammonia in Adenosine

Importance of the Topic

Adenosine is a widely used nucleoside in clinical settings for treatment of cardiac arrhythmias, blood pressure control, and pulmonary hypertension. Contamination with ammonia, an inevitable by-product of fermentation or synthesis, can affect the quality and safety of the drug substance. Precise measurement of ammonia at low levels (below 4 mg/L) is therefore essential for compliance with pharmacopeial standards and ensuring patient safety.

Objectives and Study Overview

The goal of this work was to replace a subjective, mercury‐based colorimetric test in the USP adenosine monograph with an objective, rapid, and environmentally friendly ion chromatography (IC) assay. The study encompasses method development, validation (accuracy, precision, detection limits, linearity), and robustness evaluation against small operational variations.

Methodology

Sample Preparation and Reagents

• Adenosine sample was dissolved at 50 mg/mL in water, stirred, filtered, and injected directly without final dilution.
• Ammonium standards were prepared gravimetrically from a 1000 mg/L stock, diluted to 0.025–50 mg/L.

Chromatographic Conditions

• Column: Thermo Scientific Dionex IonPac CG12A guard (3×30 mm) and CS12A-5 µm analytical (3×150 mm).
• Eluent: 33 mM methanesulfonic acid generated electrolytically.
• Flow rate: 0.7 mL/min; injection volume: 10 µL; column and detector temperature: 30 °C.
• Detection: suppressed conductivity using Dionex CSRS-300 or SC-CSRS 300 self-regenerating suppressor in autosuppression recycle mode.

Instrumentation Used

• Dionex ICS-5000+ Reagent-Free HPIC system with SP/DP pump, EG eluent generator, CD conductivity detector.
• Dionex AS-AP autosampler with 10 µL PEEK loop.
• Dionex EGC III methanesulfonic acid eluent generator cartridge.
• Dionex CR-CTC continuously regenerated cation trap column.
• Chromeleon chromatography data system.
• Dionex CSRS-300 or SC-CSRS 300 cation self-regenerating suppressor.

Main Results and Discussion

The optimized isocratic method achieved ammonium elution at ~2.1 min with resolution >2 versus sodium and peak asymmetry ~1.2. Total run time of 5 min supports high throughput. Method accuracy, evaluated over three days at spike levels of 0.05, 1, and 2.5 mg/L, yielded recoveries of 88–116%. Precision studies (nine determinations across three concentrations) showed retention time RSD <0.1% and peak area RSD <2%. Detection limit (LOD) and quantitation limit (LOQ) were 0.001 and 0.004 mg/L, respectively. Linearity was observed from 0.025 to 10 mg/L with a quadratic fit (R2=0.9992), and a linear segment up to 2 mg/L (R2=0.9984). Robustness testing against variations in eluent concentration (±2 mM), flow rate (±10%), temperature (±2 °C), and column batches confirmed consistent performance (resolution 1.96–2.32, asymmetry 1.2–1.4, RT RSD <0.2%, area RSD <2%).

Benefits and Practical Applications

This IC method eliminates subjective visual endpoints and hazardous mercury reagents, reduces waste, and improves reproducibility. Fast analysis and automated eluent generation enhance laboratory efficiency. The approach can be adopted in QC laboratories for routine release testing of adenosine drug substances and related pharmaceuticals.

Future Trends and Opportunities

Advances may include coupling IC with mass spectrometry for simultaneous profiling of multiple impurities, miniaturized high-throughput IC workflows, and further automation in sample preparation. Green chemistry initiatives could expand reagent-free techniques to other critical analytes in biopharmaceuticals.

Conclusion

The developed IC assay meets or exceeds USP/ICH validation criteria for ammonia determination in adenosine. It offers a rapid, sensitive, and robust alternative to the existing colorimetric method, with significant advantages in safety, throughput, and environmental impact.

References

1. U.S. Pharmacopeial Convention. Adenosine Monograph and Ammonia Assay, USP35-NF30.
2. International Conference on Harmonisation. Q2(R1) Validation of Analytical Procedures: Text and Methodology.
3. U.S. Pharmacopeia/NF, General Chapter <1225> Validation of Compendial Methods.
4. Hinshaw, J.V.; Dolan, J.W. Signal-to-Noise Measurements from Chromatographic Data. Pharmacopeial Forum 2012, 38(3).