Ion Chromatography Assay for Ammonia in Adenosine

Significance of the Topic

Adenosine is widely used in clinical settings for pain management, cardiac stress testing and control of blood pressure during surgery. Trace levels of ammonia, introduced during fermentation or biochemical synthesis, can compromise product safety and efficacy. Reliable quantification of ammonia in adenosine formulations is essential for quality control and regulatory compliance.

Objectives and Study Overview

This work describes development and validation of a rapid ion chromatography (IC) method to quantify ammonia in adenosine. The goal was to replace the subjective, mercury-based colorimetric assay in the USP monograph with a sensitive, reproducible and environmentally friendly technique.

Methodology and Instrumentation

An electrolytically generated 33 mM methanesulfonic acid (MSA) eluent and a self-regenerating suppressor were employed for suppressed conductivity detection. Samples were prepared by suspending 0.5 g adenosine in 10 mL water, filtering and direct injection (10 µL) without final dilution. A Dionex IonPac CG12A guard and CS12A analytical column (3 × 150 mm, 5 µm) operated at 30 °C and 0.7 mL/min achieved baseline separation of ammonium at ~2.1 min in a 5-minute run.

Results and Discussion

Accuracy was assessed over three days at three spike levels (0.05, 1, 2.5 mg/L), yielding recoveries of 88–116%. Precision studies (nine determinations) showed retention time RSD <0.1% and peak area RSD <2%. The limit of detection (LOD) was 0.001 mg/L and the limit of quantitation (LOQ) 0.004 mg/L based on a 3:1 and 10:1 signal-to-noise ratio. Linearity from 0.025–10 mg/L fitted a quadratic model (R² = 0.9992), while a linear response (R² = 0.9984) was obtained between 0.025–2 mg/L. Robustness tests varying MSA concentration, flow rate, column temperature and using columns from different batches confirmed method stability, with peak asymmetry 1.2–1.4 and resolution from sodium >1.9.

Benefits and Practical Applications

The IC method offers objective quantification with high throughput, minimal hazardous waste and no mercury use. Fast isocratic separation and reagent-free eluent generation streamline routine QA/QC workflows. The approach can be adapted to other weak-base impurity determinations in pharmaceuticals.

Future Trends and Applications

Advances may include higher-throughput UHPLC-IC hybrid systems, integration with automation platforms for online sample prep, and coupling to mass spectrometry for simultaneous multi-impurity profiling. Wider adoption of reagent-free eluents and greener suppressor technologies will enhance sustainability.

Conclusion

The proposed IC assay reliably quantifies trace ammonia in adenosine, meeting USP/ICH performance requirements. Its sensitivity, precision and speed support replacement of the legacy colorimetric test and strengthen pharmaceutical impurity control.

Used Instrumentation

• ICS-5000+ HPIC system with Reagent-Free™ HPIC
• Dionex AS-AP autosampler, 10 µL loop
• IonPac CG12A guard and CS12A analytical columns (5 µm)
• EGC III MSA eluent generator cartridge
• CR-CTC cation trap column
• CSRS 300 Cation Self-Regenerating Suppressor or SC-CSRS 300 Salt-Converter Suppressor
• Chromeleon chromatography data system

References

1. U.S. Pharmacopeial Convention. Adenosine Monograph Errata. USP35–NF30; Rockville, MD.
2. International Conference on Harmonisation (ICH). Q2(R1) Validation of Analytical Procedures: Text and Methodology; 2005.
3. U.S. Pharmacopeia. General Chapter <1225> Validation of Compendial Methods; USP–NF; Rockville, MD; 2012; pp 877–881.
4. Hinshaw JV, Dolan JW. Stimuli to the Revision Process: Signal-to-Noise Measurements from Chromatographic Data. Pharmacopeial Forum 2012, 38(3).