Development and Validation of a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Identification and Quantitation of [13N]Ammonia in PET Radiopharmaceuticals.

Ammonia N13 injection is an essential PET radiotracer for myocardial perfusion imaging due to its optimal half-life and high-resolution imaging properties. However, existing ion chromatography-based techniques for determining the molar activity of [¹³N]ammonia are limited, particularly in the presence of excess metal ions and due to the need for specialized instrumentation. To address these limitations, we developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method using dansyl chloride derivatization to convert [¹³N]ammonia into a UV-detectable dansyl amide. This allows detection using widely available UV detectors. The method was validated in accordance with International Council for Harmonisation (ICH) guidelines across multiple parameters including specificity, linearity, accuracy, precision, and robustness. Validation results confirmed excellent specificity with no significant interferences, and strong linearity over a concentration range of 0.8 µM to 100 µM (r² = 0.999). The method demonstrated high sensitivity with limits of detection (LOD) and quantitation (LOQ) determined at 0.6 µM and 1.7 µM, respectively. Precision was confirmed with a %RSD of 1.3% (n = 24), while accuracy was supported by a mean spike recovery of 95%. Robustness testing confirmed the method’s reliability under varied conditions. This RP-HPLC method was successfully applied to analyze [¹³N]ammonia product batches from an in-house cyclotron, demonstrating its practical utility. Thus, it offers a cost-effective and accessible alternative to ion chromatography for routine quality control ensuring compliance with regulatory standards.