# Labeling Peptides with Radioiodine: An Overview of Traditional and Emerging Techniques
## 5 Purification and Other Considerations for Radioiodination  
After radioiodination, purification is necessary to remove unreacted radioiodine, precursors, and byproducts. The most common method for purifying radioiodinated peptides is HPLC. HPLC allows for high separation efficiency and can separate the radiolabeled product from impurities based on their physicochemical properties [64]. It is easy to monitor with UV and gamma detection and allows for high separation efficiency. Another useful method for separating radioiodinated peptides from unreacted iodide, especially when the peptide's structure is well-defined and there is only one labeling site, is Gel Permeation Chromatography (GPC)--examples include Sephadex G-50, G-25 or LH-20 [65, 66]. Solid-Phase Extraction (SPE) with C18 cartridges can also be used to remove unreacted radioiodide, but it has limitations such as low separation efficiency [67]. SPE is generally useful for single separations based on evident lipophilicity differences. Finally, solid-state synthesis combined with simple filtration can be used to isolate radioiodinated products. For example, filtration through SiO\({}_{2}\) was used to separate [125]ISIB [58]. Several other factors must be considered for effective radioiodination such as the following: (1) Radio-chemical Yield (RCY): RCY is the percentage of the radioactive material that is incorporated into the desired product. Optimizing reaction conditions is crucial to achieve high RCY [68]. (2) Radiochemical Purity (RCP): RCP is a measure of the proportion of the total radioactivity that is associated with the desired product. High RCP is essential for accurate imaging and effective therapy [69]. (3) Molar Activity: Molar activity refers to the amount of radioactivity per mole of the radiolabeled compound. High molar activity is desired for better imaging contrast and to minimize the amount of administered substance. (4) Stability: The stability of the radiolabeled peptide is crucial for maintaining its integrity and biological activity during in vivo studies and clinical applications. (5) Scale of the Reaction: Radioiodination is typically carried out in micromolar amounts, and reoptimization is needed to adapt the technique to larger amounts. (6) Metabolic Stability: Understanding the metabolic pathways of the radiolabeled drug is crucial to prevent possible deiodination processes [13]. For clinical applications, especially where radiotracers are intended for intravenous administration, purification must comply with stringent pharmaceutical standards, including radiochemical purity, sterility, and apyrogenicity [70]. Rapid purification is essential due to the short half-lives of iodine isotopes such as \({}^{123}\)I and \({}^{124}\)I. Solid-phase extraction (SPE) is one of the most commonly employed techniques, using cartridges such as C18, alumina, or ion exchange columns to remove unreacted iodine and by-products; this method can be performed in-line and is highly amenable to automation under good manufacturing practice GMP conditions [71]. Semi-preparative HPLC offers high resolution and is often used in combination with fraction collection and sterile filtration (e.g., through 0.22 \(\upmu\)m filters) for the purification of I-labeled antibodies and larger biomolecules. Quenching agents and scavengers (e.g., sodium thiosulfate or sodium metabisulfite) are sometimes used during work-up to neutralize residual oxidants. The choice of purification strategy is guided by the nature of the radiotracer, its formulation route, and the need for rapid turnaround in clinical settings [72, 73].