Validation of HPLC and TLC analytical methods to determine radiochemical purity of 99mTc-cAbVCAM1-5, a new experimental radiotracer

Abstract

Cardiovascular diseases, including fatal myocardial infarctions from atheromatous plaques, are the primary global mortality cause. Detecting stenotic atheromatous plaques is possible through coronary angiography, but vulnerable plaques with eccentric remodeling are undetectable with current diagnostic methods. Addressing this challenge, our group developed a radiopharmaceutical drug targeting vascular cell adhesion molecule 1 (VCAM-1), radiolabeled with technetium-99m. Given the absence of a monograph in the European Pharmacopoeia, and in order to draft the investigational medicinal product documentation, analytical methods had to be validated by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) to determine the radiochemical purity (RCP) of ^99mTc-cAbVCAM1-5. This study therefore presents the results of the validation of analytical methods obtained in this context. The method validation followed the European Association of Nuclear Medicine (EANM) recommendations adapted from ICH Q2(R1), ensuring conformity with specificity, accuracy, repeatability and intermediate precision, linearity, robustness, quantification limit (LoQ), and range criteria. Regarding the results of specificity, both HPLC and TLC methods demonstrated excellent separation of ^99mTc-cAbVCAM1-5 from impurities ^99mTcO4^-. Accuracy results indicated recovery percentages within the range of 99.52-101.40% for the HPLC and 99.51-101.97% for TLC, ensuring reliable measurements for each concentration of ^99mTcO₄^-. Precision of the methods was validated by assessing repeatability and intermediate precision. Linearity was determined over the usual concentrations range and the correlation coefficient was greater than 0.99 for both methods. The limit of quantification was measured by diluting the ^99mTcO₄^- to obtain a signal-to-noise ratio of around 10:1. Under these conditions, we obtained an LOQ of 2.10 MBq/mL for HPLC and 2Mbq/mL for TLC. In conclusion, the analytical methods developed in this study comply with EANM recommendations. This therefore allows us to correctly assess the radiochemical purity of ^99mTc-cAbVCAM1-5, a new radiotracer targeting inflammation in vulnerable plaques.

Keywords: High performance liquid chromatograph; Method validation; Radiochemical purity; Radiopharmaceuticals; Single domain antibody; Thin layer chromatography.