建立和验证 HPLC 法用于放射性标记的 DOTA-TATE 和 DOTA-TOC 制剂的质量控制。

National Centre for Nuclear Research Radioisotope Centre POLATOM, 7 A. Soltana Street, 05-400 Otwock, Poland.

Department of Nuclear Medicine, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria.

Nucl Med Biol. 2021 Feb;93:63-73. doi: 10.1016/j.nucmedbio.2020.11.005. Epub 2020 Dec 3.

INTRODUCTION

The information on the presence of cold metal complexes in radiolabeled DOTA-TATE or DOTA-TOC is important in assessing the cause of the radiolabeling failure, poor radiolabeling yield and/or low effective molar activity. DOTA-peptide complexes are detectable using UV-Vis detector. The main limitation in the quantitative analysis is the limited availability of standard substances and the lack of data on their molar absorption coefficients. The aim of our study was development and validation of HPLC method enabling RCP analysis and identification and quantification of metal complexes impurities in the radiopharmaceutical preparations of DOTA-chelated peptides.

METHODS

Complexes of DOTA-TATE and DOTA-TOC with several metals, were prepared. Their molar absorption coefficients at 220 nm were determined. The developed HPLC method has been validated in terms of quantitative determination of non-complexed DOTA-TATE and DOTA-TOC and their respective complexes with metallic individuals.

RESULTS

Good chromatographic separation of the individual metal-DOTA-peptide complexes was achieved. The resolution between peaks of interest in radioactive preparations (complexes with: yttrium-90, lutetium-177, gallium-68) and metallic impurities was well above 1.5 (except gallium-68 DOTA-TOC preparations). Limits of detection and quantification were determined based on the parameters of the calibration curves. Based on the spectrophotometric and HPLC-DAD studies and statistical analysis of the results obtained, the average molar absorption coefficient was determined for studied DOTA-TATE and DOTA-TOC complexes, ε = 48 × 10M cm. With the use of the determined molar absorption coefficient the method enabled quantitative determination of non-labelled peptide in the radioactive preparation in the linearity range of 0.5-100 μg/mL for DOTA-TATE(net) and 0.5-100 μg/mL for DOTA-TOC(net).

CONCLUSION

The developed HPLC method is suitable for RCP determination of radiolabelled DOTA-TATE and DOTA-TOC preparations. Determination of the average molar absorption coefficient for DOTA-TATE and DOTA-TOC complexes allows assessment of the total content of the peptide in radiopharmaceutical preparation regardless of its chemical form (free ligand, associated with radionuclide, in the form of a complex with metal ions being the impurity) using the HPLC method with UV detection.

简介

在评估放射性标记失败、放射性标记产率低和/或有效摩尔活度低的原因时，有关冷金属配合物在放射性标记的 DOTA-TATE 或 DOTA-TOC 中的存在的信息非常重要。DOTA-肽配合物可以使用 UV-Vis 检测器检测到。定量分析的主要限制是标准物质的有限可用性以及缺乏关于它们的摩尔吸光系数的数据。我们研究的目的是开发和验证 HPLC 方法，该方法能够进行 RCP 分析，并鉴定和定量放射性药物制剂中 DOTA-螯合肽的金属配合物杂质。

方法

制备了 DOTA-TATE 和 DOTA-TOC 与几种金属的配合物，并确定了它们在 220nm 处的摩尔吸光系数。开发的 HPLC 方法已针对未络合的 DOTA-TATE 和 DOTA-TOC 及其各自与金属个体形成的配合物的定量测定进行了验证。

结果

实现了各个金属-DOTA-肽配合物的良好色谱分离。放射性制剂（与钇-90、镥-177、镓-68 形成的配合物）和金属杂质的感兴趣峰之间的分辨率远高于 1.5（除了镓-68 DOTA-TOC 制剂）。检测限和定量限是根据校准曲线的参数确定的。基于分光光度法和 HPLC-DAD 研究以及对获得的结果的统计分析，确定了所研究的 DOTA-TATE 和 DOTA-TOC 配合物的平均摩尔吸光系数，ε=48×10Mcm。使用确定的摩尔吸光系数，该方法能够在 0.5-100μg/mL 的线性范围内定量测定放射性制剂中未标记的肽，对于 DOTA-TATE（净）为 0.5-100μg/mL，对于 DOTA-TOC（净）为 0.5-100μg/mL。

结论

开发的 HPLC 方法适用于放射性标记的 DOTA-TATE 和 DOTA-TOC 制剂的 RCP 测定。测定 DOTA-TATE 和 DOTA-TOC 配合物的平均摩尔吸光系数可以使用具有 UV 检测的 HPLC 方法评估放射性药物制剂中肽的总含量，而不管其化学形式（游离配体、与放射性核素结合、与作为杂质的金属离子形成配合物的形式）。