Forschungszentrum Jülich GmbH, Institute of Neurosciences and Medicine, Nuclear Chemistry (INM-5), Wilhelm-Johnen-Str., 52428 Jülich, Germany.
Forschungszentrum Jülich GmbH, Institute of Neurosciences and Medicine, Nuclear Chemistry (INM-5), Wilhelm-Johnen-Str., 52428 Jülich, Germany; Institute of Radiochemistry and Experimental Molecular Imaging, Faculty of Medicine and University Hospital Cologne, University of Cologne, Kerpener Str. 62, 50937 Cologne, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Aug 1;1228:123847. doi: 10.1016/j.jchromb.2023.123847. Epub 2023 Aug 5.
Accurate assessment of isolated radiochemical yields (RCYs) is a prerequisite for efficient and reliable optimization of labeling reactions. In practice, radiochemical conversions (RCCs) determined by HPLC analysis of crude reaction mixtures are often used to estimate RCYs. However, incomplete recovery of radioactivity from the stationary phase can lead to significant inaccuracies if RCCs are calculated based on the activity eluted from the column (i.e. the summed integrals of all peaks). Here, we validate a simple and practical method that overcomes problems associated with retention of activity on the column by determination of the total activity in the sample using post-column injection. Post-column injections were carried out using an additional injection valve, which was placed between the outlet of the HPLC column and the inlet of the detectors. 2-[F]Fluoropyridine ([F]FPy) and 8-cyclopentyl-3-(3-[F]fluoropropyl)-1-propylxanthine ([F]CPFPX) were prepared with radiochemical purities of > 99.8% and mixed with [F]fluoride at a ratio of 1:1 to simulate reaction mixtures obtained by radiolabeling reactions with an RCC of 50%. The samples were analyzed on three different C HPLC columns using neutral and acidic mobile phases. RCCs determined using the summed area of all peaks in the chromatograms were compared with those determined using post-column injection. Additionally, RCCs determined by post-column injection were corrected for activity losses before, during and after radiosyntheses to afford analytical RCYs, which were compared with isolated RCYs. Determination of RCCs based on the summed area of all peaks gave correct results under certain chromatographic conditions, but led to overestimation of the actual RCCs by up to 50% in other cases. In contrast, determination of RCCs using post-column injection provided precise results in all cases, and often significantly reduced analysis time. Moreover, analytical RCYs calculated from RCCs determined by post-column injection showed excellent agreement with isolated RCYs (<3% deviation). In conclusion, HPLC analysis using post-column injection enables reliable determination of RCCs independent of the chromatographic conditions and, together with a simple activity balance, rapid and accurate prediction of isolated RCYs.
准确评估孤立的放射性化学收率（RCYs）是有效和可靠地优化标记反应的前提。在实践中，通常使用高效液相色谱（HPLC）分析粗反应混合物来确定放射性化学转化率（RCCs），以估计 RCYs。然而，如果基于从柱上洗脱的放射性（即所有峰的总和积分）来计算 RCCs，则从固定相上不完全回收放射性可能会导致显著的不准确。在这里，我们验证了一种简单实用的方法，通过使用柱后注射来测定样品中的总活性来克服与柱上活性保留相关的问题。柱后注射是通过在 HPLC 柱的出口和检测器的入口之间放置一个附加的注射阀来进行的。2-[F]氟吡啶([F]FPy)和 8-环戊基-3-[F]氟丙基-1-丙基黄嘌呤([F]CPFPX)的放射性化学纯度均大于 99.8%，与[F]氟化物以 1:1 的比例混合，以模拟放射性标记反应的 RCC 为 50%的反应混合物。使用中性和酸性流动相，在三种不同的 C18 HPLC 柱上分析样品。用色谱图中所有峰的总和面积确定的 RCC 与用柱后注射确定的 RCC 进行比较。此外，还对放射合成前后的放射性损失进行了校正，以获得分析 RCYs，然后将其与分离的 RCYs 进行比较。在某些色谱条件下，基于所有峰总和面积确定 RCCs 可得到正确的结果，但在其他情况下，会导致实际 RCCs 的高估高达 50%。相比之下，在所有情况下，使用柱后注射确定 RCC 都能得到精确的结果，并且通常可以显著缩短分析时间。此外，从柱后注射确定的 RCC 计算得出的分析 RCYs 与分离的 RCYs 非常吻合（偏差小于 3%）。总之，使用柱后注射的 HPLC 分析能够在不依赖于色谱条件的情况下可靠地确定 RCCs，并与简单的活性平衡一起，快速准确地预测分离的 RCYs。
