Bioanalytical Sciences, Bristol-Myers Squibb Co., Princeton, NJ 08543, United States.
Bioanalytical Sciences, Bristol-Myers Squibb Co., Princeton, NJ 08543, United States.
J Pharm Biomed Anal. 2018 Jun 5;155:141-147. doi: 10.1016/j.jpba.2018.03.064. Epub 2018 Apr 4.
Metabolite interferences represent a major risk of inaccurate quantification when using LC-MS/MS bioanalytical assays. During LC-MS/MS bioanalysis of BMS-919194, a phosphate ester prodrug, in plasma samples from rat and monkey GLP toxicology studies, an unknown peak was detected in the MRM channel of the prodrug. This peak was not observed in previous discovery toxicology studies, in which a fast gradient LC-MS/MS method was used. We found out that this unknown peak would co-elute with the prodrug peak when the discovery method was used, therefore, causing significant overestimation of the exposure of the prodrug in the discovery toxicology studies. To understand the nature of this interfering peak and its impact to bioanalytical assay, we further investigated its formation and identification. The interfering compound and the prodrug were found to be isobaric and to have the same major product ions in electrospray ionization positive mode, thus, could not be differentiated using a triple quadrupole mass spectrometer. By using high-resolution mass spectrometry (HRMS), the interfering metabolite was successfully identified to be an isobaric sulfate metabolite of BMS-919194. To the best of our knowledge, this is the first report that a phosphate prodrug was metabolized in vivo to an isobaric sulfate metabolite, and this metabolite caused significant interference to the analysis of the prodrug. This work demonstrated the presence of the interference risk from isobaric sulfate metabolites to the bioanalysis of phosphate prodrugs in real samples. It is critical to evaluate and mitigate potential metabolite interferences during method development, therefore, minimize the related bioanalytical risks and ensure assay quality. Our work also showed the unique advantages of HRMS in identifying potential metabolite interference during LC-MS/MS bioanalysis.
当使用 LC-MS/MS 生物分析测定法分析 BMS-919194（一种磷酸酯前药）在大鼠和猴子 GLP 毒理学研究的血浆样本时，检测到 MRM 通道中存在未知峰。在之前的发现毒理学研究中，未观察到该未知峰，而在该研究中使用了快速梯度 LC-MS/MS 方法。我们发现，当使用发现方法时，该未知峰会与前药峰共洗脱，从而导致在发现毒理学研究中对前药的暴露量的过高估计。为了了解该干扰峰的性质及其对生物分析测定法的影响，我们进一步研究了其形成和鉴定。发现干扰化合物和前药是等重的，并且在电喷雾电离正模式下具有相同的主要产物离子，因此，无法使用三重四极杆质谱仪进行区分。通过使用高分辨率质谱（HRMS），成功地将干扰代谢物鉴定为 BMS-919194 的等重硫酸盐代谢物。据我们所知，这是首次报道磷酸前药在体内代谢为等重硫酸盐代谢物，并且该代谢物对前药的分析造成了显著干扰。这项工作证明了在真实样本中，等重硫酸盐代谢物对磷酸前药的生物分析存在干扰风险。在方法开发过程中评估和减轻潜在代谢物干扰至关重要，因此，尽量减少相关的生物分析风险并确保测定质量。我们的工作还显示了 HRMS 在识别 LC-MS/MS 生物分析中潜在代谢物干扰方面的独特优势。
