Bioanalytical Sciences Department, Research and Development, Bristol-Myers Squibb Co.
Rapid Commun Mass Spectrom. 2013 Feb 15;27(3):481-8. doi: 10.1002/rcm.6473.
Nucleotide analogs are highly polar and ionic, which impose great challenges on bioanalysis. Ion-pairing liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the predominant reported approach for such compounds. Assay ruggedness of ion-pairing LC/MS/MS methods was often a challenge due to the potential contamination of the ion source of the mass spectrometer and LC column performance deterioration caused by ion-pairing reagents.
An ion-pairing reagent was only added to the reconstitution solution to minimize its exposure to the MS ion source. To achieve optimum sensitivity, high pH mobile phases and negative ion ESI were needed for the LC/MS/MS method. However, high pH mobile phases led to the accumulation of ion-pairing reagent on the analytical column, which was washed off with an acidic solution to restore the column performance. In addition, isopropanol was used as a mobile phase modifier to improve peak shape and sensitivity.
The limit of detection was established at 1.0 ng/mL in the cell lysate. The calibration curve showed good linearity over the range of 1.0 to 100 ng/mL. The overall accuracy was no less than 87.7% based on four levels of quality control samples. Inter-run precision and intra-run precision across four analytical runs for low, geometric, medium and high QCs were less than 12.9.
By identifying and addressing the root cause of the assay ruggedness problem, we have developed a rugged ion-pairing LC/MS/MS method for a triphosphate (TP) metabolite of BMS-986001 in peripheral blood mononuclear cells. The new method overcame challenges such as a rapid deterioration of the peak shape, increased carryover and extremely poor column life. The peak shape was well maintained throughout multiple analytical runs. This method has been successfully applied to a toxicology study in cynomolgus monkey.
核苷酸类似物具有高极性和离子性，这给生物分析带来了巨大的挑战。离子对液相色谱/串联质谱（LC/MS/MS）是此类化合物的主要报告方法。由于质谱仪的离子源可能受到污染，以及离子对试剂导致 LC 柱性能恶化，离子对 LC/MS/MS 方法的耐用性常常是一个挑战。
仅将离子对试剂添加到再溶液中，以最大程度地减少其暴露于质谱仪的离子源。为了实现最佳灵敏度，LC/MS/MS 方法需要使用高 pH 流动相和负离子电喷雾。然而，高 pH 流动相导致离子对试剂在分析柱上积累，需要用酸性溶液冲洗以恢复柱性能。此外，异丙醇被用作流动相改性剂以改善峰形和灵敏度。
在细胞裂解物中，检测限设定为 1.0ng/mL。校准曲线在 1.0 至 100ng/mL 的范围内显示出良好的线性关系。基于四个质控样品的水平，总准确度不低于 87.7%。在四个分析运行中，低、几何、中、高质控品的批内和批间精密度均小于 12.9。
通过确定和解决耐用性问题的根本原因，我们开发了一种用于 BMS-986001 的三磷酸盐（TP）代谢物在人外周血单核细胞中的耐用离子对 LC/MS/MS 方法。新方法克服了峰形迅速恶化、拖尾增加和柱寿命极差等挑战。在多个分析运行中，峰形得到了很好的保持。该方法已成功应用于食蟹猴的毒理学研究。
