Bioanalytical Sciences, Bristol-Myers Squibb, Princeton, NJ 08543, United States.
Bioanalytical Sciences, Bristol-Myers Squibb, Princeton, NJ 08543, United States.
J Pharm Biomed Anal. 2018 Aug 5;157:36-43. doi: 10.1016/j.jpba.2018.05.006. Epub 2018 May 5.
BMS-986104 is a S1PR modulator drug candidate under development and has been evaluated in Phase I clinical trials. BMS-986104 functions as a prodrug and undergoes enzymatic transformations in vivo to form the pharmacologically active phosphate drug, BMS-986104-P. Here, we report approaches to overcome the stability, solubility and extraction challenges in developing a sensitive, accurate and rugged LC-MS/MS method for the simultaneous quantification of the phosphate drug and its prodrug in blood lysate. An effective stabilization strategy using a cocktail of phosphatase and kinase inhibitors was developed to ensure the stability of both analytes during sample collection, storage, and processing. A combination of surfactant (CHAPS) and weak base (Tris) was found to be able to effectively improve the solubilization of the phosphate drug. The blood lysate samples were extracted by protein precipitation followed by solid-phase extraction using an Oasis HLB 96-well SPE plate. The method achieved acceptable matrix effect and recovery for the two analytes that have very different chemical properties. Stable-isotope labeled D6-BMS-986104 and D13-BMS-986104-P were utilized as the internal standards. Chromatographic separation was achieved using isocratic conditions on an Acquity UPLC BEH C18 analytical column. The two analytes and their internal standards were detected by positive ion electrospray tandem mass spectrometry. The calibration curves, which ranged from 2.00 to 2000 ng/mL for BMS-986104 and 4.00 to 4000 ng/mL for BMS-986104-P, were fitted to a 1/x2 weighted linear regression model. The intra-assay precision was within ±5.0% CV, inter-assay precision was within ±4.9% CV, and the assay accuracy was within ±5.8% of the nominal values for both analytes in rat blood lysate. The method was validated and successfully applied to support multiple pre-clinical toxicity studies.
BMS-986104 是一种正在开发的 S1PR 调节剂候选药物，已在 I 期临床试验中进行了评估。BMS-986104 作为前药在体内经历酶转化，形成具有药理活性的磷酸盐药物 BMS-986104-P。在这里，我们报告了克服在开发用于同时定量血液裂解物中磷酸盐药物及其前药的灵敏、准确和稳健的 LC-MS/MS 方法中的稳定性、溶解度和提取挑战的方法。开发了一种有效的稳定策略，使用磷酸酶和激酶抑制剂的混合物确保在样品收集、储存和处理过程中两种分析物的稳定性。发现表面活性剂 (CHAPS) 和弱碱 (Tris) 的组合能够有效地提高磷酸盐药物的溶解度。通过蛋白沉淀提取血液裂解物样品，然后使用 Oasis HLB 96 孔 SPE 板进行固相萃取。该方法实现了对具有非常不同化学性质的两种分析物的可接受的基质效应和回收率。稳定同位素标记的 D6-BMS-986104 和 D13-BMS-986104-P 被用作内标。使用等度条件在 Acquity UPLC BEH C18 分析柱上实现色谱分离。两种分析物及其内标通过正离子电喷雾串联质谱检测。BMS-986104 的校准曲线范围为 2.00 至 2000ng/mL，BMS-986104-P 的校准曲线范围为 4.00 至 4000ng/mL，均拟合为 1/x2 加权线性回归模型。两种分析物在大鼠血液裂解物中的日内精密度均在 ±5.0%CV 以内，日间精密度均在 ±4.9%CV 以内，测定准确度均在名义值的 ±5.8%以内。该方法经过验证，并成功应用于支持多个临床前毒性研究。
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