Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
J Pharm Biomed Anal. 2014 Feb;89:240-50. doi: 10.1016/j.jpba.2013.11.017. Epub 2013 Nov 19.
An UHPLC-MS/MS method was developed and validated to quantify BMS-927711, a drug candidate to treat migraine, in rat dried blood spots (DBS). The DBS samples were extracted using an improved liquid-liquid extraction (LLE) strategy involving in the sonication of DBS punches in 20% MeOH aqueous solution containing the internal standard, [(13)C2, D4]-BMS-927711, and then with a 100mM NH4OAc buffer solution, followed by an automated LLE with EtOAc-hexane (70:30, v/v). The presence of 20% MeOH as an organic modifier in the elution solution significantly improved the analyte elution efficiency and assay performance. A novel inter-well volume replacement dilution workflow was introduced for DBS sample dilution before LLE step. This was a simple two-step process, firstly a small portion of the DBS blank solution was discarded, and then the same volume of a concentrated DBS sample solution was spiked into the leftover blank solution to achieve a desired dilution. Chromatographic separation was achieved on an Acuity UPLC(®) BEH C18 column (2.1mm×50mm, 1.7μm) and the analyte was detected by selected reaction monitoring (SRM) with positive electrospray ionization on an AB Sciex Triple Quad 5500 mass spectrometer. The standard curve was linear from 5.00 to 5000ng/mL with assay precision ≤4.9% CV, and assay accuracy within ±3.1%Dev of the nominal values. Accurate sample dilution was achieved by using inter-well volume replacement with a precision of ≤4.2% CV and an accuracy of ±3.3% for dilution QC at 50,000ng/mL with 100-fold dilution (n=18). This robust UHPLC-MS/MS assay has been successfully applied to the non-clinical studies in rats. By using inter-well volume replacement workflow, accurate dilution was demonstrated using only one DBS blank sample for a typical dilution of <50-fold, and using only two blank DBS samples for a dilution of up to 625-fold. Moreover, this new workflow makes it easier to automate DBS sample dilution.
建立并验证了一种 UHPLC-MS/MS 方法，用于定量测定治疗偏头痛的候选药物 BMS-927711。DBS 样本通过改良的液液萃取（LLE）策略进行提取，包括在含有内标物 [(13)C2, D4]-BMS-927711 的 20%甲醇水溶液中对 DBS 打孔进行超声处理，然后用 100mM NH4OAc 缓冲液，再用 EtOAc-正己烷（70:30，v/v）进行自动 LLE。在洗脱液中加入 20%甲醇作为有机改性剂，显著提高了分析物的洗脱效率和测定性能。引入了一种新的微孔间体积替换稀释工作流程，用于 LLE 前 DBS 样本稀释。这是一个简单的两步过程，首先丢弃一小部分 DBS 空白溶液，然后将相同体积的浓缩 DBS 样本溶液加入到剩余的空白溶液中，以达到所需的稀释度。采用 Acuity UPLC(®) BEH C18 柱（2.1mm×50mm，1.7μm）进行色谱分离，通过 AB Sciex Triple Quad 5500 质谱仪的正电喷雾电离进行选择反应监测（SRM）检测分析物。标准曲线在 5.00 至 5000ng/mL 范围内呈线性，精密度≤4.9%CV，准确度在名义值的±3.1%Dev 范围内。通过使用微孔间体积替换，以≤4.2%CV 的精度和 100 倍稀释时稀释 QC 点（50,000ng/mL）的±3.3%准确度，实现了准确的样本稀释。该稳健的 UHPLC-MS/MS 测定法已成功应用于大鼠的非临床研究。通过使用微孔间体积替换工作流程，仅使用一个 DBS 空白样本即可实现典型稀释度<50 倍的准确稀释，使用两个空白 DBS 样本即可实现高达 625 倍的稀释。此外，这种新的工作流程使 DBS 样本稀释更容易实现自动化。
