Liao Debra, Chen Susan, Paton Martin, Qian Mark G
Department of Drug Metabolism and Pharmacokinetics, Takeda Pharmaceuticals International Co., 40 Landsdowne St., Cambridge, MA, 02139, USA.
Rapid Commun Mass Spectrom. 2014 Jun 15;28(11):1215-20. doi: 10.1002/rcm.6894.
Stepwise preparation of calibration standards and quality controls (QCs) is one of the most routine and laborious steps in bioanalysis. An alternative non-contact dispenser using low picoliter digitized dispensing technology is evaluated for its application in non-stepwise preparation of calibration curve and QCs in bioanalysis.
Fluorescein was initially used to assess the accuracy and precision of dispense volumes with fluorescent measurement. Various concentrations of MX-1, an in-house proprietary small molecule compound, in neat solution and in dog plasma were prepared manually with calibrated pipettors and digitally by the digital dispenser. The plasma samples were extracted by protein precipitation. The resultant extracted samples and neat solutions of MX-1 were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using an electrospray ionization (ESI) source in positive ion mode with selected reaction monitoring (SRM) of the mass transitions.
In the three-day precision and accuracy assessment of dispensing volumes between 13 pL to 411.2 nL, the intra-day precision and accuracy ranged from 1.4% to 10.3% and -12.7% to 12.8%, respectively. The inter-day precision and accuracy ranged from 3.5% to 7.8% and -6.6% to 10.4%, respectively. For real analysis of in vivo study samples, all 49 samples analyzed showed a less than 5% difference between calibrations with digital and manual curve preparations. The resultant pharmacokinetic (PK) parameters were physiologically comparable as well.
Using the digitized picoliter dispensing technology, high-speed automated precise and accurate dispense of a wide range of volumes can be achieved and tests for bioanalytical standards and QC preparations passed the stringent criteria set forth for regulated bioanalysis using LC/MS/MS-based technology. The digital dispenser has been found to be a useful tool in drug discovery for automatically preparing standards and QCs in seconds with low consumption of stock solutions and blank matrices.
校准标准品和质量控制（QC）样品的逐步制备是生物分析中最常规且最耗费人力的步骤之一。本研究评估了一种采用低皮升数字化分配技术的非接触式分配器在生物分析中用于校准曲线和QC样品非逐步制备的应用情况。
首先使用荧光素通过荧光测量来评估分配体积的准确性和精密度。使用校准移液器手动制备以及通过数字分配器以数字方式制备各种浓度的内部 proprietary 小分子化合物MX-1的纯溶液和犬血浆溶液。血浆样品通过蛋白沉淀法进行提取。所得的提取样品和MX-1的纯溶液采用液相色谱/串联质谱（LC/MS/MS）进行分析，使用电喷雾电离（ESI）源，在正离子模式下进行质量转移的选择反应监测（SRM）。
在对13皮升至411.2纳升之间的分配体积进行为期三天的精密度和准确性评估中，日内精密度和准确性分别为1.4%至10.3%和 -12.7%至12.8%。日间精密度和准确性分别为3.5%至7.8%和 -6.6%至10.4%。对于体内研究样品的实际分析，所分析的全部49个样品在数字和手动曲线制备的校准之间显示出小于5%的差异。所得的药代动力学（PK）参数在生理上也具有可比性。
使用数字化皮升分配技术，可以实现对各种体积的高速自动化精确分配，并且生物分析标准品和QC样品的测试通过了基于LC/MS/MS技术的规范生物分析所设定的严格标准。已发现数字分配器是药物研发中的一种有用工具，可在数秒内自动制备标准品和QC样品，同时储备溶液和空白基质的消耗量较低。
