Strategies for bioanalysis of an oligonucleotide class macromolecule from rat plasma using liquid chromatography-tandem mass spectrometry.
Strategies for bioanalysis of an oligonucleotide class macromolecule from rat plasma using liquid chromatography-tandem mass spectrometry.
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使用液相色谱-串联质谱法对大鼠血浆中的寡核苷酸类大分子进行生物分析的策略。
DOI：
10.1021/ac0618674
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发表时间：
2007
影响因子：
7.4
通讯作者：
J. Neil Duncan
中科院分区：
文献类型：
--
作者：
Guodong Zhang;Jian Lin;K. Srinivasan;O. Kavetskaia;J. Neil Duncan
电喷雾离子化（ESI）液相色谱-串联质谱（LC/MS/MS）分析为小分子定量提供了高通量和选择性的方法。由于缺乏灵敏度和生物基质的低分析物回收率，使用LC/MS/MS测定大分子（如寡核苷酸）具有挑战性。由于这一事实，选择用于寡核苷酸定量的方法仍然是基于杂交的配体结合测定。这些生物测定通常具有高灵敏度，但低选择性和窄的动态范围。它们还需要优化合适的“捕获和检测”探针，这在药物发现先导物优化方案中可能非常耗时且昂贵。在本文中，我们提出了一个独特的LC/MS/MS分析模型硫代磷酸骨架寡脱氧核苷酸（ODN）药物（7692 amu）从大鼠血浆。遇到了多种分析挑战。用于解决这些挑战的策略应该证明对追求质谱（MS）定量寡核苷酸的科学家有用。这些挑战包括分析物多重带电和阳离子加合（灵敏度降低）、分析物在干燥时氧化和高蛋白结合（回收率低）、ODN与暴露二氧化硅的亲和力（色谱重现性低和携带率高）、分析物与容器的非特异性结合（储存稳定性低）以及适当内标物的优化/合成（干扰和串扰）。使用缓冲液（7 mM三乙胺和3 mM甲酸铵）/甲醇（50：50（v/v））作为ESI-MS输注溶剂，并产生尖锐的多电荷状态分布。样品提取方法结合了苯酚/氯仿液-液萃取和固相萃取步骤，将绝对回收率提高到> 70%。该方法在5-2000 ng/mL范围内进行了验证，精密度（相对标准偏差百分比）<10.1%，准确度（相对误差百分比）<11.4%。
Electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC/MS/MS) assays provide high-throughput and selective methods for quantitation of small molecules. Use of LC/MS/MS assays for macromolecules, like oligonucleotides, is challenging due to lack of sensitivity and low analyte recovery from biomatrixes. Due to this fact, the method of choice for oligonucleotides quantitation remains hybridization-based ligand-binding assays. These biological assays usually possess high sensitivity but low selectivity and narrow dynamic range. They also require optimizing suitable "capture and detection" probes, which can be prohibitively time-consuming and expensive in a drug discovery lead-optimization scenario. In this paper, we present a unique LC/MS/MS assay for a model phosphorothioate backbone oligodeoxynucleotide (ODN) drug (7692 amu) from rat plasma. Multiple analytical challenges were encountered. The strategies used to solve these challenges should prove useful to scientists pursuing mass spectrometry (MS) to quantitate oligonucleotides. The challenges include analyte multiple charging and cation adduction (reduced sensitivity), oxidation of analyte on drying and high protein binding (low recovery), ODN affinity to exposed silica (low chromatographic reproducibility and high carryover), nonspecific binding of analyte to containers (low storage stability), and optimization/synthesis of an appropriate internal standard (interference and cross-talk). A buffer (7 mM triethylamine and 3 mM ammonium formate)/methanol, 50:50 (v/v), was used as an ESI-MS infusion solvent and produced a sharp multiple charge-state distribution. The sample extraction method combined a phenol/chloroform liquid-liquid extraction and solid-phase extraction steps, which improved the absolute recovery to >70%. The method was validated in the range of 5-2000 ng/mL and had precision (percent relative standard deviation)<10.1% and accuracy (percent relative error)<11.4%.