A One-Step Solid Phase Extraction Method for Bioanalysis of a Phosphorothioate Oligonucleotide and Its 3′ n-1 Metabolite from Rat Plasma by uHPLC-MS/MS
A One-Step Solid Phase Extraction Method for Bioanalysis of a Phosphorothioate Oligonucleotide and Its 3′ n-1 Metabolite from Rat Plasma by uHPLC-MS/MS
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DOI：
10.1208/s12248-012-9394-x
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发表时间：
2012-12-01
期刊：
影响因子：
4.5
通讯作者：
Bartlett, Michael
中科院分区：
文献类型：
--
作者：
Chen, Buyun;Bartlett, Michael
寡核苷酸治疗剂已成为一类有前途的药物，用于治疗由遗传异常引起的广泛疾病。用硫代磷酸酯取代磷酸二酯键是对寡核苷酸进行的最成功的修饰之一，以增强其体内稳定性。硫代磷酸酯和其他修饰的寡核苷酸的消除相较长，需要灵敏和选择性的方法来同时定量母体药物及其代谢产物。液相色谱串联质谱法在母体药物及其代谢产物之间具有良好的选择性和较宽的动态范围。然而，生物样品的提取仍然是开发寡核苷酸定量LC-MS方法的一个巨大挑战。蛋白质沉淀、蛋白质消化、液-液萃取、反相固相萃取（SPE）、强阴离子交换SPE以及它们的组合已被报道用于从生物基质中提取寡核苷酸。不幸的是，这些方法要么回收率低，要么由于所得溶液中大量的基质物质而存在色谱应用的潜在问题。本研究优化了弱阴离子交换固相萃取方法。回收率范围为60%-80%，取决于浓度。这是第一次报告的一步SPE方法，在整个方法动态范围内的回收率大于60%。该样品提取程序与超高效液相色谱-串联质谱联用。定量下限为10 ng/mL（1.3 nM），动态范围为10- 1，000 ng/mL。日内和日间精密度和准确度分别在8.4%和10.5%以内。
Oligonucleotide therapeutics have emerged as a promising class of drugs to treat a wide range of diseases caused by genetic abnormalities. Replacement of the phosphodiester linkage with a phosphorothioate is one of the most successful modifications made to oligonucleotides to enhance their in vivo stability. The longer elimination phase of phosphorothioates and other modified oligonucleotides requires sensitive and selective methods to quantify the parent drug and their metabolites simultaneously. Liquid chromatography tandem mass spectrometry has excellent selectivity between the parent drug and its metabolites and a wide dynamic range. However, the biological sample extraction remains a formidable challenge in developing quantitative LC-MS methods for oligonucleotides. Protein precipitation, protein digestion, liquid-liquid extraction, reversed phase solid phase extraction (SPE), strong anion exchange SPE, and combinations of them have been reported to extract oligonucleotides from biological matrices. Unfortunately, these methods either have low recoveries or present potential problems for applications with chromatography due to the large amount of matrix substances in the resulting solutions. In this study, a weak anion exchange SPE method was optimized. The recovery ranged from 60% to 80% depending on the concentration. This is the first report of a one-step SPE method with recoveries greater than 60% across the method dynamic range. This sample extraction procedure was used in combination with ultrahigh-performance liquid chromatography-tandem mass spectrometry. The lower limit of quantitation was 10 ng/mL (1.3 nM), and the dynamic range was 10-1,000 ng/mL. The intra-and inter-day precision and accuracy were within 8.4% and 10.5%, respectively.