Bioanalytical Science and Toxicokinetics, Platform Science and Technology, GlaxoSmithkline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 May 1;926:47-53. doi: 10.1016/j.jchromb.2013.03.004. Epub 2013 Mar 14.
A simple, selective, and sensitive quantitative method has been developed for the simultaneous determination of levodopa and carbidopa in rat and monkey plasma by protein precipitation using acetonitrile containing the derivatizing reagent, flourescamine. Derivatized products of levodopa and carbidopa were separated on a BEH C18 column (2.1 mm × 50 mm; 1.7 μm particle size) using ultra high performance liquid chromatography (UHPLC) without any further purification. Tandem mass spectrometry (MS/MS) was used for detection. The method was validated over the concentration range of 5-5000 ng/mL and 3-3000 ng/mL for levodopa and carbidopa, respectively in rat and monkey plasma. Due to the poor stability of the investigated analytes in biological matrices, a mixture of sodium metabisulfite and hydrazine dihydrochloride was used as a stabilizer. This method was successfully utilized to support pharmacokinetic studies in both species. The results from assay validations and incurred samples re-analysis show that the method is selective, sensitive and robust. To our knowledge, this is the first UHPLC-MS/MS based method that utilizes derivatization with fluorescamine and provides adequate sensitivity for both levodopa and carbidopa with 50 μL of sample and a run time of 3.5 min.
已开发出一种简单、选择性强且灵敏的定量方法，可通过使用含衍生试剂荧光胺的乙腈进行蛋白质沉淀，同时测定大鼠和猴子血浆中的左旋多巴和卡比多巴。衍生后的左旋多巴和卡比多巴产物在 BEH C18 柱（2.1mm×50mm；1.7μm 粒径）上通过超高效液相色谱（UHPLC）分离，无需进一步纯化。采用串联质谱（MS/MS）进行检测。该方法在大鼠和猴子血浆中，左旋多巴和卡比多巴的浓度范围分别为 5-5000ng/mL 和 3-3000ng/mL 时得到验证。由于在生物基质中研究分析物的稳定性差，因此使用亚硫酸钠和盐酸联氨的混合物作为稳定剂。该方法成功用于支持两种物种的药代动力学研究。来自检测验证和样品重新分析的结果表明，该方法具有选择性、灵敏性和稳健性。据我们所知，这是第一个利用荧光胺衍生化并使用 50μL 样品和 3.5 分钟运行时间提供足够灵敏度的 UHPLC-MS/MS 方法，可同时测定左旋多巴和卡比多巴。
