Strategy for the isolation, derivatization, chromatographic separation, and detection of carnitine and acylcarnitines
Strategy for the isolation, derivatization, chromatographic separation, and detection of carnitine and acylcarnitines
复制标题
DOI：
10.1021/ac0487810
复制
发表时间：
2005-03-01
影响因子：
7.4
通讯作者：
Hoppel, CL
中科院分区：
文献类型：
--
作者：
Minkler, PE;Ingalls, ST;Hoppel, CL
介绍了肉毒碱和酰基肉毒碱的检测方法。这个多功能系统有四个组成部分：（1）通过蛋白质沉淀/沉淀和阳离子交换固相萃取进行分离，（2）用五氟苯甲酰三氟甲磺酸酯对肉毒碱和酰基肉毒碱进行衍生化，（3）使用单个非封端C-8柱进行连续离子交换/反相色谱，和（4）使用离子阱质谱仪检测肉毒碱和酰基肉毒碱五氟苯甲酰酯。从分离过程中回收肉毒碱和酰基肉毒碱为77- 85%。衍生反应迅速而完全，没有酰基肉毒碱水解的迹象。顺序离子交换/反相HPLC导致试剂副产物与衍生化的肉毒碱和酰基肉毒碱分离，随后是肉毒碱和酰基肉毒碱五氟苯甲酰甲酯的反相分离。通过MS/MS的检测具有高度选择性，肉毒碱五氟苯甲酰甲酯在m/z 311处产生强产物离子，酰基肉毒碱五氟苯甲酰甲酯裂解产生两种产物离子：（1）m/z 59的损失和（2）在m/z 293处产生离子。为了证明这种分析策略，将磷酸盐缓冲血清白蛋白加标肉毒碱和15种酰基肉毒碱，并使用所述蛋白质沉淀/沉淀和阳离子交换固相萃取分离、用五氟苯甲酰三氟甲磺酸酯衍生化、使用连续离子交换/反相色谱HPLC系统进行色谱分析，并通过MS和MS/MS进行检测。示出了这种策略对大鼠肝脏中肉毒碱和乙酰肉毒碱的定量的成功应用。
A strategy for detection of carnitine and acylcarnitines is introduced. This versatile system has four components: (1) isolation by protein precipitation/desalting and cation exchange solid-phase extraction, (2) derivatization of carnitine and acylcarnitines with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/ reversed-phase chromatography using a single non-end-capped C-8 column, and (4) detection of carnitine and acylcarnitine pentafluorophenacyl esters using an ion trap mass spectrometer. Recovery of carnitine and acylcarnitines from the isolation procedure is 77-85%. Derivatization is rapid and complete with no evidence of acylcarnitine hydrolysis. Sequential ion-exchange/reversed-phase HPLC results in separation of reagent by products from derivatized carnitine and acylcarnitines, followed by reversed-phase separation of carnitine and acylcarnitine pentafluorophenacyl esters. Detection by MS/MS is highly selective, with carnitine pentafluorophenacyl ester yielding a strong product ion at m/z 311 and acylcarnitine pentafluorophenacyl ester fragmentation yielding two product ions: (1) loss of m/z 59 and (2) generation of an ion at m/z 293. To demonstrate this analytical strategy, phosphate buffered serum albumin was spiked with carnitine and 15 acylcarnitines and analyzed using the described protein precipitation/desalting and cation-exchange solid phase extraction isolation, derivatization with pentafluorophenacyl trifluoromethanesulfonate, chromatography using the sequential ion-exchange/reversed-phase chromatography HPLC system, and detection by Ms and MS/MS. Successful application of this strategy to the quantification of carnitine and acetylcarnitine in rat liver is shown.