Minkler P E, Ingalls S T, Hoppel C L
Medical Research, Veterans Administration Medical Center, Cleveland, Ohio 44106.
Anal Biochem. 1990 Feb 15;185(1):29-35. doi: 10.1016/0003-2697(90)90250-d.
A high-performance liquid chromatographic method for the separation of acylcarnitines after derivatization with 4'-bromophenacyl trifluoromethanesulfonate is presented. Derivatization of acylcarnitines was achieved at room temperature within 10 min. Separation of the acylcarnitine 4'-bromophenacyl esters was accomplished by high-performance liquid chromatography using as the analytical column a Resolve-PAK 5-microns C18 radially compressed cartridge eluted with a tertiary gradient containing varying proportions of water, acetonitrile, tetrahydrofuran, triethylamine, potassium phosphate, and phosphoric acid. Acylcarnitine 4'-bromophenacyl esters were detected spectrophotometrically at 254 nm. Baseline separation was obtained for a standard mixture (5 nmol of each injected) containing carnitine, acetyl-, propionyl-, butyryl-, valeryl-, hexanoyl-, heptanoyl-, octanoyl-, nonanoyl-, decanoyl-, lauroyl-, myristroyl-, palmitoyl-, and stearoylcarnitine. Nearly complete separation was obtained for a standard mixture containing butyryl-, isobutyryl-, isovaleryl-, and 2-methylbutyrylcarnitine. The method was applied to a normal human urine and then to this same urine spiked with the acylcarnitine standards. Urinary acylcarnitine profiles from patients having propionic acidemia, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency were performed. Urinary isovalerylcarnitine was quantified in the patient with isovaleric acidemia using heptanoylcarnitine as an internal standard.
本文介绍了一种高效液相色谱法，用于在经4'-溴苯甲酰三氟甲磺酸酯衍生化后分离酰基肉碱。酰基肉碱的衍生化在室温下10分钟内即可完成。酰基肉碱4'-溴苯甲酰酯的分离通过高效液相色谱法实现，使用Resolve-PAK 5微米C18径向压缩柱作为分析柱，用含有不同比例水、乙腈、四氢呋喃、三乙胺、磷酸钾和磷酸的三元梯度洗脱。酰基肉碱4'-溴苯甲酰酯在254 nm处通过分光光度法检测。对于含有肉碱、乙酰、丙酰、丁酰、戊酰、己酰、庚酰、辛酰、壬酰、癸酰、月桂酰、肉豆蔻酰、棕榈酰和硬脂酰肉碱的标准混合物（每种注入5 nmol），实现了基线分离。对于含有丁酰、异丁酰、异戊酰和2-甲基丁酰肉碱的标准混合物，实现了几乎完全分离。该方法应用于正常人尿液，然后应用于添加了酰基肉碱标准品的同一尿液。对患有丙酸血症、异戊酸血症和中链酰基辅酶A脱氢酶缺乏症患者的尿酰基肉碱谱进行了分析。使用庚酰肉碱作为内标，对异戊酸血症患者的尿异戊酰肉碱进行了定量。
