Müller S, Ho B, Gambus P, Millard W, Hochhaus G
University of Florida, College of Pharmacy, Gainesville 32610, USA.
J Pharm Biomed Anal. 1997 Sep;16(1):101-9. doi: 10.1016/s0731-7085(97)00010-1.
A selective HPLC/RIA procedure for the determination of dynorphin A1-13 (Dyn A1-13) and its major metabolites in human blood was developed. In order to block peptidase activity, blood samples were transferred into an aliquot of a blocking solution (5% aqueous ZnSO4 solution-acetonitrile-methanol; 5:3:2, v/v/v). After solid phase extraction, reconstituted aliquots were injected into an isocratic reversed phase HPLC system to separate Dyn A1-13 from its main metabolites (Dyn A2-13, Dyn A1-12 and Dyn A2-12). The isolated and concentrated HPLC-fractions were assayed by RIA using a commercially available antiserum. Intra-day variabilities for quality controls (0.07, 0.25, and 1 ng ml-1) of Dyn A1-13, A2-13, A1-12, A2-12 were between 9 and 41%. Accuracy was between 86 and 132%. Inter-day variability for single quality controls analyzed on five days for Dyn A1-13, A2-13, A1-12, A2-12 was between 4 and 49% for 0.07, 0.25 and 1 ng ml-1 samples, respectively. Accuracy was between 72 and 129%. Five different batches of control blood showed blood levels no different from zero. Considering the complexity of the assay, the method is selective, accurate and reproducible with a limit of detection of 0.07 ng ml-1 for Dyn A1-13, Dyn A2-13, Dyn A1-12 and 0.21 ng ml-1 for Dyn A2-12. The assay was applied to the determination of Dyn A1-13 and its metabolites in blood samples of 2 subjects receiving i.v. infusions of 250 micrograms or 1000 micrograms kg-1 Dyn A1-13 over 10 min.
开发了一种用于测定人血液中强啡肽A1-13（Dyn A1-13）及其主要代谢产物的选择性高效液相色谱/放射免疫分析（HPLC/RIA）方法。为了阻断肽酶活性，将血样转移至一份阻断溶液（5%硫酸锌水溶液-乙腈-甲醇；5:3:2，v/v/v）中。固相萃取后，将复溶的等分试样注入等度反相HPLC系统，以将Dyn A1-13与其主要代谢产物（Dyn A2-13、Dyn A1-12和Dyn A2-12）分离。使用市售抗血清通过RIA对分离并浓缩的HPLC馏分进行测定。Dyn A1-13、A2-13、A1-12、A2-12质量控制（0.07、0.25和1 ng/ml）的日内变异在9%至41%之间。准确度在86%至132%之间。在五天内对单个质量控制进行分析，Dyn A1-13、A2-13、A1-12、A2-12的0.07、0.25和1 ng/ml样品的日间变异分别在4%至49%之间。准确度在72%至129%之间。五批不同的对照血液显示血药浓度与零无差异。考虑到该分析方法的复杂性，该方法具有选择性、准确性和可重复性，Dyn A1-13、Dyn A2-13、Dyn A1-12的检测限为0.07 ng/ml，Dyn A2-12的检测限为0.21 ng/ml。该分析方法应用于2名接受静脉输注250微克或1000微克/千克体重Dyn A1-13，持续10分钟的受试者血样中Dyn A1-13及其代谢产物的测定。
