IQAC-CSIC, Jordi Girona, Barcelona, Spain.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jan 15;878(2):243-52. doi: 10.1016/j.jchromb.2009.08.027. Epub 2009 Aug 24.
A high-throughput immunosorbent solid-phase extraction (HTS-IS-SPE) procedure coupled to enzyme-linked immunosorbent assay (ELISA) has been established for the analysis of stanozolol (St) and its main metabolite in cattle, 16beta-hydroxy-stanozolol (16betaOH-St), in cow urine samples. The chemical structure of the immunizing hapten 2'H-androst-2-eno[3,2-c]-pyrazol-17-hemiglutarate 5 (hapten A) has been designed to accomplish simultaneous detection of St and 16betaOH-St. The antibodies obtained have been used to establish a microplate ELISA method able to detect these metabolites with IC(50) values of 0.57microgL(-1) and 1.46microgL(-1), respectively in PBST. Immunosorbents prepared by covalently attaching the antibodies to Sepharose, efficiently removed the matrix interferences caused by the cattle urine samples. Moreover, St and 16betaOH-St were efficiently extracted from urine samples as demonstrated by LC-MS/MS analysis. The immunosorbents are filled on small mini-columns arranges on a 96-SPE-setup compatible with the microplate based ELISA methods. Samples and standards can be run in parallel which increment considerably the speed of the screening method. The recovery values of the whole HTS-IS-SPE-ELISA procedure has found to be 112+/-10% and St can be detected in hydrolyzed urine samples with LOD of 1.26+/-0.46microgL(-1) using just 1mL of sample. As proof-of-concept the urinary excretion profile of St treated animals has been investigated by analyzing individual sampling points. Results from pooled urine samples have also been compared with the results obtained by GC-MS analysis demonstrating the StIR equiv. measured with the HTS-IS-SPE-ELISA protocol are in accordance with the St and 16betaOH-St levels found with the chromatographic method. The analytical procedure is rapid, effective and the detectability achieved is below the MPRL (minimum performance required levels) recommended by CRL (Community Reference Laboratory) to the European Community.
已建立了一种高通量免疫吸附固相萃取（HTS-IS-SPE）程序，结合酶联免疫吸附测定（ELISA），用于分析牛尿液样品中的司坦唑醇（St）及其主要代谢物 16β-羟基司坦唑醇（16βOH-St）。免疫原半抗原 2'H-雄甾-2-烯[3,2-c]-吡唑-17-戊二酸 5（半抗原 A）的化学结构旨在实现同时检测 St 和 16βOH-St。获得的抗体已用于建立微板 ELISA 方法，能够检测到这些代谢物在 PBST 中的 IC50 值分别为 0.57μg/L 和 1.46μg/L。通过将抗体共价连接到 Sepharose 上制备的免疫吸附剂，有效地去除了牛尿液样品引起的基质干扰。此外，通过 LC-MS/MS 分析证明，St 和 16βOH-St 可从尿液样品中有效提取。免疫吸附剂填充在 96-SPE 装置上的小迷你柱上，该装置与基于微板的 ELISA 方法兼容。样品和标准品可以并行运行，大大提高了筛选方法的速度。整个 HTS-IS-SPE-ELISA 程序的回收率值发现为 112+/-10%，并且仅使用 1mL 样品即可在水解尿液样品中以 LOD 为 1.26+/-0.46μg/L 检测到 St。作为概念验证，通过分析单个采样点研究了接受 St 处理的动物的尿液排泄特征。还将混合尿液样品的结果与通过 GC-MS 分析获得的结果进行了比较，证明使用 HTS-IS-SPE-ELISA 协议测量的 StIR 等效物与色谱法发现的 St 和 16βOH-St 水平一致。该分析程序快速、有效，达到的检测灵敏度低于 CRL（社区参考实验室）向欧洲共同体推荐的最低性能要求水平（MPRL）。
