In this study we describe an approach to enhance the sensitivity of an online comprehensive two-dimensional liquid chromatography (LC × LC) high-resolution mass spectrometry method for the separation and detection of trace levels of anabolic-steroid residues in complex urine matrices. Compared to one-dimensional liquid chromatography (1D-LC), LC × LC methods offer higher separation power, thanks to the combined effect of two different selectivities and a higher peak capacity. However, when using state-of-the-art LC × LC instrumentation, the price paid for the increase in separation power is a decrease in sensitivity and detectability of trace-level analytes. This can be ascribed to the sample dilution that takes place during each of the two chromatographic steps. The way in which fractions are collected and transferred from the first to the second column is also of paramount importance, especially the volume and the solvent composition of the fractions injected in the second column. To overcome the detection limitation, we present an active-modulation strategy, based on concentrating the fractions of the first-dimension effluent using a modulation interface that employs trap columns. We obtained a signal enhancement for anabolic-steroid compounds in a bovine-urine sample by a factor of 2.4-7.6 and an increase in the signal-to-noise ratio up to a factor of 7 in comparison with a standard loop-based modulation interface. In addition, thanks to the increased sensitivity of our method, a substantially larger number of peaks were detected (76 vs. 36). Moreover, we could reduce the solvent consumption by a factor of three (160 mL vs. 500 mL per run).

中文翻译：

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通过主动调制在线综合二维液相色谱 - 高分辨率质谱法提高牛尿中合成代谢类固醇残留的可检测性

在本研究中，我们描述了一种提高在线综合二维液相色谱 (LC × LC) 高分辨率质谱法灵敏度的方法，用于分离和检测复杂尿液基质中痕量水平的合成代谢类固醇残留。与一维液相色谱 (1D-LC) 相比，LC × LC 方法具有更高的分离能力，这要归功于两种不同选择性和更高峰容量的综合效果。然而，当使用最先进的 LC × LC 仪器时，为提高分离能力付出的代价是降低了痕量分析物的灵敏度和可检测性。这可以归因于在两个色谱步骤中的每一个过程中发生的样品稀释。馏分被收集并从第一个柱子转移到第二个柱子的方式也很重要，尤其是注入第二个柱子的馏分的体积和溶剂组成。为了克服检测限制，我们提出了一种主动调制策略，该策略基于使用采用陷阱柱的调制接口浓缩第一维流出物的部分。与基于循环的标准调制接口相比，我们获得了牛尿样品中合成代谢类固醇化合物的信号增强了 2.4-7.6 倍，信噪比增加了 7 倍. 此外，由于我们方法的灵敏度提高，检测到的峰数量明显增多（76 对 36）。而且，