Chair of Food Science, Institute of Nutritional Science, and Interdisciplinary Research Center, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany; TransMIT Center of Effect-Directed Analysis, Kerkrader Straße 3, 35394 Giessen, Germany.
J Chromatogr A. 2019 Sep 27;1602:458-466. doi: 10.1016/j.chroma.2019.05.043. Epub 2019 May 24.
Bioanalytical questions are more and more solved by bioassays directly in situ the planar separation. If compared to chemical derivatization in situ, several reagent applications on the same chromatogram make the workflow for enzymatic and biological assays more complex. Hence, if compared to piezoelectric spraying of chemical derivatization reagents, an assay transfer to the piezoelectric spraying technique was much more challenging. Important aspects were investigated, i.e., plate pre-wetting, spraying nozzle type and applied volumes for microorganism suspension as well as enzyme and substrate-chromogenic solutions. Finally, with the newly developed piezoelectric spraying procedures for the application of biological (Aliivibrio fischeri) and enzymatic (acetyl- and butyrylcholinesterase) assays, several obstacles of the state-of-the-art automated immersion were avoided such as the (1) required high volumes of solutions, (2) tailing of highly water-soluble zones upon slow plate withdrawal, (3) zone distortion or shift observed after previous buffer salt applications or long/slow immersion times/speeds, (4) gradual inactivation of the enzyme solution along with its ongoing re-use, and (5) lack of covering the whole plate surface. The benchmarking of both techniques also showed that simplicity remains the key argument for immersion. As proof of concept, piezoelectrically sprayed autograms were compared with those of immersion, by taking the example of Peganum harmala (P. h.) seed extract. The plate background and thus homogeneity of the applied solutions were found to be almost comparable. Three bands among the pronounced fluorescent bands were responsible for the most antibacterial activity of P. h. seed extract in the A. fischeri bioassay and were also inhibiting the AChE. These AChE and three further BChE inhibitors were detected, whereby the AChE inhibition was twice as strong as the BChE inhibition. By their in situ HRMS spectra, the active zones in the P. h. seed extract were assigned to be the AChE-inhibiting β-carboline alkaloids, harmine, harmaline and ruine, as well as the BChE-inhibiting quinazoline alkaloids, vasicine and deoxyvasicine, and the β-carboline alkaloid harmol. For the first time, the found inhibitors were calculated equivalently to the well-known ChE-inhibitor physostigmine, and thus, piezoelectric spraying was proven to be suited for quantifications.
生物分析问题越来越多地通过直接在平面分离中进行生物测定来解决。与原位化学衍生化相比，在同一色谱图上应用几种试剂会使酶和生物测定的工作流程更加复杂。因此，如果与化学衍生化试剂的压电喷雾相比，将测定法转移到压电喷雾技术更具挑战性。研究了重要方面，例如板预湿、喷雾喷嘴类型以及微生物悬浮液以及酶和底物显色溶液的应用体积。最后，使用新开发的压电喷雾程序，可应用于生物（发光菌）和酶（乙酰胆碱酯酶和丁酰胆碱酯酶）测定，避免了最新自动浸入法的几个障碍，例如（1）需要高体积的溶液，（2）缓慢提取平板时高水溶性区域的拖尾，（3）先前应用缓冲盐或长时间/缓慢浸入时间/速度后观察到的区域变形或移位，（4）随着酶溶液的不断再利用，其逐渐失活，以及（5）未能覆盖整个板表面。这两种技术的基准测试还表明，简单性仍然是浸入式技术的关键论点。作为概念验证，通过以 Peganum harmala（P. h.）种子提取物为例，比较了压电喷雾的自动图像与浸入式的自动图像。发现施加溶液的板背景和均匀性几乎可以相媲美。在发光菌生物测定中，P. h.种子提取物最具抗菌活性的三个明显荧光带中的三个带负责抑制 AChE。还检测到三种 AChE 和另外三种 BChE 抑制剂，其中 AChE 抑制作用是 BChE 抑制作用的两倍。通过原位高分辨率 MS 光谱，将 P. h.种子提取物中的活性带分配给 AChE 抑制β-咔啉生物碱，如 harmine、harmaline 和 ruine，以及 BChE 抑制喹唑啉生物碱，如 vasicine 和 deoxyvasicine，以及β-咔啉生物碱 harmol。首次发现这些抑制剂与著名的 ChE 抑制剂 physostigmine 等效，因此，证明了压电喷雾适用于定量分析。
