Fabel Susanne, Niessner Reinhard, Weller Michael G
Institute of Hydrochemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München, Germany.
J Chromatogr A. 2005 Dec 16;1099(1-2):103-10. doi: 10.1016/j.chroma.2005.08.081. Epub 2005 Sep 28.
A reversed-phase high-performance liquid chromatography system with UV-detector was equipped with an on-line acetylcholinesterase inhibition assay to achieve effect-directed analysis of potentially toxic samples. The enzyme activity was detected colorimetrically using Ellman's reagent. The inhibition and substrate conversion took place in glass capillaries at a 100 microL/min flow rate. Extra-column band spreading in the reaction coils reduces the sensitivity and separation power of biochemical detectors severely. Knitted reactors exhibited no reduction of longitudinal dispersion in the tested flow range. The implementation of air-segmentation allowed an extended inhibition and substrate conversion time without a significant loss of chromatographic resolution. The limit of detection of two model compounds carbofuran (carbamate) and paraoxon-ethyl (organophosphate) was determined to be 13 ng (injected mass) and 7.4 ng, respectively, applying an isocratic chromatography method. A mixture of five insecticides was separated by a gradient elution and the inhibitory effect on the enzyme activity could be detected with high resolution. The band width at half height of the enzyme inhibition detector signal after a reaction time of about 8 min or 4.2 m of capillary, respectively, increased only by a factor of 1.4 compared to the UV-detector signal.
配备紫外检测器的反相高效液相色谱系统与在线乙酰胆碱酯酶抑制测定法相结合，以实现对潜在有毒样品的效应导向分析。使用埃尔曼试剂通过比色法检测酶活性。抑制作用和底物转化在玻璃毛细管中以100微升/分钟的流速进行。反应盘管中的柱外谱带展宽严重降低了生化检测器的灵敏度和分离能力。编织反应器在测试的流速范围内未表现出纵向扩散的降低。空气分段的实施允许延长抑制和底物转化时间，而不会显著损失色谱分辨率。采用等度色谱法时，两种模型化合物克百威（氨基甲酸酯类）和对氧磷乙酯（有机磷类）的检测限分别确定为13纳克（进样质量）和7.4纳克。通过梯度洗脱分离了五种杀虫剂的混合物，并且能够以高分辨率检测对酶活性的抑制作用。在大约8分钟的反应时间或4.2米的毛细管长度后，酶抑制检测器信号的半高带宽与紫外检测器信号相比仅增加了1.4倍。
