Fadool D A, Cobb S J, Kass-Simon G, Brown P R
Department of Zoology, University of Rhode Island, Kingston 02881.
J Chromatogr. 1988 Oct 28;452:491-501. doi: 10.1016/s0021-9673(01)81472-2.
High-performance liquid chromatography, with serial electrochemical and ultraviolet detectors, was used with a reduced activity catecholamine C18 column to separate and quantify compounds important in the serotonergic and octopamine pathways in lobster hemolymph. The chromatographic mobile phase was composed of potassium dihydrogenphosphate buffer, trichloroacetic acid, sodium dodecyl sulfate, the sodium salt of ethylenedinitrilotetraacetic acid and the organic solvents, acetonitrile and methanol. The compounds serotonin, 5-hydroxyindoleacetic acid, tryptophan, 5-hydroxytryptophan, tryptamine, melatonin, octopamine and tyrosine were well resolved within 13 min. Good electrode maintenance, the use of a silica gel precolumn and careful sample preparation were necessary to give a stable baseline, high resolution of these compounds and reproducibility of retention times and peak heights. The electrochemical detector extended the range of detection to the picogram level. Because of the instability of the solutes and of the chromatographic baseline, sample preparation procedures were investigated. Deproteinization with ammonium sulfate gave the best recovery of the compounds of interest and the most stable baseline with the electrochemical detector. Peaks in the hemolymph were characterized by addition of standards, dual detection (electrochemical and ultraviolet) and the enzyme peak shift technique. With this methodology, important endogenous neurohormones in the hemolymph of lobsters can be quantitatively determined with respect to the molt cycle.
采用配有串联电化学和紫外检测器的高效液相色谱法，使用活性降低的儿茶酚胺C18柱，对龙虾血淋巴中血清素能和章鱼胺途径中的重要化合物进行分离和定量。色谱流动相由磷酸二氢钾缓冲液、三氯乙酸、十二烷基硫酸钠、乙二胺四乙酸钠盐以及有机溶剂乙腈和甲醇组成。血清素、5-羟吲哚乙酸、色氨酸、5-羟色氨酸、色胺、褪黑素、章鱼胺和酪氨酸等化合物在13分钟内得到了很好的分离。要获得稳定的基线、这些化合物的高分辨率以及保留时间和峰高的重现性，需要进行良好的电极维护、使用硅胶预柱并仔细进行样品制备。电化学检测器将检测范围扩展到皮克水平。由于溶质和色谱基线的不稳定性，对样品制备程序进行了研究。用硫酸铵进行脱蛋白处理能使目标化合物的回收率最高，并使电化学检测器的基线最稳定。通过添加标准品、双检测（电化学和紫外）以及酶峰移技术对血淋巴中的峰进行了表征。采用这种方法，可以根据蜕皮周期对龙虾血淋巴中的重要内源性神经激素进行定量测定。
