Hashimoto K, Ohno N, Hattori T, Takahara J, Ofuji T
Nihon Naibunpi Gakkai Zasshi. 1982 Mar 20;58(3):167-76. doi: 10.1507/endocrine1927.58.3_167.
High performance liquid chromatography (HPLC) was used for the separation of many neuropeptides. Chromatography was carried out using a Hitachi Model 638 high performance liquid chromatograph. Peptides and samples from tissue dissolved in an aqueous buffer were injected into a stainless-steel column (4 X 250mm) packed with Hitachi #3053 (octadecylsilane). The aqueous buffer consisted of NaH2PO4 and H3PO4. After a loading phase (0% organic solvent) of 1 min, the peptides were sequentially eluted at room temperature using a gradient of organic solvent (acetonitrile or methanol, 0-60%). The eluted polypeptides were detected by UV absorbance at 220nm, and then they were collected for subsequent bio and radioimmunoassay using a fraction collector. The gradient of methanol or acetonitrile in 0.02M NaH2PO4, 0.1% H2PO4 was useful for separating small molecular peptides. The gradient of acetonitrile in 0.05-0.1M NaH2PO4, 0.1% H2PO4 was useful for separating many neuropeptides including ACTH related peptides. Retention times of chromatographed polypeptides showed good reproducibility. Good reproducibility was also found in peak areas of these peptides. A linear relationship was observed between the doses of peptides and their peak areas. The extracts of rat pituitary neurointermediate lobe showed several peaks of UV absorbance on PHLC; some of them coincided with AVP, oxytocin, alph-MSH, CLIP and beta-endorphin but others were unidentified. AVP immunoreactivity showed one peak which coincided with the AVP peak of UV absorbance, but ACTH immunoreactivity showed 5-6 peaks. Thus, many polypeptides were well separated using HPLC by changing the eluting condition. The simplicity, speed, good reproducibility and good quality of the separations render this technique suitable for purification and quantitative analysis of neuropeptides, and the combination of HPLC, radioimmunoassay and bioassay gives very fine analysis of neuropeptides.
高效液相色谱法（HPLC）被用于多种神经肽的分离。色谱分析使用日立638型高效液相色谱仪进行。将溶解于水性缓冲液中的肽和组织样品注入填充有日立#3053（十八烷基硅烷）的不锈钢柱（4×250mm）中。水性缓冲液由磷酸二氢钠和磷酸组成。在1分钟的上样阶段（0%有机溶剂）后，在室温下使用有机溶剂（乙腈或甲醇，0 - 60%）梯度依次洗脱肽。通过在220nm处的紫外吸光度检测洗脱的多肽，然后使用部分收集器收集它们用于后续的生物和放射免疫测定。在0.02M磷酸二氢钠、0.1%磷酸中的甲醇或乙腈梯度对于分离小分子肽很有用。在0.05 - 0.1M磷酸二氢钠、0.1%磷酸中的乙腈梯度对于分离包括促肾上腺皮质激素相关肽在内的多种神经肽很有用。色谱分析多肽的保留时间显示出良好的重现性。在这些肽的峰面积方面也发现了良好的重现性。观察到肽的剂量与其峰面积之间存在线性关系。大鼠垂体神经中间叶提取物在高效液相色谱上显示出几个紫外吸光度峰；其中一些与精氨酸加压素、催产素、α - 促黑素、促肾上腺皮质激素样中叶肽和β - 内啡肽重合，但其他峰未鉴定。精氨酸加压素免疫反应性显示出一个与紫外吸光度的精氨酸加压素峰重合的峰，但促肾上腺皮质激素免疫反应性显示出5 - 6个峰。因此，通过改变洗脱条件，使用高效液相色谱法可以很好地分离许多多肽。该技术的简单性、速度、良好的重现性和分离质量使其适用于神经肽的纯化和定量分析，并且高效液相色谱法、放射免疫测定法和生物测定法的结合能够对神经肽进行非常精细的分析。
