DETERMINATION OF OPIOID PEPTIDES BY ELECTROSPRAY LC/MS

该计划旨在发展和完善联合在线 电喷雾液相色谱/质谱（ES-LC/MS） 生物样品中阿片肽的灵敏度和特异性测定 矩阵已知阿片类药物的分子量范围很广 肽，这些肽在复合物中的浓度极低， 生物材料，以及不断增加的个体数量 肽挑战了当前的分析方法。此程序将 通过研究电喷雾（ES）电离来解决这些问题 其已被证明可检测1-5 fM范围内的肽。 电喷雾电离的灵敏度远优于其他质谱的上级 用于非挥发性物质的电离技术，（例如，快原子 轰击或热喷涂），实现与 放射免疫测定（RIA）方法。该计划将调查 影响ES电离的各种参数（即施加的电压， 溶剂、缓冲剂、pH、流速），以获得对阿片样物质最佳敏感性 肽测定 ES是一种软电离技术， 表示肽分子量的离子。取得更多 针对化合物鉴定的特异性，拟议的计划将 研究使用固定化酶生物反应器和串联MS。 固定化酶生物反应器将允许某些肽的水解 键，对所用的特定酶具有特异性。这些水解 产品是非常具体的肽提供信息， 结构确认。串联MS应提供结构 片段，这取决于肽的键强度和稳定性。 离子形成。多电荷隆恩中的强库仑排斥 允许高分子量肽的片段化， 碰撞激活 ES-LC/MS将在临床情况下进行测试，以确定影响 麻醉、手术和镇痛对血浆 狗中的前阿黑皮素阿片肽。血浆浓度 将通过RIA和ES-LC/MS评价两种对照犬的阿片类药物 （仅麻醉，客户犬接受麻醉和手术。 麻醉前评价阿片类药物的血浆浓度 直至拔管后48小时。血浆阿片类物质 注意力和疼痛或压力相关的行为将被用来 确定镇痛治疗的效果

The proposed program seeks to develop and perfect combined on-line electrospray-liquid chromatography/mass spectrometry (ES-LC/Ms) for the sensitive and specific detemination of opioid peptides in biological matrices. The wide range of molecular weights of the known opioid peptides, the extremely low concentrations of these peptides in complex biological materials, and the ever-increasing number of individual peptides challenge current analytical methodology. This program will address these problems by investigating electrospray (ES) ionization which has been demonstrated to detect peptides at the 1-5 fM range. Sensitivity of the electrospray ionization is far superior to other MS ionization techniques used for nonvolatile species, (e.g., fast atom bombardment or thermospray), achieving comparable sensitivity to radioimmunoassay (RIA) methods. The proposed program will investigate various parameters that effect ES ionization (i.e. applied voltages, solvent, buffer, pH, flowrate) to achieve optimal sensitivity for opioid peptide determination. ES is a soft ionization technique, producing single and multicharged ions indictative of a peptide's molecular weight. To achieve more specificity for compound identification, the proposed program will investigate the use of ilmnobilized enzyme bioreactors and tandem MS. Immobilized enzyme bioreactors will permit hydrolysis of certain peptide bonds, specific for the particular enzyme employed. These hydrolysis products are very specific for a peptide providing information for structure confirmation. Tandem MS should provide structural fragmentation, depending on the peptide's bond strength and stability of the ion formed. The strong coulombic repulsion in multicharged lons permits the fragmentation of high molecular weight peptides by collisional activation. ES-LC/MS will be tested in a clinical situation to determine the effects of anesthesia, surgery and analgesia on the concentration of plasma pro-opiomelanocortin opioid peptides in dogs. Plasma concentration of oiioids will be evaluated by RIA and ES-LC/MS for both control dogs (anesthesia only and client dogs undergoing anesthesia and surgery. Plasma concentrations of opioids will be evaluated prior to anesthesia and up to 48 hours after extubation. Correlations between plasma opioid concentration and pain or stress-related behavior will be used to detemine the effect of analgesia therapy