DETERMINATION OF OPIOID PEPTIDES BY ELECTROSPRAY LC/MS

建议的方案旨在发展和完善联合在线 电喷雾-液-质联用(ES-LC/MS)测定 生物样品中阿片肽的灵敏和特异检测 矩阵。已知阿片类药物的广泛分子量范围 多肽，这些多肽在复合体中的浓度极低 生物材料，以及不断增加的个体数量 多肽对当前的分析方法提出了挑战。这一计划将 通过研究电喷雾电离来解决这些问题 它已被证明可以在1-5fM范围内检测多肽。 电喷雾电离的灵敏度远远优于其他质谱仪 用于非挥发性物质(例如快原子)的电离技术 轰击或热喷雾)，获得与之相当的敏感性 放射免疫分析(RIA)方法。拟议中的计划将调查 影响ES电离的各种参数(即施加的电压， 溶剂、缓冲液、pH、流速)以实现对阿片类药物的最佳敏感性 多肽测定。 ES是一种软电离技术，产生单电荷和多电荷 指示多肽分子量的离子。要取得更多成就 对于化合物鉴定的特异性，拟议的计划将 研究固定化酶生物反应器和串联MS的使用。 固定化酶生物反应器将允许某些多肽的水解性 键，特定于所使用的特定酶。这些水解液 产品对一种提供信息的多肽是非常特异的 结构确认。Tandem MS应提供结构 碎裂，取决于多肽的结合强度和稳定性 离子就形成了。多电荷离子中的强库仑斥力 允许通过以下方式对高分子量多肽进行碎裂 碰撞激活。 ES-LC/MS将在临床情况下进行测试，以确定效果 麻醉、手术和镇痛对血浆浓度的影响 狗的前阿片黑素皮质素阿片肽。血药浓度 类油类物质将通过RIA和ES-LC/MS对两只对照犬进行评估 (仅限麻醉和接受麻醉和手术的客户犬。 麻醉前将评估阿片类药物的血浆浓度 拔管后最多48小时。血浆阿片类药物的相关性 注意力和疼痛或与压力相关的行为将被用来 确定止痛治疗的效果

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