DETERMINATION OF OPIOID PEPTIDES BY ELECTROSPRAY LC/MS

通过电喷雾 LC/MS 测定阿片类肽

• 批准号: 3212981
• 负责人: ROBERT D VOYKSNER
• 金额: $ 11.87万
• 依托单位: RESEARCH TRIANGLE INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-30 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3212981
• 关键词: adrenocorticotropic hormone; analgesics; anesthesia; bioreactors; cerebrospinal fluid; dogs; dynorphins; endorphins; enkephalins; enzyme mechanism; high performance liquid chromatography; hydrogen bond; hydrolysis; ionization; neuropeptides; pain; peptide structure; plasma; postoperative complications; proopiomelanocortin; radioimmunoassay

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

拟议的计划旨在开发和完善联合在线 电喷雾液相色谱/质谱 (ES-LC/Ms) 生物中阿片肽的灵敏和特异测定 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。该计划将 通过研究电喷雾 (ES) 电离来解决这些问题 已被证明可以检测 1-5 fM 范围内的肽。 电喷雾电离的灵敏度远远优于其他 MS 用于非挥发性物质的电离技术（例如，快原子 轰击或热喷雾），达到与 放射免疫测定（RIA）方法。拟议的计划将调查 影响 ES 电离的各种参数（即施加的电压、 溶剂、缓冲液、pH 值、流速）以实现阿片类药物的最佳灵敏度 肽测定。 ES 是一种软电离技术，可产生单电荷和多电荷 指示肽分子量的离子。为了实现更多 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肽的水解 键，特定于所使用的特定酶。这些水解 产品对于肽非常具体，可提供以下信息： 结构确认。串联 MS 应提供结构 断裂，取决于肽的键强度和稳定性 形成的离子。多电荷离子中的强库仑斥力 允许通过以下方式裂解高分子量肽 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 狗中的阿片黑皮质素原阿片肽。血浆浓度 将通过 RIA 和 ES-LC/MS 对两只对照狗进行类固醇评估 （仅限麻醉和接受麻醉和手术的客户狗。 麻醉前将评估阿片类药物的血浆浓度 以及拔管后 48 小时内。血浆阿片类药物之间的相关性 注意力集中和疼痛或压力相关的行为将被用来 确定镇痛治疗的效果