We seek to develop and perfect on-line high performance liquid chromatography/mass spectrometry (HPLC/MS) techniques for the sensitive and specific determination 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: (1) ionization techniques such as thermospray and fast atom bombardment (FAB), which are particularly useful with high molecular weight compounds, for on-line HPLC/MS determination of opioid peptides, and (2) new mass spectrometric methods such as MS/MS and high resolution/MS combined with HPLC/MS, which help to reduce chemical noise, improve sensitivity, and provide additional structural information. Using FAB and thermospray ionization techniques, sample molecules need not be in the gas phase for ionization to occur, thus high molecular weight peptides (molecular weight approximately 10,000) can be analyzed. Accurate molecular weight determinations in the high molecular weight ranges should be possible using these soft ionization techniques together with the instruments' ability to efficiently transmit high mass ions. We will investigate the use of high resolution/MS detection, MS/MS (in the form of mass analyzed ion kinetic energy spectrometry [MIKES]), and shift reagents. These techniques should provide reduced chemical noise and thereby increased sensitivity, and should also provide additional structural information for definitive structure determination. All HPLC/MS development will be performed using standard solutions of the various opioid peptides. Attention will be given to sample processing and cleanup, relying primarily on previously published procedures. Validation of the developed methodologies will be performed using spiked tissues, cerebrospinal fluid, and urine. Methods developed under this program, procedures for the profiling of the opioid peptides in various biological tissues and fluids should prove valuable in aiding the study of the relationships between opioid profiles and certain psychiatric and metabolic disorders.
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