The experience gathered during the first three years of this program project grant clearly indicates the need for improvement in three separate areas for this analytical chemistry component. A. The detection sensitivity for each peptide will be improved 100-fold to 1000 fold, from the picomole to the femtomole level. Recent preliminary data show a limit of detection of 60 fmol. That improvement will enable us to detect any synthetic peptide that may have been transferred to the fetus, and will allow the infection of a smaller amount of peptide into the mother. B. After Drs. Szeto and Clapp screen each synthetic opioid peptide analog to determine whether that opioid peptide analog has appropriate physiologic activity, plasma samples will be collected and the peptide measured. The HPLC-purified peptide will be quantified with mass spectrometry (MS). Data are discussed below on the MS quantitation of the opioid peptide analogs DALDA and DAMGO. The corresponding deuterated synthetic analog of each peptide (synthesized by Dr. Schiller) will be used as the internal standard for MS quantification, and also as a carrier to increase the recovery of that synthetic peptide from the biological matrix. Those analytical data will be used to derive the corresponding pharmacokinetic parameters. C. The rate of analyzing ovine plasma samples will be improve 10-fold to 100-fold by employing new solid phase extraction (SPE) cartridges. Ovine plasma will be applied directly, and protein precipitation will be avoided.
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