Sample preparation, high-performance liquid chromatographic separation and determination of substance P-related...

This review deals with the determination of low levels of substance P and peptide fragments derived from the undecapeptide, i.e. covers the whole amount of so-called substance P-like immunoreactivity (SPLI) in biological samples. First an overview of the most currently used sample pretreatment procedures is given, followed by a description of the most effective high-performance liquid chromatographic (HPLC) separation methods. Special attention is paid to the choice of the appropriate column and the possible pitfalls encountered in separation of fmol amounts of peptide material. Subsequently the most important techniques of detection are discussed. This section primarily focuses on the coupling of HPLC with radioimmunoassay (RIA), which is indispensable for detection of components in the fmol range at present. Finally, some aspects of preparation and chromatographic separation of radiolabelled antigens for use in RIA are discussed.