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Reversed-phase high-performance liquid chromatography for the determination of haemorphin-like immunoreactivity in
K Sanderson1, M Thörnwall, G Nyberg
1Department of Pharmaceutical Biosciences, Uppsala University, Sweden.
The haemorphins are opioid peptides derived form the blood protein haemoglobin. This study was focused on the detection and determination of haemorphin-like immunoreactivity in human cerebrospinal fluid (CSF) by reversed-phase HPLC. For this purpose a SMART System, optimized for micropurification, was applied. Prior to application to HPLC, the peptide fraction of the CSF sample was extracted using a reversed-phase silica gel cartridge (Sep-Pak C18). In the HPLC separation, the peptide-like material associated with haemorphin-7 immunoreactivity was recovered and determined using a UV detector. The tryptophan residue present in the haemorphin sequence allowed UV detection at wavelengths (e.g., 276 nm) where interference with other co-eluting peptides lacking this residue is minimized. Recorded levels of haemorphin-like immunoreactivity were compared with those detected by radioimmunoassay.
The haemorphins are opioid peptides derived form the blood protein haemoglobin. This study was focused on the detection and determination of haemorphin-like immunoreactivity in human cerebrospinal fluid (CSF) by reversed-phase HPLC. For this purpose a SMART System, optimized for micropurification, was applied. Prior to application to HPLC, the peptide fraction of the CSF sample was extracted using a reversed-phase silica gel cartridge (Sep-Pak C18). In the HPLC separation, the peptide-like material associated with haemorphin-7 immunoreactivity was recovered and determined using a UV detector. The tryptophan residue present in the haemorphin sequence allowed UV detection at wavelengths (e.g., 276 nm) where interference with other co-eluting peptides lacking this residue is minimized. Recorded levels of haemorphin-like immunoreactivity were compared with those detected by radioimmunoassay.