A high-recovery extraction procedure for quantitative analysis of substance P and opioid peptides in human cerebrospinal fluid.

This study reports an improved approach for the determination of neuropeptide levels in human cerebrospinal fluid (CSF). The method is based on sample acidification followed by liquid-liquid extraction (LLE) combined with radioimmunoassay. It was applied to study the recovery and level of some opioid peptides (Met-enkephalin-Arg(6)-Phe(7) and Leu-enkephalin-Arg(6)), substance P and the substance P(1-7) fragment, which are all compounds known to be present in human CSF. The results indicated that the use of LLE highly improved the recovery of these peptides compared to current liquid-solid-phase extraction methods by using silica gel cartridges or mini-columns for ion-exchange chromatography. Peptides added to CSF in concentrations down to 10 fmol/ml were recovered in yields exceeding 80%. The mean recovery of synthetic peptides as recorded by radioimmunoassay in the LLE procedure was significantly improved when HCl was added to the sample. In contrast, when the (125)I-labeled analogues of the peptides were added to CSF samples, the mean recovery of the four labeled peptides using the LLE procedure was markedly reduced in acidified samples. We also found that the inclusion of HCl effectively improved the removal of proteins present in the samples. As an application the levels of substance P and Met-enkephalin-Arg(6)-Phe(7) in CSF samples from patients with chronic pain (fibromyalgia syndrome) were measured using the new procedure. It was possible to confirm a significant difference in the CSF levels of both peptides when comparing patients and controls.