Quantitative analysis of the opioid peptide DAMGO in rat plasma and microdialysis samples using liquid chromatography-tandem mass spectrometry.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of the opioid peptide DAMGO in rat plasma, as well as DAMGO and the microdialysis recovery calibrator [(13)C(2),(15)N]-DAMGO in microdialysis samples, is described. The microdialysis samples consisted of 15 μL Ringer solution containing 0.5% bovine serum albumin. Pretreatment of the samples involved protein precipitation with acetonitrile followed by dilution with 0.01% formic acid. The lower limits of quantification were 0.52 ng/mL and 0.24 ng/mL for DAMGO and [(13)C(2),(15)N]-DAMGO respectively and the response was linear up to 5000 fold higher concentrations. The plasma samples (50 μL) were precipitated with acetonitrile containing the isotope labeled analog [(13)C(2),(15)N]-DAMGO as internal standard. The method was linear in the range of 11-110,000 ng/mL. The separations were conducted on a HyPurity C18 column, 50×4.6 mm, 3 μm particle size, with a mobile phase consisting of acetonitrile, water and formic acid to the proportions of 17.5:82.5:0.01. Low energy collision dissociation tandem mass spectrometric (CID-MS/MS) analysis was carried out in the positive ion mode using multiple reaction monitoring (MRM) of the following mass transitions: m/z 514.2→453.2 for DAMGO and m/z 517.2→456.2 for [(13)C(2),(15)N]-DAMGO. The intra-day precision and accuracy did not exceed 5.2% and 93-104% for both compounds and sample types described. The inter-day precision an accuracy were <6.8% and 95-105% respectively. The method described is simple, reproducible and suitable for the analysis of small sample volumes at low concentrations.