Rapid quantification of the δ-opioid receptor selective enkephalin DPDPE in canine cerebrospinal fluid by liquid chromatography–mass spectrometry

An atmospheric pressure ionization liquid chromatographic–mass spectrometric assay was developed and validated for the determination of d-penicillamine2,5 enkephalin (DPDPE) in cerebrospinal fluid (CSF) from dog. DPDPE and internal standard (d-Ala2,d-Leu5 enkephalin=DADLE) were isolated from CSF by reversed-phase C18 solid-phase extraction with ZipTip micro-cartridges. Aliquots of extracted eluate were injected onto an Agilent Zorbax SB C18 column (30×2.2 mm; 3.5 μm) at a flow-rate of 0.4 ml/min. The isocratic mobile phase of methanol–10 mM ammonium formate (pH 3) (75:25, v/v) was then diverted to waste for 45 s after injection, after which time flow was directed to the single quadrupole mass spectrometer. DPDPE was detected by positive mode selected ion monitoring. Standard curves were linear (r2≥0.991) over the concentration range 1–1000 ng/ml. The efficiency of extraction recovery was greater than 97%, and the intra-assay and inter-assay precisions were within 9% relative standard deviation. DPDPE and the internal standard were stable in the injection solvent at 4 °C for at least 48 h. The assay was applied to the pharmacokinetic study of intrathecal DPDPE administration in the dog animal model.