Screening for urinary methadone by capillary electrophoretic immunoassays and confirmation by capillary
W Thormann1, M Lanz, J Caslavska
1Department of Clinical Pharmacology, University of Bern, Switzerland. wolfgang.thormann@ikp.unibe.ch
Abstract:
This paper characterizes competitive binding, electrokinetic capillary-based immunoassays for urinary methadone using reagents which were commercialized for a fluorescence polarization immunoassay. After incubation of 25 microL urine with the reactants, a small aliquot of the mixture is applied onto a fused-silica capillary and the unbound fluorescein-labeled methadone tracer is monitored by capillary electrophoresis with on-column laser-induced fluorescence detection. Configurations in presence and absence of micelles were investigated, found to be capable of recognizing urinary methadone concentrations > or = 10 ng/mL, and shown to be suitable for rapid methadone screening of patient urines. Based upon shorter run times and a much better separation of free tracer and antibody-tracer complex, conditions without micelles are preferred. For confirmation analysis of urinary methadone and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), capillary electrophoresis in a pH 4.6 ammonium acetate-acetic acid buffer was interphased to an atmospheric pressure ionization triple quadrupole mass spectrometry system. Using positive ion electrospray ionization and the tandem mass spectrometry mode with collision-induced dissociation in the collision cell, fragmentation of the two substances was determined. For confirmation via direct urine injection or application of a urinary extract, in-source fragmentation was employed and the first quadrupole was operated in the selected ion monitoring mode by switching between the masses of relevant precursor/product ion sets for methadone (m/z = 310, 265) and EDDP (m/z = 278, 249, 234). This capillary electrophoresis-mass spectrometry approach is shown to permit the confirmation of methadone and EDDP in patient urines that tested positive for methadone using electrokinetic capillary-based immunoassays, a fluorescence polarization immunoassay, and capillary electrophoresis with UV absorption detection.
