Analysis of codeine, dihydrocodeine and their glucuronides in human urine by electrokinetic capillary immunoassays and capillary electrophoresis-ion trap mass spectrometry

Screening for and confirmation of illicit, abused and banned drugs in human urine is a timely topic in which capillary separation techniques play a ke y role. Capillary electrophoresis (CE) represents the newest technology emp loyed in this field of analysis. Two rapid competitive binding, electrokine tic capillary-based immunoassays are shown to be capable of recognizing the presence, but not the identity, of urinary opioids, namely codeine (COD), codeine-6-glucuronide, dihydrocodeine (DHC), dihydrocodeine-6-glucuronide, morphine (MOR), morphine-3-glucuronide and ethylmorphine (EMOR). In these a pproaches, aliquots of urine and immunoreagents of a commercial, broadly cr oss-reacting fluorescence polarization immunoassay for opiates were combine d and analyzed by capillary zone electrophoresis or micellar electrokinetic capillary chromatography with laser induced fluorescence detection. With t he fluorescent tracer solution employed, the former method is shown to prov ide simple electropherograms which are characterized by an opioid concentra tion dependent magnitude of the free tracer peak. In presence of dodecyl su lfate micelles, however, two tracer peaks with equal opioid concentration s ensitivity are monitored. These data suggest the presence of two fluorescen t tracers which react competitively with the urinary opioids for the bindin g sites of the antibody. Assay sensitivities for COD and MOR are comparable (10 ng/ml), whereas those for DHC and EMOR are about four-fold lower. Furt hermore, glucuronides are shown to react like the corresponding free opioid s. Analysis of urines that were collected after administration of 7 mg COD and 25 mg DHC tested positively in both assay formats. The presence of the free and conjugated codeinoids in these urines and their identification was accomplished by capillary electrophoresis-ion trap mass spectrometry (CE-M S). This confirmatory assay is based upon solid-phase extraction using a mi xed-mode polymer cartridge followed by CE hyphenated to the LCQ mass spectr ometer with electrospray ionization in the positive ion mode. With this tec hnology, MS? is employed for proper identification of COD (m/z 300.4) and D HC (m/z 302.4) whereas MS3 provides unambiguous identification of the glucu ronides of COD (m/z 476.5) and DHC (m/z 478.5) via their fragmentation to C OD and DHC, respectively. MSn (n greater than or equal to2) is shown to be capable of properly identifying the urinary codeinoids on the 100-200 ng/ml concentration level. (C) 2000 Elsevier Science B.V. All rights reserved.