Head-column field-amplified sample stacking in presence of siphoning. Application to capillary electrophoresis-electrospray ionization mass spectrometry of opioids in urine.

Capillary electrophoresis (CE) with head-column field-amplified sample stacking (FASS) in presence of a water plug inserted at the capillary tip is a robust approach providing a more than 1000-fold sensitivity enhancement when applied to low-conductivity samples that are analyzed in an integrated instrument. Employing modular systems comprising a small hydrodynamic buffer flow (siphoning) towards the capillary end and featuring UV absorption or electrospray ionization mass spectrometric (MS) detection, insertion of a water plug is demonstrated to deteriorate the performance of head-column FASS or making it unfunctional. Electroinjection in the absence of the water plug can be employed instead and is shown to provide a ng/ml sensitivity when applied to low conductivity samples. With some suction of sample into the capillary during electroinjection, contamination of the sample vial with buffer is thereby largely avoided. Electroinjection applied to the CE-ion trap MS-MS and MS-MS-MS analysis of twofold diluted urines, urinary solid-phase extracts and urinary liquid-liquid extracts is shown to provide much improved sensitivity compared to hydrodynamic injection of these samples. With electroinjection from diluted urine and urinary solid-phase extracts, the presence of free opioids and their glucuronic acid conjugates can be unambiguously confirmed in urines that were collected after single-dose administration of small amounts of opioids (tested with about 7 mg codeine and 25 mg dihydrocodeine, respectively). Thus, CE-multiple MS with direct electroinjection of opioids from untreated urines could prove to become a rapid and simple approach for unambiguous urinary testing of drug abuse. Procedures leading to the reduction of siphoning in modular CE setups are briefly discussed as well.

Capillary electrophoresis-electrospray ionization ion trap mass spectrometry for analysis and confirmation testing of morphine and related compounds in urine.

Using an aqueous background electrolyte containing 25 mM ammonium acetate and NH3 (pH 9), CE-tandem MS and CE-triple MS with atmospheric pressure electrospray ionization in the positive ion mode are shown to represent attractive approaches for analysis and confirmation testing of morphine (MOR) and related opioids in human urine. Injection of plain or diluted urine permits monitoring of solutes at concentrations above 2-5 microg/ml. For the recognition of lower concentrations, solute extraction and concentration is required. Liquid-liquid extraction at alkaline pH is shown to be suitable for analysis of free opioids only whereas solid-phase extraction using a mixed-mode polymer phase is demonstrated to permit analysis of both free and glucuronidated opioids. The former sample preparation approach, however, requires about half of the time only. Commencing with 2 ml of urine, reconstitution to provide a sample volume of 0.2 ml and hydrodynamic sample injection, detection limits for free opioids are shown to be on the 100-200 ng/ml drug level. Much improved (ppb) sensitivity is obtained by infusing the extract directly into the source of the MS system. However, solutes that produce equal fragments (such as the two glucuronides of MOR) can thereby not be distinguished. CE-tandem MS and CE-triple MS are demonstrated to be suitable to confirm the presence of MOR, MOR-3-glucuronide, 6-monoacetylmorphine, codeine, codeine-6-glucuronide, dihydrocodeine, methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in a toxicological quality control urine. The same is shown for selected metabolites of codeine and dihydrocodeine in urines collected after administration of pharmaceutical preparations.

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 key role. Capillary electrophoresis (CE) represents the newest technology employed in this field of analysis. Two rapid competitive binding, electrokinetic 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 approaches, aliquots of urine and immunoreagents of a commercial, broadly cross-reacting fluorescence polarization immunoassay for opiates were combined and analyzed by capillary zone electrophoresis or micellar electrokinetic capillary chromatography with laser induced fluorescence detection. With the fluorescent tracer solution employed, the former method is shown to provide simple electropherograms which are characterized by an opioid concentration dependent magnitude of the free tracer peak. In presence of dodecyl sulfate micelles, however, two tracer peaks with equal opioid concentration sensitivity are monitored. These data suggest the presence of two fluorescent tracers which react competitively with the urinary opioids for the binding 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. Furthermore, glucuronides are shown to react like the corresponding free opioids. 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-MS). This confirmatory assay is based upon solid-phase extraction using a mixed-mode polymer cartridge followed by CE hyphenated to the LCQ mass spectrometer with electrospray ionization in the positive ion mode. With this technology, MS2 is employed for proper identification of COD (m/z 300.4) and DHC (m/z 302.4) whereas MS3 provides unambiguous identification of the glucuronides of COD (m/z 476.5) and DHC (m/z 478.5) via their fragmentation to COD and DHC, respectively. MSn (n > or = 2) is shown to be capable of properly identifying the urinary codeinoids on the 100-200 ng/ml concentration level.

Head-column field-amplified sample stacking in binary system capillary electrophoresis. Preparation of extracts for determination of opioids in microliter amounts of body fluids.

Head-column field-amplified sample stacking (head-column FASS) is an efficient, on-line sample concentration technique that can easily provide a sensitivity enhancement of three orders of magnitude. Application of head-column FASS to the capillary electrophoretic analysis of opioid extracts prepared from 20 to 100 microliters of human plasma, serum or urine is reported. In the described approach, efficient concentration of cationic opiates from low conductivity extracts of body fluids is effected across a water plug, with separation taking place in a binary buffer comprising 60% (v/v) ethylene glycol, 75 mM Na2HPO4 and 25 mM NaH2PO4 (pH 7.9), and detection is effected at 210 nm. Sample extracts are prepared in 55% (v/v) ethylene glycol containing 100 microM H3PO4. Application of mixed-mode polymer solid-phase resins is shown to provide extracts that are either too salty or contain quite a large number of endogenous substances that could interfere with certain opioids. Liquid-liquid extraction with hexane, dichloromethane, ethyl acetate and dichloromethane-isopropanol is shown to provide extracts that are sufficiently clean. At a given pH, however, only closely related opioids can be extracted. Using ethyl acetate at alkaline pH, dihydrocodeine and nordihydrocodeine can reproducibly be recovered from 20-100 microliters of plasma, serum and urine. Application of head-column FASS and UV absorption detection thereby leads to the determination of ppb concentrations (> or = 1 ng/ml) of these compounds, an approach that only requires microliter amounts of sample and organic solvents.

Identification of new oxycodone metabolites in human urine by capillary electrophoresis-multiple-stage ion-trap mass spectrometry.

Capillary electrophoresis-electrospray ionization multiple-stage ion-trap mass spectrometry (CE-MSn) and computer simulation of fragmentation are demonstrated to be effective tools to detect and identify phase I and phase II metabolites of oxycodone (OCOD) in human urine. OCOD is a strong analgesic used for the management of moderate to severe mainly postoperative or cancer-related pain whose metabolism in man is largely unknown. Using an aqueous pH 9 ammonium acetate buffer and CE-MSn (n < or = 5), OCOD and its phase I metabolites produced by O-demethylation, N-demethylation, 6-ketoreduction and N-oxidation (such as oxymorphone, noroxycodone, noroxymorphone, 6-oxycodol, nor-6-oxycodol, oxycodone-N-oxide and 6-oxycodol-N-oxide) and phase II conjugates with glucuronic acid of several of these compounds could be detected in alkaline solid-phase extracts of a patient urine that was collected during a pharmacotherapy episode with daily ingestion of 240-320 mg of OCOD chloride. The data for three known OCOD metabolites for which the standards had to be synthesized in-house, 6-oxycodol, nor-6-oxycodol and oxycodone-N-oxide, were employed to identify two new metabolites, the N-oxidized derivative of 6-oxycodol and an O-glucuronide of this compound. CE-MSn and computer simulation of fragmentation also led to the identification of the N-glucuronide of noroxymorphone, another novel OCOD metabolite for which no standard compound or mass spectra library data were available.

Capillary electrophoresis in clinical and forensic analysis: recent advances and breakthrough to routine applications.

This paper is a comprehensive review article on capillary electrophoresis (CE) in clinical and forensic analysis. It is based upon the literature of 1997 and 1998, presents CE examples in major fields of application, and provides an overview of the key achievements encountered, including those associated with the analysis of drugs, serum proteins, hemoglobin variants, and nucleic acids. For CE in clinical and forensic analysis, the past two years witnessed a breakthrough to routine applications. As most coauthors of this review are associated with diagnostic or forensic laboratories now using CE on a routine basis, this review also contains data from routine applications in drug, protein, and DNA analysis. With the first-hand experience of providing analytical service under stringent quality control conditions, aspects of quality assurance, assay specifications for clinical and forensic CE and the pros and cons of this maturing, cost-and pollution-controlled age technology are also discussed.