Feasibility study of a drug immunoassay based on micellar electrokinetic capillary chromatography with laser induced fluorescence detection: Determination of theophylline in serum

Abstract

This paper presents principle and first results of a novel competitive binding immunoassay for monitoring of theophylline in human serum. The assay is based upon short time incubation of a mixture of antiserum, containing the antibody raised against theophylline, fluorescein labelled theophylline (tracer) and serum prior to injection of a few nanoliter of this mixture onto a fused-silica capillary for subsequent separation and analysis of free tracer and the antibody-tracer-complex by micellar electrokinetic capillary chromatography with laser induced fluorescence detection. Quantitation based upon multi-level calibration using the height of the peak produced by the free tracer is shown to provide theophylline serum levels which are in agreement with those obtained by a commercial fluorescence polarization immunoassay and with those determined by micellar elektrokinetic capillary chromatography with direct serum injection and on-column UV absorption detection.

Stereoselective screening for and confirmation of urinary enantiomers of amphetamine, methamphetamine, designer drugs, methadone and selected metabolites by capillary electrophoresis

“…Liquid chromatography [11,12] and capillary electrophoresis (CE) [13,14] with multiwavelength solute detection represent other confirmatory methods. Independent of the method used, the customary assays are typically not enantioselective.…”

Capillary electrophoresis in clinical and forensic analysis

“…The spectral information obtained in that approach is employed for identification of substances. The most comprehensive protocol elucidated thus far for the screening and confirmation of illicit and abused drugs uses urine pretreatment via a two-step solid-phase extraction with a copolymeric sorbent which provides, from the very same aliquot of urine, barbiturates, some benzodiazepines, ll-nor-A9-tetrahydrocannabinol-9-carboxylic acid and methaqualone in the first eluate, as well as opioids, cocaine and benzoylecgonine, selected benzodiazepines or their metabolites, methadone and its primary metabolite, diphenhydramine and amphetamines in the second eluate [89]. With the exception of methadone and its metabolite which have to be determined by CZE, all these extracted compoundls can effectively be analyzed by MECC.…”

“…Furthermore, CE assays were determined to be capable of recognizing false-negative and false-positive results from an immunological screening process [92]. Based upon these developments, a concept of an automated capillary analyzer featuring sample cleanup, multiple capillaries for simultaneous analysis of the fractions in one or several buffers and computer-aided peak identification has been proposed [89]. The exploration of CE-MS for the confirmation analysis of illicit drugs and their metabolitesis in body fluids is also underway.…”

Analysis of the biotin‐binding protein actinavidin using affinity capillary electrophoresis

“…Arentoft et al [12] used MEKC, with cholate as detergent, to separate immuno-complexes and, most recently, Steinmann et al [13] determined theophylline in serum using SDS as buffer additive. These authors did not evaluate the denaturing effect of the detergent, although previous work [14] has shown that SDS binding (even at submillimolar concentration) results in protein denaturation with potential loss of affinity.…”