Capillary electrophoresis with laser-induced fluorescence in clinical drug development routine application and future aspects.

The clinical bioanalytical setting is characterized by sample volumes of < 1 ml biological fluid (e.g. plasma, urine), a range of 3-4 decades of concentrations to be quantified and a limit of quantitation in the microg/l-ng/l range for sets of 100-5000 individual samples. Setup of capillary electrophoresis (CE) for routine application in this analytical field was successful for analytes accessible to fluorescence detection by using laser-induced fluorescence (LIF) detection. Empowerment of CE-LIF for routine serial analysis of thousands of samples includes improvement in autosampler techniques, thorough procedures for capillary treatment and particularly more advanced detection technology. Introduction of multi-capillary systems with charge-coupled device cameras and frequency doubled Ar-ion laser (lambda = 257 nm) offers this technique the chance of superiority over classical analytical assays - especially in the field of (new) low volume samples e.g. capillary blood or microdialysate encouraging clinicians to search for meaningful non-invasive samples.

Direct chiral assay of tramadol and detection of the phase II metabolite O-demethyl tramadol glucuronide in human urine using capillary electrophoresis with laser-induced native fluorescence detection.

A chiral separation using carboxymethyl-beta-cyclodextrin and methyl-beta-cyclodextrin for the direct assay of tramadol in human urine by capillary electrophoresis (CE) with laser-induced native fluorescence detection was developed. Furthermore, the phase II metabolite O-demethyl tramadol glucuronide was determined from the urine samples and the ratio of the diasteromers was determined. The chiral method was validated. Correlation coefficients were higher than 0.999. Within day variation showed accuracy in the range 96.1-105.8% with a RSD less than 6.00%. Day to day variation present an accuracy ranging from 100.2 to 103.5% with a RSD less than 5.4%. After oral administration of 150 mg tramadol hydrochloride to a healthy volunteer, the urinary excretion was monitored during 24 h. About 11.4% of the dose was excreted as 1S,2S-tramadol, 16.4% as 1R,2R-tramadol and 23.7% as O-demethyl tramadol glucuronide. The amount of 1S,2S O-demethyl tramadol glucuronide was more than three fold higher as IR,2R-O-demethyl tramadol glucuronide. The enantiomeric ratio of tramadol and the diastereomeric ratio of O-demethyl tramadol glucuronide was deviated from 1.0 showing that a stereoselective metabolism of tramadol occurs.

Assay of tramadol in urine by capillary electrophoresis using laser-induced native fluorescence detection.

Capillary electrophoresis (CE) with UV laser-induced native fluorescence detection was developed as a sensitive and selective assay for the direct determination of tramadol in human urine without extraction or preconcentration. The main problem in CE is the small inner diameter of the capillary which causes a low sensitivity with instruments equipped with a UV detector. Laser-induced native fluorescence with a frequency doubled argon ion laser at an excitation wavelength of 257 nm was used for the direct assay of tramadol in urine to enhance the limit of detection about 1,000-fold compared to UV absorption detection. The detection system consists of an imaging spectrograph and an intensified CCD camera, which views an illuminated 1.5 mm section of the capillary. This set-up is able to record the whole emission spectra of the analytes to achieve additionally wavelength-resolved electropherograms. In the concentration range of 20 ng/ml-5 microg/ml in human urine coefficients of correlation were better than 0.998. Within-day variation determined on four different concentrations showed accuracies ranging from 90.2 to 108.4%. The relative standard deviation (RSD) was determined to be less than 10%. Day-to-day variation presented accuracies ranging from 90.9 to 103.1% with an RSD less than 8%.

Simultaneous quantification of etoposide and etoposide phosphate in human plasma by capillary electrophoresis using laser-induced native fluorescence detection.

A method using capillary electrophoresis with UV laser-induced native fluorescence detection was developed as a sensitive and selective assay for the simultaneous determination of etoposide and etoposide phosphate in human plasma. Laser-induced native fluorescence detection with a frequency-doubled argon ion laser at an excitation wavelength of 257 nm was used for the simultaneous assay of etoposide and etoposide phosphate in plasma to improve the sensitivity compared to that obtained with UV absorption. The detection system consists of an imaging spectrograph and an intensified CCD camera which views an illuminated 1.5-mm section of the capillary. This setup is able to record the whole emission spectra of the analytes to achieve additional wavelength-resolved electropherograms. In the concentration range of 200 microg/L-50 mg/L in plasma for etoposide and 100 microg/L-20 mg/L for etoposide phosphate, coefficients of correlation were better than 0.998. Within-day variation determined with three different concentrations showed accuracies ranging from 91.0 to 109.3% for etoposide and from 91.2 to 109.9% for etoposide phosphate (n = 6) with a precision of about 8%. Day-to-day variation presented accuracies ranging from 91.8 to 107.9% for etoposide and from 94.4 to 109.3% for etoposide phosphate with a relative standard deviation less than 6% (n = 5). To our knowledge, this is the first method for the simultaneous quantification of etoposide and etoposide phosphate in plasma samples.