HPLC with laser-induced native fluorescence detection for morphine and morphine glucuronides from blood after immunoaffinity extraction.

A new immunoaffinity solid phase extraction of morphine and its phase II metabolites, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide is described. An immunoadsorber was applied which was created for the first time by the immobilisation of specific antibodies (polyclonal, host: rabbit) by the sol-gel method. The extraction method in combination with high performance liquid chromatography-fluorescence determination has been validated and shown to be applicable to blood samples of heroin victims in a low concentration range. Blood extracts were essentially free of interfering matrix components when compared to C8-extracts. Additionally, a novel, sensitive and selective detection system for wavelength-resolved analysis of laser-induced fluorescence coupled to HPLC was developed. The analytes were excited with a frequency tripled Ti:Sa laser (lambda=244 nm quasi cw). The total emission spectrum was recorded with a detection system consisting of an imaging spectrograph and a back-illuminated CCD camera. This technique of detection, combined with an extended optical path (at least 6 mm could be illuminated by the laser), resulted in an optimal fluorescence intensity of the analytes. The method permitted the analysis of morphine, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide in a low concentration range and could be applied to a complex matrix such as postmortem blood samples because analyte peaks could be discriminated from matrix peaks by their characteristic emission spectra.

Recent results of biotransformation of drugs: investigation of the in vitro biotransformation of thalidomide using a dual cyclodextrin system in capillary electrophoresis.

A previously developed capillary electrophoresis method for the simultaneous separation and enantioseparation of thalidomide (TD) and its hydroxylated metabolites was extended to one additional biotransformation product. The dual chiral selector system using native beta-cyclodextrin (beta-CD) and the negatively charged sulfobutyl-beta-CD (SBE-beta-CD) was slightly modified up to a concentration of 12 mg/ml running buffer of each CD. The carrier mode in which these buffer additives transport the neutral compounds to the detector as well as the use of a polyacrylamide-coated capillary were necessary to achieve reproducible enantioseparations of all eight analytes.

Validated methods for direct determination of hydroquinone glucuronide and sulfate in human urine after oral intake of bearberry leaf extract by capillary zone electrophoresis.

Bearberry leaf extracts are used in herbal medicinal products for the treatment of lower urinary tract infections. Two metabolites of the major phenolic constituent in the extract, arbutin (hydroquinone-1-O-beta-D-glucoside), must be assumed to be precursors of the active disinfectant principle hydroquinone. In order to assay the renal elimination of these two metabolites. i.e., hydroquinone conjugates with glucuronic and sulfuric acid, two separate capillary electrophoresis methods have been developed. Both methods were validated according to the criteria for validation of pharmaceutical bioanalytical methods as drafted by the US Department of Health and Human Services. 1998. As there is little sample preparation necessary, both methods are very suitable for urine analysis with large sample numbers as frequently coming up in the course of pharmaceutical bioavailability, bioequivalence and pharmacokinetic studies.

Enantioseparations by capillary electrochromatography.

The review summarizes recent developments in enantioseparations by capillary electrochromatography (CEC). Selected fundamental aspects of CEC are discussed in order to stress those features which may allow the success of this technique in the competitive field of enantioseparations. In addition, the comparative characteristics of the different modes of chiral CEC and the stationary phases are presented. The effects of the characteristics of the stationary and liquid phases and operational conditions on the separation results are discussed. Finally, some future trends are briefly addressed.

Mechanistic study on the opposite migration order of clenbuterol enantiomers in capillary electrophoresis with beta-cyclodextrin and single-isomer heptakis(2,3-diacetyl-6-sulfo)-beta-cyclodextrin.

Opposite migration order was observed for the enantiomers of the chiral beta2-adrenergic drug clenbuterol (CL) in capillary electrophoresis (CE) when resolved with native beta-cyclodextrin (beta-CD) and heptakis (2,3-diacetyl-6-sulfo)-beta-CD (HDAS-beta-CD). The possible mechanisms of the affinity reversal of the CL enantiomers depending on the structure of the CD were studied using 1H-nuclear magnetic resonance (1H-NMR) spectrometry and one-dimensional rotating frame nuclear Overhauser and exchange spectrometry (1-D ROESY). Significant differences were observed between the structure of the (+/-)-CL complexes with beta-CD and HDAS-beta-CD.

Enantioseparation of the anticoagulant drug phenprocoumon in capillary electrophoresis with UV and laser-induced fluorescence detection and application of the method to urine samples.

The enantioseparation of phenprocoumon (PhC) in capillary electrophoresis (CE) has been studied using various cyclodextrins (CDs) such as native alpha, beta and gamma-CD and several neutral and randomly, as well as selectively substituted charged CD derivatives. Reversal of the enantiomer migration order was observed when using heptakis(2,3,6-tri-O-methyl (TM)-beta-CD as a chiral selector compared to all other CDs used. The detection of PhC was performed using either UV or laser-induced fluorescence (LIF) detection. The limit of detection (LOD) observed with LIF detection was ca. 20 times lower compared to UV. The method has been applied to the analysis of urine samples of the patient under treatment with PhC in combination with other drugs such as ramipril, hydrochlorothiazide, and nifedipine.

Effect of organic solvent, electrolyte salt and a loading of cellulose tris (3,5-dichlorophenyl-carbamate) on silica gel on enantioseparation characteristics in capillary electrochromatography.

The effect of the amount of the chiral selector, cellulose tris(3,5-dichlorophenylcarbamate) (CDCPC) on the separation characteristics of enantiomers of some charged and neutral analytes was studied in capillary electrochromatography (CEC). For better understanding of the effect of the loading of the chiral selector on the particles and pore size of the packing material, laser-beam particle size analyzer and scanning electron microscopy (SEM) were used. As shown in this study, CDCPC can be used for CEC enantioseparations of a wide range of chiral charged and neutral analytes with high efficiency. The loading of the polysaccharide derivative on the surface of silica materials even in high amounts does not markedly affect the particle size and porous structure of the packing material. The separation characteristics are strongly affected by the loading of CDCPC onto silica gel in both CEC and capillary liquid chromatography (CLC).

Isolation and identification of 8-hydroxypraziquantel as a metabolite of the antischistosomal drug praziquantel.

Praziquantel, a broad spectrum anthelmintic drug, is extensively metabolized in the liver, yielding mainly monohydroxylated and dihydroxylated phase-I metabolites. However, the exact chemical structure of most metabolites is still unknown. One of these unidentified phase-I metabolites was isolated from human urine by high performance liquid chromatography using an isocratic separation method. This metabolite was identified as 8-hydroxypraziquantel. For the structure elucidation, electrospray ionization-mass spectrometry, 1H and 13C NMR spectroscopy have been used.

Investigation of praziquantel metabolism in isolated rat hepatocytes.

Isolated rat hepatocytes were used to elucidate the metabolism of praziquantel (PZQ). Our studies were designed to investigate mainly qualitative differences in the biotransformation of PZQ enantiomers. Additionally, the main metabolites cis- and trans-4-hydroxypraziquantel were determined semiquantitatively. For this purpose, racemic PZQ and both enantiomers were incubated with isolated rat hepatocytes. The incubation mixtures were investigated using high-performance liquid chromatography/mass spectrometry. Hepatocytes prepared from male Wistar rats were incubated in Krebs-Ringer buffer at 37 degrees C for 4 h. Aliquots were withdrawn hourly throughout 4 h of incubation. We found that hepatocytes converted both enantiomers of PZQ to the major metabolites cis- and trans-4-hydroxypraziquantel. Additional metabolites were detected after incubating the S-(+)-enantiomer. These minor metabolites were identified by means of their mass/charge ratio as monohydroxypraziquantel metabolites of different, unknown structure.

Chiral separation of verapamil and some of its metabolites by HPLC and CE.

HPLC and CE assays were developed for chiral separations of verapamil and its metabolites in serum samples. Three chiral HPLC columns (Chiralcel OJ, Chiralpak AD and Chiralcel OD-R) were tested in normal and reverse-phase modes. All HPLC analyses were performed with fluorescence detection at 276 and 310 nm. CE was realized using CM-beta-CD as a chiral selector for the enantiomeric analysis. The results of HPLC and CE studies were compared and the possibilities for the applications in therapeutic drug monitoring were discussed.

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.

Separation of carvedilol enantiomers in very small volumes of human plasma by capillary electrophoresis with laser-induced fluorescence.

A sensitive capillary electrophoretic method for the determination of carvedilol enantiomers in 100 microl of human plasma has been developed and validated. Carvedilol and the internal standard carazolol are isolated from plasma samples by liquid-liquid extraction using diethylether. A sensitive and selective detection is provided by helium-cadmium laser-induced fluorescence. The total analysis time is 17.5 min, about 30 min are needed for the sample preparation. The linearity of the assay ranges from 1.56 to 50 ng/ml per carvedilol enantiomer. The limits of quantification (LOQ) for the carvedilol enantiomers in 100 microl of human plasma are 1.56 ng/ml. The inter-day accuracy for R-carvedilol is between 95.8 and 103% (104% at LOQ) and for S-carvedilol between 97.1 and 103% (107% at LOQ); the inter-day precision values are between 3.81 and 8.64% (10.9% at LOQ) and between 5.47 and 7.86% (7.91% at LOQ) for R- and S-carvedilol, respectively. The small sample volume needed is especially advantageous for the application in clinical studies in pediatric patients. As an application of the assay concentration/time profiles of the carvedilol enantiomers in a 5-year-old patient receiving a test dose of 0.09 mg/kg carvedilol are reported.

Enantioseparations using nonaqueous capillary electrochromatography on cellulose and amylose tris(3,5-dimethylphenylcarbamates) coated on silica gels of various pore and particle size.

Enantioseparations of chiral compounds were studied in nonaqueous capillary electrochromatography (NAQ CEC) with cellulose and amylose tris(3,5-dimethylphenylcarbamates) (Chiralcel OD and Chiralpak AD, respectively) coated on the silica gels of various pore and particle size. Increasing intraparticle perfusive transport with increasing pore size of silica favorably affected peak efficiency and resolution of enantiomers, although some decrease of separation factor was observed in the pore size range 60-200 A. Further improvement of peak efficiency was observed when the particle size of silica was reduced from 5 to 3 microm. The effects of a separation medium and temperature are also reported and the data obtained in the same capillaries in CEC and capillary liquid chromatography (LC) mode are compared.

Isolation of an unknown metabolite of the non-steroidal anti-inflammatory drug etodolac and its identification as 5-hydroxy etodolac.

The non-steroidal anti-inflammatory drug etodolac is extensively metabolized in the liver. Renal elimination of etodolac mainly as glucuronide and its other phase I and phase II metabolites is the primary route of excretion. High-performance liquid chromatography assays of human urine after application of etodolac indicated the existence of a further monohydroxylated metabolite (metabolite X) that was identified as 5-hydroxy etodolac. For the identification, electrospray ionization mass spectrometry (ESI-MS) as well as 1H-nuclear magnetic resonance (1H-NMR) and 13C-NMR spectroscopy have been used.

Stereoselective determination of ofloxacin and its metabolites in human urine by capillary electrophoresis using laser-induced fluorescence detection.

A capillary electrophoresis method for the simultaneous separation and enantioseparation of the antibacterial drug ofloxacin and its metabolites desmethyl ofloxacin and ofloxacin N-oxide in human urine has been developed and validated. Enantioseparation was achieved by adding sulfobutyl beta-cyclodextrin to the running buffer. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd-laser with an excitation wavelength of 325 nm. In comparison with conventional UV detection, LIF detection provides higher sensitivity and selectivity. The separation can be performed after direct injection of urine into the capillary without any sample preparation, because no matrix compounds interfere with the assay. Additionally, the high sensitivity of this method allows the quantification of the very low concentrations of enantiomers of both metabolites. The limit of quantification was 250 ng/ml for ofloxacin enantiomers and 100 ng/ml for each metabolites' enantiomers. This method was applied to the analysis of human urine samples collected from a volunteer after oral administration of 200 mg of (+/-)-ofloxacin to elucidate stereoselective differences in the formation and excretion of the metabolites. It could be demonstrated that the renal excretion of the S-configured metabolites, especially S-desmethyl ofloxacin, within the first 20 h after dosage, is significantly lower than that of the R-enantiomers.

High-performance liquid chromatographic determination of the beta2-selective adrenergic agonist fenoterol in human plasma after fluorescence derivatization.

A sensitive high-performance liquid chromatographic method has been developed for the determination of the beta2-selective adrenergic agonist fenoterol in human plasma. To improve the sensitivity of the method, fenoterol was derivatized with N-(chloroformyl)-carbazole prior to HPLC analysis yielding highly fluorescent derivatives. The assay involves protein precipitation with acetonitrile, liquid-liquid-extraction of fenoterol from plasma with isobutanol under alkaline conditions followed by derivatization with N-(chloroformyl)-carbazole. Reversed-phase liquid chromatographic determination of the fenoterol derivative was performed using a column-switching system consisting of a LiChrospher 100 RP 18 and a LiChrospher RP-Select B column with acetonitrile, methanol and water as mobile phase. The limit of quantitation in human plasma was 376 pg fenoterol/ml. The method was successfully applied for the assay of fenoterol in patient plasma.

Enantioseparations in capillary electromigration techniques: recent developments and future trends.

This review summarizes the current status of enantioseparations using capillary electromigration techniques and gives the authors insights on the selected fundamental aspects and future trends in this field. The most recent developments in the field of chiral separations using capillary electrophoresis (CE) and capillary electrochromatography (CEC) are summarized. The status of chiral electromigration techniques is evaluated tacking into account the most recent developments in related techniques such as chiral HPLC, GC and SFC.

Development of a Bright Futures curriculum for pediatric residents.

OBJECTIVE: To develop a standardized case-based curriculum for pediatric residents on child growth, development, behavior, and adolescent medicine that incorporates the Bright Futures health supervision guidelines. DESIGN: This project included a needs assessment, development of a list of important topics, writing and revising of standardized cases, formative evaluation of cases, and efficacy pilot testing of 2 cases. SETTING: A large pediatric teaching hospital continuity clinic. PARTICIPANTS: Pediatric residents, fellows, and faculty. INTERVENTIONS: Preparation of standardized cases, facilitator training, and resident-led teaching conferences. OUTCOME MEASURES: Learner and facilitator evaluation forms and two 10-item diagnostic skills assessments. RESULTS: During the project, faculty-fellow teams wrote 29 case-teaching modules. All participants gave high ratings to cases, and resident facilitators reported increased comfort with the case discussion method. Resident learners' ability to accurately interpret developmental screening tests and growth charts improved following sessions on those topics. CONCLUSIONS: Further evaluation is required, but these standardized cases appear promising for teaching pediatric residents. The curriculum is now freely available to faculty nationwide.

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.

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.