Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine.

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66µM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26µM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

Stereoselective biotransformation of ketamine in equine liver and lung microsomes.

Stereoselectivity has to be considered for pharmacodynamic and pharmacokinetic features of ketamine. Stereoselective biotransformation of ketamine was investigated in equine microsomes in vitro. Concentration curves were constructed over time, and enzyme activity was determined for different substrate concentrations using equine liver and lung microsomes. The concentrations of R/S-ketamine and R/S-norketamine were determined by enantioselective capillary electrophoresis. A two-phase model based on Hill kinetics was used to analyze the biotransformation of R/S-ketamine into R/S-norketamine and, in a second step, into R/S-downstream metabolites. In liver and lung microsomes, levels of R-ketamine exceeded those of S-ketamine at all time points and S-norketamine exceeded R-norketamine at time points below the maximum concentration. In liver and lung microsomes, significant differences in the enzyme velocity (V(max)) were observed between S- and R-norketamine formation and between V(max) of S-norketamine formation when S-ketamine was compared to S-ketamine of the racemate. Our investigations in microsomal reactions in vitro suggest that stereoselective ketamine biotransformation in horses occurs in the liver and the lung with a slower elimination of S-ketamine in the presence of R-ketamine. Scaling of the in vitro parameters to liver and lung organ clearances provided an excellent fit with previously published in vivo data and confirmed a lung first-pass effect.

Modeling of electroosmotic and electrophoretic mobilization in capillary and microchip isoelectric focusing.

Our dynamic capillary electrophoresis model which uses material specific input data for estimation of electroosmosis was applied to investigate fundamental aspects of isoelectric focusing (IEF) in capillaries or microchannels made from bare fused-silica (FS), FS coated with a sulfonated polymer, polymethylmethacrylate (PMMA) and poly(dimethylsiloxane) (PDMS). Input data were generated via determination of the electroosmotic flow (EOF) using buffers with varying pH and ionic strength. Two models are distinguished, one that neglects changes of ionic strength and one that includes the dependence between electroosmotic mobility and ionic strength. For each configuration, the models provide insight into the magnitude and dynamics of electroosmosis. The contribution of each electrophoretic zone to the net EOF is thereby visualized and the amount of EOF required for the detection of the zone structures at a particular location along the capillary, including at its end for MS detection, is predicted. For bare FS, PDMS and PMMA, simulations reveal that EOF is decreasing with time and that the entire IEF process is characterized by the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic transport and electroosmotic zone displacement are opposite and of equal magnitude. The location of immobilization of the boundary between anolyte and most acidic carrier ampholyte is dependent on EOF, i.e. capillary material and anolyte. Overall time intervals for reaching this state in microchannels produced by PDMS and PMMA are predicted to be similar and about twice as long compared to uncoated FS. Additional mobilization for the detection of the entire pH gradient at the capillary end is required. Using concomitant electrophoretic mobilization with an acid as coanion in the catholyte is shown to provide sufficient additional cathodic transport for that purpose. FS capillaries dynamically double coated with polybrene and poly(vinylsulfonate) are predicted to provide sufficient electroosmotic pumping for detection of the entire IEF gradient at the cathodic column end.

Analysis of ethyl glucuronide in human serum by capillary electrophoresis with sample self-stacking and indirect detection.

Ethyl glucuronide (EtG), a metabolite of ethanol, is a marker of recent alcohol consumption. In the past few years, its analysis in body fluids has attracted considerable attention because it closes a gap between short time and long time alcohol markers such as ethanol and carbohydrate-deficient transferrin, respectively. The capillary zone electrophoresis (CZE) analysis of EtG in model mixtures and human serum is reported using uncoated and coated fused-silica capillaries together with acidic buffers in the pH range between 3.2 and 4.4 and indirect detection. In these approaches, separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) is based upon transient isotachophoretic stacking referred to as sample self-stacking. The selection of a favorable bufferco-ion and pH is shown to be crucial for optimized sensitivity. Abuffercomposed of 10 mM nicotinic acid and epsilon-aminocaproic acid (pH 4.3) is demonstrated to provide a detection limit for EtG in serum of 0.1 microg/ml, a value that is relevant for clinical and forensic purposes.

Isolated lung perfusion: single-pass system versus recirculating blood perfusion in pigs.

BACKGROUND: Cytostatic isolated lung perfusion has been advocated for treating pulmonary metastasis of soft tissue sarcoma. Different techniques of isolated lung perfusion have been developed. METHODS: Isolated lung perfusion with and without doxorubicin was performed on white pigs during 15 minutes either by a single-pass system (n = 7) or by a recirculating-blood perfusion system (n = 7). Three animals with endovenous drug application served as controls. Leakage was assessed using isotopic tracers. Perfusion-induced lung tissue injury was determined by postperfusion chest radiographs, by angiotensin-converting enzyme-to-protein ratio in the plasma and in the bronchioalveolar lavage fluid, and by wet-to-dry weight ratio and histologic examination of lung biopsy specimens at 20 and 50 minutes. Doxorubicin concentration in lung tissue and plasma was compared between the three study groups. RESULTS: All isolated lung perfusion studies were successfully performed without significant systemic leakage (< 0.6%). Wet-to-dry weight ratio was significantly lower after single-pass as compared with recirculating-blood perfusion and endovenous drug application at both time points (5.0 +/- 1.1 and 5.3 +/- 0.8 for single-pass versus 6.6 +/- 1.1 and 6.9 +/- 0.5 for recirculating-blood versus 6.6 +/- 0.2 and 5.9 +/- 0.7 for the control group, respectively; p < 0.05). Angiotensin-converting enzyme-to-protein plasma ratio in the single-pass group was significantly lower only at 20 minutes (6.3 +/- 2.4 versus 9.3 +/- 1.0 versus 9.7 +/- 1.9, respectively; p < 0.05) but not at 50 minutes. Angiotensin-converting enzyme-to-protein ratio in bronchoalveolar lavage fluid, histology of lung biopsy specimens, and chest radiographs did not differ significantly between the three groups. Doxorubicin lung tissue concentration was not significantly different after single-pass (17.5 micrograms/g) and recirculating-blood perfusion (21.9 micrograms/g), but was significantly higher than after endovenous drug application (3.0 micrograms/g; p < 0.01). CONCLUSIONS: Both isolated lung perfusion techniques resulted in a sixfold to sevenfold higher doxorubicin lung tissue concentration than after endovenous application. Isolated lung perfusion-induced lung injury was similar for both techniques, but recirculating-blood perfusion appeared to result in more acute lung injury and was technically more demanding than single-pass perfusion.

Cytostatic lung perfusion by use of an endovascular blood flow occlusion technique.

BACKGROUND: Different modalities of cytostatic lung perfusion were compared regarding plasma and tissue drug concentrations to assess the efficacy of an endovascular blood flow occlusion technique. METHODS: A cytostatic lung perfusion study with doxorubicin hydrochloride was performed on large white pigs (n = 12). Plasma and tissue concentrations of doxorubicin were compared for isolated lung perfusion with open cannulation (ILP), blood flow occlusion perfusion with open cannulation of the pulmonary artery alone (BFO), and intravenous drug administration (i.v.). In a fourth group, thoracotomy-free BFO perfusion was performed by endovascular balloon catheterization of the pulmonary artery (endovascular BFO). The 3 animals in this group were used to compare the doxorubicin-perfused pulmonary tissue with the contralateral nonperfused lobes after 1 month. RESULTS: The mean lung tissue doxorubicin concentration at the end of perfusion was 19.8 +/- 1.6 microg/g after ILP, 27.6 +/- 2.2 microg/g after BFO (p = not significant), and 3.0 +/- 0.8 microg/g after i.v. perfusion (p < 0.01). Whereas doxorubicin was not detectable in the plasma in the ILP group, concentrations ranged from not detectable to 0.44 microg/mL in the BFO group and from 0.31 to 0.84 microg/mL in the i.v. group (p < 0.05). Mean myocardial tissue concentration was not significantly different after BFO than i.v. perfusion (1.1 +/- 0.5 microg/g and 1.8 +/- 0.1 microg/g, respectively). In the endovascular BFO group, balloon-blocked pulmonary artery perfusion was successfully performed in all animals, and after 1 month, lung tissue showed no cytostatic-induced histologic changes. CONCLUSIONS: Compared with ILP, BFO cytostatic lung perfusion produced an insignificantly higher lung-tissue concentration, corresponding to a sixfold to ninefold higher level than after i.v. perfusion. Plasma drug levels during BFO perfusion were lower than during i.v. perfusion. Endovascular BFO may be a promising technique for repeated cytostatic lung perfusion.

Enantioselective determination of drugs in body fluids by capillary electrophoresis.

During the past decade, chiral capillary electrophoresis (CE) emerged as a promising, effective and economic approach for the enantioselective determination of drugs in body fluids, hair and microsomal preparations. This review discusses the principles and important aspects of CE-based chiral bioassays, provides a survey of the assays developed and presents an overview of the key achievements encountered. Applications discussed encompass the pharmacokinetics of drug enantiomers, the elucidation of the stereoselectivity of drug metabolism and bioanalysis of drug enantiomers of toxicological and forensic interest.

Determination of naproxen in liver and kidney tissues by electrokinetic capillary chromatography with laser-induced fluorescence detection.

Pharmacotherapy through the targeting of drugs is a promising new approach that requires adequate analytical methods capable of monitoring the free drug, the drug carrier and metabolites in body fluids and organs. A micellar electrokinetic capillary chromatography (MECC) based assay for analysis of naproxen (NAP) in extracts of hydrolyzed liver and kidney tissue homogenates using salicylate as internal standard and solute detection by laser-induced fluorescence is reported. The assay described uses 100 microliters of hydrolyzed tissue homogenate and has a detection limit of 0.07 microgram/ml. It is shown to be selective, reproducible (at a NAP level of 0.25 microgram/ml, intra-day and inter-day R.S.D. values are 3.73% and 6.39%, respectively), simple and economical (operates with inexpensive separation columns and small amounts of chemicals). It has been successfully applied to the assessment of the total NAP content within liver and kidney tissues of male Sprague Dawley rats that have been treated with NAP conjugated to human serum albumin (the drug targeting carrier) and free NAP. Compared to previously applied techniques, including high-performance liquid chromatography, MECC offers the advantage of having lower running costs and lower consumption of organic solvents.

Analysis of carnitine and acylcarnitines in urine by capillary electrophoresis.

A capillary electrophoresis method is described for the simultaneous analysis of carnitine and short-chain acylcarnitines in aqueous standard solutions and urine samples. Samples were worked up using silica gel extraction and derivatization with 4'-bromophenacyl trifluoromethanesulfonate. Separation was performed in less than 8 min using a binary buffer system containing phosphate/phosphoric acid and sodium dodecyl sulfate. 3-(2,2,2-Trimethylhydrazinium)propionate (mildronate) was used as an internal standard. The method was developed with aqueous standard solutions and then applied successfully to spiked and unspiked human urine samples. The limit of detection for both carnitine and acetylcamitine is 3 microM.

Modification of a tunable UV-visible capillary electrophoresis detector for simultaneous absorbance and fluorescence detection: profiling of body fluids for drugs and endogenous compounds.

Using fused-silica optical fibres for fluorescence light collection and bandpass filters for selection of emission wavelengths, a capillary electrophoresis detection cell of a conventional, tunable UV-Vis absorbance detector was adapted for simultaneous fluorescence (at selected emission wavelength) and absorbance (at selected excitation wavelength) detection. Detector performance is demonstrated with the monitoring of underivatized fluorescent compounds in body fluids by micellar electrokinetic capillary chromatography with direct sample injection. Compared with UV absorption detection, fluorescence detection is shown to provide increased selectivity and for selected compounds also up to tenfold higher sensitivity. Examples studied include screening for urinary indole derivatives (tryptophan, 5-hydroxytryptophan, tyrosine, 3-indoxyl sulfate and 5-hydroxyindole-3-acetic acid) and catecholamine metabolites (homovanillic acid and vanillylmandelic acid) and the monitoring of naproxen in serum, quinidine in serum and urine and of salicylate and its metabolites in serum and urine.

Analysis of urinary drugs of abuse by a multianalyte capillary electrophoretic immunoassay.

This paper characterizes a novel multianalyte competitive binding, electrokinetic capillary-based immunoassay for urinary methadone, opiates, benzoylecgonine (cocaine metabolite) and amphetamines. After incubation of 25 microliters urine with the reactants for several minutes in the presence of an internal standard, a small aliquot of the mixture is applied onto a fused-silica capillary and the unbound fluorescein labelled drug tracers are monitored by capillary electrophoresis with on-column laser induced fluorescence detection. The multianalyte assay is shown to be rapid, simple, quantitative, capable of recognizing urinary drug concentrations > or = 30 ng/ml and suitable for screening of patient urines. Data are demonstrated to compare well with those obtained by routine screening methods based on enzyme multiplied immunoassay techniques and fluorescence polarization immunoassays. The electrokinetic capillary assay has been validated via analysis of external quality control urines and confirmation analysis of patient urines using GC-MS.

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.

Chiral capillary electrophoresis as predictor for separation of drug enantiomers in continuous flow zone electrophoresis.

Separation of the enantiomers of chlorpheniramine and methadone in acidic buffers containing carboxymethyl-betacyclodextrin (CMCD) as chiral selector was investigated by capillary zone electrophoresis. For a range of pH and CMCD concentrations, the mobility difference and resolution of the enantiomers were determined. Then, conditions known to provide well resolved enantiomers and optimized chiral separation were applied to chiral continuous flow electrophoresis. In that approach, a thin film of fluid flowing between two parallel plates is employed as carrier for electrophoresis. The electrolytes and the sample are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. The number of pure enantiomeric fractions obtained by chiral continuous flow electrophoresis was found to be directly dependent on the enantiomeric mobility difference. For racemic chlorpheniramine separated in a betaine-acetic acid buffer at a total throughput of 5 mg/h, complete enantiomeric separation is shown to require a mobility difference of about 3 x 10(-9) m2/V s. Furthermore, compared to the previous investigations with hydroxypropyl-beta-cyclodextrin, CMCD was found to permit improved fractionation of methadone enantiomers. With a total racemic drug throughput of about 15 mg/h, continuous flow zone electrophoresis processing with CMCD as chiral selector is shown to have the potential of providing pure enantiomers on a mg/h scale. The results indicate that chiral capillary zone electrophoresis data can be employed as predictor for preparative scale chiral separations based upon continuous flow zone electrophoresis.

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.

Assessment of automated capillary electrophoresis for therapeutic and diagnostic drug monitoring: determination of bupivacaine in drain fluid and antipyrine in plasma.

In an effort to evaluate the use of electrokinetic capillary technology for therapeutic and diagnostic drug monitoring, samples were analysed batchwise with an automated, high-throughput capillary electrophoretic instrument coupled to an inexpensive PC data acquisition and evaluation system. Examples studied included the capillary electrophoretic (HPCE) determination of bupivacaine in drain fluid collected after pulmonary surgery and the micellar electrokinetic capillary chromatographic (MECC) determination of antipyrine in human plasma. Analyses for antipyrine could be accomplished without any sample pretreatment whereas bupivacaine required extraction prior to analysis. Antipyrine determination was effected through external calibration using either peak areas, relative peak areas or peak heights. The intraday and interday reproducibilities (n = 15) of the evaluated concentrations were 1.5-3% and 5-6%, respectively. For bupivacaine, determination based on internal and external calibration employing peak areas and peak heights was investigated. The intraday and interday reproducibilities (n = 5) of bupivacaine concentrations were about 1% and 2%, respectively, for internal calibration and both about 5% for external calibration. The electrokinetic capillary data compared well with data obtained by gas chromatography (bupivacaine) and high-performance liquid chromatography (antipyrine).

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.

Characterization of the stereoselective metabolism of thiopental and its metabolite pentobarbital via analysis of their enantiomers in human plasma by capillary electrophoresis.

Using capillary zone electrophoresis (CZE) with a 75 mM phosphate buffer at pH 8.5 containing 5 mM hydroxypropyl-gamma-cyclodextrin (OHP-gamma-CD) as chiral selector, the separation of the enantiomers of thiopental and its oxybarbiturate metabolite, pentobarbital, is reported. Enantiomer assignment was performed via preparation of enantiomerically enriched fractions using chiral recycling isotachophoresis (rITP) processing of racemic barbiturates and analysis of rITP fractions by chiral CZE and circular dichroism spectroscopy. Thiopental and pentobarbital enantiomers in plasma were extracted at low pH using dichloromethane and extracts were reconstituted in acetonitrile or 10-fold diluted, achiral running buffer. The stereoselectivity of the thiopental and pentobarbital metabolism was assessed via analysis of 12 plasma samples that stemmed from patients undergoing prolonged or having completed long-term racemic thiopental infusion. The data obtained revealed a modest stereoselectivity with R-(+)-thiopental/S-(-)-thiopental and R-(+)-pentobarbital/S-(-)-pentobarbital plasma ratios being < 1 (P < 0.05 compared to data obtained with racemic controls) and > 1 (P < 0.001), respectively. The total S-(-)-thiopental plasma concentration was found to be on average about 24% higher compared to the concentration of R-(+)-thiopental, whereas the total R-(+)-pentobarbital plasma level was observed to be on average 29% higher compared to the S-(-)-pentobarbital concentration.

Therapeutic drug monitoring of antiepileptics by capillary electrophoresis. Characterization of assays via analysis of quality control sera containing 14 analytes.

Quality assurance is an important aspect in therapeutic drug monitoring (TDM). Capillary electrophoresis (CE) assays for determination of (i) ethosuximide via direct injection of serum or plasma, (ii) lamotrigine after protein precipitation by acetonitrile and analysis of an aliquot of the acidified supernatant, and (iii) carbamazepine and carbamazepine-10,11-epoxide after solute extraction followed by analysis of the reconstituted extract are characterized via analysis of a large number of commercial quality control sera containing up to 14 analytes (9 of them are anticonvulsants) in sub-therapeutic, therapeutic and toxicologic concentration levels. CE data obtained in single determinations are shown to compare well with the spike values and the mean of data determined in other laboratories using immunoassays and/or high-performance liquid chromatography, values that are reported by the external quality control scheme. Carbamazepine and ethosuximide drug levels are also shown to agree well with those determined in our departmental drug assay laboratory using automated immunoassays. The presented data reveal the effectiveness of assay assessment via analysis of quality control sera and confirm the robustness of the assays for TDM in a routine setting.

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.