Analysis of fullerene-based nanomaterial in serum matrix by CE

With the increasing interest in using nanoparticles as vehicles for drug delivery and image contrast agents, there is a need to develop assays for their detection and quantitation in complex matrices to facilitate monitoring their biodistribution. In this study, we developed a CE approach for the analysis of two nanoparticles: carboxyfullerene (C3) and dendrofullerene (DF1) in both standard solutions and a serum matrix. These highly soluble, charged C(60) derivatives were characterized by CZE using either a bare or dynamically coated fused-silica capillaries. The resolution of both nanoparticles was slightly lower with the coated capillary; however, their migration times were faster. While separation of the DF1 nanoparticles using MEKC resulted in a greater number of observable peaks, the peak profile of C3 was basically unchanged regardless of whether SDS micelles were added to the running buffers or not. The MEKC and/or CZE assays were then used to quantitate the C3 and DF1 nanoparticles in spiked human serum samples. The quantitation of the nanoparticles was linear from 0-500 microg/mL with detection limits ranging from 0.5 to 6 microg/mL.     

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.     

Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis

Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or cardiovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time- and labour-consuming, and generally they have not been proven to be compatible with capillary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interferents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chromatography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated.     

Capillary zone electrophoresis with a dynamic double coating for analysis of carbohydrate-deficient transferrin in human serum. Precision performance and pattern recognition

Capillary zone electrophoresis (CZE) with a dynamic double coating permits the simultaneous, individual, quantitative determination of transferrin (Tf) isoforms in human serum and thus carbohydrate-deficient transferrin (CDT), the most specific marker available today for the detection of chronic, excessive alcohol intake. CZE of serum Tf was carefully evaluated using the P/ACE MDQ with fused-silica capillaries of 50 microm I.D. and 60.2 cm total length, the CEofix CDT kit and the instrumental conditions recommended by the kit manufacturer. The precision performance assessed over a 20-day period according to the internationally accepted NCCLS EP5-A guidelines revealed the CZE assay as being highly reproducible with within-run and total precision being dependent on the Tf isoform level and RSD values ranging between 2.2 and 17.6%. Inter-day RSD values for asialo-Tf were noted to be between 9.8 and 11.5% and for disialo-Tf between 3.8 and 8.6%, whereas those for CDT levels of 0.87 and 4.31% of total Tf were determined to be 8.6 and 3.4%, respectively. The RSD values for trisialo-Tf, tetrasialo-Tf, pentasialo-Tf and hexasialo-Tf were found to be between 0.4 and 4.1%. Tf patterns are recognized and identified via detection times of Tf isoforms (intra-day and inter-day RSD values < 1.0% and < 1.7%, respectively), immunosubtraction of Tf and enzymatic sequential cleavage of sialic acid residues. Furthermore, heterozygous Tf BC and Tf CD variants are assigned via spiking with a known mixture of Tf isoforms (e.g. the serum of a healthy Tf C homozygote). Among the non-Tf peaks monitored, the CRP peak detected shortly before disialo-Tf was identified by immunosubtraction and peak magnitudes were found to correlate well with immunochemically determined CRP serum levels. The CZE assay with dynamic double coating could thereby be shown to be sensitive enough to determine elevated CRP levels in human serum. Furthermore, unusual peaks in the gamma-region were identified by customary serum protein CZE, immunosubtraction CZE and immunofixation.     

Determination of ethyl sulfate in human serum and urine by capillary zone electrophoresis

The use of capillary zone electrophoresis (CZE) with indirect absorbance detection for the analysis of ethyl sulfate (EtS) in serum and urine was investigated. EtS is a direct metabolite of ethanol employed as marker for recent alcohol consumption. Fused-silica capillaries of 60 cm total length were either coated with cetyltrimethylammonium bromide (CTAB, 50 microm I.D. capillary) or poly(diallyldimethylammonium chloride) (PDADMAC, 100 microm I.D. capillary) to allow CZE analyses to be performed with reversed polarity. At pH 2.2 with a maleic acid/phthalic acid background electrolyte, both approaches provided reliable EtS serum levels down to 0.2 mg L(-1) (1.6 microM) for the analysis of solid-phase extracts that were prepared after chloride precipitation. Analysis of urines diluted to a conductivity of 5 S m(-1) and analyzed in the two capillary formats resulted in limits of quantification (LOQs) of 2 and 1 mg L(-1), respectively. With urines adjusted to 10 S m(-1) via dilution or condensation, an LOQ of 0.6 mg L(-1) (4.8 microM) was obtained in the CTAB coated capillary whereas in the PDADMAC-coated capillary of equal length not all matrix components were resolved from EtS. The developed assays are robust and suitable to monitor EtS in samples of individuals who consumed as little as one standard drink of an alcoholic beverage containing about 14 g of ethanol.     

Simultaneous multiresponse optimization applied to epinastine determination in human serum by using capillary electrophoresis

Experimental design and optimization techniques were implemented for the development of a rapid and simple capillary zone electrophoresis method (CZE) for the determination of epinastine hydrochloride in human serum. The effects of five factors were studied on the resolution between the peaks for the target analyte (epinastine hydrochloride) and lidocaine hydrochloride, used as internal standard, as well as on the analysis time. The factors were the concentration and pH of the buffer, the injection time, the injection voltage and the separation voltage. The separation was carried out by using an uncoated silica capillary with 50 microm i.d. and total length 64.5 cm (150 microm of path length) and UV detection (200 nm). Multiple response simultaneous optimization by using the desirability function was used to find experimental conditions where the system generates desirable results. The optimum conditions were: sodium phosphate buffer solution, 16.0 mmol L(-1); pH 8.50; injection voltage, 20.0 kV; injection time, 30 s; separation voltage, 26.7 kV. The method was confirmed to be linear in the range of 2.0-12 ng mL(-1). The injection repeatability of the method was evaluated by six injections at three concentration levels, while intra-assay precision was assessed by analysing a single concentration level, yielding a CV's of ca. 1% for standard and 2% for serum samples. Accuracy was evaluated by recovery assays and by comparing with an HPLC method, the results being acceptable according to regulatory agencies. The rudgeness was evaluated by means of an experimental Plackett-Burman design, in which the accuracy was assessed when small changes were set in the studied parameters. Clean-up of human serum samples was carried out by means of a liquid-liquid extraction procedure, which gave a high extraction yield for epinastine hydrochloride (93.00%).     

Determination of ethyl glucuronide in human serum by hyphenation of capillary isotachophoresis and zone electrophoresis

The determination of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in human serum by hyphenation of capillary ITP (CITP) and CZE is reported. For CITP step, 1 x 10(-2) M hydrochloric acid adjusted with epsilon-aminocaproic acid (EACA) to pH 4.4 was used as the leading electrolyte, and 1 x 10(-2) M nicotinic acid with EACA, pH 4.4, was used as the terminating electrolyte (TE). All electrolytes contained 0.2% hydroxypropylcellulose to suppress electroosmosis. In CITP, EtG was separated from fast serum macrocomponents chloride, phosphate, lactate, and acetate. Zones of microcomponents including EtG that migrated between acetate and nicotinate were forwarded to the second capillary filled with a BGE identical with the TE. Conductivity detection was used in the CITP step. Sensitive detection in the CZE step was performed using indirect spectrophotometric detection at 254 nm. The assay is based on a 1:5 dilution of serum with deionized water and has a concentration LOD for EtG in diluted sample of 9.8 x 10(-9) M. The method was used for the determination of EtG in sera of volunteers consuming alcohol.     

The analysis of human amniotic fluid using capillary electrophoresis

This study has investigated the composition of amniotic fluid (AF) using capillary electrophoresis (CE). A detailed optimisation investigation was undertaken to obtain the best resolution of the major peaks in amniotic fluid. In the final method, capillary zone electrophoresis (CZE) of AF was performed on a Hewlett Packard3D CE instrument using a fused-silica capillary of 44 cm total length (36 cm to the detector) with in internal diameter of 50 microm. The background electrolyte was 20 mM sodium tetraborate containing 0.8 mM EDTA adjusted to pH 9.0. AF was diluted 1 plus 1 with deionised water prior to hydrodynamic injection for 3 s at 50 mbar. The separation was performed at +22.5 kV and resulted in a current of 65 microA. The capillary temperature was 28 degrees C. Using this CZE method, some eight peaks were consistently resolved in AF samples and several other more transient peaks have been separated from AF in less than 10 min. A scheme for the identification of peaks once they had been separated was also developed. Four peaks have been identified as proteins, i.e., gamma-globulin, alpha1-antitrypsin, transferrin and albumin. Surprisingly, one major peak was shown to be the purine catabolite, xanthine.     

Evaluation and optimization of capillary zone electrophoresis with different dynamic capillary coatings for the determination of carbohydrate-deficient transferrin in human serum

Serum transferrin (Tf) comprises several isoforms with up to two complex oligosaccharide chains containing zero to eight sialic acid residues and neutral sugars. The major glycoform, known as tetrasialo-Tf, contains four sialic acid residues and accounts for about 80% of whole Tf in human serum. Carbohydrate-deficient transferrin (CDT) encompasses isoforms that are deficient in carbohydrate chains and consequently in sialic acid residues (including asialo-, monosialo- and disialo-Tf) and is a well known marker for chronic alcohol abuse. Recently capillary zone electrophoresis (CZE) has been reported as a tool extremely effective for the simultaneous, individual, quantitative determination of CDT isoforms. Three CZE methods that feature different dynamic capillary coatings were evaluated and optimized for CDT determination in human serum of alcohol abusers and control subjects. CZE separation was performed in alkaline borate buffers after serum sample saturation with iron, electropherograms were detected at 200 nm, data were evaluated as % area of disialo-Tf in relation to tetrasialo-Tf and peak identification was accomplished via relative migration times to tetrasialo-Tf, immunosubtraction and enzymatic sequential cleavage of sialic acid residues. Dynamic capillary coatings with diaminobutane, spermine and a double coating produced by commercially available proprietary agents were investigated and found to be suitable for determination of CDT in human serum. For all three approaches, best results were obtained in 50 microm I.D. fused-silica capillaries of 50 cm effective length and a capillary cartridge temperature of 20-25 degrees C. Using 3 mM 1,4-diaminobutane or 0.02 mM spermine in a borate-based running buffer of pH 8.3 provided data of remarkable similarity with resolution of di-, tri-, tetra- and pentasialo-Tf within 15-18 min. With the double coating, asialo-Tf and Tf isoforms with two to six sialic acid residues were baseline separated. Compared to the two amine-based procedures, the run times were found to be somewhat shorter, the detector signals higher, the applied power level significantly lower and the reproducibility better.     

Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing

A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 microm inner diameter (ID) were employed in CZE and 100 microm ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Resolution of indobufen enantiomers by capillary zone electrophoresis. Pharmacokinetic studies of human serum

A direct and stereospecific method was worked out to quantify indobufen enantiomers in human serum using capillary zone electrophoresis (CZE). The indobufen enantiomers and (+)-S-ketoprofen (internal standard, IS) were separated in a fused silica capillary, filled with heptakis 2,3,6-tri-O-methyl-beta-cyclodextrin as a chiral selector in a buffer of pH 5.0. Indobufen enantiomers and other non-steroidal anti-inflammatory drugs: flurbiprofen, ketoprofen and (+)-S-naproxen were also separated during one analytical run. UV absorbances of indobufen enantiomers were measured at 282 nm. Influence of temperature on resolution of the enantiomers, and the electrophoretic parameters: electrophoretic (muep) and electroosmotic (muEOF) mobilities were also determined. Validation of the method was carried out. Calibration curves of indobufen enantiomers were linear in the range of 0.2-20.0 microg/ml. Percent recovery of both enantiomers from acidified serum was calculated after extraction with methylene chloride. Intra- and inter-day measurement precision and accuracy were below 15.0%. Limits of quantitation and detection were also estimated. The elaborated method was tested in vivo after administration of a single dose of 200 mg rac-indobufen tablets to healthy volunteers. Calculated parameters confirmed usefulness of the method in human pharmacokinetic studies on indobufen enantiomers. The direct CZE method can provide an alternative to HPLC, where enantiomers used to be derivatised before determination.     

Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.     

Effects of lactate and acetate on the determination of serum ethyl glucuronide by CZE

The analysis of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in serum with an optimized CZE assay is reported. The method uses a 0.1-mm id fused-silica capillary of 50 cm effective length that is coated with linear polyacrylamide, a pH 4.4 nicotinic acid/epsilon-aminocaproic acid (EACA) BGE, reversed polarity and indirect analyte detection. The assay is based on a 1:1 dilution of serum with deionized water and has LODs for EtG, lactate and acetate of 3.8 x 10(-7) M, 2.60 x 10(-6 )M and 2.18 x 10(-6 )M, respectively. Separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) and its quantification are shown to be possible for a wide range of lactate (stacker) and acetate (destacker) concentrations, macrocomponents that have an impact on the CZE behavior of EtG and that change after intake of ethanol. The assay has been successfully applied to the analysis of EtG, lactate and acetate in (i) sera of volunteers that ingested known amounts of alcohol and (ii) samples of patients that were classified (teetotalers and social drinkers vs. alcohol abusers) via analysis of carbohydrate-deficient transferrin.     

A capillary zone electrophoresis method for determining N-acetylneuraminic acid in glycoproteins and blood sera

A simple capillary zone electrophoresis (CZE) method for the determination of the content of the major sialic acid form N-acetylneuraminic acid (Neu5Ac) in glycoproteins was established. The present method utilizes a simplified hydrolysis-purification procedure consisting of mild acid hydrolysis (25 mM trifluoroacetic acid for 2h at 80 degrees C) to release Neu5Ac and ultrafiltration on Centricon-3 membrane to remove the obtained asialoglycoproteins and other macromolecules present in biologic samples. Derivatization with benzoic anhydride at 80 degrees C for 20 min resulted in complete conversion of Neu5Ac to per-O-benzoylated Neu5Ac. CZE analysis was performed using the operating buffer 25mM phosphate, pH 3.5, containing 50% (v/v) acetonitrile as organic modifier at 30 kV, and detection of the per-O-benzoylated Neu5Ac at 231 nm. The method showed excellent repeatability (RDS<1.98%) and a linearity range from 5 microg/mL to 5mg/mL with a detection limit of 2 microM. Application of the method to microanalysis of human alpha(1)-acid glycoprotein and blood serum samples showed excellent agreement with previously published values, suggesting a high precision for the developed CZE method.     

A data treatment method for detecting fluorescence anisotropy peaks in capillary electropherograms

A data treatment method is presented to detect fluorescence anisotropy (FA) peaks in capillary electrophoresis electropherograms. The data treatment method converts plots of fluorescence anisotropy vs. time that contain no peaks that are distinguishable from the noise of the anisotropy background into plots that show distinct fluorescence anisotropy peaks. The method was demonstrated using laser-induced fluorescence anisotropy data from individual Aβ (1–42) aggregates separated using capillary electrophoresis. Applying this data treatment method enabled the detection of anisotropy peaks for individual Aβ aggregate fluorescence peaks that were not observed prior to the data treatment method. The data treatment method is not specifically designed for Aβ aggregate analysis or capillary electrophoresis, and it should be applicable to other applications and other separation methods with FA detection.     

Immunoassay of serum alpha(1)-antitrypsin by affinity-probe capillary isoelectric focusing using a fluorescence-labeled recombinant antibody fragment

An immunoassay for human alpha(1)-antitrypsin based on affinity-probe capillary isoelectric focusing (AP-CIEF) is described. The method is based on the separation of free and bound labeled antibody fragments by CIEF with laser-induced fluorescence detection. A recombinant Fab' fragment of mouse immunoglobulin G1 (IgG1) against human alpha(1)-antitrypsin was labeled with tetramethylrhodamine on the single cysteine residue at the hinge region. A single pI isomer of the labeled Fab' was purified by IEF in a slab of agarose gel and was then used as the affinity probe for alpha(1)-antitrypsin. The use of recombinant Fab' considerably simplified the labeling process. Although there was some difficulty in the quantification of native alpha(1)-antitrypsin with the affinity probe, carbamylation of the antigen sample by heat treatment with urea made the complex peaks appear reproducibly and more distinct, thus facilitating the identification and quantification of the complex. The system provided an almost linear response to a pure sample of alpha(1)-antitrypsin over a concentration range of 5-1000 ng/mL and the detection limit extended down to around 2 ng/mL. Alpha(1)-antitrypsin in a serum sample was determined using this system to be 1.2 mg/mL which is comparable to the reported value for the protein.     

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms.     

Analysis of 5-methyltetrahydrofolate in human blood, serum and urine by on-line coupling of capillary isotachophoresis and zone electrophoresis

The predominant circulating folate coenzyme in plasma/serum, 5‐methyltetrahydrofolate (5‐MTHF) was determined in human blood, serum and urine using a method based on the hyphenation of capillary ITP and zone electrophoresis. Measurements were done with a commercially available instrument for capillary isotachophoresis equipped with a column‐switching system. The choice of electrolytes was limited by the instability of 5‐MTHF and volatility of electrolytes for the potential coupling of the instrumentation with MS detector. To get an insight into the separability of individual sample components in an isotachophoretic analysis, we constructed zone existence diagrams for isotachophoretic electrolyte systems having a leading electrolyte composed of acetate and ammonium of pH 4.5 and 7.0, hydrocarbonate and ammonium, pH 7.8, chloride and ammonium, pH 5.6, and chloride and creatinine, pH 5.0, with hydroxide ion as the terminator. For isotachophoretic preseparation, the non‐volatile leading electrolyte with good buffering capacity composed of 1×10⁻² M HCl and 2.5×10⁻² M creatinine, pH 5.0, and terminating electrolyte composed of 1×10⁻² M MES was selected as the most suitable. The optimum BGE for CZE analysis from the standpoint of analyte stability, separability and volatility for MS coupling was 1×10⁻² M acetate with 3.5×10⁻² M ammonium, pH 4.5. Using this combination of electrolytes, LODs reached with optical detection at 220 nm were 1.6×10⁻⁷ M in human blood, 1.1×10⁻⁷ M in human serum and 4.7×10⁻⁶ M in human urine. Estimated content of 5‐MTHF in blood and serum samples of women following oral daily administration of 0.8 mg of folic acid was 1.2×10⁻⁵ and 5.8×10⁻⁶ M, respectively.     

Analysis of gadobenate dimeglumine by capillary zone electrophoresis coupled with electrospray-mass spectrometry

Highly reliable and accurate analytical methods are needed for the determination of magnetic resonance imaging (MRI) contrast agents in complex matrices of clinical interest. We demonstrate the reliability of capillary zone electrophoresis (CZE) coupled with electrospray ionization-mass spectrometry (ESI-MS) for the analysis of MultiHance (gadobenate dimeglumine), a gadolinium-based MRI agent. A sheath liquid interface connected the CE system with an electrospray mass spectrometer equipped with an ion-trap analyzer. CZE with ultraviolet (CZE-UV) and with mass detection (CZE-MS) were compared by analyzing gadobenate dimeglumine and the free ligand diluted in water and in biological fluids (i.e., human serum and urine). The optimization of some relevant CZE-MS parameters was accomplished, like CE buffer composition, sheath liquid composition and flow, and type and length of the separation capillary. CZE-UV was highly influenced by the biological sample components, which hindered a reliable quantification of both gadobenate and free ligand in serum and urine. In CZE-MS, on the other hand, the electrophoretic runs turned out to be independent of the clinical matrices, due to the informative potential and to the selectivity of MS detection.     

Application of capillary zone electrophoresis with off-line solid-phase extraction to in vitro metabolism studies of antifungals

A simple and robust solid-phase extraction (SPE) procedure for the cleanup and sample preconcentration of antifungals (ketoconazole, clotrimazole, itraconazole, fluconazole, and voriconazole) and their metabolites after incubation with human liver microsomes, as well as a simplified capillary zone electrophoresis (CZE) method for their rapid analysis, have been developed to determine the stability of these compounds in in vitro samples. Three different sample pretreatment procedures using SPE with reversed-phase sorbents (100 mg C8, 100 mg C18, and 30 mg Oasis-HLB) were studied. The highest and most reproducible recoveries were obtained using a 30 mg Oasis-HLB sorbent and methanol containing 2% acetic acid as eluent. Enrichment by a factor of about four times was achieved by reconstituting the final SPE eluates to a small volume. For the CZE separation, good separations without interfering peaks due to the in vitro matrix were obtained with a simple running electrolyte using a fused-silica capillary. The best separation for all components originated by each tested drug after incubation with human liver microsomes (unmetabolized parent drug and its metabolites) was obtained using a 0.05 M phosphate running buffer (pH 2.2) without additives. The effect of the injection volume was also investigated in order to obtain the best sensitivity. Performance levels in terms of precision, linearity, limits of detection, and robustness were determined.