Therapeutic drug monitoring of albendazole: determination of albendazole, albendazole sulfoxide, and albendazole sulfone in human plasma using nonaqueous capillary electrophoresis

A nonaqueous capillary electrophoretic method (NACE) for the fast determination of plasma levels of albendazole (ABZ), albendazole sulfoxide (ABZSO), and albendazole sulfone (ABZSO2) is described. The assay is based upon liquid/liquid extraction of these compounds using dichloromethane at pH 10.2 (recovery between 63 and 98%), followed by a NACE separation performed within 8 min employing a 0.036 M borate buffer (apparent pH 9.9) in a mixture of methanol and N-methylformamide (1:3) and on-column absorbance detection at 280 nm. Using 0.5 mL of plasma and extract reconstitution in 200 microL N-methylformamide, drug levels between 1.0-10 microM were found to provide linear calibration graphs. Intraday and interday imprecisions evaluated from peak area ratios (n = 5) were <10% and <12%, respectively. Corresponding imprecisions of detection times (n = 5) were <1% and <6%, respectively. The limit of detection (LOD) for ABZ, ABZSO and ABZSO2 was 8 x 10(-7) M. The reliability of the method developed was verified via analysis of 45 plasma samples obtained from patients treated with ABZ. Good agreement was obtained between the levels of ABZSO and those determined by routine HPLC. ABZ was found to be undetectable in all patient samples, whereas the levels of ABZSO2 were below or close to LOD.     

Enantioselective analysis of cetirizine in pharmaceuticals by cyclodextrin-mediated capillary electrophoresis

The present paper demonstrates the potential of cyclodextrin (CD)-mediated CE for the chiral analysis of a drug of zwitterionic nature, viz. cetirizine (CET). Various separation mechanisms were applied and several parameters affecting the separation were studied, including the type and concentration of chiral selector, coselector, and carrier ion, and pH of buffer. The optimal separation conditions were based on a medium buffer pH (approximately 5.2) (migration velocity of CET molecule was near to zero) and a highly substituted CD derivative, sulfated-beta-CD, serving as an analyte carrier in the anionic regime of the separation with suppressed electroosmotic flow. In this way, a baseline enantioseparation, reasonable separation efficiency, and short analysis time could be easily achieved. Acceptable validation criteria for sensitivity, linearity, precision, accuracy, and robustness were obtained using a hydrodynamically closed CE separation system. The proposed method was successfully applied to the enantioselective assay of CET in pharmaceutical formulations using fexofenadine (FEX) as an internal standard.     

Enantioselective analysis of primaquine and its impurity quinocide by capillary electrophoresis

A capillary electrophoretic (CE) method for the baseline separation of the enantiomers of primaquine diphosphate (PQ) and quinocide (QC) (a major contaminant) in pharmaceutical formulations is proposed. Both components were separated under the following conditions: 50 mm tris phosphate buffer (pH 3.0) containing 15 mm hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as background electrolyte; applied voltage, 16 kV; capillary temperature, 25 degrees C; detection wavelength, 254 nm; hydrostatic injection, 10 s. The separations were conducted using a 35 cm length and 50 microm i.d. uncoated fused silica capillary column. Under the optimized conditions, the components were successfully separated in about 5 min. Intraday precision of migration time and corrected peak areas when expressed as relative standard deviation ranged from 0.17 to 0.45 and 2.60 to 3.94%, respectively, while the interday precision ranged from 2.59 to 4.20 and 3.15 to 4.21%, respectively. After the validation exercise, the proposed method was applied for the determination of QC impurity in PQ formulations.     

Enantioselective analysis of primaquine and its metabolite carboxyprimaquine by capillary electrophoresis

An enantioselective capillary electrophoresis method for the simultaneous determination of primaquine (PQ) and carboxyprimaquine (CPQ) in rat liver mitochondrial fraction, suitable for in vitro metabolism studies is presented. The drug and metabolite were extracted by liquid-liquid extraction using ethyl ether. The enantiomers were resolved in a fused-silica capillary, 50 microm inside diameter (ID) and 24 cm of effective length, using an electrolyte solution consisting of a 20 mmol/L sodium phosphate solution, pH 3.0, and 10% w/v maltodextrin. Hydrodynamic sample injection was used with a 10 s injection time at 50 mbar pressure. The applied voltage was 22 kV and the capillary temperature was controlled at 20 degrees C. Detection was carried out at 264 nm. Under these conditions, the enantiomeric fractions of the drug and of its metabolite were analyzed within 6 min. The extraction procedure was efficient in removing endogenous interferents and low values (<10%) for the coefficients of variation and deviation from theoretical values were demonstrated for both within-day and between-day assays. The method described allows the determination of PQ and CPQ enantiomers as low as 100 and 40 ng/mL, respectively. After validation, the method was used for an in vitro metabolism study of PQ. The results showed that the enantiomer (-)-PQ was preferentially metabolized to (-)-CPQ.     

Determination of enterostatin in human cerebrospinal fluid by capillary electrophoresis with laser induced fluorescence detection

A capillary electrophoresis (CE) method with laser induced fluorescence (LIF) detection is described for quantification of enterostatin (Val-Pro-Asp-Pro-Arg), a pentapeptide involved in appetite regulation and insulin secretion. Enterostatin and two other pentapeptides belonging to the enterostatin family (i.e. Ala-Pro-Gly-Pro-Arg and Val-Pro-Gly-Pro-Arg) were well separated from each other. The peptides were fluorescently tagged with naphthalene-2,3- dicarboxaldehyde (NDA) and separated by micellar electrokinetic chromatography (MEKC) in the presence of methanol as an organic modifier. Coupled with LIF detection, the method had a detection limit of 4.8 x 10(-6) M for enterostatin. The relative standard deviation was to be 4.0% from five determinations of enterostatin at 37.2 microM in a human cerebrospinal fluid (CSF) sample. Twenty-three human CSF samples were analyzed. The level of enterostatin ranged from 24 microM to 51 microM with a mean (+/- SEM) value of 41.7 +/- 2.0 microM.     

Determination of enterostatin in human cerebrospinal fluid by capillary electrophoresis with laser induced fluorescence detection

A capillary electrophoresis (CE) method with laser induced fluorescence (LIF) detection is described for quantification of enterostatin (Val-Pro-Asp-Pro-Arg), a pentapeptide involved in appetite regulation and insulin secretion. Enterostatin and two other pentapeptides belonging to the enterostatin family (i.e. Ala-Pro-Gly-Pro-Arg and Val-Pro-Gly-Pro-Arg) were well separated from each other. The peptides were fluorescently tagged with naphthalene-2,3- dicarboxaldehyde (NDA) and separated by micellar electrokinetic chromatography (MEKC) in the presence of methanol as an organic modifier. Coupled with LIF detection, the method had a detection limit of 4.8 × 10^–6 M for enterostatin. The relative standard deviation was to be 4.0% from five determinations of enterostatin at 37.2 μM in a human cerebrospinal fluid (CSF) sample. Twenty-three human CSF samples were analyzed. The level of enterostatin ranged from 24 μM to 51 μM with a mean (± SEM) value of 41.7 ± 2.0 μM.     

A simple LC-MS/MS method to determine plasma and cerebrospinal fluid levels of albendazole metabolites (albendazole sulfoxide and albendazole sulfone) in patients with neurocysticercosis

The development and validation of an LC-MS/MS method for the simultaneous determination of albendazole metabolites (albendazole sulfoxide and albendazole sulfone) in human plasma are described. Samples of 200 μL were extracted with ether-dichloromethane-chloroform (60:30:10, v/v/v). The chromatographic separation was performed using a C(18) column with methanol-formic acid 20 mmol/L (70:30) as the mobile phase. The method was linear in a range of 20-5000 ng/mL for albendazole sulfoxide and 10-1500 ng/mL for albendazole sulfone. For both analytes the method was precise (RSD < 12%) and accurate (RE <7%) with high recovery (>90%). The method was successfully applied to determine the plasma and cerebrospinal fluid levels of albendazole sulfoxide and albendazole sulfone in patients with subarachnoidal neurocysticercosis who received albendazole at 30 mg/kg per day for 7 days. This LC-MS/MS method yielded a quick, simple and reliable protocol for determining albendazole sulfoxide and albendazole sulfone concentrations in plasma and cerebrospinal fluid samples and is applicable to therapeutic monitoring.     

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.     

Analysis of cerebrospinal fluid proteins by electrophoresis

The cerebrospinal fluid (CSF) is a specific ultrafiltrate of plasma, which surrounds the brain and spinal cord. The study of its proteins and their alteration may yield useful information on several neurological diseases. By using various electrophoretic separation techniques, several CSF proteins have been identified derived from plasma or from brain. Different one-dimensional methods, such as agarose gel electrophoresis and isoelectric focusing, are of similar value in identifying the non-specific oligoclonal bands, which are mainly helpful in the diagnosis of multiple sclerosis and other inflammatory diseases. Isoelectric focusing has a greater resolution than other one-dimensional methods, and it yields additional data about disease-associated proteins occurring in Alzheimer's disease, Huntington's chorea and amyotrophic lateral sclerosis. Silver-stained two-dimensional gels provide more information about the complex protein composition of CSF, particularly about proteins produced in the brain, such as apolipoprotein E and neuron-specific enolase. For the detection of oligoclonal antibodies, the investigation of protein changes revealed by Parkinson's disease, schizophrenia and Creutzfeldt-Jakob disease, and the analysis of CSF immune complexes, two-dimensional electrophoresis has a greater sensitivity.     

Enantioselective separation of rivastigmine by capillary electrophoresis with cyclodextrines

Different beta-cyclodextrines (beta-cyclodextrin, heptakis (2,3,6-tri-O-methyl)-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, and sulfated beta-cyclodextrin) were investigated as additives for the enantioselective separation of the R-form from rivastigmine ((S)-N-ethyl-3-[(1-dimethylamino) ethyl]-N-methyl-phenyl carbamate), contained as impurity in this drug, which is used for the treatment of Alzheimer's disease. Electrophoresis was performed in an acidic background electrolyte (triethanolammonium phosphate, 75 mM, pH 2.5) with various concentrations of the additives. The electrophoretic mobilities measured are typical functions of the additive concentrations, with complex constants (obtained by fitting the appropriate binding curve on the data) ranging between about 180 and 770 M(-1). Best separation was obtained with 7.5 mM beta-cyclodextrin, with the R-enantiomer as impurity migrating before the main S-compound. Intra- and interday reproducibility (n = 6 and 18, respectively) of migration time and peak area was in the low percentage range, linearity of the calibration line for the quantitation of the impurity in the range between 2.3 and 50 microg/ml, expressed by the linear correlation coefficient, was 0.9998. The limits of detection and quantitation, respectively, were 0.7 and 2.3 microg/ml, corresponding to 0.05 and 0.15%, m/m of the R- relative to the S-compound. Analysis can be carried out at 18 degrees C in less than 19 min.     

Enantioselective analysis of ofloxacin enantiomers by partial-filling capillary electrophoresis with bacteria as chiral selectors

The enantiomeric separation of ofloxacin enantiomers (OFLX) was achieved by using capillary electrophoresis partial-filled with Escherichia coli, Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive) as chiral selectors. Experimental parameters, including the concentration of background electrolyte, applied voltage, length of the filled bacteria plug, and pH of the buffer, were intensively investigated. Baseline separation of OFLX could be achieved within 7 min by using E. coli and P. aeruginosa as chiral selectors under the following conditions: electrophoretic buffer composed of 10 mM phosphate buffer at pH 7.4, applied voltage at 15 kV, and the bacteria (6.0 × 10(8) cells/mL) were injected into the capillary by gravity with injection height of 17.5 cm for 180 s (E. coli), 300 s (P. aeruginosa), and 300 s (S. aureus), respectively. E. coli and P. aeruginosa had better chiral selectivity for OFLX than S. aureus, which was in good agreement with OFLX having better antimicrobial activity on Gram-negative rather than Gram-positive bacteria. A novel method was developed for the enantioselective separation of enantiomers using bacteria as chiral selectors, which provides a new approach for antimicrobials enantioselective analysis, chiral pharmacodynamics, and chiral pharmacokinetics studies.     

Analysis of methotrexate and its eight metabolites in cerebrospinal fluid by solid-phase extraction and triple-stacking capillary electrophoresis

We establish a triple-stacking capillary electrophoresis (CE) separation method to monitor methotrexate (MTX) and its eight metabolites in cerebrospinal fluid (CSF). Three stacking methods with different mechanisms were combined and incorporated into CE separation. Complete stacking and sharp peaks were achieved. Firstly, the optimized buffer (60 mM phosphate containing 15% THF and 100 mM SDS) was filled into the capillary, which was followed by the higher conductivity buffer (100 mM phosphate, 2 psi for 45 s). The analytes extracted from CSF were injected at 2 psi for 99.9 s, which provided long sample zones and pH junction for focusing. Finally, the stacking step was performed by sweeping, and separation was achieved by micellar electrokinetic chromatography. The results of the linear regression equations indicated high linearity (r ≥ 0.9981) over the range of 0.5–7 μM. In intra- and inter-batch results, all data of RSD and RE were below 11%, indicating good precision and accuracy of this method. The LODs (S/N = 3) were 0.1 μM for MTX, 7-hydroxymethotrexate (7-OHMTX) and MTX-polyglutamates (MTX-(Glu) _{n, n = 2–5}), 0.2 μM for MTX-(Glu)₆, and 0.3 μM for 2,4-diamino-N ¹⁰-methylpteroic acid (DAMPA) and MTX-(Glu)₇. Our method was implemented for analysis of MTX and its metabolites in the CSF, and could be used for evaluation of its curative effects of acute lymphoblastic leukemia patients. The data were also confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results showed good coincidence.     

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.     

Enantioselective determination by capillary electrophoresis with cyclodextrins as chiral selectors

This review surveys the separation of enantiomers by capillary electrophoresis using cyclodextrins as chiral selector. Cyclodextrins or their derivatives have been widely employed for the direct chiral resolution of a wide number of enantiomers, mainly of pharmaceutical interest, selected examples are reported in the tables. For method optimisation, several parameters influencing the enantioresolution, e.g., cyclodextrin type and concentration, buffer pH and composition, presence of organic solvents or complexing additives in the buffer were considered and discussed. Finally, selected applications to real samples such as pharmaceutical formulations, biological and medical samples are also discussed.     

Pesticide analysis by capillary electrophoresis

In this work, a critical and updated revision of the current situation of the analysis of pesticides by Capillary Electrophoresis (CE) is presented. The review has been written in two main sections. The first one presents a thorough revision of the various offline and on-line sample preconcentration procedures that have been used in conjunction with CE to analyze these compounds. The second part reviews the various detection strategies (i.e., UV, LIF, MS, and electrochemical) and CE modes that have been applied to the analysis of pesticides. Future trends that can be expected from this hot research area are also discussed.     

Determination of amino acid neurotransmitters in human cerebrospinal fluid and saliva by capillary electrophoresis with laser-induced fluorescence detection

A CE-LIF detection method has been developed to identify and quantitate six amino acid neurotransmitters including glutamic acid, aspartic acid, gamma-aminobutyric acid, glycine, taurine, and glutamine. N-hydroxysuccinimidyl fluorescein-O-acetate, a fluorescein-based dye, was employed for the derivatization of these neurotransmitters prior to CE-LIF analysis. Different parameters which influenced separation and derivatization were optimized in detail. Under optimum conditions, linearity was achieved within concentration ranges of up to three orders of magnitudes for those analytes with correlation coefficients from 0.9989 to 0.9998. The LODs ranged from 0.06 nM to 0.1 nM, and are thus superior or equivalent to those previously reported in the literature using CE-LIF detection. The proposed method has been successfully applied to the determination of amino acid neurotransmitters in biological samples such as human cerebrospinal fluid and saliva with satisfactory recoveries.     

Capillary electrophoresis and hollow fiber liquid-phase microextraction for the enantioselective determination of albendazole sulfoxide after biotransformation of albendazole by an endophytic fungus

Hollow fiber liquid-phase microextraction and CE were applied for the determination of albendazole sulfoxide (ASOX) enantiomers in liquid culture medium after a fungal biotransformation study. The analytes were extracted from 1 mL of liquid culture medium spiked with the internal standard (rac-hydroxychloroquine) and buffered with 0.50 mol/L phosphate buffer, pH 10. The analytes were extracted into 1-octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the polypropylene hollow fiber. The electrophoretic separations were carried out in 0.05 mol/L tris(hydroxymethyl)aminomethane buffer, pH 9.3, containing 3.0% w/v sulfated-β-CD (S-β-CD) with a constant voltage of +15 kV and detection at 220 nm. The method was linear over the concentration range of 250-5000 ng/mL for each ASOX enantiomer. Within-day and between-day assay precision and accuracy for the analytes were studied at three concentration levels and the values of RSD% and relative error % were lower than 15%. The developed method was applied for the determination of ASOX after a biotransformation study employing the endophytic fungus Penicillium crustosum (VR4). This study showed that the endophytic fungus was able to metabolize the albendazole to ASOX enantioselectively. In addition, it was demonstrated that hollow fiber liquid-phase microextraction coupled to CE can be an excellent and environmentally friendly technique for the analysis of samples obtained in biotransformation studies.     

Determination of L-arginine in amniotic fluid by capillary zone electrophoresis

Conditions for the determination of L-arginine in a biological fluid using capillary zone electrophoresis were optimized. The effects of the pH of a running buffer, the time of sample injection into a capillary, the operating voltage, the temperature, and the wavelength on the results of the determination were studied. The procedure made it possible to evaluate the concentration of L-arginine over a range of 6–1000 μg/mL (c = 3 μg/mL; RSD = 2%). The duration of analysis including sample preparation was no longer than 30 min. The analysis of amniotic fluid samples in the cases of physiological pregnancy and pregnancy complicated by placental insufficiency demonstrated that the arginine content of amniotic fluid increased in the case of placental insufficiency, as compared to normal values.     

Rapid analysis of inflammatory cytokines in cerebrospinal fluid using chip-based immunoaffinity electrophoresis

A chip-based capillary electrophoresis system has been designed for rapidly measuring the concentrations of inflammatory cytokines in the cerebrospinal fluid of patients with head trauma. Isolation of the reactive cytokines was achieved by immunoaffinity capture using a panel of six immobilized antibodies, directly attached to the injection port of the chip. The captured cytokines were labeled in situ with a red light-emitting laser dye and electroeluted into the separation channel. Separation of the isolated cytokines was achieved by electrophoresis in under 2 min with quantification of the resolved peaks being achieved by on-line laser-induced fluorescence and integration of each peak area. Comparison of the results to commercially available high-sensitivity immunoassays demonstrates that the chip-based assay provides a fast, accurate procedure for studying the concentrations of these analytes in complex biological materials. The degree of accuracy and precision achieved by the chip-based CE is comparable to conventional immunoassays, the system being able to analyze between 10-12 samples per hour. With the ever-expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different analyses.