Determination of theophylline and its metabolites in rat liver microsomes and human urine by capillary electrophoresis

A capillary electrophoretic (CE) method has been developed for the determination of theophylline and all of its identified and potential metabolites. The method is rapid, resolves all metabolites to baseline, and requires extraction of only some biological fluids. It has been applied to the analysis of theophylline metabolism by hepatic microsomes from rats treated with a variety of inducing agents for different forms of P450 enzymes which metabolize theophylline, and to human urine spiked with theophylline and its metabolites, and concentrated by solid-phase extraction.     

High performance thin layer chromatography as a method to authenticate Hoodia gordonii raw material and products

Hoodia gordonii which contains the perceived active molecule, P57, is a plant used in many weight loss products that are highly susceptible to adulteration due to increased public demand and limited availability. Rapid and simple methods for authentication and confirmation of the presence of P57 are desirable for the quality control of H. gordonii raw material and products. High performance thin layer chromatography (HPTLC) analysis of several H. gordonii raw material samples collected from different locations as well as weight loss products was carried out on silica gel plates and developed in a mobile phase of toluene:chloroform:ethanol (40:40:12.5 v/v/v). Liebermann–Burchard (LB) reagent was used as derivatising agent since it is specific for glycosides and triterpenes (such as P57) and the plates were viewed under UV light at 365 nm. This method produced good separation of the compounds in complex mixtures with well-defined bands including that of the P57 band (R_f 0.42), which was confirmed by liquid chromatography coupled to mass spectrometry (LC–MS) after preparative thin layer chromatography (TLC). All the HPTLC results obtained for the H. gordonii raw materials and products were confirmed with quantitative LC–MS analyses, which confirmed the qualitative reliability of the HPTLC method. The HPTLC method was used successfully to develop a chemical fingerprint for authentication and reliable confirmation of the presence of P57 in H. gordonii raw material and products.     

High-speed microchip electrophoresis method for the separation of (R,S)-naproxen

In this research, a capillary electrophoretic method for the fast enantiomeric resolution of (R,S)-naproxen was investigated. Method development involved variation of applied potential, buffer concentration, buffer pH, and cyclodextrin concentration. The optimum electrophoretic separation conditions were 110 mM sodium acetate run buffer (pH 6.0), 30 mM methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, 25 degrees C. The total length of capillary was 48 cm, (50 microm I.D.) with ultra violet (UV) detection at 232 nm. Using these conditions, the number of theoretical plates was close to one million (896,000/m). The possibility of achieving a fast chiral separation of (R,S)-naproxen on a microchip of 2.5 cm in length was investigated. Complete enantiomeric resolution of naproxen was achieved in less than 1 min, on this microchip platform, with linear imaging UV detection. This system had the advantage of real-time separation monitoring, so that enantiomeric resolution could be visually observed, and high-speed chiral analysis was realized. The microchip electrophoresis (MCE) separation was compared with the capillary electrophoresis (CE) separation with regards to speed, efficiency, separation platform, and precision. This work highlights the potential of CE and MCE in future chiral separations.     

Direct determination of paracetamol and its metabolites in urine and serum by capillary electrophoresis with ultraviolet and mass spectrometric detection

The use of capillary electrophoresis (CE) for the determination of paracetamol and its main metabolites in urine and serum is described. Due to its high efficacy, CE enables the analysis of drugs directly in complex matrices. Thus, simple, rapid and reliable assays could be developed that made use of some of the main advantages of this analytical technique. In order to prevent the peaks from tailing, a water zone was injected behind the sample. Occasionally occurring peak splittings of paracetamol were investigated and methods to suppress these splittings were developed. Paracetamol, its main metabolites, paracetamol glucuronide, paracetamol sulfate as well as paracetamol cysteinate and paracetamol mercapturate, as metabolites of the oxidative pathway were identified in urine using diode-array detection and coupling of the CE instruments to electrospray–mass spectrometry. The assays were validated. Their usefulness was demonstrated by applying them to the analysis of urine and serum samples of healthy volunteers as well as to urine samples from children under anticancer therapy.     

Development of a capillary electrophoretic method for the separation of the macrolide antibiotics, erythromycin, josamycin and oleandomycin

Capillary electrophoresis (CE) provides high separation efficiency and thus is suitable for the analysis of complex mixtures of structurally similar compounds. The versatile nature of CE can be realised by controlling the chemistry of the inner capillary wall, by modifying the electrolyte composition and by altering the physicochemical properties of the analyte. A CE method has been developed for the separation of three macrolide antibiotics, erythromycin, oleandomycin and josamycin. A systematic approach was used to maximise analyte differential electrophoretic mobility by manipulating electrolyte pH, molarity and composition. In addition, some instrumental parameters such as capillary length and diameter and applied voltage were varied. The effect of the sample solvent and on-capillary concentrating techniques such as field amplified sample injection were investigated. Also, the influence of the injection of a water plug on the quantity of sample injected was demonstrated. The macrolides were completely resolved in less than 30 min in a 100 cm×75 μm I.D. fused-silica uncoated capillary with a Z-shaped flow cell of path-length 3 mm. The analysis was performed in a 75 mM phosphate buffer (pH 7.5) with 50% (v/v) methanol and an applied voltage of 25 kV was selected to effect the separation.     

Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization

Capillary electrophoresis (CE) represents a versatile platform for integrating sample pretreatment with chemical analysis because of its ability to tune analyte electromigration and band dispersion properties in discontinuous electrolyte systems. In this article, a single-step method that combines on-line sample preconcentration with in-capillary chemical derivatization is developed for rapid, sensitive, and enantioselective analysis of micromolar levels of amino acids that lack intrinsic chromophores by CE with UV detection. Time-resolved electrophoretic studies revealed two distinct stages of amino acid band narrowing within the original long sample injection plug occurring both prior to and after in-capillary labeling via zone passing by ortho-phthalaldehyde/N-acetyl l-cysteine (OPA/NAC). This technique enabled direct analysis of d-amino acids in a 95% enantiomeric excess mixture with sub-micromolar detection limits and minimal sample handling, where the capillary functions as a preconcentrator, microreactor, and chiral selector. On-line sample preconcentration with chemical derivatization CE (SPCD-CE) was applied to study the enantioselective amino acid flux in Escherichia coli bacteria cultures, which demonstrated a unique l-Ala efflux into the extracellular medium. New strategies for high-throughput analyses of low-abundance metabolites are important for understanding fundamental physiological processes in bacteria required for screening the efficacy of new classes of antibiotics as well as altered metabolism in genetically modified mutant strains.     

Enantioselective determination of zopiclone and its metabolites in urine by capillary electrophoresis

A method has been developed for the stereoselective determination of zopiclone and its main metabolites in urine. After the addition of the internal standard zolpidem the urine samples were extracted at pH 8 with chloroform-isopropanol (9:1). Analyses were carried out using capillary electrophoresis (CE) with β-cyclodextrin as the chiral selector. The analytes were detected using UV laser-induced fluorescence detection with a He-Cd laser operated at 325 nm. Urine samples of two volunteers after oral administration of 7.5 mg zopiclone were investigated. The S-(+)-enantiomers of zopiclone and its metabolites were always excreted in higher amounts than the R-(−)-enantiomers. With the same method the zopiclone enantiomers were quantified in saliva. Compared to high-performance liquid chromatography, the CE method is very fast and simple.     

Determination of three major catecholamines in human urine by capillary zone electrophoresis with chemiluminescence detection

A sensitive simple method is presented for the determination of three major catecholamines in human urine by capillary electrophoresis (CE) with on-line chemiluminescence (CL) detection. This was also the first time that the luminol-Ag(III) complex CL system was used for CE detection. This method was based on the enhancing effect of epinephrine (EP), norepinephrine (NE), and dopamine (DA) on the CL reaction between luminol and the Ag(III) complex in alkaline solution. The separations and determinations were performed with an electrophoretic buffer consisting of 20.0mM sodium borate and 1.0mM luminol. Under optimized conditions, the three catecholamines were baseline separated and detected in less than 8 min. Detection limits of 7.9 × 10(-8)M, 1.0 × 10(-7)M, and 6.9 × 10(-8)M were observed for EP, NE, and DA, respectively. Relative standard deviation (RSD) values for the peak height were 4.7% to 5.4% (n = 5). Our proposed method was applied to the determinations of the catecholamines in urine samples from 12 healthy individuals and 26 pheochromocytoma patients. Our results suggest that this method might be useful to monitor the catecholamine levels in routine screening and to diagnose pheochromocytoma.     

An enzyme immobilized microassay in capillary electrophoresis for characterization and inhibition studies of alkaline phosphatases

A simple and fast dynamically coated capillary electrophoretic method was developed for the characterization and inhibition studies of alkaline phosphatases (EC 3.1.3.1). An inside capillary enzymatic reaction was performed, and hydrolysis of the substrate 4-nitrophenylphosphate to 4-nitrophenol was measured. Fused-silica capillary surface was dynamically modified with polycationic polybrene coating. By reversal of the electroosmotic flow (EOF), analysis time was reduced up to 3 min as the anionic analytes were migrated in the same direction as the EOF. Furthermore, the sensitivity of the method was increased using electroinjection through high-field amplified injection. The baseline separation of 4-nitrophenylphosphate and 4-nitrophenol was achieved by employing 50 mM sodium phosphate as the running buffer (pH 8.5), 0.0025% polybrene, and a constant voltage of −15 kV, and the products were detected at 322 nm. Under the optimized conditions, a good separation with high efficiency was achieved. The new method was applied to study enzyme kinetics and inhibitor screening. K_m and K_i values obtained with the new CE method were compared well with the standard spectrophotometric method. Dynamic coating of fused-silica capillary gave fast and reproducible separation of substrate and product. The method can be easily optimized for inhibition studies of other isozymes.     

Site-specific sampling of taurine from rat brain followed by on-line sample pre-concentration, throughout in-capillary derivatization and capillary electrophoresis

A method of pinpoint-sampling followed by on-line pre-concentration of the sample, throughout in-capillary derivatization and capillary electrophoretic separation was evaluated by demonstrating the detection of taurine, 2-aminoethanesulfonic acid at a specific location of a rat brain. The direct sampling of taurine from the rat brain was accomplished by using voltage injection associated with two kinds of driving forces, electrophoretic flow and electroosmotic flow (EOF). The capillary tube (75 microm of inner diameter x 375 microm of outer diameter) of the capillary electrophoresis (CE) apparatus was already filled with a CE run buffer, viz., 40 mM phosphate-borate buffer (pH 10) containing 2mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as the derivatization reagent. One end of a platinum wire (0.5mm o.d.), used as the anode, and the inlet end of capillary tube (from which a 1.0 cm long polyimide coating was removed), were pricked down onto the surface of either the cerebrum or cerebellum of a rat brain at a location of very small dimension. When a low voltage (5 kV, 30s) was applied, taurine began to move from the rat brain into the capillary tube, and, simultaneously, electric focusing of taurine occurred by the action of "the pH-junction effect" at the inlet end of the capillary tube. After completing the injection, both the platinum wire and capillary tube were detached from the brain and dipped into the run buffer in an anode reservoir filed with the same solution as that in the capillary tube for the CE apparatus. Then, by applying a high voltage (20 kV) between the ends of the capillary tube, taurine was automatically derivatized to yield the fluorescent derivative, separated and detected with fluorescence (E(x)=340 nm, E(m)=455 nm) during migration throughout the capillary tube. The migration profiles obtained from cerebrum and cerebellum appeared to be different, but the peak corresponding to taurine was identified on both electropherograms. The efficacy of the present method including sample on-line pre-concentration prior to throughout in-capillary derivatization CE was first verified with several preliminary experiments by using samples of taurine in water, saline and a piece of 1.5% agar-gel block, as an alternate standard for the rat brain used in this study.     

Simultaneous determination of the main metabolites in rice leaves using capillary electrophoresis mass spectrometry and capillary electrophoresis diode array detection

The study of the metabolomics of primary metabolites using conventional chemical analyses requires a high-throughput method. Chemical derivatizations are a prerequisite for gas-chromatographic separation, and a large sample quantity is needed for liquid-chromatographic separation and nuclear magnetic resonance detection systems. Recently, we have developed a capillary electrophoresis-mass spectrometry (CE-MS) technology that can simultaneously quantify a large number of primary metabolites, using only a small quantity of samples, and without any chemical derivatizations. Parallel use of a capillary electrophoresis-diode array detector (CE-DAD) system further enables almost all water-soluble intracellular metabolites to be analyzed. We demonstrate, with rice leaves, a simple and rapid method of sample preparation for CE analysis; using this method, we have successfully measured the levels of 88 main metabolites involved in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, photorespiration, and amino acid biosynthesis.     

A chiral enantioseparation generic strategy for anti‐Alzheimer and antifungal drugs by short end injection capillary electrophoresis using an experimental design approach

The present study describes a generic strategy using capillary electrophoretic (CE) method for chiral enantioseparation of anti‐Alzheimer drugs, namely, donepezil (DON), rivastigmine (RIV), and antifungal drugs, namely, ketoconazole (KET), Itraconazole (ITR), fluconazole (FLU), and sertaconazole (SRT) in which these drugs have different basic and acidic properties. Several modified cyclodextrins (CDs) were applied for enantioseparation of racemates such as highly sulfated α, γ CDs, hydroxyl propyl‐β‐CD, and Sulfobutyl ether‐β‐CD. The starting screening conditions consist of 50‐mM phosphate‐triethanolamine buffer at pH 2.5, an applied voltage of 15 kV, and a temperature of 25°C. The CE strategy implemented in the separation starts by screening prior to the optimization stage in which an experimental design is applied. The design of experiment (DOE) was based on a full factorial design of the crucial two factors (pH and %CD) at three levels, to make a total of nine (3²) experiments with high, intermediate, and low values for both factors. Evaluation of the proposed strategy pointed out that best resolution was obtained at pH 2.5 for five racemates using low percentages of HS‐γ‐CD, while SBE‐β‐CD was the most successful chiral selector offering acceptable resolution for all the six racemates, with the best separation at low pH values and at higher %CD within 10‐min runtime. Regression study showed that the linear model shows a significant lack of fit for all chiral selectors, anticipating that higher orders of the factors are most likely to be present in the equation with possible interactions.     

Enantiomeric separation of N‐protected amino acids by non‐aqueous capillary electrophoresis using quinine or Tert‐butyl carbamoylated quinine as chiral additive

A capillary electrophoretic (CE) method for the enantioseparation of N‐protected chiral amino acids was developed using quinine and tert‐butyl carbamoylated quinine as chiral selectors added to nonaqueous electrolyte solutions (NACE). A series of various N‐derivatized amino acids were tested as chiral selectands, and in order to optimize the CE enantioseparation of these compounds, different parameters were investigated: the nature of the organic solvent, the combination of different solvents, the nature and the concentration of the background electrolyte, the selector concentration, the capillary temperature, and the applied voltage. The influence of these factors on the separation of the analyte enantiomers and the electroosmotic flow was studied. Generally, with tert‐butyl carbamoylated quinine as chiral selector, better enantioseparations were achieved than with unmodified quinine. Optimum experimental conditions were found with a buffer made of 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM tert‐butyl carbamoylated quinine in an ethanol–methanol mixture (60:40 v/v). Under these conditions, DNB‐Leu enantiomers could be separated with a selectivity factor (α) of 1.572 and a resolution (Rs) of 64.3; a plate number (N) of 127,000 and an asymmetry factor (As) of 0.93 were obtained for the first migrating enantiomer. Chirality 11:622–630, 1999. © 1999 Wiley‐Liss, Inc.     

Design and applications of a self-aligning liquid junction-electrospray interface for capillary electrophoresis-mass spectrometry

A simple self-aligning liquid junction-electrospray interface for coupling a capillary electrophoresis (CE) system to an atmospheric pressure ionization (API) mass spectrometer (CE-MS) was developed. In contrast to previous liquid junction interfaces, the self-aligning liquid junction interface simplifies the precise alignment of the CE capillary and the sprayer needle and uses a positive make-up flow. Several capillary CE-MS applications were run using both the self-aligning liquid junction interface and the widely used sheath flow interface for comparison purposes. The electrospray stability of the self-aligning liquid junction interface is consistently better even when non-volatile electrolyte solutions are used. At first, some band broadening was obtained with the self-aligning liquid junction interface. Experiments with different CE buffer systems suggested that this band broadening was caused by the materials used in constructing the interface. By using a more inert material for the sprayer needle, the self-aligning liquid junction exhibits excellent electrophoretic resolution, comparable sensitivity, and higher signal-to-noise ratios when run under the same conditions as the sheath flow interface.     

Capillary electrophoretic behavior of milk proteins in the presence of non-ionic surfactants

The electrophoretic behavior of α-lactalbumin and β-lactoglobulins (A and B) in the presence of non-ionic surfactants was studied by capillary electrophoresis (CE), using a poly(ethylene glycol) coated capillary column. The surfactants (Tween 20, Brij 35 and 78) were used as buffer additives. The separation is based on the difference in the strength of protein–surfactant association complexes, which results in a change of the effective electrophoretic mobility. The modification of the electrophoretic mobilities of proteins was observed and this variation permitted the estimation of the interaction between protein and surfactant. The effect of surfactant type and concentration on the migration behavior of protein in CE is discussed. It is found that the retention behavior of the milk proteins (the α-lactalbumin and the β-lactoglobulins) in CE is very different. The pH of the buffer and the surfactant type influence significantly the protein–surfactant interactions.     

Optimization of a capillary electrophoretic method to detect and quantify the Gly-Pro dipeptide in complex matrices from long term cultured prolidase deficiency fibroblasts

A capillary electrophoresis (CE) method has been developed and optimized for the detection of Gly-Pro dipeptide in complex biological samples: medium, cell layer and matrix obtained from long term cultured human fibroblasts of control and prolidase deficiency patients. The influence of different detergents in the sample preparation and electrophoretic conditions were investigated. The method was validated for cellular extracts with respect to limits of detection and quantitation, precision, linearity, accuracy and robustness. The optimized method was applied to real samples and revealed a significant increase of intracellular Gly-Pro dipeptide in prolidase deficiency fibroblasts with respect to the control.     

Simultaneous determination of 19 intracellular nucleotides and nucleotide sugars in Chinese Hamster ovary cells by capillary electrophoresis

Twelve nucleotides and seven nucleotide sugars in Chinese Hamster ovary (CHO) cells were determined by capillary electrophoresis (CE). The CE operating conditions of buffer pH value, ion strength, capillary temperature, polymer additive and cell extraction method were investigated. Optimum separation was achieved with 40 mM sodium tetraborate buffer (pH 9.5) containing 1% (w/v) polyethylene glycol (PEG) at a capillary temperature of 22 degrees C. Acetonitrile and chloroform were used for intracellular extraction. This method can be used to monitor intracellular carbohydrate metabolism.     

Capillary electrophoresis with laser-induced native fluorescence detection for profiling body fluids

Laser-induced native fluorescence detection with a KrF excimer laser (λ=248 nm) was used to investigate the capillary electrophoretic (CE) profiles of human urine, saliva and serum without the need for sample derivatization. All separations were carried out in sodium phosphate and/or sodium tetraborate buffers at alkaline pH in a 50-μm I.D. capillary. Sodium dodecyl sulfate was added to the buffer for micellar electrokinetic chromatography (MEKC) analysis of human urine. Although inherently a pulsed source, the KrF excimer laser was operated at a high pulse repetition rate of 553, 1001 or 2009 Hz to simulate a continuous wave excitation source. Detection limits were found to vary with pulse rate, as expected, in proportion to average excitation power. The following detection limits (3σ) were determined in free solution CE: tryptophan, 4 nM; conalbumin, 10 nM; α-lactalbumin, 30 nM. Detection limits for indole-based compounds and catecholamine urinary metabolites under MEKC separation conditions were in the range 7–170 nM.     

Picomolar analysis of flavins in biological samples by dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection

Sensitive capillary electrophoresis (CE) methods are required for emerging areas of biochemical research such as the metabolome. In this report, dynamic pH junction-sweeping CE with laser-induced fluorescence (LIF) detection is applied as a robust single method to analyze trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein, and biological fluids. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of Bacillus subtilis. Significant differences in flavin concentration were measured in cell extracts derived from either glucose or malate as the carbon source in the culture media. Quantitative assessment of FAD and FMN content from selected flavoenzymes was demonstrated after heat denaturation to release noncovalently bound coenzymes and deproteinization. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration. Picomolar detectability of flavins by CE-LIF detection was realized with on-line preconcentration (up to 15% capillary length used for injection) by dynamic pH junction-sweeping, resulting in a limit of detection (S/N = 3) of about 4.0 pM for FAD and FMN. This represents over a 60-fold improvement in concentration sensitivity compared to those of previous techniques using conventional injections. The method was validated in terms of reproducibility, sensitivity, linearity, and specificity. Flavin analysis by dynamic pH junction-sweeping CE-LIF offers a simple, yet sensitive way to analyze trace levels of flavin metabolites from complex biological samples.     

Simultaneous enantioselective separation of azelastine and three of its metabolites for the investigation of the enantiomeric metabolism in rats. I. Liquid chromatography-ionspray tandem mass spectrometry and electrokinetic capillary chromatography

Enantioselective separation methods and the enantioselective determination of the anti-allergic drug azelastine and of three of its main phase I metabolites in a biological matrix underwent chromatographic and electrophoretic investigations. An enantioselective assay of a coupling of HPLC using a beta-cyclodextrin chiral stationary phase to ionspray tandem mass spectrometry is presented. Additionally, this assay is compared to another enantioselective assay using electrokinetic capillary chromatography with beta-cyclodextrin and carboxymethyl-beta-cyclodextrin in polyacrylamide-coated capillaries. For capillary electrophoresis (CE) the importance of polyacrylamide coating for the validation of this separation method is highlighted. Extracted rat plasma samples of enantioselective metabolism studies were measured by both validated assays. Differences in the pharmacokinetics and pharmacodynamics were evaluated for the main substance azelastine and its main metabolite demethylazelastine. So, a first hint about the enantioselectivity of biotransformation of azelastine in rats was seen after oral application of either enantiomer or the racemate to rats.