Study of recombinant cytochrome P450 2C9 activity with diclofenac by MEKC

Cytochrome P450 2C9 (CYP2C9) is one of the most important isoforms in human liver involved in the metabolism of a large number of therapeutic agents. The aim of this paper is to demonstrate the applicability of CE for the determination of the enzymatic activity of CYP2C9 with diclofenac as a probe substrate. MEKC with SDS as a pseudostationary phase was used for this purpose. Compared to other assays, the MEKC-based method is rapid, can be automated and requires only a small quantity of enzymes and substrate. Moreover, the enzymatic reaction can be monitored with high sensitivity and repeatability even when the reaction mixture is used for the analysis without any pretreatment. The kinetic study on the given enzymatic reaction was also performed since the basic characterization of drug biotransformation generally begins with the enzyme kinetic analysis of metabolite formation. As a result, the Michaelis constant and maximum reaction velocity were evaluated, the values 3.44 +/- 0.45 microM and 19.78 +/- 0.76 nmol min(-1) nmol(-1), respectively, were in agreement with the literature data. On the other hand, a slight deviation from typical Michaelis-Menten kinetics with a weak positive cooperativity was found at diclofenac concentrations below 2 microM. The same atypical kinetic behavior of CYP2C9 was also observed by other authors.     

Development of an ultrasensitive stacking technique for 5‐nitroimidazole determination in untreated biological fluids by micellar electrokinetic chromatography

Cation‐selective exhaustive injection and sweeping followed by a MEKC separation is evaluated for the sensitive analysis of 5‐nitroimidazoles in untreated human serum and urine. Deproteinized serum and urine samples were diluted 76 and 143 times, respectively, in a low‐conductivity solvent (5.00 mM orthophosphoric acid containing 5.0% v/v methanol). Samples were electrokinetically injected at 9.8 kV for 632 s in a previously conditioned fused‐silica capillary (65.0 cm × 50 μm id). Separation was performed at –30 kV and 20°C using 44 mM phosphate buffer (pH 2.5), 123 mM SDS, and 8% v/v tetrahydrofurane as BGE. Signals were monitored at 276 nm and peak area was selected as analytical response. Good linearity (R² ≥ 0.988) and LODs lower than 1.5 and 1.8 μg/mL were achieved in serum and urine, respectively.     

Determination of malachite green in fish water samples by cloud‐point extraction coupled to cation‐selective exhaustive injection and sweeping‐MEKC

We have employed a high‐sensitivity off‐line coupled with on‐line preconcentration method, cloud‐point extraction (CPE)/cation‐selective exhaustive injection (CSEI) and sweeping‐MEKC, for the analysis of malachite green. The variables that affect CPE were investigated. The optimal conditions were 250 g/L of Triton X‐100, 10% of Na₂SO₄ (w/v), heat‐assisted at 60°C for 20 min. We monitored the effects of several of the CSEI‐sweeping‐MEKC parameters – including the type of BGE, the concentrations of SDS, the injection length of the high‐conductivity buffer, and the injection time of the sample – to optimize the separation process. The optimal BGE was 50 mM citric acid (pH 2.2) containing 100 mM SDS. In addition, electrokinetic injection of the sample at 15 kV for 800 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancement for malachite green was 1.9×10⁴ relative to CZE; the coefficients of determination exceeded 0.9928. The LOD, based on an S/N of 3:1, of CSEI‐sweeping‐MEKC was 0.87 ng/mL; in contrast, when using off‐line CPE/CSEI‐sweeping‐MEKC the sensitivity increased to 69.6 pg/mL. This proposed method was successfully applied to determine trace amounts of malachite green in fish water samples.     

Simultaneous separation of jasmonic acid conjugates with amino acids by MEKC

Jasmonic acid (JA) conjugates with amino acids (AAs) are a group of plant hormone in the family of jasmonates. The separation of stereoisomers of JA‐AA conjugates is a very challenging work since these stereoisomers have similar chromatographic and electrophoretic behavior. Simultaneous separation of ten (±)‐JA conjugates with five AAs including l‐ Tyr (tyrosine), l‐ leucine, l‐ Ile (isoleucine), l‐ valine, and l‐ phenylalanine and their stereoisomers has been achieved by MEKC with diode array detector in this work. Optimum separation of the analytes was obtained on a 61.5 cm × 75 μm id capillary using a running buffer containing 80 mM SDS and 50 mM phosphates (pH 7.0) at +18 kV applied voltage and capillary temperature of 35°C. Ten stereoisomers of JA conjugates with five AAs are completely separated in 13 min. The RSDs of the migration times and peak areas of the ten stereoisomers were in the range of 0.48–1.03% and 1.03–2.07%, respectively. In the tested concentration range, good linear relationships (correlation coefficients above 99%) between peak areas and concentrations of the analytes were observed. The proposed method has been successfully applied to the analysis of spiked rice floret sample and original reaction solution of (±)‐JA‐Ile conjugate and (±)‐JA‐Tyr conjugate. The recoveries ranged from 91.7 to 107.6% for the rice floret sample and 92.9 to 107.2% for the original reaction solution.     

Preliminary study on the monitoring of glutathione S-tranferase activity toward styrene oxide by electromigration methods

A new method has been developed for the monitoring of glutathione S-tranferase (GST) detoxification activity toward styrene oxide (SO). The enzymatic reaction was carried out directly in a thermostatted autosampler vial and the formation of conjugates between glutathione (GSH) and SO was monitored by sequential MEKC runs. The determinations were performed in a 50-microm fused silica capillary using 50 mM SDS in 20 mM phosphate 20 mM tetraborate buffer (pH 8.3) as a background electrolyte; separation voltage 28 kV (positive polarity), temperature of capillary 25 degrees C, and detection at 200 nm. The method is rapid, amenable to automation, and requires only small amounts of samples, which is especially important in the case of GST isoenzyme analyses.     

Determination of lumiracoxib by a validated stability‐indicating MEKC method and identification of its degradation products by LC‐ESI‐MS studies

A stability‐indicating MEKC method was developed and validated for the analysis of lumiracoxib (LMC) in pharmaceutical formulations using nimesulide as the internal standard (IS). Optimal conditions for the separation of LMC and degradation products were investigated. The method employed 50 mM borate buffer and 50 mM anionic detergent SDS solution at pH 9.0. MEKC method was performed on a fused‐silica capillary (50 μm id; effective length, 40 cm) maintained at 30°C. The applied voltage was 20 kV and photodiode array (PDA) detector was set at 208 nm. The method was validated in accordance with the International Conference on Harmonisation requirements. The stability‐indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using PDA detection. The degradation products formed under stressed conditions were investigated by LC‐ESI‐MS and the two degraded products were identified. MEKC method was linear over the concentration range of 5–150 μg/mL (r²=0.9999) of LMC. The method was precise, accurate, with LOD and LOQ of 1.34 and 4.48 μg/mL, respectively. The robustness was proved by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of LMC in tablets to support the quality control.     

MEKC Determination of Nilutamide in Human Serum Using Sweeping in High Salt Sample Matrix

The determination of the antiandrogen drug nilutamide in human serum by MEKC using flutamide as an internal standard is described. Several parameters influencing the separation, such as the running electrolyte composition concerning the micelle concentration and pH, are discussed. MEKC separation was achieved within 7 min using 50 mM sodium borate pH 9.0 with the addition of 50 mM sodium dodecylsulfate at +20 kV. The proposed method was applied to determination of nilutamide in spiked human serum samples after protein precipitation with acetonitrile. The increasing of sensitivity for determination of nilutamide in serum was done by sweeping in a high salt concentration sample matrix when the injection of a larger volume of sample diluted in 150 mM NaCl was applied. The limit of detection after the preconcentration step for nilutamide was 26 ??g L^?1.     

Determination of doxycycline in pharmaceuticals and human urine by micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MEKC) was performed at 25 °C and 30 kV (under a pressure of 15 mbar), using 30 mM borate buffer containing 60 mM sodium dodecysulfate (SDS) and 5% (v/v) methanol as background electrolyte (pH 9.0) to determine doxycycline. UV detection was at 350 nm. The method was shown to be specific, accurate (recovery was 100.3 ± 1.0%), linear over the tested range (correlation coefficient 0.9995) and precise (RSD <1.9%). The method was used to determine doxycycline in tablets, capsules and human urine after oral application.     

Sample stacking by field-amplified sample injection and sweeping for simultaneous analysis of acidic and basic components in clinic application

In this study, online sample concentration method, which coupled field-amplified sample injection (FASI) and sweeping technology with micellar electrokinetic chromatography (MEKC), was used to detect and analyze acidic and basic components in a single run. In order to concentrate the acidic and basic components simultaneously in a single run sweeping step, a combination of successive anion- and cation-selective injections were used. Before sample loading, a rinse buffer containing 50 mM Tris buffer (pH 3) with 41% MeOH and 0.1% polyethylene oxide (PEO) was injected in order to suppress the electroosmotic flow (EOF). Sample loading of anionic components was achieved by electrokinetic injection at a negative voltage of -2.5 kV for 80 s, and then the cationic components were injected at a positive voltage of +5 kV for 120 s. Finally, sweeping with SDS micelles from the separation buffer (25 mM Tris buffer with 60 mM SDS, pH 3) was performed at a negative voltage of -20 kV. This capillary electrophoretic methodology was applied to the quantification of acidic and basic drugs in commercial tablets and in plasma samples. The precision and accuracy of the proposed method at different concentrations ranging from high, medium, to low were evaluated on spiked plasma samples. The intra and interday precision and accuracy values at three concentrations were all below 6.1%. The method was also successfully applied to monitor the tested drugs in the plasma of nine elderly cardiovascular and/or Alzheimer's disease patients after oral administration of the commercial products.     

Determination of 5-nitroimidazoles and metabolites in environmental samples by micellar electrokinetic chromatography

A method based on micellar electrokinetic chromatography (MEKC) with UV detection has been developed for the determination of nine 5-nitroimidazoles (5-NDZs), including metabolites in river water samples. Due to the relative insensitivity of UV detection in MEKC, a solid-phase extraction (SPE) method has been proposed that preconcentrates water samples fiftyfold and cleans them up off-line. An on-line preconcentration approach based on sweeping and the use of an extended light path fused-silica capillary (64.5?cm?×?50?μm i.d., 56?cm effective length) was also found to improve the sensitivity of the method. Separation was carried out in <21?min using 20?mM phosphate buffer (pH 6.5) and 150?mM SDS as the background electrolyte (BGE). The temperature of the capillary was kept constant at 20°C, a voltage of 25?kV was applied (normal mode), and a detected wavelength of 320?nm was utilized. Hydrodynamic injection (50?mbar for 15?s) of the samples, which were dissolved in 20?mM phosphate (pH 6.5), was employed. The limits of detection were lower than 1.1?μg?L(-1). Recoveries of >80% from spiked river water samples were obtained for most of the analytes at three different concentration levels with acceptable precision. This method could provide an efficient and economical alternative to the use of chromatographic methods to monitor nitroimidazole residues, thus supplementing the relatively few methods available for the analysis of these compounds in environmental samples.     

Green methodology based on dispersive liquid–liquid microextraction and micellar electrokinetic chromatography for 5‐nitroimidazole analysis in water samples

Dispersive liquid–liquid microextraction has been proposed as an extraction technique combined with micellar electrokinetic chromatography (MEKC) for the analysis of eight 5‐nitroimidazole compounds, including some metabolites, in water samples. Determination has been carried out using a diode array detector, employing 20 mM sodium phosphate and 150 mM SDS as separation buffer. Separation has taken place under a voltage of 25 kV and a temperature of 20°C. Samples were prepared in a buffer without micelles and they were hydrodynamically injected at 50 mbar for 25 s, producing a sweeping effect on the analytes for increasing sensitivity. Different factors involved in the dispersive liquid–liquid microextraction procedure were optimized, such as sample pH, nature, and volume of extraction and dispersive solvents in the mixture, percentage of NaCl added to sample and shaking time after the injection of the extraction and dispersive solvents. The method was characterized for water samples, achieving detection limits lower than 2.4 μg/L. Trueness was checked in river, tap, and bottled water. Dispersive liquid–liquid microextraction combined with MEKC constitutes an easy, cheap, and green alternative for 5‐nitroimidazole analysis in environmental water samples.     

Analysis of Carbamazepine in Tablet and Human Serum by Sweeping‐Micellar Electrokinetic Chromatography Method

Abstract

A sensitive and simple micellar electrokinetic chromatography (MEKC) method was developed for the determination of the antiepileptic drug carbamazepine (CBZ) using a sweeping on‐line concentration method with photodiode array detection. The effect of pH, concentration of the running buffer solution, organic modifier, applied voltage and injection time on the concentration efficiency and separation was investigated. An untreated fused‐silica capillary was used (50 cm; effective length, 40 cm, 75 µm i.d.) for the analysis. The background solution (BGS) was 50 mmol · L^?1 NaH₂PO₄ (pH 3.0) containing 100 mmol · L^?1 SDS and 20% acetonitrile (5.82 ms · cm^?1) with an applied voltage of ?20 kV at 25°C. Sample introduction was performed at 0.5 psi for 90 s with diode array detection at 214 nm. For the method, the calibration curve was linear over a range of 0.5–40 µg · mL^?1 for CBZ with a correlation coefficient of 0.998. The detection limit (S/N=3∶1) of CBZ was 0.10 µg · mL^?1. About 100‐fold improvement in concentration sensitivity was achieved in terms of peak height by the sweeping method compared to conventional injection method. The sweeping‐MEKC method has been successfully applied to the analysis of CBZ in tablet and human serum.     

Selective extraction of alkaloids in human urine by on-line single drop microextraction coupled with sweeping micellar electrokinetic chromatography

A novel method of on-line single drop microextraction (SDME) coupled with sweeping micellar electrokinetic chromatography (MEKC) for the selective extraction and dual preconcentration of alkaloids was developed. In this technique, analytes of three alkaloids were firstly extracted from 4.0 mL basic aqueous sample solution (donor phase, 500 mM NaOH) into a layer of n-octanol at temperature 30 °C with the stirring rate of 1150 rpm, then back-extracted into the acidified aqueous acceptor (acceptor phase, 50 mM H₃PO₄) suspended at the tip of a capillary at 650 rpm. Then, the aqueous acceptor was introduced into capillary by hydrodynamic injection with a height difference of 15 cm between the inlet and outlet of capillary for 300 s, and analyzed directly by on-line sweeping MEKC. With the selective SDME, we were able to extract three alkaloids without any interfering components in human urine samples. Under the optimum conditions, the proposed method achieved limits of detections (LOD) of between 0.2 ng mL⁻¹ and 1.5 ng mL⁻¹ with 1583–3556-fold increases in detection sensitivity for three analytes, which indicated that it was a promising method for analysis of alkaloids in human urine.     

Screening of aromatase inhibitors in traditional Chinese medicines by electrophoretically mediated microanalysis in a partially filled capillary

An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in‐capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced β‐nicotinamide adenine dinucleotide 2′‐phosphate hydrate dissolved in the reaction buffer was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer, enzyme, and substrate solution. The reaction was initiated with a voltage of 5 kV applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced β‐nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver–Burk equation, the Michaelis–Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.     

Analysis of red inks by micellar electrokinetic capillary chromatography with laser‐induced fluorescence detection

A combination of MEKC with a—highly sensitive but not commonly used—LIF detector was tested regarding the possibility of differentiation of red inks. The separation process was conducted in a fused silica capillary (id 50 μm, 60/50 cm total/effective length) in BGE consisting of 40 mM sodium borate, 20 mM SDS and 10% v/v ACN with 30 kV applied. The optimized temperature of storage and capillary was 10 and 25°C, respectively. The samples were prepared using 20 dots (ø 0.5 mm), extracted in 35 μL BGE and diluted with 30 μL of water. The proposed method showed excellent repeatability and reproducibility (RSD (t_m) < 0.8 and < 2.5%, respectively). It was applied to group identification and differentiation of different brands, models, and batches of red printing, stamp, and pen inks. It was demonstrated that differentiation can be performed effectively on the basis of migration times and ratios of peak areas. The high efficiency of the developed method was indicated by discriminating power ranging from 87.3 to 98.8%, for stamp and pen inks, respectively. The results showed that the proposed procedure can be valuable for an objective examination of the red parts of questioned documents.     

Strategies for the on-line preconcentration and separation of hypolipidaemic drugs using micellar electrokinetic chromatography

Three strategies were investigated for the simultaneous separation and on-line preconcentration of charged and neutral hypolipidaemic drugs in micellar electrokinetic chromatography (MEKC). A background electrolyte (BGE) consisting of 20 mM ammonium bicarbonate buffer (pH 8.50) and 50 mM sodium dodecyl sulfate (SDS) was used for the separation and on-line preconcentration of the drugs. The efficiencies of sweeping, analyte focusing by micelle collapse (AFMC), and simultaneous field-amplified sample stacking (FASS) and sweeping, were compared for the preconcentration of eight hypolipidaemic drugs in different conductivity sample matrices. When compared with a hydrodynamic injection (5 s at 50 mbar, 0.51% of capillary volume to detection window) of drug mixture prepared in the separation BGE, improvements of detection sensitivity of 60-, 83-, and 80-fold were obtained with sweeping, AFMC and simultaneous FASS and sweeping, respectively, giving limits of detection (LODs) of 50, 36, and 38 μg/L, respectively. The studied techniques showed suitability for focusing different types of analytes having different values of retention factor (k). This is the first report for the separation of different types of hypolipidaemic drugs by capillary electrophoresis (CE). The three methods were validated then applied for the analysis of target analytes in wastewater samples from Hobart city.     

Cation-selective exhaustive injection and sweeping MEKC for direct analysis of methamphetamine and its metabolites in urine

Direct analysis of methamphetamine, amphetamine, and p-hydroxymethamphetamine in urine was achieved by cation-selective exhaustive injection and sweeping micellar EKC. A bare fused-silica capillary (40 cm, 50 microm id) was filled with phosphate buffer (80 mM, pH 3, containing 20% ACN). Then a high-conductivity buffer (100 mM phosphate, pH 3; 6.9 kPa for 2.5 min) was injected. Samples were loaded using electrokinetic injection (10 kV, 600 s) which created long zones of cationic analytes. To enhance sensitivity by sweeping, the stacking step was performed using a phosphate buffer (50 mM, pH 3, containing 20% ACN and 100 mM SDS) at -20 kV before separation by MEKC. This method was capable of detecting the analytes at ppb levels. The calibration plots were linear (r(2) >or= 0.9948) over a range of 100-5000 ng/mL for methamphetamine, and 100-2000 ng/mL for amphetamine and p-hydroxymethamphetamine. The LODs (S/N = 3) were 20 ng/mL for methamphetamine, and 15 ng/mL for amphetamine and p-hydroxymethamphetamine. The method was applied to analysis of 14 urine samples of addicts and is suitable for screening suspected samples for forensic purposes. The results showed good agreement with fluorescence polarization immunoassay and GC-MS.     

Study of aldehyde oxidase by micellar electrokinetic chromatography separation of O6‐benzylguanine and 8‐oxo‐O6‐benzylguanine

A separation method for O⁶‐benzylguanine (O⁶‐BG) and 8‐oxo‐O⁶‐benzylguanine (8‐oxo‐O⁶‐BG) is developed by using MEKC. This study includes the optimization of separation and incubation parameters for both off‐line and on‐line procedures. The BGE consisted of 25 mM sodium phosphate buffer‐methanol (70:30, v/v), apparent pH 7.4, in which SDS and methyl‐β‐cyclodextrin were dissolved yielding final concentrations of 50 and 15 mM, respectively. Separations were performed at 15 kV using an untreated fused‐silica capillary (40 cm length, effective length is 30 cm) with the detection wavelength at 195 nm. The capillary was kept at 15°C. Good performances were demonstrated for the repeatability and linearity. The LOQ was determined to be 14 μM for 8‐oxo‐O⁶‐BG (S/N = 10). The accuracy values showed a bias of +7.9% for 50 μM and –7.0% for 100 μM. Premix and transverse diffusion of laminar flow profiles (TDLFP) methods were used for on‐line mixing and reaction of the substrate O⁶‐BG with aldehyde oxidase. Both procedures were successful in mixing as well as subsequent separation of the substrate and the metabolite, while the repeatability of TDLFP (14.7% (n = 3)) was much better than the premix technique.     

Analysis of artichoke (Cynara cardunculus L.) extract by means of micellar electrokinetic capillary chromatography

The main constituents of artichoke extract were separated by micellar electrokinetic chromatography (MEKC), using a buffer consisting of 100 mM sodium dodecyl sulfate (SDS) in 20 mM sodium dihydrogen phosphate, 20 mM disodium tetraborate (pH 8.6) as background electrolyte. Optimum separation voltage of 28 kV (positive polarity) and a capillary temperature of 25 degrees C gave the best analysis. The UV detection was performed at 200 nm. The method was successfully used to analyze plant and drug samples as well as for the study of artichoke antioxidant activity. The quantitative MEKC results were in good agreement to those obtained previously by reversed-phase high-performance liquid chromatography (RP-HPLC).     

Development of a CD‐MEKC method for investigating the metabolism of tamoxifen by flavin‐containing monooxygenases and the inhibitory effects of methimazole,nicotine and DMXAA

A selective and low‐cost CD‐MEKC method under acidic conditions was developed for investigating the N‐oxygenation of tamoxifen (TAM) by flavin‐containing monooxygenases (FMOs). The inhibitory effects of methimazole (MMI), nicotine and 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) on the given FMO reaction were also evaluated; 100 mM phosphate buffer (pH 8.6) was used for performing the enzymatic reaction and the separation of TAM and its metabolite tamoxifen N‐oxide (TNO) was obtained with a BGE consisting of 100 mM phosphoric acid solution adjusted to pH 2.5 with triethanolamine containing 50 mM sodium taurodeoxycholate, 20 mM carboxymethyl β‐CD and 20% ACN. The proposed method was applied for the kinetics study of FMO1 using TAM as a substrate probe. A Michaelis–Menten constant (K_m) of 164.1 μM was estimated from the corrected peak area of the product, TNO. The calculated value of the maximum reaction velocity (V_max) was 3.61 μmol/min/μmol FMO1; 50% inhibitory concentration and inhibition constant (K_i) of MMI, the most common alternate substrate FMO inhibitor, were evaluated and the inhibitory effects of two other important FMO substrates, nicotine and DMXAA, a novel anti‐tumour agent, were investigated.