Derivatizing assay for the determination of aldehydes using micellar electrokinetic chromatography

In this work, the use of a novel derivatization agent for the determination of aldehydes (in this particular case: formaldehyde, acetaldehyde, propionaldehyde, and valeraldehyde) using micellar electrokinetic chromatography is reported. The derivatization reaction is based on the reaction of aldehydes with benzhydrazide to form the corresponding derivates with maximum absorbance at 250 nm. The experimental conditions of the derivatization reaction as well of the separation were optimized. The adducts were separated with a +22 kV voltage at a temperature of 29°C. The adducts’ separation was performed in less than 14 min using as the running buffer a mixture containing 110 mmol/L of sodium dodecyl sulfate and 27 mmol/L of sodium tetraborate at pH 9.45. Samples were injected using hydrodynamic mode (50 mbar × 5 s). The calibration curves were linear up to 15.0 mg/L with r ² above 0.99. Intra and inter‐day precisions were in average 3 and 4%, respectively, and recoveries were in average of 95%. Limits of detection and quantification were around 0.5 and 1.5 mg/L, respectively. The developed method was successfully applied in the analysis of low molar weight aldehydes in yogurt and vinegar samples.     

Optimization strategies in micellar electrokinetic capillary chromatography

Summary Computer-assisted procedures for the one-parameter optimization of the surfactant concentration and the concentration of urea or D-glucose as modifiers in micellar electrokinetic capillary chromatography have been developed. These procedures permit a rapid optimization of one parameter on the basis of only two experiments. Predicted values are compared to empirically obtained optimum values. The influence of the modifier concentration on the critical micelle concentration of sodium dodecyl sulfate was experimentally determined in buffers commonly employed in micellar electrokinetic chromatography. The alteration of retention factors of solutes caused by the influence of urea addition on the critical micelle concentration of sodium dodecyl sulfate was calculated under the assumption of constant distribution coefficients and compared to experimentally obtained values. It was demonstrated that the addition of urea or of D-glucose does not alter the phase ratio substantially.     

毛细管区带电泳用于多种类兴奋剂的同时快速分离检测

建立了一种同时分离检测包括利尿剂、蛋白同化剂、β-阻断剂、麻醉剂、β2-激动剂、刺激剂等6类8种兴奋剂的毛细管区带电泳-紫外检测法。优化的色谱条件为：以50 mmol/L甲酸铵-氨水(pH 7.8)缓冲液为运行液,于3 kPa下进样10 s,分离电压为20 kV,检测波长为214 nm。在此条件下,8种兴奋剂在7 min内实现了快速的基线分离。在相应的浓度范围内,8种组分的浓度与峰高呈良好的线性关系,检出限达为0.2~0.7 μg/mL。该方法快速,分析成本低,无污染,非常适用于多种类兴奋剂的同时快速检测。     

Development of a micellar electrokinetic capillary chromatography method for the determination of four naphthalenediols in cosmetics and a comparison with a HPLC method

A micellar electrokinetic capillary chromatography (MEKC) method with ultraviolet visible (UV) detection was used for the determination of 1,7-naphthalenediol, 2,3-naphthalenediol, 1,5-naphthalenediol, and 2,7-naphthalenediol in cosmetics. The current method for their determination in various cosmetics is high-performance liquid chromatography (HPLC). Separation conditions affecting the MEKC method were optimized as 20 mM Na₂B₄O₇–50mM SDS, pH 9.8, with 22 kV applied voltage and UV detection at 230 nm. Under optimal conditions, electrophoretic analysis was completed in less than 6 min, with limit of detection (LOD) of 0.070–0.19 μg/mL and limit of quantitation (LOQ) of 0.23–0.63 μg/mL. A good linear relationship (r² > 0.99) was obtained at the range of 0.75–20 μg/mL. Recoveries for the four naphthalenediols in lotion, loose powder, and sun cream are between 91.2–107.2% with relative standard deviation (RSD) less than 4.04%. The method has been successfully applied to the determination of the four naphthalenediols in different kinds of cosmetics. A comparison with HPLC-UV method was also carried out according to the National Standards of the People's Republic of China. The results obtained by MEKC and HPLC methods are comparable, but the proposed MEKC method can help us obtain a much shorter detection time and low cost.     

A first approach using micellar electrokinetic capillary chromatography for the determination of fipronil and fipronil-sulfone in eggs

Fipronil is an insecticide that is not approved in the European Union in food. In 2017, fipronil was involved in a European health alert due to its presence in fresh hen eggs because of an illicit use in poultry farms, so reliable methods are needed to determine fipronil and its main metabolites in these matrixes. In this work, we report the first approach to the study of fipronil and two metabolites, fipronil-sulfone and fipronil-sulfide by CE. MEKC mode was employed using a solution of 50 mM ammonium perfluorooctanoate pH 9.0 with 10% (v/v) methanol as background electrolyte. The proposed method was combined with a simple sample treatment based on salting-out assisted LLE (SALLE) using acetonitrile as extraction solvent and ammonium sulfate as salt. The SALLE–MEKC–UV method allowed the simultaneous quantification of fipronil and fipronil-sulfone. Validation parameters yielded satisfactory results, with precision, expressed as relative SD, below 14% and recoveries higher than 83%. Limits of detection were 90 µg/kg for fipronil and 150 µg/kg for fipronil-sulfone, so in terms of sensitivity further studies of sample treatments allowing extra preconcentration or the use of more sensitive detection, such as MS, would be needed.     

Separation and determination of anthraquinones in Cassia obtusifolia (Leguminosae) by micellar electrokinetic capillary electrophoresis

An MEKC method was developed for the determination of the five pharmaceutically important anthraquinones: chrysophanol (1), physcion (2), emodin (3), aloe-emodinin (4), and rhein (5) in Cassia obtusifolia (Leguminosae). A buffer solution (pH 9.00) composed of 20 mM sodium borate, 20 mM sodium deoxycholate (DOC), and 15% ACN was found to be the most suitable electrolyte for this separation. Regression equations revealed linear relationships (correlation coefficients: 0.9993, 0.9992, 0.9996, 0.9989, and 0.9991) between the peak area of each compound (1, 2, 3, 4, and 5) and its concentration. The RSDs of migration times and peak areas were <1.23 and 2.72% within 1 day, respectively. The effects of pH value, surfactant (DOC) concentration, and organic modifier on the migration were also studied. By this way, the contents of five anthraquinones in the extracts of the seed of C. obtusifolia (Leguminosae) from different sources were successfully determined within 14 min.     

Micellar electrokinetic chromatography for simultaneous determination of six corticosteroids in commercial pharmaceuticals

We have developed a micellar electrokinetic chromatography (MEKC) method using bile salts for the simultaneous determination of six corticosteroids, including betamethasone, cortisone, prednisolone, 6alpha-methylprednisolone, triamcinolone, and prednisone. The separation was performed using borate buffer containing sodium cholate and sodium deoxycholate. Several parameters were studied, including bile salt concentrations, concentrations and pH of borate buffer, and analytical voltages. In method validation, calibration curves were linear over a range of 10-100 microM for each corticosteroid. The RSD (relative standard deviation) and RE (relative error) were all less than 5% for intra- and interday assays. The limit of detection of each analyte was 5 microM. The recoveries were greater than 95%. Application of this method for quality control of commercial tablets also proved to be feasible. All analytical values fall within the labeled amount of 90-110% for betamethasone and prednisolone, and of the labeled amount of 92.5-107.5% for 6alpha-methylprednisolone, as required by the United State Pharmacopeia 25 (USP 25).     

A sulfonated capillary that gives reproducible migration times for capillary zone electrophoresis and micellar electrokinetic chromatography

To obtain reproducible migration times and rapid analyses of analytes, sulfonate groups were chemically introduced to the inner wall of untreated fused-silica capillary with 2-(4-chlorosulfonylphenyl)ethyltrichlorosilane. The sulfonated capillary showed relatively constant electroosmotic mobility which was greater than that obtained by an untreated fused-silica capillary over the pH range studied (pH 2-9). In both CZE and MEKC, the RSDs of the migration times of analytes with the sulfonated capillary were less than 0.2% which were significantly lower than those obtained with an untreated fused-silica capillary (0.5-3.5%). When BGE were set at pH 7.0 for CZE and MEKC, the analysis times with the sulfonated capillary were about half those obtained with an untreated fused-silica capillary. These results indicate that the sulfonated capillary can provide highly reproducible and rapid analyses in CE.     

Ultrafast capillary electrophoresis method for the simultaneous determination of ammonium and diphenhydramine in pharmaceutical samples

Ammonium and diphenhydramine are active ingredients commonly found in the same pharmaceutical preparations. We report, for the first time, a sub‐minute method for the simultaneous determination of ammonium and diphenhydramine. The method is based on capillary electrophoresis with capacitively coupled contactless conductivity detection. Both analytes can be quantified in a single run (∼80 injections/h) using 30 mmol/L 2‐(N‐morpholino)ethanesulfonic acid and 15 mmol/L lithium hydroxide (pH 6.0) as background electrolyte. The separation by capillary electrophoresis was achieved on a fused‐silica capillary (50 cm total length, 10 cm effective length, and 50 μm inside diameter). The limits of detection were 0.04 and 0.02 mmol/L for ammonium and diphenhydramine, respectively. The proposed method also provided adequate recovery values for spiked samples (100–106 and 97–104% for ammonium and diphenhydramine, respectively). The results obtained with the new capillary electrophoresis method were compared with those of the high‐performance liquid chromatography method for diphenhydramine and the Kjeldahl method for ammonium and no statistically significant differences were found (95% confidence level).     

Simple and rapid HPLC method for simultaneous determination of multiple antiepileptic drugs in human serum

Summary A very rapid, sensitive and reproducible HPLC method was developed for simultaneous determination of eight anti-epileptic drugs (AEDs): lamotrigine, primidone, ethosuximide, sulthiame, felbamate, phenobarbital, carbamazepine, phenytoin and oxcarbazepine-metabolite (10-hydroxy-carbazepine) in human serum. Sample purification requires only protein precipitation with an appropriate reagent. Separation was by reversed-phase HPLC, using a C18 column, 20% acetonitrile and 40 mM phosphoric acid buffer as mobile phase. Column temperature was set at 50°C, and measurement was by UV detection at 205 nm. The inter and intra-assay coefficients of variation (CV) ranged 1.13–7.10% and 1.14–8.49%, respectively. The absolute (measured) and relative (analytic) recoveries of the drugs ranged 96.7%–104.4% and 97.3%–106.1%, respectively. No interference with other common antiepileptic drugs and analgesics were observed. The method requires only 100 μl serum or less. It is very fast (sample preparation and analysis time approx. 23 min for all 9 AEDs), and suitable for routine clinical use, especially for epileptic patients on polytherapy.     

Quantitative determination of glucoraphanin in Brassica vegetables by micellar electrokinetic capillary chromatography

Glucoraphanin, a glucosinolate, is found naturally in plants and is present in relatively high concentrations in broccoli. Glucosinolates have received much attention as studies have indicated that a diet rich in them may provide some protection from certain cancers. A micellar electrokinetic chromatography (MEKC) method using sodium cholate as the micellar phase has been developed to quantify for glucoraphanin in broccoli (seeds and florets) and Brussels sprouts. The glucoraphanin peak elutes just under 5 min with a theoretical plate number of 380,000 per metre of capillary. The method is suitable for crude extracts of broccoli and Brussels sprouts. Glucoraphanin in broccoli seeds (1330 mg/100 g) broccoli florets (89 mg/100 g) and Brussels sprouts (3 mg/100 g) was determined and agreed with the data obtained by high performance liquid chromatography. The LODs were 10-100 times below the levels typically found in broccoli seeds (4 mg/100 g), broccoli florets (0.9 mg/100 g) and Brussels sprouts (0.1 mg/100 g).     

Development and validation of a micellar electrokinetic capillary chromatography method for the determination of goserelin and related substances

An MEKC method for the analysis of goserelin and related substances has been developed using a combination of additives including CTAB, β‐CD, and sodium hexanesulfonate. For this assay, the running buffer (pH and additives) and separation conditions (voltage and temperature) were optimized. The optimized system was the following: 200 mM 6‐aminocaproic acid buffer (pH 4.2) supplemented with 175 mM CTAB, 3.0% w/v β‐CD, and 20 mM sodium hexanesulfonate; the voltage was 10 kV in reverse polarity mode, the temperature was 20°C, and UV detection was measured at 220 nm. The method was qualified by evaluating the specificity, precision, linearity, accuracy, LOD, and LOQ. According to validation experiments, the optimized method was specific, accurate, and repeatable and satisfied the requirements for the analysis of goserelin and related substances. Compared with the RP‐HPLC method, the MEKC method better solved the problem of overlapping impurity signals, and the migration time required was shorter. This method can be used for quality control and for the analysis of goserelin and its related substances.     

Simultaneous determination of creatinine and uric acid in human plasma and urine by micellar electrokinetic chromatography

High performance capillary electrophoresis using a buffer solution containing micelles of ionic surfactant (e.g. sodium dodecyl sulfate), called micellar electrokinetic chromatography, has been applied to the separation and simultaneous determination of creatinine and uric acid in human plasma and urine. The sample was introduced into the capillary by siphoning an appropriate volume of untreated plasma or urine spiked with an internal standard (antipyrine). Creatinine, uric acid, and antipyrine were separated mutually, and from other endogeneous components within 18 min. The calibration plots showed good linearity (correlation coefficient > 0.999) over the concentration range needed for clinical analysis. Standard addition tests indicated that the recoveries of creatinine and uric acid from urine samples ranged, respectively, from 97 % to 106 % and 97.4 % to 108 % with a coefficient of variation (C.V.) of 3.3 % (n = 5), and that those from plasma samples ranged, respectively, from 100 % to 112 % and 101 % to 107 % with a C.V. of 4.7 % (n = 5). The results were in agreement with those obtained by conventional methods.     

Separation and determination of acrylamide in potato chips by micellar electrokinetic capillary chromatography

A simple and rapid method using micellar electrokinetic capillary chromatography (MEKC) was developed for the separation and determination of acrylamide in potato chips at low levels for the first time. The experimental conditions for the separation and quantification of acrylamide were optimized at first. The optimized conditions were: 50 mmol/L Na₂B₄O₇ and 40 mmol/L SDS at pH 10.0, 12 kV applied voltage, 76 cm total length (67 cm effective length) and 75 μm i.d. capillary, 198 nm wavelength, 15 cm high 25 s hydrodynamics sample injection, 20 °C air-cooling. The linear response of acrylamide concentration ranges from 0.5 to 100 μg/mL with high correlation coefficient (r = 0.9986, n = 9). The LOD and LOQ were estimated to be 0.1 and 0.33 μg/mL based on S/N = 3 and 10. The precision values (expressed as R.S.D.) of intra- and inter-day were 0.86-4.35% and 2.61-9.65%, respectively. Recoveries spiked at levels 2, 20, 60 μg/mL ranged between 90.86% and 99.6% with R.S.D. less than 6.5%. Finally, the developed method has been applied to the analysis of real samples and has achieved satisfactory results. All of these indicated that it was a reliable method for the quantification of acrylamide in potato chips.     

Quality by design-guided development of a capillary electrophoresis method for the simultaneous chiral purity determination and impurity profiling of tamsulosin

Analytical Quality by Design principles using the design of experiments were applied for the development of a capillary electrophoresis method for the determination of enantiomeric purity and chemically related impurities of tamsulosin. From initial scouting experiments, a dual cyclodextrin (CD) system composed of sulfated β-CD and carboxymethyl-α-CD was selected as the chiral selector. A fractional factorial resolution V+ design was used for the identification of the critical process parameters, while a face-centered central composite design and Monte Carlo simulations were employed for final optimization and defining the design space of the method. The experimental conditions of the working point were: 30 mM sodium phosphate buffer, pH 3.0, containing 40 mg/mL sulfated β-CD and 7 mg/mL carboxymethyl-α-CD, capillary temperature 18°C, applied voltage -23 kV. Following the assessment of robustness by applying a Plackett-Burman design, the method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guideline Q2(R1). The method allowed the quantification of the chiral impurity and three other related impurities at the 0.1 % level with acceptable accuracy and precision.     

A green capillary zone electrophoresis method for the simultaneous determination of piperacillin,tazobactam and cefepime in pharmaceutical formulations and human plasma

A green, novel, rapid, accurate and reliable capillary zone electrophoresis method was developed and validated for the simultaneous determination of piperacillin, tazobactam and cefepime in pharmaceutical preparations. Separation was carried out using fused silica capillary (50 µm i.d. × 48.6 cm and 40.2 cm detection length) and applied potential of 20 kV (positive polarity) and a running buffer containing 15 m m sodium borate buffer adjusted to pH 9.3 with UV detection at 215 nm. Amoxicillin was used as an internal standard. The method was suitably validated according to International Conference on Harmonization guidelines. The method showed good linearity in the ranges of 10–100, 20–400 and 10–400 µg/mL with limits of quantitation of 1.87, 3.17 and 6.97 µg/mL and limits of detection of 0.56, 0.95 and 2.09 µg/mL for tazobactam, piperacillin and cefepime, respectively. The proposed method was successfully applied for the analysis of these drugs in their synthetic mixtures and co‐formulated injection vials. The method was extended to the in vitro determination of the two drugs in spiked human plasma. It is considered a ‘green’ method as it consumes no organic solvents. Copyright © 2015 John Wiley & Sons, Ltd.     

Separation and determination of isoflavones in red clover by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method has been developed for the determination of the four isoflavones, i.e. biochanin A, formononetin, genstein and daidzein in red clover (Trifolium Pratense L.). The effect of running buffer pH and concentration were investigated. An electrolyte composed of 30 mm borate, 20 mm sodium dodecyl sulfate (SDS) and 4 mg/mL HP-beta-CD containing 5% (v/v) ethanol at pH 10.1 provides a satisfactory separation for all the analytes. The applied voltage was 25 kV, and the capillary temperature was kept constant at 25 degrees C with a UV detection at 254 nm. The relative standard deviations (RSD) of the migration time and peak area were less than 1.73 and 3.94% (intra-day), and 2.29 and 4.38% (inter-day), respectively, under the optimized separation conditions. Regression equations revealed a good linear relationship between the peak area of each compound and its concentration. The contents of the four compounds in red clover were successfully determined with satisfactory repeatability and recovery.     

Monitoring of cefepime in human serum and plasma by micellar electrokinetic capillary chromatography: Improvement of sample preparation and validation by liquid chromatography coupled to mass spectrometry

Cefepime monitoring in deproteinized human serum and plasma by micellar electrokinetic capillary chromatography and liquid chromatography coupled to mass spectrometry in presence of other drugs is reported. For micellar electrokinetic capillary chromatography, sample preparation comprised dodecylsulfate protein precipitation at pH 4.5 using an increased buffer concentration compared to that of a previous assay and removal of hydrophobic compounds with dichloromethane. This provided robust conditions for cefepime analysis in the presence of sulfamethoxazole and thus enabled its determination in samples of patients that receive cotrimoxazole. The liquid chromatography assay is based upon use of a column with a pentafluorophenyl‐propyl modified and multiendcapped stationary phase and the coupling to electrospray ionization with a single quadrupole detector. The performances of both assays with multilevel internal calibration were assessed with calibration and control samples and both assays were determined to be robust. Cefepime levels monitored by micellar electrokinetic capillary chromatography in samples from patients that were treated with cefepime only and with cefepime and cotrimoxazole were found to compare well with those obtained by liquid chromatography coupled to mass spectrometry. Cefepime drug levels determined by micellar electrokinetic capillary chromatography could thereby be validated.     

Micellar electrokinetic capillary chromatographic method for simultaneous determination of drugs used to treat advanced breast cancer

Summary A micellar electrokinetic capillary chromatography (MEKC) method has been developed for quantification of four drugs-tamoxifen, anastrozole, letrozole, and methotrexate—used to treat advanced breast cancer. Separation was performed at 25°C and 25kV, with 20mm borate buffer (pH 9.2) containing 40mm sodium dodecylsulfate as electrolyte solution. Under these conditions analyses were performed in 12 min. The linearity of the response was investigated for the concentration range 2.0–20.0 mg L⁻¹. The intra-day residual standard deviation (n=4 graphs) between the slopes of the calibration graphs was acceptable for the four drugs studied. Detection limits (signal-to-noise ratio=3) were below 1 mg L⁻¹ for all the compounds. The simplicity, precision, and sensitivity of MEKC proved suitable for quality control of pharmaceutical preparations used to treat advanced breast cancer. Six different pharmaceutical preparations, each containing one of the above-mentioned drugs, were successfully analyzed.