Development and validation of a capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C(4) D) for the analysis of amikacin and its related substances

Amikacin is a semisynthetic aminoglycoside antibiotic derived from kanamycin A that lacks a strong UV absorbing chromophore or fluorophore. Due to the physicochemical properties of amikacin and its related substances, CE in combination with capacitively coupled contactless conductivity detection (CE-C(4) D) was chosen. The optimized separation method uses a BGE composed of 20 mM MES adjusted to pH 6.6 by l-histidine and 0.3 mM CTAB that was added as flow modifier in a concentration below the CMC. Ammonium acetate 20 mg.L(-1) was used as internal standard. 30 kV was applied in reverse polarity on a fused silica capillary (73/48 cm; 75 μm id). The optimized separation was obtained in less than 6 min with good linearity (R(2) = 0.9996) for amikacin base. It shows a good precision expressed as RSD on relative peak areas equal to 0.1 and 0.7% for intraday and interday, respectively. The LOD and LOQ are 0.5 mg.L(-1) and 1.7 mg.L(-1) , respectively.     

Determination of pharmaceuticals classified as emerging pollutants using capillary electrophoresis with capacitively coupled contactless conductivity detection

A study on the simultaneous separation of 13 pharmaceutical products by capillary electrophoresis with capacitively coupled contactless conductivity detection was presented. The parameters of the background electrolyte, such as pH, organic additives as well as types and concentrations of cyclodextrins (CD) were studied. The optimal separation conditions were achieved with a background electrolyte consisting of 9 mM Tris/5 mM lactic acid at pH 8.0, containing 5% n-propanol, 0.025% gamma-CD, 0.075% hydroxyl-beta-CD and 0.15% dimethyl-beta-CD. Limits of detections ranged from 61 to 1676 microg/L (S/N=3) and the relative standard deviations for migration time and peak area were below 2 and 6%, respectively. This demonstrated the potential of the capillary electrophoresis-capacitively coupled contactless conductivity detection method for biomedical and environmental analysis, as shown in the determination of pharmaceuticals identified as emerging pollutants in water samples.     

Quantification of plasma lactate concentrations using capillary electrophoresis with contactless conductivity detection

The quantification of plasma lactate and evaluation of the lactate threshold by CE with capacitively coupled contactless conductivity is demonstrated. The only sample preparation needed was deproteinization with a ACN/methanol mixture. A solution of 10 mmol/L 2-morpholinoethanesulfonic acid monohydrate, 10 mmol/L DL-histidine, 70 μmol/L hexadecyltrimethylammonium bromide, pH 6.0 was found suitable as running buffer. Linearity was achieved for the concentration range of 10-1000 μmol/L with a correlation coefficient of 0.9994. The limit of detection (3 S/N) was determined as 3.2 μmol/L. Intra- and inter-day variabilities were less than 7% RSD. The suitability of the method could be demonstrated by analyzing various clinical samples, where the results correlated satisfactorily with those of an established enzymatic method.     

Capillary electrophoresis with contactless conductivity detection for uric acid determination in biological fluids

The suitability of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) for the direct determination of uric acid in human plasma and urine was investigated. It was found that a careful optimization of the buffer composition and pH was necessary to achieve selective determination in the complex sample matrices. An electrolyte solution consisting of 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM histidine and 0.1 mM hexadecyltrimethylammonium bromide (CTAB), pH 6.0, was finally found suitable for use as running buffer for both sample matrices. The limit of detection (3 S/N) was determined as 3.3 μM. The linearity of the response was tested for the range between 10 and 500 μM and a correlation coefficient of 0.9996 was obtained. Intra- and inter-day variabilities were <10%. Quantitative analysis of urine and plasma samples showed a good correlation with the routine enzymatic method currently used at the University Hospital of Basel.     

Fast determination of paracetamol and its hydrolytic degradation product p-aminophenol by capillary and microchip electrophoresis with contactless conductivity detection

Paracetamol (PAC) is one of the most extensively used analgesics and antipyretic drugs to treat mild and moderate pain. P-aminophenol (PAP), the main hydrolytic degradation product of PAC, can be found in environmental water. Recently, CE has been developed for the detection of a wide variety of chemical substances. The purpose of this study is to develop a simple and fast method for the detection and separation of PAC and its main hydrolysis product PAP using CE and microchip electrophoresis with capacitively coupled contactless conductivity detection. The determination of these compounds using microchip electrophoresis with capacitively coupled contactless conductivity detection is being reported for the first time. The separation was run for all analytes using a BGE (20 mM β-alanine, pH 11) containing 14% (v/v) methanol. The RSDs obtained for migration time were less than 4%, and RSDs obtained for peak area were less than 7%. The detection limits (S/N = 3) that were achieved ranged from 0.3 to 0.6 mg/L without sample preconcentration. The presented method showed rapid analysis time (less than 1 min), high efficiency and precision, low cost, and a significant decrease in the consumption of reagents. The microchip system has proved to be an excellent analytical technique for fast and reliable environmental applications.     

Simple in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity method for the determination of colistin

An in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity detection method was developed for the direct measurement of colistin in pharmaceutical samples. The flow injection and capillary electrophoresis systems are connected by an acrylic interface. Capillary electrophoresis separation is achieved within 2 min using a background electrolyte solution of 5 mM 2‐morpholinoethanesulfonic acid and 5 mM histidine (pH 6). The flow‐injection section allows for convenient filling of the capillary and sample introduction without the use of a pressure/vacuum manifold. Capacitively coupled contactless conductivity detection is employed since colistin has no chromophore but is cationic at pH 6. Calibration curve is linear from 20 to 150 mg/L, with a correlation coefficient (r²) of 0.997. The limit of quantitation is 20 mg/L. The developed method provides precision, simplicity, and short analysis time.     

Determination of tobramycin in human serum by capillary electrophoresis with contactless conductivity detection

A study on the determination of the antibiotic tobramycin by CE with capacitively coupled contactless conductivity detection is presented. This method enabled the direct quantification of the non-UV-absorbing species without incurring the disadvantages of the indirect approaches which would be needed for optical detection. The separation of tobramycin from inorganic cations present in serum samples was achieved by optimizing the composition of the acetic acid buffer. Field-amplified sample stacking was employed to enhance the sensitivity of the method and a detection limit of 50 microg/L (S/N = 3) was reached. The RSDs obtained for migration time and peak area using kanamycin B as internal standard were typically 0.12 and 4%, respectively. The newly developed method was validated by measuring the concentration of tobramycin in serum standards containing typical therapeutic concentrations of 2 and 10 mg/L. The recoveries were 96 and 97% for the two concentrations, respectively.     

Separation and quantification of the urinary enantiomers of 2-hydroxyglutaric acid by capillary electrophoresis with capacitively coupled contactless conductivity detection: Application to the diagnosis of D- and L-2-hydroxyglutaric aciduria

2-hydroxyglutaric aciduria is an inherited neurometabolic disorder with two major types: D-2-hydroxyglutaric aciduria and L-2-hydroxyglutaric aciduria. An easy and fast capillary electrophoresis system combined with a capacitively coupled contactless conductivity detection method was developed for the enantioseparation and determination of D- and L-2-hydroxyglutaric acid in urine. D- and L-2-hydroxyglutaric acids were separated using vancomycin as the chiral selector. The optimal separation conditions for enantiomers were achieved by the use of a buffer containing 50 mM 4-(N-morpholino) butane sulfonic acid solution (pH 6.5), an electroosmotic flow modifier (0.001% [w/v] polybrene), and 30 mM vancomycin as chiral selector. The analysis time was 6 min under optimal conditions. The optimized and validated method was successfully implemented for quantifying D- and L-2-hydroxyglutaric aciduria in patients’ urine, without any pretreatment step. The linearity of the method was determined to be in the range of 2–100 mg/L for D- and L-2-hydroxyglutaric acid in urine. The precision (relative standard deviation%) was obtained at about 7%. For D- and L-2-hydroxyglutaric acids, the limits of detection were 0.567 and 0.497 mg/L, respectively.     

Direct analysis of formate in human plasma,serum and whole blood by in-line coupling of microdialysis to capillary electrophoresis for rapid diagnosis of methanol poisoning

A simple method using direct injection of human blood samples and quantitative analysis of formate was developed for rapid diagnosis of methanol poisoning. A sample pretreatment device including a 500 Da molecular weight cut-off dialysis membrane was in-line coupled to capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C⁴D). 50 μL of 1:9 diluted blood samples and 50 μL of DI water were filled into the donor and the acceptor chamber, respectively, and small ionic species in blood samples were electrokinetically injected across the dialysis membrane directly into the separation capillary. Matrix components, such as red blood cells, proteins, lipids and other high molecular weight compounds, were retained by the dialysis membrane and did not interfere with subsequent CE separation. Formate was separated from other small anions in an optimized background electrolyte solution consisting of 20 mM l-histidine and 25 mM l-glutamic acid at pH 4.8. The method showed excellent analytical parameters in terms of repeatability and linearity; RSD values for migration times and peak areas at a formate concentration typical for methanol poisoning were below 0.3% and 7.4%, respectively, and linear calibration curves with correlation coefficients better than 0.999 were obtained. Limit of detection and limit of quantification were 15 and 50 μM formate in original (undiluted) blood samples, respectively. The method was applied to determination of formate in serum samples of a patient diagnosed with acute methanol poisoning.     

Trace determination of perchlorate using electromembrane extraction and capillary electrophoresis with capacitively coupled contactless conductivity detection

Electromembrane extraction (EME) and CE with capacitively coupled contactless conductivity detection (CE‐C⁴D) was applied to rapid and sensitive determination of perchlorate in drinking water and environmental samples. Porous polypropylene hollow fiber impregnated with 1‐heptanol acted as a supported liquid membrane (SLM) and perchlorate was transported and preconcentrated in the fiber lumen on application of electric field. High selectivity of perchlorate determination and its baseline separation from major inorganic anions was achieved in CE‐C⁴D using background electrolyte solution consisting of 7.5 mM L ‐histidine and 40 mM acetic acid at pH 4.1. The analytical method showed excellent parameters in terms of reproducibility; RSD values for migration times and peak areas at a spiked concentration of 15 μg/L of perchlorate (US EPA recommended limit for drinking water) were below 0.2 and 8.7%, respectively, in all examined water samples. Linear calibration curves were obtained for perchlorate in the concentration range 1–100 μg/L (r²≥0.999) with limits of detection at 1 μg/L for tap water and at 0.25–0.35 μg/L for environmental and bottled potable water samples. Recoveries at 15 μg/L of perchlorate were between 95.9 and 106.7% with minimum and maximum recovery values for snow and bottled potable water samples, respectively.     

Determination of inorganic ions in mineral water by gradient elution moving boundary electrophoresis

A sensitive method was developed for the determination of the major inorganic ions in commercial mineral waters using gradient elution moving boundary electrophoresis with capacitively coupled contactless conductivity detection. This application was the first to demonstrate the separation of cations and anions simultaneously using gradient elution moving boundary electrophoresis. Seven ionic analytes (calcium, chloride, magnesium, nitrate, potassium, sodium, and sulfate) were separated in less than 7 min with detection values in the low μmol/L to sub-μmol/L range. Calculated values of the major ions in three commercial mineral waters were compared to reported values with good correlation. In another application, phosphate and arsenate were separated in less than 2 min with limits of detection of 300 and 140 nmol/L, respectively. For all standard analyses, the RSD for migration times and peak areas were under 3%.     

Simultaneous determination of atenolol and amiloride in pharmaceutical preparations by capillary zone electrophoresis with capacitively coupled contactless conductivity detection

Capillary zone electrophoresis coupled with a capacitively coupled contactless conductivity detector (CE‐C⁴D) has been employed for the determination of atenolol and amiloride in pharmaceutical formulations. Acetic acid (150 mm ) was used as background electrolyte. The influence of several factors (detector excitation voltage and frequency, buffer concentration, applied voltage, capillary temperature and injection time) was studied. Non‐UV‐absorbing L‐valine was used as internal standard; the analytes were all separated in less than 7 min. The separation was carried out in normal polarity mode at 28°C, 25 kV and using hydrodynamic injection (25 s). The separation was effected in an uncoated fused‐silica capillary (75 μm, i.d. × 52 cm). The CE‐C⁴D method was validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. Calibration curves were linear over the range 5–250 μg/mL for the studied analytes. The relative standard deviations of intra‐ and inter‐day migration times and corrected peak areas were less than 6.0%. The method showed good precision and accuracy and was successfully applied to the simultaneous determination of atenolol and amiloride in different pharmaceutical tablet formulations. Copyright © 2010 John Wiley & Sons, Ltd.     

毛细管电泳柠檬酸-Zn2+体系对异亮氨酸对映体的手性分离

应用毛细管电泳/电容耦合非接触式电导（CE-C⁴D）分离检测技术,研究了柠檬酸-Zn²⁺体系对异亮氨酸对映体的手性识别行为。结果表明,采用未涂层石英毛细管（45 cm×50 μm,L_eff=40 cm）,以2.8 mmol/L NaOH+0.8 mmol/L柠檬酸+2.0 mmol/L乙酸锌为非手性介质电泳运行液,分离电压+13 kV,D,L-异亮氨酸对映体得到了良好的手性识别,对映体分离度达到2.0。线性检测范围为1.0~20 mg/L,检出限（S/N=3）为0.40 mg/L。对影响分离度的因素（Zn²⁺的浓度、电泳运行液的组成、分离电压以及其他氨基酸的干扰情况等）进行了详细的讨论,并对手性识别机理作了初步探讨。     

Determination of ciclopirox olamine in pharmaceutical products by capillary electrophoresis with capacitively coupled contactless conductivity detection

This paper describes the determination of ciclopirox olamine in pharmaceutical formulations using capillary electrophoresis with capacitively coupled contactless conductivity detection. In an alkaline medium, ciclopirox olamine is converted into an anionic species and its detection is possible in capillary electrophoresis with capacitively coupled contactless conductivity detection without an electroosmotic flow modifier, because it is a low-mobility species. A linear working range from 2.64 to 264 μg/mL in sodium hydroxide electrolyte as well as low detection limit (0.39 μg/mL) and a good repeatability (RSD = 3.4% for 264 μg/mL ciclopirox solution (n = 10)) were achieved. It was also possible to determine olamine in its cationic form when acetic acid was used as the electrolyte solution. The results obtained include a linear range from 26.4 to 184.8 μg/mL and a detection limit of 2.6 μg/mL olamine. The proposed methods were applied to the analysis of commercial pharmaceutical products and the results were compared with the values indicated by the manufacturer as well as those obtained using a titrimetric method recommended by American Pharmacopoeia.     

Determination of muscle relaxants pancuronium and vecuronium bromide by capillary electrophoresis with capacitively coupled contactless conductivity detection

A novel capillary electrophoretic method for the separation of pancuronium (PM) and vecuronium (VM) ions utilizing capacitively coupled contactless conductivity detection was devised and validated. The separation was carried out in bare fused-silica capillaries (50 μm id, 75/45 cm) at 25°C. Optimal BGE was 50 mM borate buffer of pH 9.5 containing 12.5 mg/mL of (2-hydoxypropyl)-γ-CD. The samples were injected hydrodynamically at 1000 mbar for 3 s. Separation was performed at +30 kV. Under such conditions the PM and VM were base-line resolved and the separation took < 4 min. For quantification phenyltrimethylammonium iodide was used as internal standard. Calibration curves were linear for both pancuronium bromide (PMB) and vecuronium bromide (VMB) in the range 25-250 μg/mL with r> 0.9968. The limits of detection were 7 and 6?μg/mL for PMB and VMB, respectively. The accuracy tested by recovery experiment at three concentration levels of added PMB and VMB was satisfactory (95.7-102.7%, n =3, with RSD < 2.61%). The method was successfully applied to the assay of PMB and VMB in commercial injection solutions.     

Determination of gamma-hydroxybutyric acid in clinical samples using capillary electrophoresis with contactless conductivity detection

A method for the determination of gamma-hydroxybutyric acid (GHB) in urine and serum samples with capillary electrophoresis using capacitively coupled contactless conductivity detection (CE-C(4)D) was developed. The optimized separation buffer consisting of 20 mM of arginine, 10 mM of maleic acid and 30 microM of cetyltrimethylammonium bromide (CTAB) contained 5 mM vancomycin to facilitate the separation of gamma-hydroxybutyric acid from beta-hydroxybutyric acid (BHB), which is also present in clinical samples. The detection limits in the clinical samples were found to be about 2 microg/ml, which is well below the required sensitivity for the recognition of overdosage and adequate for the determination of endogenous concentrations in urine. The determination of GHB in both types of samples was carried out directly after a fourfold dilution without requiring any derivatization or extraction procedures.     

Anion-exchange capillary electrochromatography with indirect UV and direct contactless conductivity detection

Conductivity detection is applied to ion-exchange capillary electrochromatography (IE-CEC) with a packed stationary phase, using a capacitively coupled contactless conductivity detector with detection occurring through the packed bed. Columns were packed with a polymeric latex-agglomerate anion-exchanger (Dionex AS9-SC). A systematic approach was used to determine suitable eluants for IE-CEC separations using simultaneous indirect UV and direct conductivity detection. Salicylate and p-toluenesulfonate were identified as potential eluant competing anions having sufficient eluotropic strength to induce changes in separation selectivity, but salicylate was found to be unsuitable with regard to baseline stability. It was also found for both indirect UV and direct conductivity detection that homogenous column packing was imperative, and monitoring of the baseline could be used to assess the homogeneity of the packed bed. Using a p-toluenesulfonate eluant, the separation of eight common anions was achieved in 2.5 min. Direct conductivity detection was found to be superior to indirect UV detection with regard to both baseline stability and detection sensitivity with detection limits of 4-25 microg/L being obtained. However, the calibration for each anion was not linear over more than one order of magnitude. When using conductivity detection, the concentration of the eluant could be varied over a wider range (2.5-50 mM p-toluenesulfonate) than was the case with indirect UV detection (2.5-10 mM), thereby allowing greater changes in separation selectivity to be achieved. By varying the concentration of p-toluenesulfonate in the eluant, the separation selectivity could be manipulated from being predominantly ion-exchange in nature (2.5 mM) to predominantly electrophoretic in nature (50 mM).     

Determination of vitamin C and preservatives in beverages by conventional capillary electrophoresis and microchip electrophoresis with capacitively coupled contactless conductivity detection

The separation and detection of commonly used preservatives (benzoate, sorbate) and vitamin C by both conventional CE and microchip electrophoresis with capacitively coupled contactless conductivity detection is presented. The separation was optimized by adjusting the pH-value of the buffer and the use of hydroxypropyl-beta-CD (HP-beta-CD) and CTAB as additives. For conventional CE, optimal separation conditions were achieved in a histidine/tartrate buffer at pH 6.5, containing 0.025% HP-beta-CD and 0.1 mM CTAB. LOD ranged from 0.5 to 3 mg/L (S/N = 3) and the RSDs for migration time and peak area were less than 0.1 and 2%, respectively. A considerable reduction of analysis time can be accomplished by using microchip electrophoresis without significant loss in sensitivity under optimal separation conditions. A histidine/tartrate buffer at pH 6.5, incorporating 0.06% HP-beta-CD and 0.25 mM CTAB, gave detection limits ranging between 3 and 10 mg/L and satisfactory reproducibilities of < or =0.4% for the migration time and < or =3.5% for the peak area. The methods developed are useful for the quantitative determination of food additives in real samples such as soft drinks and vitamin C tablets.     

Separation of carbohydrates on electrophoresis microchips with controlled electrolysis

This study reports the separation of fructose, galactose, glucose, lactose and sucrose on glass microchip electrophoresis (ME) devices using a microfluidic platform adapted with external reservoirs for controlling the electrolysis phenomenon. The connections between external reservoirs and microfluidic platform were performed by saline bridges created using silicone tubing filled with BGE. The separation conditions were optimized and the best results were achieved using a BGE containing 75 mmol/L NaOH and 15 mmol/L trisodium phosphate. Electrophoretic separations were monitored using a capacitively coupled contactless conductivity detection system. The controlled electrolysis has successfully allowed the application of a higher voltage on the separation channel promoting the baseline separation of five carbohydrates within 180 s with great run‐to‐run repeatability (RSD < 1%). The achieved efficiencies ranged from 45 000 ± 6000 to 70 000 ± 3000 plates/m demonstrating a performance better than ME devices without controlled electrolysis. The proposed system offered good linearity from 1 to 10 mmol/L and LODs between 150 and 740 μmol/L. The use of external tubes for controlling the electrolysis phenomenon on ME devices has solved common problems associated to run‐to‐run repeatability and analytical reliability required for routine and quantitative analysis.     

Method development and validation for trifluoroacetic acid determination by capillary electrophoresis in combination with capacitively coupled contactless conductivity detection (CE-C4D)

A method was developed to determine traces of trifluoroacetic acid as impurity in synthetic or semi-synthetic drugs as antibiotics, macropeptides, etc. Capillary electrophoresis in combination with capacitively coupled contactless conductivity detection (CE-C⁴D) was used due to lack of UV absorbance property of trifluoroacetic acid (TFA). The optimized method took less than 1 min with good linearity (R² = 0.9995) for trifluoroacetic acid concentration from 2 to 100 ppm. It also has a good repeatability expressed by the relative standard deviation (% RSD) which is 1.2 and 2.1% for intraday and interday precision, respectively, at 50 ppm TFA, and good sensitivity with 0.34 ppm, 1.2 ppm LOD and LOQ, respectively. In addition, the content of TFA in synthetic drug, was determined using the validated method which gave good linearity (R² = 0.9996) for trifluoroacetic acid spiked into drug in a concentration range of 2-80 ppm, with good intraday repeatability of 2.0%.The analysis is performed in a background electrolyte composed of 20 mM morpholinoethane-sulfonic acid (Mes) and 20 mM l-histidine (l-His) pH 6.1. Cetyltrimethylammonium bromide (CTAB) was added as flow modifier in a concentration (0.2 mM) lower than the critical micellar concentration. Ammonium formate 6 ppm was used as internal standard. The applied voltage was 30 kV in reverse polarity. A fused silica capillary with 75 μm internal diameter and total length 47 cm (31 cm to C⁴D detector and 37 cm to DAD detector) was used.