High-speed chiral separations on a microchip with UV detection

Fast chiral separations of a variety of basic and acidic compounds could be realized on microfluidic quartz chips. A microchip electrophoresis instrument equipped with a linear imaging UV-detector was used. The usually applied but troublesome fluorescence tagging in order to enable fluorescence detection could be omitted. Using sulfated cyclodextrins as chiral selectors baseline separation of 19 compounds could be achieved in less than 1 min with high reproducibility. The relative standard deviation of migration time was below 7%. The fastest separation could be performed in 2.5 s which is to date the fastest separation of enantiomers reported. It was possible to apply microchip electrophoresis (MCE) for the determination of high enantiomeric excess (ee) values, as exemplarily shown for pseudoephedrin where 2% of the minor enantiomer could reliably be determined beside high amount of the other isomer. Successful separation of a mixture of 3 chiral drugs could be performed in a single run in less than 11 s utilizing a separation length of only 12 mm. These results show that MCE has great potential for fast chiral analysis and high-throughput screening.     

Stereospecific analysis of lorazepam in plasma by chiral column chromatography with a circular dichroism-based detector

The chiral separation of lorazepam was achieved on a chiral column with UV and circular dichroism (CD) detection. The good resolution of lorazepam enantiomers was obtained on the column of beta-cyclodextrin derivative immobilized silica gel under reversed-phase conditions. The enantiomeric separation and identification of lorazepam were succeeded by CD detector. The method described allows the quantitation of the stereoisomers of lorazepam in human plasma following the administration of a therapeutic dose of the racemic drug. Chiroptical detection is useful for the pharmacokinetic study of chiral drugs.     

New triazine spectroscopic reagent for the separation of DL-amino acids by micellar electrokinetic chromatography

An approach to the chiral separation of racemic mixtures of amino acids by means of micellar electrokinetic chromatography after derivatization with a new triazine spectroscopic reagent, 3-(4,6-dichloro-1,3,5-triazinylamino)-7-dimethylamino-2-methylphenazine (DTDP), has been evaluated. It was found that the derivatives of the aliphatic amino acids such as serine, valine and arginine, could produce a strong UV absorption at 282 nm, whose apparent molar absorptivities are of 10(-4) M(-1) cm(-1), and thus the concentration of the amino acids down to 3 x 10(-7) M can still give a detectable signal (S/N = 3). Beta-Cyclodextrin (beta-CD) added to the buffer system was used as a chiral selector, and separation conditions were optimized. The presence of an organic modifier (2-propanol) was also a prerequisite for the chiral separation. The best results for the chiral separation of DTDP-amino acids were achieved in a mixed sodium dodecylsulfate-beta-CD-borate-2-propanol medium at pH 9.0. Compared to some of the commonly used derivatization methods, the present one offers a relatively stable derivative and strong UV absorption for the spectroscopically inert amino acids, thus enabling amino acids to be separated and detected by CE even with a simpler UV detector.     

Stereospecific analysis of loxoprofen in plasma by chiral column liquid chromatography with a circular dichroism-based detector

The chiral separation of loxoprofen was achieved on a chiral column with UV and circular dichroism (CD) detection. The good resolution of four loxoprofen stereoisomers was obtained. The column used for the chiral separation was Chiralcel OJ column (250 x 4.6 mm) using hexane-2-propanol-trifluoroacetic acid (95:5:0.1), as an eluent. The flow-rate was 1.0 ml/min and the detection was at 225 nm. In addition, CD and UV spectra were obtained by stopped flow scanning. The method allows the determination of the stereoisomers of loxoprofen in human plasma after the administration of therapeutic dose of the racemic drug, thus HPLC with CD detector is useful for the stereospecific determination of loxoprofen products in biological samples.     

Microchip electrophoresis for chiral separations

Microchip electrophoresis (MCE) is a promising new technique for the separation of enantiomers. This recently introduced technique enables chiral separations to be performed in seconds on tiny micromachined devices. This review is intended to give a brief introduction into the principles of chiral separations with MCE with regard to methodology and instrumentation. Different approaches to realize chiral separations in microfluidic devices are described and discussed. This review gives an overview of original work done in this field with emphasis on approaches to improve detection and resolution in chiral MCE.     

Design and performance of a microchip electrophoresis instrument with sensitive variable-wavelength fluorescence detection

A modular instrument for high-speed microchip electrophoresis (MCE) equipped with a sensitive variable-wavelength fluorescence detection system was developed and evaluated. The experimental setup consists mainly of a lamp-based epifluorescence microscope for variable-wavelength fluorescence detection and imaging and a programmable four-channel bipolar high-voltage source capable of delivering up to +/- 10 kV per channel. The optical unit was equipped with a high-sensitivity photomultiplier tube and an adjustable aperture. The system was applied to MCE separations of flurescein isothiocyanate (FITC)-labelled amines utilizing blue light (450-480 nm) for excitation as well as for the separation of rhodamines utilizing excitation light in the green spectral region (531-560 nm). At optimized conditions baseline separation of four FITC-labelled amines could be obtained in less than 50 s at a detection limit of 460 ppt (1 nM) with a signal-to-noise ratio of 3:1. Three rhodamines could be baseline-separated in less than 6 s at a detection limit of 240 ppt (500 pM). The relative standard deviations of absolute migration times determined in repetitive MCE separations of FITC-labelled amines were below 2.5% (n= 25). By the application of cyclodextrin-modified electrolytes, chiral separation of FITC-labelled amines could be performed in seconds demonstrating the potential of microchip electrophoresis for chiral high-throughput screening.     

Chiral separation of polychlorinated biphenyls using a combination of hydroxypropyl-gamma-cyclodextrin and a polymeric chiral surfactant

Chiral separation of moderately to highly hydrophobic polychlorinated biphenyls (PCBs) using a conventional chiral micelle or a polymeric chiral surfactant, as the single chiral selector is very difficult since the hydrophobic interactions between the chiral PCB and the monomeric or polymeric surfactant is very strong. Combined use of a polymeric chiral surfactant, polysodium N-undecanoyl-D-valinate (poly-D-SUV) with hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) was successful in cyclodextrin modified electrokinetic chromatography (CD-EKC) enantioseparation of PCB congeners. Addition of HP-gamma-CD to the background electrolyte containing poly-D-SUV functioned to improved chiral resolution for the PCBs and reduce the analysis time for these congeners. In addition, concentration of methanol, concentration of 2-(N-cyclohexylamino) ethanesulfonic acid (CHES) buffer and separation voltage was also varied to optimize multicomponent separation of five chiral PCBs. Simultaneous separation and enantioseparation of all five PCBs was possible in less than 50 min under optimized conditions that requires a 5 mM CHES solution buffered at about pH 10 with 1.5% w/v (ca. 60 mM) poly-D-SUV and 16 mM HP-gamma-CD. In addition, 1 M urea and 20% v/v methanol should be added as organic modifier and the capillary temperature maintained at 45 degrees C. As expected the polymeric surfactant showed improved chiral resolution of PCBs over conventional micelles of SUV. Under optimized conditions, when CD-EKC of chiral PCBs using poly-D-SUV was compared to sodium dodecyl sulfate (SDS), better resolution, higher efficiency and shorter analysis time was achieved with poly-D-SUV.     

Enantioseparation of warfarin and its metabolites by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method with direct ultraviolet (UV)-absorbance detection is presented for the simultaneous enantiomeric separation of warfarin and its main metabolites, including warfarin alcohols, 4'-, 6-, and 7-hydroxywarfarin, using highly sulfated beta-cyclodextrin (HS-beta-CD) as the chiral selector. This chiral separation method was optimized in terms of the electrophoretic parameters, which included the concentration of HS-beta-CD used, the type and composition of organic modifier added to the background electrolyte (BGE) buffer, and the BGE buffer pH. Chiral separation of warfarin and its major metabolites was achieved with high resolution, selectivity, efficiency, repeatability, and reproducibility. This optimized chiral analysis of warfarin along with its metabolites was completed within a satisfactory electrophoresis time of 20 min.     

Separation of chiral primary amino compounds by forming a sandwiched complex in reversed-phase high performance liquid chromatography

Separation of chiral primary amino compounds was efficiently achieved under reversed-phase high performance liquid chromatography (RP-HPLC) conditions using a mixture of non-chiral crown ether (18-crown-6) and dimethyl-β-cyclodextrin (DM-β-CD) in the mobile phase. Under these conditions, the amino group of the chiral compound was protonated in a low pH mobile phase, and then interacted with 18-crown-6 and DM-β-CD to form a sandwiched complex [18-crown-6 + amine + CD]. Enantiomers of the compound in the sandwiched complex were separated with good enantioselectivity. Formation of the sandwiched complex among the chiral compound and additives in the mobile phase is a key step of the chiral separation. Four different chiral amino compounds namely, 1-aminoindan (AI), 1,2,3,4-tetrahydro-1-naphthylamine (THNA), tyrosine (Tyr), and phenylalanine (Phe), were selected to demonstrate the separation using the sandwiched complex mechanism in RP-HPLC.     

Chiral amines as reagents for HPLC-MS enantioseparation of chiral carboxylic acids

Mass spectrometry (MS) has become a popular analytical technique because of its high sensitivity and specificity. Therefore, the use of a chiral derivatization reagent for the MS detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis is very limited. The applicability of commercially available chiral amines as the derivatization reagents for the enantiomeric separation of chiral carboxylic acids is reported in this paper by using non-steroidal anti-inflammatory drugs (NSAIDs), i.e. ibuprofen, flurbiprofen, and loxoprofen. The efficiency of the chiral reagents was evaluated in terms of tagging easiness, separation by reversed-phase chromatography, and detection sensitivity by electrospray ionization (ESI)-MS/MS. Among the tested eight chiral amines, i.e. (R)-(+)-4-(3-aminopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-APy), (S)-(+)-1-(2-pyrrolidinylmethyl)-pyrrolidine (PMP), L-prolinamide, (3R)-(-)-1-benzyl-3-aminopyrrolidine, (S)-(+)-1-cyclohexyl-ethylamine, (3R)-(+)-3-(trifluoroacetamido)-pyrrolidine (TFAP), (R)-(-)-1-aminoindan (AI), and (S)-(+)-tetrahydrofurfuryl-amine, DBD-APy, PMP, AI, and TFAP could be used as the chiral reagents for the enantiomeric separation of the NSAIDs. The Rs values and the detection limits of the derivatives were in the range of 1.29-3.85 and 0.57-0.96 fmol, respectively. These four reagents were applied for the determination of the NSAIDs in rat plasma.     

High-performance liquid chromatography applications of optical rotation detection with compensation for scattering and absorbance at the laser wavelength.

Use of instrumentation developed to enable simultaneous monitoring of optical rotation (OR) and transmittance allows OR measurements to be made in the presence of high levels of absorbance, scattering or other effects that change the intensity of the plane-polarised light at the photodiode detector. This extends the application of OR detection to areas where it was previously difficult. Examples of the application of high-performance liquid chromatography (HPLC) with the improved OR detector include (i) the analytical scale separation of fructose and sucrose and (ii) the semi-preparative separation of enantiomers of warfarin and Tr?gers base. A signal-to-noise improvement of up to 150% is found when comparing signals with and without correction for transmittance changes. The improved OR detector has been used in series with a UV detector and the system shown to be suitable for on-line measurement of peak purity in separations using a chiral column under overload conditions.     

Computational modeling of capillary electrophoretic behavior of primary amines using dual system of 18-crown-6 and β-cyclodextrin

Using capillary electrophoresis (CE) three chiral primary amine compounds 1-aminoindan (AI), 1-(1-naphthyl)ethylamine (NEA) and 1,2,3,4-tetrahydro-1-naphthylamine (THAN), exhibited only partial or no separation when β-cyclodextrin (βCD) was used as chiral selector. The use of 18-crown-6 (18C6) as a second additive with βCD resulted in an enhanced separation. A molecular modeling study, using molecular mechanics and the semiempirical PM6 calculations, was used to help explaining the mechanism of the enantiodifferentiation and to predict the separation process. Optimization of the structures of the complexes by the PM6 method indicate that the poor separation obtained in the presence of the βCD chiral selector alone is due to the small binding energy differences (ΔΔE) of 4.7, 1.1 and 1.2 kcal mol(-1) for AI, NEA and THAN, respectively. In the presence of 18C6 it was suggested that a sandwich compound between 18C6, amine and βCD is formed. Theoretical calculations show that a significant increase in the binding energy is obtained for the sandwich compounds indicating strong hydrophobic and van der Waals interactions that show enhanced enantiodifferentiation.     

Rapid analysis of atorvastatin calcium using capillary electrophoresis and microchip electrophoresis

In this work, a capillary electrophoretic method for the rapid quantitation of atorvastatin (AT) in a lipitor tablet was investigated and developed. Method development included studies of the effect of applied potential, buffer concentration, buffer pH, and hydrodynamic injection time on the electrophoretic separation. The method was validated with regard to linearity, precision, specificity, LOD, and LOQ. The optimum electrophoretic separation conditions were 25 mM sodium acetate buffer at pH 6, with a separation voltage of 25 kV using a 50 microm capillary of 33 cm total length. Sodium diclofenac was used as an internal standard. Analysis of AT in a commercial lipitor tablet by electrophoresis gave quite high efficiency, coupled with an analysis time of less than 1.2 min in comparison to LC. Once the separation was optimized on capillary, it was further miniaturized to a microchip platform, with linear imaging UV detection using microchip electrophoresis (MCE). Linear imaging UV detection allowed for real-time monitoring of the analyte movement on chip, so that the optimum separation time could be easily determined. This microchip electrophoretic method was compared to the CE method with regard to speed, efficiency, precision, and LOD. This work represents the most rapid and first reported analysis of AT using MCE.     

Development of chiral HPLC for selenoamino acids with ICP-MS detection:application to selenium nutritional supplements

The enantiomeric separation of three underivatized seleno-amino acids, D,L-selenocystine, and D,L-selenomethionine, and D,L-selenomethionine, with UV and ICP-MS detection is described. An HPLC column with a chiral crown ether stationary phase and a mobile phase of 0.10 M HCIO4 was used. Absolute detection limits obtained with UV detection ranged from 34.5 to 47.1 ng whereas those obtained with the plasma detector were ca. 40-400 times better. The separations with either detector were good, with the little detector effect on the resolution. Ten commercially available dietary selenium supplements were analyzed using the chiral column to identify and quantify the selenium species present with both detection modes. Selenium species were easily identified using ICP-MS detection, whereas UV detection was not viable because of interferences from the sample matrix and inadequate sensitivity. Selenium species that were unretained using the chiral column were identified using anion exchange chromatography. Total amounts in the samples were also measured using a conventional digestion and enzymatic digestion with ICP-MS detection.     

Enantiomeric separation of basic drugs with partially filled serum albumin as chiral selector in capillary electrophoresis.

A reliable method is presented for the chiral separation of three basic drugs (mexiletine, chlorpheniramine and propranolol) with serum albumins (human and porcine, HSA and PSA) as chiral selectors by capillary electrophoresis in combination with the partial filling technique. Based on the systematic optimization of operation variables, the chiral separation of mexiletine, chlorpheniramine and propranolol was achieved in the pH 7.4 phosphate buffer by using HSA, PSA and PSA as selectors, respectively. The chiral recognition ability of HSA and PSA was compared. HSA and PSA show a different chiral recognition ability for each of the three drugs. In addition, the association constants between enantiomeric drugs and proteins were determined to be 2.00 and 3.80 x 10(2) M(-1) for mexiletine and HSA, 0.59 and 1.12 x 10(3) M(-1) for chlorpheniramine and PSA, and 0.87 and 1.42 x 10(3) M(-1) for propranolol and PSA. The method for the chiral separation and determination of association constants possesses the advantages of simple performance, effective avoiding of the interference of the UV detection from protein, and lowering of the reagent consumption.     

Direct Enantiomeric Separation of Chiral Pesticides by LC on Amylose Tris(3,5-dimethylphenylcarbamate) Stationary Phase under Reversed Phase Conditions

Amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase was used by liquid chromatography under reversed-phase conditions for the chiral separation of 20 pesticides, of which ten samples were separated directly under suitable conditions. The influence of mobile phase composition and column temperature from 0 to 40 °C on the separation was investigated. The mobile phases were methanol/water or acetonitrile/water at a flow rate of 0.5 mL min^?1 with UV detection at 230 nm. The two enantiomers of fenamiphos, terallethrin, fenoxaprop-ethyl, benalaxyl and lactofen could obtain base separation under optimized conditions, while the enantiomers of quizalofop-ethyl, metalaxyl, napropamide, fluroxypyr-meptyl and 2,4-D-ethylhexyl got partial separation. The retention factors (k) and selectivity factor (α) for the enantiomers of most investigated pesticides decreased with increasing the temperature. The lnα–1/T plots for enantiomers of chiral pesticides were linear at the range of 0–40 °C except for that of metalaxyl, fenoxaprop-ethyl and 2,4-D-ethylhexyl enantiomers in methanol/water. The thermodynamic parameters calculated based on linear Van’t Hoff plots showed the chiral separation was controlled by enthalpy. Better separation was not always at low temperature. The chiral recognition mechanisms were discussed. The elution orders of the eluting enantiomers were determined by a circular dichroism detector.     

Stability evaluation of tramadol enantiomers using a chiral stability-indicating capillary electrophoresis method and its application to pharmaceutical analysis

In this study, a chiral stability-indicating CE assay was developed for the stability evaluation of tramadol (TR) enantiomers in commercial tablets using maltodextrin as chiral selector. To investigate the stability-indicating power of the analytical method as well as stability evaluation of TR enantiomers, active pharmaceutical ingredient and TR tablets were subjected to photolysis, heat, oxidation and hydrolysis to conduct stress testing. Best separation for the TR enantiomers was achieved on an uncoated fused-silica capillary at 20 °C using borate buffer (50 mM, pH 10.2) containing 10% m/v maltodextrin. All determinations were performed by a UV detector at 214 nm. A constant voltage of 20 kV was applied to obtain the separation. The range of quantitation for both enantiomers was 5-100 μg/mL (R>0.996). Intra- and inter-day RSD (n=6) were less than 10%. The percent relevant errors were obtained to be less than 4.0 for both enantiomers. The limits of quantitation and detection for both enantiomers were 5 and 1.5 μg/mL, respectively. Degradation products resulting from the stress studies were the same for both enantiomers and did not interfere with the detection of the enantiomers.     

Optimization of high-performance liquid chromatographic parameters for the determination of capsaicinoid compounds using the simplex method.

A high-performance liquid chromatographic method was developed for the analysis of capsaicinoid compounds, the pungent principles of capsicum fruits. A sequential simplex method was applied to optimize the chromatographic response function used to assess the quality of separation by varying the chromatographic parameters. The separation was achieved in 11 min using a C-8 column of 15-cm length and 4.6 mm diameter using a UV detector. A flow rate of 1.15 ml min(-1) at a column temperature of 43.5 degrees C using 63.7% methanol in water gave the most efficient separation. The method was found to be suitable for the determination of the major capsaicinoid compounds in the capsicum samples.     

Enantioseparation of dihydrofurocoumarin derivatives by various separation modes of capillary electrophoresis

Chiral dihydrofurocoumarin compounds are currently the focus of industrial and pharmacological research. These derivatives have been shown to possess many physiological properties that could be medically beneficial. This work proposes four different chiral separation methods using capillary electrophoresis and micellar capillary electrophoresis (MCE). Several different cyclodextrin chiral selectors were examined to evaluate their effectiveness in the enantioseparation of dihydrofurocoumarins. In addition, the effects of the chiral selector concentration, the presence of an organic modifier, run buffer pH, and in two cases, the ratio between the chiral selector and an additional charged pseudophase were investigated. Overall, the best separations for this class of chiral compounds were achieved using sulfated beta-cyclodextrins at low pH in the reversed polarity mode.     

毛细管电泳微芯片在临床尿蛋白检测中的应用研究

用微芯片毛细管电泳法对临床患者尿蛋白进行了分离, 初步探讨了用于判断肾损伤的应用前景. 以pH 10.3, 75 mmol•L^－1的硼酸盐缓冲液作为芯片电泳缓冲体系, 利用蛋白质的紫外吸收特性, 在210 nm波段检测吸光度并进行信号收集和分析. 研究两种添加剂对提高尿蛋白分离效率的影响, 分析了肾病综合症、妊娠高血压症、风湿性心脏病和多发性骨髓瘤等患者尿样本, 并与美国Helena电泳系统分析结果对比, 得到了较一致的结果.