Separation methods for pharmacologically active xanthones

Xanthones, as a kind of polyphenolic natural products with many strong bioactivities, are attractive for separation scientists due to the similarity and diversity of their structures resulting in difficult separation by chromatographic methods. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are traditional methods to separate xanthones. Recently, capillary electrophoresis (CE), as a micro-column technique driven by electroosmotic flow (EOF), with its high efficiency and high-speed separation, has been employed to separate xanthones and determine their physicochemical properties such as binding constants with cyclodextrin (CD) and ionization constants. Since xanthones have been used in clinic treatment, the development of chromatographic and CE methods for the separation and determination of xanthones plays an essential role in the quality control of some herbal medicines containing xanthones. This article reviewed the separation of xanthones by HPLC, TLC and CE, citing 72 literatures. This review focused on the CE separation for xanthones due to its unique advantages compared to chromatographic methods. The comparison of separation selectivity of different CE modes including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC) and capillary electrochromatography (CEC) was discussed. Compared with traditional chromatographic methods such as HPLC and TLC, CE has higher separation efficiency, faster separation, lower cost and more flexible modes. However, because of low sensitivity of UV detector and low contents of xanthones in herbal medicines, CE methods have seldom been applied to the analysis of real samples although CE showed great potential for xanthone separation. The determination of xanthones in herbal medicines has been often achieved by HPLC. Hence, how to enhance CE detection sensitivity for real sample analysis, e.g. by on-line preconcentration and CE-MS, would be a key to achieve the quantitation of xanthones.     

Investigation of the Enantioselectivity of Tetramethylammonium L‐hydroxyproline Ionic Liquid as a Novel Chiral Ligand in Ligand‐Exchange CE and Ligand‐Exchange MEKC

Chiral ionic liquids (ILs) have drawn more and more attention in separation science; however, only a few papers focused on the application of chiral ILs as chiral ligands in LE‐CE. In this article, a novel amino acid ionic liquid (AAIL), tetramethylammonium L‐hydroxyproline ([TMA][L‐OH‐Pro]), was first applied as a chiral ligand to evaluate its enantioselectivity towards several aromatic amino acids in ligand‐exchange capillary electrophoresis (LE‐CE) and ligand‐exchange micellar electrokinetic capillary chromatography (LE‐MEKC). In the LE‐CE system, excellent separations were achieved for tryptophan (Rs = 3.03) and 3, 4‐dihydroxyphenylalanine (DOPA) (Rs = 4.35). Several parameters affecting the enantioseparation were systematically investigated, including AAIL concentration, type and concentration of central metal ion, buffer pH, as well as applied voltage. The optimum separation was obtained with 60 mM AAIL containing 30 mM Cu (II) at pH 4.5. Additionally, an LE‐MEKC system was established to further study the enantioselectivity of [TMA][L‐OH‐Pro] towards selected analytes. As observed, the separations of the enantiomers of tryptophan, phenylalanine, and histidine were all improved compared to the LE‐CE system. The results indicated that the application of AAILs as chiral ligands is a promising method in chiral separation science. Chirality 27:58–63, 2015. © 2014 Wiley Periodicals, Inc.     

Separation and enantiomer determination ofOPA-derivatised amino acids by using capillary zone electrophoresis

Summary Amino acids react with OPA and chiral mercaptans to give diastereomeric isoindole derivatives. The resolution of these diastereomers was investigated by micellar electrokinetic chromatography (MECC) and free solution capillary electrophoresis. MECC with SDS as micellar phase allows to separate the amino acid derivatives and to resolve the diastereomers. The separation is influenced by the amount of detergent and the organic modifier added. Capillary zone electrophoresis offers a valuable alternative to the traditional methods for amino acid analysis and enantiomer determination.     

Enantioselective separations using capillary electrophoresis

The preconditions are outlined for enantioselective separations in capillary electrophoresis (CE) with chiral selectors as additives to the background electrolyte. Free solution capillary electrophoresis conditions are characterised by a single solution phase. Chiral separations are reviewed by selector type (chiral ligand exchange, cyclodextrins, crown ethers, glycoproteins) with the extensive studies on cyclodextrins grouped into sections on amino acids, pharmaceuticals, and speciality chemicals, optimisation, biological fluids, and quantitative aspects. In micellar electrokinetic capillary chromatography, enantioselective discrimination occurs by partition in a two-phase system, with a chiral micellar phase as selector. Optimum separation conditions can be readily predicted for a given selector–selectand combination, and absolute values of binding constants determined by CE. Advantages of CE in comparison with HPLC using a chiral stationary phase include robust, rapid assays and the use of small volumes of aqueous solutions; disadvantages include less favourable detection limits. © 1994 Wiley-Liss, Inc.     

Determination of free aspartic acid enantiomers in rat brain by capillary electrophoresis with laser-induced fluorescence detection

Quantification of aspartic acid enantiomers in rat brain by using a chiral capillary electrophoresis procedure is described. Amino acids were pre-column derivatized with naphthalene-2,3-dialdehyde. Enantiomeric separation was achieved by micellar electrokinetic chromatography in the presence of methanol and β-cyclodextrin as chiral selector. The chiral separation was coupled with laser-induced fluorescence detection. Contents of d- and l-aspartic acids in rats at different stages of growth (from 1 day before birth to 90 days after birth) were determined. d-Aspartic acid was detected in all the brain tissue samples tested, but at different levels. In the cerebrum of rats 1 day before birth, d-aspartic acid was found to be at the highest concentration of 81 nmol/g wet tissue. The level of d-aspartic acid in rat brain falls rapidly after birth, while the l-aspartic acid level increases with age.     

Clinical applications of capillary electrophoresis

Capillary electrophoresis (CE) is an extremely sensitive technique, which has been used in the clinical laboratory for almost 10 yr. The components of CE instrumentation are described, as are injection modes, buffers, and effects of electroosmotic flow. The modes of separation used in CE, namely, capillary zone electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic capillary chromatography, are explained. References for 26 different clinical applications of CE are included, among them assays that are used routinely as well as niche assays for specialized applications of CE. Verification of CE assays, current instrumentation, and future development of CE in the clinical laboratory are addressed.     

Chiral separation of amino acids in biological fluids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A method is presented for the chiral analysis of amino acids in biological fluids using micellar electrokinetic chromatography (MEKC) and laser-induced fluorescence (LIF). The amino acids are derivatized with the chiral reagent (+/−)-1-(9-anthryl)-2-propyl chloroformate (APOC) and separated using a mixed micellar separation system. No tedious pre-purification of samples is required. The excellent separation efficiency and good detection capabilities of the MEKC-LIF system are exemplified in the analysis of urine and cerebrospinal fluid. This is the first time MEKC has been reported for chiral analysis of amino acids in biological fluids. The amino acids -alanine, -glutamine, and -aspartic acid have been observed in cerebrospinal fluid, and -alanine and -glutamic acid in urine. To the best of our knowledge no measurements of either -alanine in cerebrospinal fluid or -glutamic acid in urine have been presented in the literature before.     

Separation of the enantiomers of some racemic nonsteroidal aromatase inhibitors and barbiturates by capillary electrophoresis

High-performance capillary electrophoresis (HPCE) and micellar electrokinetic capillary chromatography (MECC) were applied to the resolution of racemic nonsteroidal antiaromatase drugs and intermediates. Successful results were obtained in both modes using α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), or 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD) as chiral selectors. Depending on the structure of the solute, one of the cyclodextrins was generally better suited for resolution of the racemate. The basic solutes were analyzed under HPCE conditions, whereas the nonionizable compounds such as glutethimide (Doriden®) were analyzed in MECC mode. For the azole-type antiaromatase Fadrozole, both HPCE and MECC modes could be used to achieve the separation of the enantiomers. The influence of experimental factors such as pH, the presence of organic modifier, temperature, the micelle concentration, and the concentration of the chiral selector is also discussed on the basis of the results obtained with some chiral barbiturates. The possibility of analyzing the enantiomers directly in plasma samples was also demonstrated. © 1993 Wiley-Liss, Inc.     

Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

The application of capillary electrophoresis (CE) methods in forensic toxicology for the determination of illicit and/or misused drugs in biological samples is reviewed in the present paper. Sample pretreatments and direct injection modes used in CE for analysis of drugs in biological fluids are briefly described. Besides, applications of separation methods based on capillary zone electrophoresis or micellar electrokinetic chromatography with UV absorbance detection to (i) analysis of drugs of abuse, (ii) analysis of other drugs and toxicants of potential forensic interest and (iii) for metabolism studies are reviewed. Also, alternative CE methods are briefly discussed, including capillary isotachophoresis and separation on mixed polymer networks. High sensitivity detection methods used for forensic drug analysis in biological samples are then presented, particularly those based on laser induced fluorescence. A glimpse of the first examples of application of CE–mass spectrometry in forensic toxicology is finally given.     

Tropane alkaloid analysis by chromatographic and electrophoretic techniques: An update

Tropane alkaloids like atropine are antidotes applied against organophosphorus intoxications. Atropine is toxic itself and should be closely monitored during treatment. Hence, simple, fast, and sensitive determination methods for tropane alkaloids in serum are desirable. Mostly adopted methods of analysis are gas chromatography (GC); high performance liquid chromatography (HPLC), and capillary electrophoresis (CE). Various liquid and solid capillary fillings used in micellar electrokinetic chromatography, microemulsion electrokinetic chromatography, capillary electrochromatography, and enantioseparation provide high versatility to CE applications. In HPLC, specialised columns enhance separation efficacy. Ultraviolet light detection is common practise, but recently sensitivity and analyte identification were enhanced by coupling GC, HPLC, and CE to mass spectrometry. Apart from medical treatment, tropane alkaloids, cocaine in particular, are abused with various intentions. Forensic analysis of tropane alkaloids and their metabolites comprises the additional difficulty of unequivocal drug identification. Because of severe legal consequences, sophisticated analytical methods were developed and may provide additional techniques for therapeutic drug monitoring. Examples from forensic cocaine analysis and from doping analysis are included in this review.     

Cyclodextrins and enantiomeric separations of drugs by liquid chromatography and capillary electrophoresis: basic principles and new developments

Investigation of individual drug enantiomers is required in pharmacokinetic and pharmacodynamic studies of drugs with a chiral centre. Cyclodextrins (CDs) are extensively used in high-performance liquid chromatography as stationary phases bonded to a solid support or as mobile phase additives in HPLC and capillary electrophoresis (CE) for the separation of chiral compounds. We describe here the basis for the liquid chromatographic and capillary electrophoretic resolution of drug enantiomers and the factors affecting their enantiomeric separation. This review covers the use of CDs and some of their derivatives in studies of compounds of pharmacological interest.     

Chiral derivatization for separation of racemic amino and thiol drugs by liquid chromatography and capillary electrophoresis

Separation of racemic amino drugs (α-methylbenzeneethanamine, 6-amino-2-methyl-2-heptanol and 1-aminoethyl-benzenemethanol) and thiol drugs [N-(2-mercapto-1-oxopropyl) glycine, 2-mercaptopropanoic acid, and N-acetyl-3-mercaptovaline] has been evaluated after derivatization. ortho-Phthalaldehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA) were used with either homochiral thiols (N-acetyl-L-cysteine and N-acetyl-D-penicillamine) or amines [(-)-(1R,2S)-norephedrine, L-phenylalanine, L-tyrosine, and 3-hydroxy-L-tyrosine] as chiral selectors according to the analyte reactive group. The resulting 36 diastereoisomeric derivatives were studied using reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Of the CE modes, micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) as surfactant, β-cyclodextrin (β-CD)-modified capillary zone electrophoresis (β-CD-CZE), and β-CD-MEKC were applied. Results highlight respective performance of the reagents and separative techniques. All OPA derivatives of racemic amino drugs were resolved either by MEKC or β-CD-MEKC. In the case of racemic thiol drugs, 10 of the 12 OPA derivatives were resolved in β-CD-CZE. © 1995 Wiley-Liss, Inc.     

Capillary electrophoresis enantioselective separation of vigabatrin enantiomers by precolumn derivatization with dehydroabietylisothiocyante and UV-vis detection

A simple and reliable capillary electrophoresis (CE) method with UV-vis detection is presented for the enantioselective separation and determination of vigabatrin enantiomers. Dehydroabietylisothiocyante (DHAIC), a novel chiral derivatizing reagent, was used for precolumn derivatization of vigabatrin enantiomers. Optimal separation was obtained with a running buffer consisting of 50 mM Na2HPO4 (pH 9.0), 17 mM sodium dodecyl sulfate (SDS) and 25% acetonitrile. The enantiomeric separation of vigabatrin derivatives was achieved within 25 min, and the resolution was found to be 2.1. Detection was followed by direct UV absorptiometric measurements at 202 nm. A calibration curve ranging from 0.3 to 6.0 microg/ml was shown to be linear, and the limit of detection was 0.15 microg/ml. The developed method has been applied to the determination of vigabatrin enantiomers spiked in human plasma, no interferences were found from endogenous amino acids.     

Chiral separation principles in chromatographic and electromigration techniques

Almost half of the drugs in use today are chiral. It is well established that the pharmacological activity is mostly restricted to one of the enantiomers (eutomer). There can be qualitative and quantitative differences in the activity of the enantiomers. In many cases, the inactive enantiomer (distomer) shows unwanted side effects or even toxic effects. Even if the side effects are not that drastic, the distomer has to be metabolized and this represents an unnecessary burden for the organism. Therefore, the development of methods for the separation of enantiomers, both on analytical and preparative scale, has become increasingly important. Chromatographic techniques such as thin layer chromatography (TLC), gas chromatography (GC), supercritical fluid chromatography (SFC), and above all high-performance liquid chromatography (HPLC) have been used for enantiomer separation for about two decades. More recently, electromigration techniques, such as capillary electrophoresis and capillary electrochromatography, have been shown to be powerful alternatives to chromatographic methods. This review gives a short overview of different chiral separation principles and their application. Several new developments are discussed.     

A technique for the determination of enantiomeric amino acids in biological samples.

Racemic amino acids can be separated into their enantiomers by means of gas-liquid chromatography. The most applied technique, today, is the conversion of chiral compunds into diastereoisomers with optically active reagents and subsequent chromatography on conventional optically inactive stationary phases. In previous studies it has been realized that this technique is associated with various problems. We studied the use of optically active stationary phases for separating enantiomers directly via a diastereoisomeric association complex. The optically acitve stationary phases employed are N- and C-terminal substituted dipeptides of the type N-trifluoroacetyl-dipeptide-cyclohexyl esters and have been synthesised by the I-hydroxibenztriazole dicyclohexylcarbodiimide method. The quality of these phases with respect to separation factors, resolution factors, and thermodynamical properties have been evaluated. All synthetic phases show excellent properties; however, when attempting separation of mixtures of naturally occurring amino acids extensive overlap in the elution diagram was detected. Only one phase - N-TFA-L-chi-amino-n-butyryl-L-chi-amino butyric acid cyclohexyl ester - gave complete resolution of the naturally occurring amino acids alanine, valine, glycine, threonine, leucine, isoleucine, serine and proline on a 400 ft x 0.02 in capillary column. Less volatile amino acids such as aspartic acid, phenylalanine, methionine, glutamic acid, tyrosine, arginine, and tryptophan can be resolved at a 100 ft x 0.02 in column.     

Separation and quantitation of azimilide and its putative metabolites by capillary electrophoresis

Reliable methods based on capillary electrophoresis (CE) have been developed for the separation and quantitation of azimilide, an antiarrhythmic drug under development at Procter & Gamble Pharmaceuticals (P&GP). Both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were employed in the separation of azimilide from its impurities, degradants and/or metabolites. Separation of azimilide from NE-11178, F-410, F-1054 and F-1292 was obtained by MECC at pH 9 with 50 mM sodium dodecyl sulfate (SDS). The separation of azimilide and NE-10171, a key metabolite of azimilide, was difficult because their structures differ by only a single methyl group. The best separation was achieved under acidic pH conditions with cetyltriethyl ammonium chloride (CTAC) additive in the buffer. All of the CE separations were completed within a substantially shorter time and with better resolution than the corresponding high-performance liquid chromatography (HPLC) separations. Quantitation was done with azimilide and NE-10171. Calibration curves ranging from 10 to 1000 μg/ml were obtained with R² greater than 0.997 for both azimilide and NE-10171. The back-calculated concentrations of the calibration standards and the recoveries of the quality control (QC) samples were within the acceptance range currently used for HPLC methods. These results demonstrated the viability of CE as an alternative technique for drug metabolism studies in support of pharmaceutical development.     

Multiplexed on-column protein digestion and capillary electrophoresis for high-throughput comprehensive peptide mapping

A novel scheme based on multiplexed capillary electrophoresis (CE) has been developed for high-throughput, low-cost and comprehensive peptide mapping. Orthogonal peptide maps of the protein of interest were obtained by using multiple reaction conditions with three different enzymes (trypsin, pepsin, and chymotrypsin), and multiple separation conditions with six zone electrophoresis buffers and two micellar electrokinetic chromatography (MEKC) buffers. Fifteen nanoliters of two protein samples (beta-lactoglobulin A and beta-lactoglobulin B) were separately mixed on-column and digested independently at 37 degrees C for 10 min to produce peptides in a 20-capillary system. The resulting peptides were detected simultaneously at 214 nm by a photodiode array detector. The overall analysis time from reaction to detection was about 40 min.     

Use of mathematically enhanced spectral analysis and spectral contrast techniques for the liquid chromatographic and capillary electrophoretic detection and identification of pharmaceutical compounds

The use of mathematically enhanced ultraviolet/visible (UV/VIS) absorbance spectral analysis and spectral contrast software techniques in high performance liquid chromatography (HPLC) and micellar electrokinetic capillary electrophoresis (MECC) as an aid for the determination of peak homogeneity, identification, and tracking during method development was investigated. Various structurally similar pharmaceutical compounds, and compounds present as either cis/trans isomers, diastereomers, or enantiomers were used as test compounds to probe the limits of this technique. Two tricyclic antidepressants, nortriptyline and imipramine, were employed to study the effects of HPLC mobile phase composition and pH on the ability to identify and track peaks during method development. It was found that method changes altered the spectral matches used for identification, but not enough to cause incorrect peak identification. It was also shown using HPLC that the cis/trans isomers of doxepin and the diastereomers ephedrine and pseudoephedrine could be distinguished. The mathematically enhanced spectral analysis and spectral contrast software techniques were also employed with MECC. Peaks tracking during method development as pH and the concentration of surfactant changes is shown for a separation of various penicillin type antibiotics. It was shown that during chiral MECC (CMECC) analyses ephedrine/pseudoephedrine diastereomers as well as ephedrine enantiomers could be distinguished. The determination of enantiomers is possible in CMECC since enantiomers are eluted as diastereomeric complexes, as opposed to HPLC where they are eluted in their native state. © 1996 Wiley-Liss, Inc.     

Separation methods used for Scutellaria baicalensis active components

Scutellaria baicalensis Georgi is one of the most widely used traditional Chinese herbal medicines. Its roots have been used for anti-inflammation, anticancer, antiviral and antibacterial infections of the respiratory and the gastrointestinal tract, cleaning away heat, moistening aridity, purging fire, detoxifying toxicosis, reducing the total cholesterol level and decreasing blood pressures. Baicalin, baicalein, wogonin and oroxylin A are its main active components. This review provides an overview of various separation, detection, and identification techniques employed for the quantitative and qualitative determination of these active components. Applications of high-performance liquid chromatography, high-speed counter-current chromatography, thin layer chromatography, capillary electrophoresis, and micellar electrokinetic capillary chromatography to the separation and determination of these active components are described. Examples of identification of these active components and their metabolites in complex matrices by high-performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry are also presented. The advantages and limitations of these separation and identification methods are assessed and discussed.     

Use of organic modifiers to enhance chiral selectivity in capillary electrophoresis

Summary Using capillary electrophoresis, the enantiomers of a number of dansyl amino acids were resolved using native-cyclodextrin. The neutral chiral host resolved analytes possessing a negative charge at pH 9, the conditions employed in this study. Organic modifiers added to the running buffer were particularly adept at enhancing chiral recognition between the guest and host molecule in capillary electrophoresis. This work examined the effects of methanol, dimethylformamide, and acetonitrile on the resolution, migration time, and efficiency of twelve dansyl amino acids. Examples are given of the separation of racemic dansyl amino acids utilizing this technique and conditions necessary to achieve enantioselectivity.